27th Annual Conference of the United Mitochondrial Disease Foundation Abstracts - Mitochondrial Medicine 2024 - Cleveland, Ohio – June 26-29, 2024

Abstract #	Abstract Title	Presenter Name
2024PA-0000000001	Cardiac involvement: the fatal dance of mitochondrial disease.	Mousa	Jehan
2024PA-0000000006	Understanding mtDNA variant segregation in single cells: How T cell activation contributes to purifying selection against the MELAS-associated m.3243A>G pathogenic variant in blood.	Walker	Melissa
2024PA-0000000011	Using a Pre-visit Questionnaire for Initial Visits in a Pediatric Mitochondrial Clinic: Perspectives of Parents, a Specialty Physician, and a Clinical Coordinator.	Sepulveda	Coral
2024PA-0000000018	Delineating the effects of mTORC1 dysregulation on energy production in mitochondrial disease.	Navarro	Kristen
2024PA-0000000022	Somatic mitochondrial mutation detection approach.	Bonner	Joseph
2024PA-0000000026	Postnatal neurogenic niches are disrupted in the subventricular zone of a mouse model of Leigh syndrome.	Biswas	Sahitya Ranjan
2024PA-0000000028	Investigating AAV9-MECR Gene Therapy for Mitochondrial Enoyl CoA Reductase Protein-Associated Neurodegeneration (MEPAN) Syndrome.	Saha	Madhurima
2024PA-0000000030	Defining a Major Regulatory Step in the Function of the Mitochondrial Acyl Carrier Protein in Mammals.	Wedan	Riley
2024PA-0000000031	Novel CRISPR/Cas9 generated ndufs2-/- zebrafish larvae have short lifespan, developmental anomalies, and globally disrupted intermediary metabolism.	Iadarola	Donna
2024PA-0000000033	mTOR inhibition as potential treatment for Leigh syndrome: report of three cases.	Emmanuele	Valentina
2024PA-0000000035	Patient experiences of thymidine kinase 2 deficiency: preliminary results from an online survey conducted in partnership with the patient community.	Balcells	Cristy
2024PA-0000000036	Sarm1 knockout rescues age-dependent retinal ganglion cell (RGC) degeneration in a novel mouse model of Autosomal Dominant Optic Atrophy (ADOA)	Ding	Chen
2024PA-0000000037	Expanded clinical phenotype and the role of untargeted metabolomics analysis in confirming the diagnosis of sodium-dependent multivitamin transporter deficiency	Walimbe	Ameya
2024PA-0000000039	Intrauterine growth restriction due to idiopathic placental insufficiency causes long-term postnatal mitochondrial pathophysiology and severe neurodevelopmental defects	Chiaramello	Anne
2024PA-0000000043	Untargeted Metabolomics as a Potential Screening Tool for 3-Methylglutaconic Aciduria Syndromes	Difalco	Charles
2024PA-0000000044	Biallelic variants in POLG2 provide a rare molecular diagnosis in a patient with hepatocerebral syndrome	Rossi	Vittoria
2024PA-0000000046	Detection and quantification of large scale mtDNA deletions using PacBio long read sequencing	Wang	Jing
2024PA-0000000047	Long-Term Elamipretide Treatment in a Patient with Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP) Syndrome: A Case Study	Miller	Lindsey
2024PA-0000000048	Natural History of Mitochondrial Myopathy objective measures reveals significantly slow, progressive decline	Zolkipli-Cunningham	Zarazuela
2024PA-0000000049	Pyrimidine Deoxynucleoside Treatment for POLG-Related Disorders: Open-Label Human Trial	Myers	Kenneth
2024PA-0000000051	Guanylate Kinase 1 Deficiency: A Novel and Potentially Treatable Mitochondrial DNA Depletion/Deletions Disease	Hidalgo-Gutierrez	Agustin
2024PA-0000000053	Reduction of mutant mtDNA by LNP/mRNA delivery of mitoARCUS to skeletal muscle	Bacman	Sandra R.
2024PA-0000000055	Leber Hereditary Optic Neuropathy (LHON) Scientific Retreat	Marsh	Malinda
2024PA-0000000058	Elamipretide via Open Label Expanded Access Program in Patients with Genetically Confirmed Primary Mitochondrial Disorders	MacMullen	Laura
2024PA-0000000059	Digital gait and balance sensors characterize fatigue and enhance MM-COAST data in primary mitochondrial disease	Rahaman	Imon
2024PA-0000000062	Elucidating the role of specific immune populations in CNS and peripheral disease pathogenesis in the Ndufs4(-/-) model of Leigh syndrome	Hanaford	Allison
2024PA-0000000063	Manipulating the Nuclear Epigenome: A Novel Approach for Shifting Mitochondrial DNA Heteroplasmy	Mayorga	Lia
2024PA-0000000064	Preclinical evaluation of dichloroacetate in two C. elegans models of pyruvate dehydrogenase deficiency	Remes	Cristina
2024PA-0000000065	Characterization of 19 mitochondrial aminoacyl-tRNA synthetases in C. elegans and the effect of specific amino acid treatment on mt-ARS deficiencies	Remes	Cristina
2024PA-0000000066	The mitoDdCBE system as a mitochondrial gene therapy approach	Barrera-Paez	Jose Domingo
2024PA-0000000068	Fishing for Cures: Zebrafish as a Pioneering In Vivo Model to Help Solve Mitochondrial Medicine Odysseys	Sabharwal	Ankit
2024PA-0000000069	Validation of the Mitochondrial Myopathy Function Scale	Flickinger	Jean
2024PA-0000000071	Data optimization-MMFP data warehouse usage for guidance in therapeutic developments and clinical studies	George-Sankoh	Ibrahim
2024PA-0000000072	Apomorphine and derived synthetic aporphine alkaloids are potential therapeutics for mitochondrial diseases with anti- ferroptotic effect	Kobayashi	Mizuki
2024PA-0000000073	The mitochondrial fission protein Fis1 safeguards from sterile inflammation in vivo	Branco	Tiago
2024PA-0000000074	Deoxyguanosine kinase deficiency: natural history and liver transplant outcome	Garone	Caterina
2024PA-0000000075	Developing in vivo models for RRM2B mitochondrial encephalomyopathy	Garone	Caterina
2024PA-0000000077	Making A Comeback: A Case Study of an Adolescent with TK2d Treated with Doexecitine and Doxribtimine	Miller	Lindsey
2024PA-0000000078	Environmental contaminant 6PPD-quinone has metabolic effects consistent with redox cycling activity in C. elegans	Jameson	Laura
2024PA-0000000079	Relevance of mitochondrial biogenesis and mitochondria-lysosome crosstalk in neurodevelopmental and neurodegenerative disorders	Amin	Shahreen
2024PA-0000000080	Aberrant glycosylation in mitochondrial disease enhances influenza A virus pathogenesis	Jetmore	Jillian
2024PA-0000000082	Restoration of defective oxidative phosphorylation in a subset of neurons is sufficient to prevent the development of mitochondrial encephalopathy in mice	Walker	Brittni
2024PA-0000000083	Dopaminergic Neurodegeneration is Induced by Inhibition of Mitochondrial Complex III in Caenorhabditis elegans	Huayta	Javier
2024PA-0000000085	Standardized assessment of the relationship between 37 mitochondrial DNA genes and Primary Mitochondrial Disease using the ClinGen Clinical Validity Framework	McCormick	Elizabeth
2024PA-0000000086	Expert panel curation of the MITOMAP-Confirmed disease-associated variants according to the mitochondrial DNA variant interpretation specification guidelines: Outcomes, strengths, limitations, and plans for optimization	McCormick	Elizabeth
2024PA-0000000087	Transcriptional analysis and metabolic characterization of uveal melanoma suggest an altered epigenome	Kopinski	Piotr
2024PA-0000000088	Pearson Syndrome Natural History Study	Lucas	Stephanie
2024PA-0000000089	Immunoglobulin Therapy in the Mitochondrial Disease Community	Gordon-Lipkin	Eliza
2024PA-0000000090	Interferon-gamma contributes to disease progression in the Ndufs4(-/-) model of Leigh syndrome	Hanaford	Allison
2024PA-0000000091	MRS2 N-Glycosylation serves as a physiologic brake to reduce rapid Mg2+ influx capacity into mitochondria and dynamically communicate relative cellular nutrient status and bioenergetic capacity	Falk	Marni
2024PA-0000000092	Deoxyribonucleoside supplementation rescues mtDNA depletion in neuronal models of POLG mutations	Zarate Mendez	Mariana
2024PA-0000000094	Modulating Mitochondrial Function through Nutrition as a Therapeutic Strategy in Pediatric Osteosarcoma	Keith	Kelsey
2024PA-0000000095	A Disease-Agnostic High-Throughput Screening Platform for PMD Candidate Drug Therapies and Mitotoxicants	Keith	Kelsey
2024PA-0000000096	Phenotypic Analysis of Mitochondrial Perturbation in Human Cells, a Path to Uncovering Differential Susceptibilities in Neurodegenerative Disease	Kremitzki	Colin
2024PA-0000000097	Curating Patient-Provided Genetic Diagnostic Lab Reports in a Mitochondrial Disease Registry	Wilson	Nicole
2024PA-0000000099	Next-generation sequencing, genomic data analysis, utility, and diagnostic rate of a patient advocacy group-led no-cost genetic testing program for primary mitochondrial diseases	McGinn	Daniel
2024PA-0000000100	SPIMD-301 (NuPower) Target Patient Population and Demographics	Karaa	Amel
2024PA-0000000101	Using genome-scale metabolic modeling to prioritize genetic variants in patients with rare mitochondrial disorders	Mitchell	Dana
2024PA-0000000102	PROMIS® for MELAS: Measuring What’s Important from the Patient and Clinician Perspectives	Glasser	Chad
2024PA-0000000103	Barth Syndrome and the brain: A novel bioenergetic phenotype in TAFAZZIN deficient neural progenitor cells	Harris	Kodi
2024PA-0000000104	The Champ Foundation Registry (CFR): Single Large-Scale Mitochondrial Deletion Syndrome Natural History Study (SLSMDS-NHS)	Stanley	Katelynn
2024PA-0000000105	Modeling the metabolic flux capacity of mitochondrial disease patient cell using transcriptomics data in galactose growth media	Mitchell	Dana
2024PA-0000000106	Harnessing resilience in adults with primary mitochondrial disease: A pilot study investigating the feasibility of the Promoting Resilience in Stress Management (PRISM) and clinical-focused narrative (CFN) interventions	McCormick	Elizabeth
2024PA-0000000107	Harmonization of Phenotype Terminology: Identifying Common Human Phenotype Ontology (HPO) Terms for Primary Mitochondrial Disease Across Six Mitochondrial Disease Centers	Mitchell	Dana
2024PA-0000000109	Interrogating mechanisms of brain cell death induction upon acute complex IV inhibition in a surf1-/- zebrafish model of Leigh Syndrome	Burg	Leonard
2024PA-0000000110	Facilitating community-based genomic data analysis in primary mitochondrial disease: mitoSHARE patient registry and Mitochondrial Disease Sequence Data Resource (MSeqDR) collaboration to accelerate genomic data discoveries	Bogush	Emily
2024PA-0000000111	Clinical Utility in Hospital-Wide Use of GDF15 as a Biomarker for Mitochondrial DNA Encoded Primary Mitochondrial Disorders	Cortes Fernandez	Andrea
2024PA-0000000112	Avoidant/Restrictive Food Intake Disorder (ARFID) in Mitochondrial Disease – a Dangerous Diagnosis	Tormey	Cassandra
2024PA-0000000113	Cockayne syndrome cells display pronounced mitochondrial function and acute stress sensitivity that is rescued by N-acetylcysteine	Kose	Melis
2024PA-0000000115	Leigh Syndrome Induced pluripotent stem cells exhibit defects in neural and cardiac differentiation	Iyer	Shilpa
2024PA-0000000117	Toward targeted therapies: the diverse pharmaceutical and nutritional responses of Leigh syndrome patient derived induced pluripotent stem cell-cardiomyocytes	Dinchman	Amber
2024PA-0000000118	SARS-COV-2 ORF10 Induces Mitochondrial Dysfunction	Haltom	Jeffrey
2024PA-0000000120	Identification of FDA-approved compounds that rescue mitochondrial stress in a heteroplasmic single large-scale mtDNA deletion (SLSMD) C. elegans animal model	Mendel	Ryan
2024PA-0000000123	Absence of MgmeI results in an increase rate of mtDNA deletion formation in dopaminergic neurons in vivo but does not contribute to accumulation of deletions	Arguello	Tania
2024PA-0000000124	Leigh Syndrome Roadmap Project: A Natural History Study	MacMullen	Laura
2024PA-0000000125	MitoTEMPO improves colonic mitochondrial dysfunction in the hyperandrogenemic rat model of PCOS	Hoang	Ngoc
2024PA-0000000126	mtDNA mutation heteroplasmic zebrafish models created by mitochondrial base editor tools	Nakamaru-Ogiso	Eiko
2024PA-0000000129	High-throughput drug screening in a humanized C. elegans model identified potential therapeutic compounds for OPA1 disease.	Haroon	Suraiya
2024PA-0000000130	Burden, coping, and resilience in caregivers of individuals with primary mitochondrial disease: Exploration, assessment, and implications for an intervention trial	McCormick	Elizabeth
2024PA-0000000131	Use of dichloroacetate as novel therapy in ECHS1 deficiency: A comparison of two cases	Elsharkawi	Ibrahim
2024PA-0000000132	Colonic mitochondrial dysfunction and sub-acute gastrointestinal inflammation in the hyperandrogenemic rat model of PCOS is exacerbated by metformin	Edwards	Kristin
2024PA-0000000135	Antibody response to the pneumococcal vaccine in children with mitochondrial disease	Schlein	Melissa
2024PA-0000000138	Low- Level Large Deletions in Mitochondria Genomes: A Potential Diagnosis of Mitochondrial Diseases	Wang	Yue
2024PA-0000000140	Patient Perspectives on Myopathy Associated With the m.3243A>G Mitochondrial Disorders Variant: A Qualitative Research Study	Moore	Margaret
2024PA-0000000142	Heterozygosity for HOGA1 variants is associated with an increased risk for kidney stones	Purao	Sohum
2024PA-0000000143	Succinic semialdehyde dehydrogenase deficiency: Disease prevalence, plasma biomarkers, and a proposed integrated metabolomic and genomic approach to rapid diagnosis	Elsea	Sarah
2024PA-0000000145	MARS2 Deficiency: Where are the missing patients?	Webb	Bryn
2024PA-0000000146	Induced Pluripotent Stem Cell Based Cardiac Model of MELAS Disease	Preston	Graeme
2024PA-0000000147	Modeling Endocrine Dysfunction in Mitochondrial Disease: The Promise of Stem Cell-Derived Beta Cells	Peterson	Quinn
2024PA-0000000152	FBXL4-associated mtDNA Depletion Syndrome is characterized by elevated BNIP3/NIX-mediated mitophagy	Kozul	Keri-Lyn

Abstract#: 2024PA-0000000001

Presenter: Jehan Mousa, MD

Cardiac involvement: the fatal dance of mitochondrial disease

Mousa J^1*, Nayfeh T², Kozicz T³, Morava E³

¹Department of Internal Medicine, Medstar Health, Baltimore, MD, USA, ²Evidence-based Practice Center, Mayo Clinic, Rochester, MN, USA, ³Department of Clinical Genomics, Mayo Clinic, Rochester, MN, USA

*jehan.mousa@medstar.net (Corresponding author’s email)

Abstract: Background: The link between mitochondrial disease and cardiac involvement (CI) has been frequently reported in the literature. We conducted this systematic review to study the prognosis of CI in mitochondrial disease.

Methods: We searched MEDLINE, Embase, Scopus, Central and Web of Science, from each database’s inception to October 2023. Search terms covered the concepts of mitochondrial diseases, CI, prognosis, and survival. We included studies comparing the prognosis of patients with mitochondrial disease with and without CI. Meta-analysis was conducted using the restricted maximum likelihood random-effects model. Fixed effects were used when the number of studies was small. Effect estimates were reported as proportion, odds ratio (OR) and hazards ratio (HR) along with 95% confidence intervals (95%CI). Heterogeneity was quantified using the i squared statistic (i squared >50% was considered substantial).

Results: After screening 2769 references, we included 12 studies (3 USA, 2 France, 2 Sweden, 1 England, 1 Italy, 1 Germany, 1 Australia, and 1 Japan), involving a total of 1195 patients with mitochondrial disease (43.3% female), with a median age of 5.2 years (range intrauterine to 55 years). Most common reported syndromes were 11% Leigh, 11% progressive external ophthalmoplegia, 7% Barth syndrome, and 6.7% MELAS. CI was found in 39.1%, of whom, 15.7% had ACAD9, 15.7% Barth, 11.5% Sengers syndrome, 6.3% Kearns-Sayre syndrome, and 5.5% MELAS. Most common EKG findings were 83 LVH, 47 QTC ⩾ 450 ms, and 44 conduction block. Most common echocardiogram findings were 119 HCM, 67 DCM, 14 HCM+DCM, 33 LVH, and 29 LV dilation. In total, 391 patients died, and 7 patients underwent heart transplantation. The estimated proportion of patients with a combined outcome of mortality or heart transplant (36%, 95%CI 28% to 43%; i2 = 83%). Patients with CI had higher odds of mortality or heart transplant (OR 4.65, 95%CI 1.62 to 13.32; i2 = 80%; p<.01). Predictors of mortality were arrhythmia (HR 2.74, 95%CI 1.64 to 4.58; i2 = 0%), and LVH (HR 2.95, 95%CI 2.01 to 4.33; i2 = 89%).

Conclusion: CI in patients with mitochondrial disease carries poor prognosis. LVH and conduction abnormalities are independent risk factors of mortality. Physicians should regularly screen for early signs of cardiovascular involvement in patients with mitochondrial disease.

Abstract #: 2024PA-0000000006

Presenter: Melissa A. Walker MD, PhD

Understanding mtDNA variant segregation in single cells: How T cell activation contributes to purifying selection against the MELAS-associated m.3243A>G pathogenic variant in blood

Walker MA^{1, 2, 3}*

¹Department of Neurology, Division of Child Neurology, Massachusetts General Hospital, Boston, Massachusetts, USA, ²Howard Hughes Medical Institute and the Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA, ³Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

*walker.melissa@mgh.harvard.edu

Abstract: Unlike nuclear variants, mitochondrial (mt)DNA variants can coexist with the wildtype allele in a percentage across hundreds or thousands of copies of mtDNA present in a cell, a state called heteroplasmy, and a course of great variability between and within affected kindreds and tissues within the same individual. While the somatic segregation of the m.3243A>G pathogenic variant (MT-TL1, associated with Maternally Inherited Diabetes and Deafness [MIDD], Mitochondrial Encephalomyopathy and Lactic Acidosis [MELAS]) is notoriously variable, trends have historically been observed with apparent concordance with clinical phenotypes: the most severely affected tissues (brain, muscle) often have the highest heteroplasmy burden. Intriguingly, these tissues are also largely composed of non-dividing cells. Conversely, hematologic symptoms are comparatively rare and blood has long been recognized to maintain lower m.3243A>G heteroplasmy levels compared to other tissues. We have shown at the single cell level that T cells maintain a lower percentage (heteroplasmy) of the m.3243A>G. The mechanism(s) underlying this purifying selection, however, remain unknown. We now report data supporting a model of cell fitness in the maintenance of lower heteroplasmy distributions in the T cell compartment. Specifically, we have observed that purified patient memory CD4+ T cells have lower bulk m.3243A>G heteroplasmy compared to naïve CD4+ T cells. In vitro activation of naïve CD4+ m.3243A>G patient T cells results in lower bulk m.3243A>G heteroplasmy after proliferation. Finally, m.3243A>G patient T Cell Receptor (TCR) repertoire sequencing reveals relative oligoclonality compared to controls. These data support a role for T cell activation as a bottleneck for cell level selection against high heteroplasmy cells, in a likely cell-autonomous fashion. This model is consistent with findings of relative T cell oligoclonality in other genetically encoded deficits of cell fitness, mouse models of distinct mtDNA variants, and in cell lines edited to introduce mtDNA variants. Further work will be required to elucidate precise metabolic underpinnings and potential clinical consequences of this finding.

Abstract #: 2024PA-0000000011

Presenter: Coral Sepulveda

Using a Pre-visit Questionnaire for Initial Visits in a Pediatric Mitochondrial Clinic: Perspectives of Parents, a Specialty Physician, and a Clinical Coordinator

Coral Sepulveda, RN, MSN, CNL, CPN*, Elaine Walsh, PhD, RN, PMHCNS-BC, FAAN, Kristin Carlin, MPH, and Russell P. Saneto, DO, PhD

Seattle Children’s Hospital

*coral.sepulveda@seattlechildrens.org

Abstract: In this study we assessed the usefulness and effects of a pre-visit questionnaire for children who were referred for an initial evaluation in the mitochondrial subspecialty of a neurology clinic. We explored the themes regarding parent’s questions, concerns, and goals. We aimed to add to existing knowledge about the usefulness of pre-visit questionnaires in a pediatric specialist setting from the perspective of parents, the specialist, and the clinical coordinator. We enrolled 25 patients and their parent(s) over a 25-month period between 2021 and 2023, focusing on the initial appointment with the mitochondrial specialist in an ambulatory neurology clinic at a pediatric hospital. A pre-visit questionnaire was completed by the parent(s) and a post- visit questionnaire was completed by the parent(s), the principal investigator (who is the clinical coordinator for the mitochondrial clinic) and the mitochondrial specialist. Questionnaires were sent via an e-mailed link using REDCap. For participants that spoke a language other than English, questionnaires were completed verbally by phone or in person with an interpreter. Descriptive statistics were used to summarize questionnaire results. Thematic analysis was used to summarize open-ended responses. Twenty-five families consented to participate and 22 completed the post-visit questionnaires. Over half of children had a genetically confirmed mitochondrial disorder. Parents’ responses suggested that they are most concerned about their child’s clinical problems, disease progression and prognosis, understanding mitochondrial disease, quality of life, physical challenges including muscle and energy problems, communication and language skills, and developmental delays. On a scale of 0 - 10, parents felt the pre-visit questionnaire was very helpful for both the doctor (Median=10) and for themselves (Median=10) to be prepared for their visit. The specialist also felt that the pre-visit questionnaire was very helpful (Median=9). Out of 22 parents, 21 felt that the pre-visit questionnaire should continue to be used in the clinic. Parent’s comments suggested that they felt that writing down the story of their child’s life was helpful for the provider, allowed time for reflection, and improved the appointment experience. Some felt it was a difficult or redundant activity. Parents were willing, and often pleased, to complete the pre-visit questionnaire. This allowed them to highlight concerns and share information that they wanted the team to know about their child. We will continue to use the tool, which was helpful for families and for the team.

Abstract #: 2024PA-0000000018

Presenter: Kristen G. Navarro, PhD

Delineating the effects of mTORC1 dysregulation on energy production in mitochondrial disease

Kristen G. Navarro, PhD¹*, Rebecca D. Ganetzky, MD¹

¹Children’s Hospital of Philadelphia, Division of Human Genetics

*navarrok@chop.edu

Abstract: Primary mitochondrial disorders (PMD) are an umbrella of genetic diseases caused by errors in cellular metabolism and energy production. The biggest challenge with treating PMDs is the lack of precision therapies. Our work focuses on mitochondrial complex V (CV; ATP Synthase) - the primary enzyme responsible for producing energy in the cell. Pathogenic variants in multiple CV subunit genes have been implicated in PMD. In particular, CV deficiency due to mutations in the mtDNA-encoded subunit, MT-ATP6, is among the most common mtDNA disorders in children. Recent investigations in invertebrate model organisms have suggested a regulatory relationship between CV and mTORC1 signaling. Therefore, we hypothesized that mTORC1 signaling is dysregulated in CV deficiency, and that this may be a potential avenue for developing CV-specific therapies. We measured expression of phosphorylated mTORC1 proteins in multiple CV-deficient PMD and isogenic control cell lines. Three pathogenic variants affecting CV: m.8993tT>C, m.8993T>G, and m.9185T>C, were selected for study as the most known pathogenic variants in MT-ATP6. Transmitochondrial cybrids containing each known pathogenic variant as well as corresponding isogenic controls, were created. mTORC1-mediated phosphorylation (including phospho-S6, phospho-S6K1, phospho-mTOR, phospho-AKT, and phospho-PI3K) was measured using semi-quantitative western blotting in three nutrient conditions: normal cell-media, serum starvation and serum rescue following serum starvation. All western blot expressions were normalized to a house-keeping gene. Our analysis has shown that phosphorylation of Ribosomal Protein S6 (p-S6[Ser235/236]), the last step of the mTORC1 pathway, is significantly decreased at baseline in the 8993T>G and 8993T>C cell lines (p=0.04, 0.02, respectively). S6 is a component of the 40S ribosomal subunit and is involved in protein translation. Along with this observation, we found that all CV variant cells have abnormally persistent S6 phosphorylation during starvation (8993T>G p=0.009, 8993T>C p=0.0003, 9185T>C, p=0.04) and 8993 variants had reduced capacity to restore homeostatic S6phosphorylation post-serum starvation (8993T>G p=0.009, 8993T>C p=0.03), suggesting that the nutrient sensing abilities of mTORC1 are impaired. Preliminary experiments measuring expression of p-S6K1(Thr421/Ser424) and p-mTOR(Ser2448) have also shown similar patterns of abnormal phosphorylation. Overall, these results suggest that the CV-mTORC1 interaction seen in invertebrate PMD models is conserved, and that nutrient sensing may be impaired in CV deficient cells. Ongoing transcriptomic and proteomic analysis will further characterize pathway dysregulation. In conclusion, dysregulation of the mTORC1 pathway in response to nutrient manipulation is a hallmark for cells with pathogenic variants in MT-ATP6. Differences in signaling pattern among known pathogenic variants, further emphasizing the importance of precision-based treatment for patients with PMD.

Abstract #: 2024PA-0000000022

Presenter: Joseph D. Bonner

Somatic mitochondrial mutations in cancer

Bonner JD¹, Seuylemezian A¹, Flores M², Gray S^1,3, Samuels DC⁴, Gruber SB¹

¹City of Hope, Center for Precision Medicine, ²Leonard Davis School of Gerontology, University of Southern California, ³City of Hope, Clinical Cancer Genomics, ⁴Vanderbilt University.

*joedonbonner@gmail.com

Abstract: A century ago, Otto Warburg observed that cancer cells shift their energy production away from oxidative phosphorylation. The mitochondrial genome principally encodes for essential OXPHOS polypeptides. A large pan-cancer study of mitochondrial genomes finds at least one mutation in 85% of samples, with 3 SNVs on average per tumor and an extreme of one breast case showing 33 mutations. Yet little is known about mitochondrial mutations and their role in cancer risk or prognosis.

Clinical DNA sequencing of paired tumor and normal tissues routinely informs precision oncology decisions. Paired sequencing often relies on deep sequencing of tumor DNA and shallow sequencing of normal tissue and typically ignores mitochondrial mutations. We present an analytical approach to detect tumor mutations by salvaging mitochondrial DNA from clinical sequencing.

We processed whole-exome sequencing BAM files derived from OncoExTRa analyses of paired tumor-normal DNA & RNA, a commercial, clinical grade, precision oncology platform. Mitochondrial DNA sequences were isolated from BAM files using MitoHPC, filtering out potential nuclear mitochondrial sequences while accommodating the circular molecule. Heteroplasmy of single nucleotide variants (SNVs) was identified using the mutserve algorithm at a threshold minimum depth of 2 reads per position. The workflow merges SNVs from both tumor and normal to isolate tumor-only SNVs. We calculated variant allele count per read depth in the tumor-only SNVs. We then performed continuity-corrected normalized Z-test to find statistically significant heteroplasmy differences between tumor and normal mitochondrial DNA and applied a multiple testing correction.

We analyzed paired tumor and normal DNA sequencing from 5,462 patients across 38 cancer sites. The median coverage depth per position was 12x in normal and 54x in tumor tissue. The mutserve algorithm identified 376,019 QC-filtered SNVs, with 188,723 in normal and 187,296 from tumor tissue. When merged by patient and position, 163,307 SNVs intersected both tissue types, 25,426 were found solely in normal tissue, and 23,989 only in tumor tissue. Of the tumor-only SNVs, 19,580 had adequate normal sequencing depth for comparison. The continuity-corrected Z-test finds 10,374 tumor-only SNVs, 9,286 of which passed multiple comparison correction. Of the 9,286 mitochondrial SNVs, 4,054 were in coding DNA, including 2,327 missense variants and 75 start/stop mutations. The top five genomic ranges with SNVs were the D-loop, 16S-rRNA, 12S-rRNA, CYB, and ND2.

Of the 5,462 pairs, 2,784 (51%) had at least one SNV. The median count of SNVs in tumor-normal pairs was 3. There are two tumors with 33 or more SNVs. One prostate tumor had 68 SNVs. Among common cancer sites, breast had the highest SNV rate (61%), prostate (58%), colorectal (54%), ovary (52%), uterus (49%).

Our work is one of the largest studies on somatic mitochondrial mutations, revealing significant variation in the prevalence in mitochondrial SNVs. Compared to existing literature, we found more non-mutated tumors, an equivalent number of hyper-mutated tumors, and among those mutated, a similar count of mutations per tumor. This work highlights how mitochondrial DNA salvaged from clinical genomic sequencing may provide crucial insights into its role in cancer.

Abstract #: 2024PA-0000000026

Presenter: Sahitya Ranjan Biswas

Postnatal neurogenic niches are disrupted in the subventricular zone of a mouse model of Leigh syndrome

Biswas S R¹, Brindley S², DeFoor N¹, Henry S N³, Soto Y², Kelly C¹, Pickrell A M^2*

¹Graduate Program in Translational Biology, Medicine, and Health, Virginia Tech, Blacksburg, VA 24061, ²School of Neuroscience, Virginia Tech, Blacksburg, VA 24061, ³ Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Blacksburg, VA, USA, 24061

*alicia.pickrell@vt.edu

Abstract: Leigh syndrome (LS) is a complex, genetic mitochondrial disorder with pediatric manifestation. The disorder is characterized by spongiform lesions in multiple brain regions and most research focuses on understanding the neurodegenerative and neuroinflammatory aspects of the disease. However, considering that the genetic mutations are inherited, and recent patient studies implicate primary developmental delays occur prior to psychomotor regression, this motivated the study to examine postnatal neurodevelopmental abnormalities in a well-established preclinical mouse model of LS. Proper mitochondrial function is important for the survival of postmitotic neurons and for maintaining neurogenesis and differentiation, the process by which mature neurons are generated from neural stem and progenitor cells. Postnatal neurogenesis occurring in the subventricular zone (SVZ) and dentate gyrus (DG) results in the production of interneurons and granular neurons, respectively, both of which play critical roles in normal brain development. This has led us to test whether chronic mitochondrial dysfunction in LS negatively affects early postnatal neurogenesis and differentiation that could contribute to the disease pathogenesis prior to degenerative phenotypes. Using the NDUFS4 knockout mouse, which is required for mitochondrial complex I assembly, we performed immunohistochemistry (IHC) on postnatal day P14, P24, and P30 brain sections to label SOX2 (uncommitted neural stem and progenitors) and DCX (neuroblasts) cells in the SVZ. At P14, we have observed a significant decline in the density of neural stem/progenitors and neuroblasts in NDUFS4 KO as compared to wildtype. But, at later timepoints during development, this decline in these two cell populations were absent at P24 and P30. However, at P24 and P30, there was a significant reduction in neural stem cell commitment in the NDUFS4 KO group as demonstrated by the decreased density of double positive and double positive/SOX2+ cells. Body weight at this time points was unchanged between groups, and brain weight normalized to body weight and half hemispheric brain measurements were unchanged between groups. These results suggest that this neurodevelopmental defect was generalized to the SVZ. Moreover, NDUFS4 KO animals steadily increased the number of SOX2+ cells throughout the developmental timeline but displayed no alterations in DCX+ cells suggesting that at P24 and P30 when neural cell commitment is defective, the number of NSCs and progenitors are unable to mature in KO mice. In conclusion, complex I deficiency negatively impacts the neurogenic niche in the SVZ in the early postnatal period. This data is insightful in understanding developmental delays seen in LS patients and encourages future studies into understanding the neurodevelopmental aspects of those suffering from mitochondrial disease.

Abstract #: 2024PA-0000000028

Presenter: Madhurima Saha, PhD

Investigating AAV9-MECR Gene Therapy for Mitochondrial Enoyl CoA Reductase Protein-Associated Neurodegeneration (MEPAN) Syndrome

Madhurima Saha*, PhD, Radhika Bhake, MS, Mulan Yin, Manuela Corti, PT, PhD, Barry J Byrne, MD, PhD

University of Florida, Department of Pediatrics, College of Medicine

*madhurima.saha@ufl.edu (*Corresponding author email)

Abstract: MEPAN syndrome arises from a rare autosomal recessive disorder triggered by a loss-of-function mutation within the gene encoding mitochondrial enoyl CoA reductase (MECR). This pivotal enzyme orchestrates the final step of mitochondrial fatty acid synthesis, recently linked to sustaining neuronal function. Clinically, MEPAN syndrome is characterized by early childhood dystonia, basal ganglia anomalies, and progressive optic atrophy, underscoring MECR’s significance in neuronal health. This study aims to enhance the transduction efficiency of the AAV9 viral vector within central nervous system (CNS) tissues by investigating isoform 1 of the human MECR gene. Employing the pTR dual backbone, we cloned MECR gene variants using Synapsin and Desmin promoters renowned for maximizing transduction efficacy in CNS. Subsequent transfection into human embryonic kidney 293T (HEK293T) cells and mouse neuroblasts (Neuro-2a) allowed evaluation of MECR protein expression. MECR gene variants were cloned into the pTR (pUC vector backbone with AAV2 ITRs) dual (consisting of Kanamycin and Ampicillin resistance genes) plasmid backbone, utilizing promoters for enhancing transduction efficiency in CNS tissues. Lipofection performed transfection experiments in HEK293T and Neuro-2a cells to assess MECR protein expression. Western blotting confirmed successful protein expression. Successful MECR protein expression was observed in transfected HEK293T and Neuro-2a cells, indicating the potential viability of AAV9-MECR as a gene therapy vector. These findings underscore the prospective utility of this vector in further in vitro investigations aimed at understanding MEPAN syndrome and its underlying molecular mechanisms. Our study showcases the promise of the AAV9-MECR vector for potential gene therapy applications targeting MEPAN syndrome. The demonstrated expression of MECR protein in cellular models lays a foundation for future explorations toward developing therapeutic interventions for this debilitating neurodegenerative disorder.

Abstract #: 2024PA-0000000030

Presenter: Riley Wedan

Defining a Major Regulatory Step in the Function of the Mitochondrial Acyl Carrier Protein in Mammals

Wedan RJ^1,2*, Norden PR¹, Longenecker JZ¹, Graber N¹, Canfield M¹, McLaughlin B¹, Nownski SM¹

¹Department of Metabolism and Nutritional Programming, Van Andel Institute, Grand Rapids, MI 49503, USA, ²College of Human Medicine, Michigan State University, Grand Rapids, MI 49503, USA

*riley.wedan@vai.org

Abstract: Recent advances in cell biology have implicated metabolism in diseases ranging from cancer to heart failure. The cellular hubs of metabolism, mitochondria, have become an attractive therapeutic target for these common diseases. Patients suffering from mitochondrial disease also stand to benefit from mitochondrially-targeted therapeutics. However, currently, our ability to influence mitochondrial function therapeutically is limited. Uncovering how mitochondria regulate energy production in normal conditions will provide the groundwork for the development of future treatment strategies. The mitochondrial acyl carrier protein (mtACP) sits at the intersection of nutrient inputs and energy expenditure in the mitochondria. The acylated form of mtACP both serves as a source for citric acid cycle (TCA) enzyme activation via lipoylation, as well as a stabilization factor in electron transport chain assembly, controlling the two major arms of mitochondrial oxidative metabolism. Despite its central role in oxidative metabolism, how acyl-mtACP is formed in the mitochondria is poorly defined. Acyl-mtACP formation requires a 4’-phosphopantetheine (4’-PP) modification, followed by the addition of 2-carbon units from de novo mitochondrial fatty acid synthesis (mtFAS) that underlies patient disorders including MEPAN syndrome. The enzyme responsible for mtACP 4’-phosphopantetheinylation in vivo is not known, and the subcellular compartment where this occurs is undefined yet has major implications for substrate (CoA) availability and mtACP regulation. We set out to define the enzyme responsible for this reaction in mammalian cells, and to demonstrate the subcellular location where the reaction takes place. To screen for candidate enzymes, we used a CRISPR library screen and identified the co-essentiality of mtACP activity with the only known mammalian 4’-phosphopantetheinyl transferase: aminoadipate-semialdehyde dehydrogenase-phosphopantetheinyl transferase (AASDHPPT). We further generated CRISPR knock-out single-cell clones of AASDHPPT and found that a loss of AASDHPPT resulted in a loss of mtACP activity endpoints. We have also screened several patient variants from ClinVar for their ability to rescue mtACP activity in the AASDHPPT knockout cells. Next, we sought to demonstrate the localization of this reaction. To date, the only known localization of AASDHPPT was in the cytoplasm, yet we have subcellular fractionation samples that support a secondary localization to the mitochondria, where 4’-phosphopantetheinylation of mtACP occurs. By demonstrating that AASDHPPT catalyzes this key step in mtACP activation, we provide the foundation for future studies that seek to modulate mtACP activity. We also anticipate that these results will help identify further patients with defects in mtACP activity due to loss of the previously unassociated AASDHPPT.

Abstract #: 2024PA-0000000031

Presenter: Donna M. Iadarola

Novel CRISPR/Cas9 generated ndufs2^-/- zebrafish larvae have short lifespan, developmental anomalies, and globally disrupted intermediary metabolism

Mitchell DV^3*, Iadarola DM^1*, Mathew ND¹, Keith K^1,3, Seiler C⁴, Nakamaru-Ogiso E^1,2, Taylor DM^2,3, and Falk MJ^1,2

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA, ² Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, ³Department of Biomedical and Health Informatics, The Children’s Hospital of Philadelphia, Philadelphia, PA, ⁴Aquatics Core Facility, The Children’s Hospital of Philadelphia, Philadelphia, PA. *Equal contribution

falkm@chop.edu (*Corresponding author email)

Abstract: Background: Mitochondrial complex I deficiency manifests with complex multi-system dysfunction, commonly in the Leigh syndrome spectrum (LSS), with early-onset metabolic strokes, lactic acidemia, and early mortality. To facilitate pre-clinical screening of novel therapeutic candidates for complex I diseases including LSS disorder, we developed a stable genetic knockout zebrafish animal model for a nuclear-encoded complex I subunit, Ndufs2.

Methods: CRISPR/Cas9 was used to generate a 16 base pair deletion in ndufs2; which are maintained as heterozygotes and in-crossed to generate homozygous mutants and wild-type (WT) siblings for larval analyses. Larvae were phenotypically characterized for survival, swimming activity, and gross morphologic defects. RNA-Seq-based transcriptome profiling was performed to evaluate pathway adaptations in ndufs2^-/- larvae at 6 vs 7 dpf, originating from same or different clutches, and relative to sibling (mixed ndufs2^+/+ and ndufs2^+/-) or AB WT controls. RNA-Seq zebrafish results were compared to transcriptome profiling in ndufs2^-/- missense mutant C. elegans (gas-1(fc21)) and complex I disease patient fibroblasts. Targeted biochemical analyses (ETC enzyme activity) and untargeted metabolomic profiling (LC-MS/MS) were performed to characterize global intermediary metabolic adaptations and confirm pathway alterations suggested by transcriptome analyses. Finally, therapies previously identified in the C. elegans ndufs2^-/- missense model are being screened for their ability to rescue impaired swimming activity in this severe zebrafish ndufs2^-/- knockout model.

Results: ndufs2^-/- zebrafish had severely reduced survival to a median of 11 days post fertilization (dpf) relative to wild-type (WT) animals that live greater than 2 years. ndufs2^-/- zebrafish had marked neuromuscular dysfunction with ~50% decreased swimming activity. Morphological analysis showed an uninflated swim bladder, decreased yolk absorption, a dark liver phenotype, and small eyes. RNA-Seq transcriptome profiling confirmed ~90% knockdown of ndufs2 expression and revealed many dysregulated pathways in the ndufs2^-/- zebrafish larvae, with increased expression of the electron transport chain, TCA cycle, eye development, fatty acid beta oxidation, and one carbon metabolism. Many of these dysregulated pathways were similarly dysregulated in C. elegans and human cell line models of complex I disease. Targeted biochemical analyses revealed an 80% reduction in complex I enzyme activity, but no reduction in other ETC enzyme complex activities. Unbiased metabolomic studies identified significantly increased lactate, TCA cycle intermediates, carnitine species, formyl-methionine, and nucleotides. Together, expected changes were seen in central metabolism to shunt glucose utilization away from respiration towards lactate production along with significant changes in one carbon metabolism and associated pathways that likely contribute to pathology. Screening of therapies identified in a C. elegans model of complex I disease are ongoing in the ndufs2^-/- knockout zebrafish model.

Conclusions: We have established and validated a novel vertebrate animal model of severe complex I deficiency using CRISPR/Cas9 technology to knock-out ndufs2 in zebrafish. This model recapitulated many phenotypes associated with mitochondrial disease and LSS. This model enables the investigation of organ specific mechanisms of disease and high-throughput treatment studies.

Our research study is the first to use CRISPR/Cas9 technology to generate a stable genetic complex I deficiency model in zebrafish and rigorously characterize the impact on animal survival, activity, morphology, and biochemistry. We further developed novel approaches to enable robust multi-omics profiling of mitochondrial disease effects in zebrafish. Indeed, global metabolic disruption was identified and new insights into disrupted one carbon metabolism in complex I disease evident by transcriptome profiling and validated by metabolomics. Ongoing studies in this novel complex I disease zebrafish model are focused on uncovering organ dysfunction, metabolic flux profiling, and screening of candidate therapies.

Abstract#: 2024PA-0000000033

Presenter: Valentina Emmanuele

mTOR inhibition as potential treatment for Leigh syndrome: report of three cases

Emmanuele V^1*, Engelstad K¹, Katus LE¹, Bain JM¹, Garvin JH², De Vivo, DC^1,2, Hirano M¹

¹Department of Neurology, Columbia University Irving Medical Center, New York, NY, USA, ²Department of Pediatrics, Columbia University Irving Medical Center, New York, NY, USA

*ve2129@cumc.columbia.edu

Abstract: Leigh syndrome (LS) is a neurodegenerative disorder due to mitochondrial dysfunction, characterized by developmental delay/regression accompanied by symmetrical involvement of central nervous system, in particular basal ganglia and brain stem, but also cerebellum and spinal cord. Clinical manifestation includes dystonia, weakness, dysphagia, ataxia, and seizures. The course of the disease is usually characterized by early onset and rapid progression, with deterioration often associated with metabolic stressors. Genetically, LS can be caused by defects in either the mitochondrial or nuclear genomes. The clinical and genetic heterogeneity of this disorder has hindered the development of effective therapies. Studies in animal models of LS have shown that rapamycin, an inhibitor of the mTOR signaling pathway, can delay onset and progression of symptoms. Based on these observations, two children with mitochondrial disorders were treated with everolimus, a rapamycin analogue approved for the treatment of cancer. The responses were disparate, with improvement in the patient with LS. We now update our experience with everolimus in that child and two additional children with LS.

Medical records of three genetically confirmed patients with LS treated with everolimus were reviewed. All were treated with everolimus targeting trough levels of 5-15ng/ml. Variables considered were age at onset of symptoms, sex, symptoms, genetic variants, duration of disease before treatment, everolimus dose/level, duration of treatment, side effects, impact on clinical course, imaging, concomitant medications.

Patient A, previously published, currently is a 9 year-old girl. The onset of symptoms was at age 11 months with loss of milestones, hypotonia, and seizures. MRI at age 1 year showed symmetric lesions of thalami, cerebral peduncles, pons and medulla. Genetic analysis revealed a homozygous pathogenic variant in NDUFS4 gene. Everolimus treatment was started at age 2 years. She has been followed for 7 years. After initial clinical improvement, her condition stabilized for about 3 years. She is alive but now with clinical deterioration and MRI evidence of disease progression.

Patient B is a 16 year-old boy with LS due to compound heterozygous variants in NDUFAF6 gene. Symptoms started at age 3 years with speech and motor regression, and dystonia. A brain MRI at age 3.5 years revealed increased T2/FLAIR signal in the putamen. He started everolimus at age 14 years and has been stable for 2 years.

Patient C is an 11 year old boy with LS due to m.8344 A>G in MT-TK (95% heteroplasmy in blood). His complex clinical syndrome includes upper cervical/medullary lesions causing hypoventilation and dysphagia requiring PEG-dependence, myoclonic epilepsy, myopathy, cardiac hypertrophy, optic neuropathy, sensorineural hearing loss. Symptom onset was at age 8 years with myoclonic epilepsy. Everolimus was started at age 10 years. Initial follow up after 6 months showed mild improvement and stabilization of symptoms. His course has been complicated by diabetes mellitus and clinical worsening at time of intercurrent infection.

In conclusion, everolimus is a promising treatment for LS which may prolong survival by delaying onset of respiratory failure, with acceptable side effects. This preliminary experience justifies a prospective trial examining safety, timing, and effectiveness of everolimus treatment.

Abstract #: 2024PA-0000000035

Presenter: Cristy Balcells

Patient experiences of thymidine kinase 2 deficiency: preliminary results from an online survey conducted in partnership with the patient community

Balcells C^1*, Karaa A², Waller K³, Yeske P⁴, Morrison A⁵, Ross M⁵ and Hareendran A⁶

¹UCB Pharma, Smyrna, GA, USA, ²Massachusetts General Hospital, Genetics Division, Harvard Medical School, Boston, MA, USA, ³The Lily Foundation, Warlingham, Surrey, UK, ⁴United Mitochondrial Disease Foundation, Pittsburgh, PA, USA, ⁵Rare Disease Research Partners, Amersham, Buckinghamshire, UK, ⁶UCB Pharma, Slough, Berkshire, UK

*Cristy.Balcells@ucb.com

Abstract: Thymidine kinase 2 deficiency (TK2d) is an ultra-rare, autosomal recessive, mitochondrial disease associated with progressive, life-threatening myopathy that affects feeding, breathing, and ambulation. Currently, there are no approved treatments for TK2d. Understanding the lived experience of patients and their caregivers is crucial for characterizing the burden of TK2d and tailoring disease management. The Assessment of TK2d Patient Perspectives (ATP) Study aims to capture the lived patient and caregiver experience of symptoms, and the impact of those symptoms on health-related quality of life (HRQoL). This cross-sectional, mixed-methods study uses an online survey to collect data from patients and caregivers in countries where >3 TK2d patients have previously been identified. The survey, which includes multiple-choice and open-text questions, and visual analog scales, was co-created in partnership between UCB Pharma, Rare Disease Research Partners, and patient expert advisors to meet the study objectives and ensure relevance for the target population. The survey was distributed by patient organizations and clinicians to adult patients or caregivers of patients (including current or bereaved) with a self-reported, genetically confirmed diagnosis of TK2d. Patients enrolled in active clinical trials were excluded. Five months after the original survey launch in September 2023, an interim analysis of quantitative and qualitative survey data to describe patients’ experiences of their symptoms and their impact on HRQoL was completed. In total, 24 patients (14 females) with a median age of 31.0 years (mean [standard deviation]: 30.1 [13.63] years; range: 2–54 years) from Europe (n=11), North America (n=8), and South America (n=5) were included in this analysis. Most (21/24) required at least some assistance with daily activities, with 10 patients requiring full-time support. Most patients (22/24) experienced fatigue, low energy levels, and upper-body muscle weakness, and many (20/24) reported lower-body muscle weakness, consistent with the myopathic component of TK2d. Psychological symptoms were commonly reported, including anxiety (15/24 patients), irritability (11/24 patients), and depression (10/24 patients). The psychological burden was greatest for patients who first experienced TK2d symptoms at >12 years of age (n=5); all 5 of these patients reported psychological symptoms, with 4/5 reporting anxiety, depression, and irritability, 2/5 reporting mood swings, and 1/5 reporting feelings of isolation. Respondents reported the biggest impact on HRQoL to be caused by breathing difficulties (11/24 patients); muscle weakness (8/24 patients) and fatigue (7/24 patients) were also reported as impacting HRQoL. Specific words used by respondents to describe their experiences of living with TK2d included “exhausting”, “overwhelming”, “frustrating”, “debilitating”, “limiting”, “dependence”, and “hopeless”. The degenerative nature of the condition was particularly challenging for patients, who in their qualitative open-text responses, described experiencing emotional distress as their functional abilities and independence diminished. This study expands on existing findings and highlights the unmet needs and burden of disease in TK2d. The final results of the ATP study will provide valuable insights into these areas and the role of caregivers for patients with TK2d, leading to an improved understanding of the patient’s experience.

Study funded by UCB Pharma.

Abstract #: 2024PA-0000000036

Presenter: Chen Ding

Sarm1 knockout rescues age-dependent retinal ganglion cell (RGC) degeneration in a novel mouse model of Autosomal Dominant Optic Atrophy (ADOA)

Ding CD¹, Ndiaye PN¹, Campbell SC¹, Gong JG¹, Gibbs WG¹, Do MTHD¹, Schwarz TLS^1*

¹Kirby Neurobiology Center, Boston Children’s Hospital, Harvard Medical School, USA

*Thomas.Schwarz@childrens.harvard.edu

Abstract: Autosomal Dominant Optic Atrophy (ADOA) is the most common form of inherited optic neuropathy. ADOA patients typically show vision loss during the first decade of life due to selective degeneration of RGCs, which may then slowly progress to legal blindness over the next decade or more. Most of the ADOA cases are caused by mutations in the OPA1 gene, which encodes a conserved GTPase that mediates fusion of the inner mitochondrial membrane and regulates cristae remodeling. Homozygous for loss of OPA1 are embryonic lethal, and ADOA human patients are heterozygous. Cells with defective OPA1 may have fragmented mitochondria, abnormal cristae structure, reduced ATP production, increased oxidative stress and loss of mitochondrial DNA. So far, no therapies are available for ADOA and the disease mechanism remains poorly understood. We have built a novel mouse model of ADOA that carries the pathogenic OPA1^R290Q/+ mutation. We show that the OPA1^R290Q/+ mice recapitulate many aspects of the human ADOA symptoms, including RGC death, optic nerve degeneration, demyelination, and declined RGC responses. At the cellular level, the OPA1^R290Q/+ cells have a highly fragmented mitochondria network, reduced respiration, and signs of oxidative stress. We then tested whether Sarm1, a conserved NADase and a major pro-degenerative factor in neurons, mediates RGC degeneration in ADOA. Sarm1 knockout has been shown to prevent neurodegeneration in many pathological conditions, and of particular interest are glaucoma and mitochondrial stress models. We show that knocking out Sarm1 in OPA1^R290Q/+ mice strongly suppresses the age dependent RGC loss. Therefore, our results suggest that Sarm1 activation mediates RGC degeneration in ADOA. We are currently investigating how OPA1-induced mitochondrial dysfunctions activate Sarm1. Overall, our work show that the OPA1^R290Q/+ mouse model recapitulates human ADOA and that therapies targeting Sarm1 may be effective for ADOA patients.

Abstract #: 2024PA-0000000037

Presenter: Ameya Walimbe MD PhD MSE

Expanded clinical phenotype and the role of untargeted metabolomics analysis in confirming the diagnosis of sodium-dependent multivitamin transporter deficiency

Walimbe AS^1,2, Waskow E^2,3, Mackay L⁴, Miller M⁵, Gijavanekar C³, Elsea SH³, Scaglia F^2,3,6*

¹Division of Pediatric Neurology and Developmental Neuroscience, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA, ²Texas Children’s Hospital, Houston, TX, USA, ³Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA, ⁴Department of Pediatrics, UT Southwestern Medical Center, Dallas, TX, USA, ⁵Indiana University School of Medicine, Indianapolis, IN, USA, ⁶BCM-CUHK Center of Medical Genetics, Prince of Wales Hospital, Hong Kong SAR, China

*fscaglia@bcm.edu

Abstract: The Sodium Multivitamin Transporter (SMVT) is a ubiquitously expressed sodium-solute symporter that is required for the absorption of the water-soluble vitamins pantothenic acid (B5) and biotin (B7) and the vitamin-like compound α-lipoic acid across the intestinal epithelia and through the blood-brain barrier. Biallelic pathogenic variants in SLC5A6 (NM_021095.4) lead to the ultra-rare autosomal recessive disorder, Sodium-Dependent Multivitamin Transporter Deficiency (SMVTD, MIM # 618973). The clinical spectrum of SMVTD includes severe feeding difficulties, failure-to-thrive, developmental delay, brain atrophy, microcephaly, epilepsy, sensorineural hearing loss, and optic and peripheral neuropathy, as well as gastrointestinal, immunological, respiratory, and cutaneous abnormalities. In severe cases, patients can develop infantile-onset epileptic encephalopathy with progressive neurodegeneration. Here, we describe a 23-year-old female with agenesis of the corpus callosum, a history of failure to thrive, focal epilepsy, intellectual disability, and autism spectrum disorder (ASD) who presented at 15 years of age with altered mental status in the setting of metabolic acidosis (pH 7.15, HCO₃: 6 mEq/L, normal 21-28 mEq/L), elevated lactate (6.8 mmol/L, normal <2 mmol/L), hyperammonemia of 365 µmol/L (normal <35 µmol/L), transaminitis (maximum AST: 323 U/L, normal <36 U/L, and ALT: 151 U/L, normal < 36 U/L), and rhabdomyolysis (maximum creatine kinase: 21,000 U/L, normal < 170 U/L). She required intravenous sodium benzoate and sodium phenylacetate, an insulin drip, intravenous hydration, and urine alkalinization for clinical improvement. Trio Whole Exome Sequencing identified compound heterozygous variants in SLC5A6- NM_021095.4: c.1865_1866del (p.Gln622ArgfsTer51), a maternally inherited pathogenic variant, and NM_021095.4: c.814G>A (p.Gly272Arg), a previously unpublished, paternally inherited variant of uncertain significance. Brain MRI was notable for mildly dysmorphic lateral ventricles secondary to the patient’s known callosal agenesis. An EEG demonstrated diffuse, generalized background slowing and the absence of an occipital dominant rhythm. Plasma untargeted metabolomics analysis demonstrated deficiencies in pantothenate (z-score -2.6) and CoA metabolism subpathways, with elevated medium and long-chain fatty acids and low levels of multiple long-chain glycerophospholipids, indicating impaired fatty acid oxidation and lipid metabolism. Acylcarnitine analysis indicated a deficiency of free carnitine (C0: 21 µM/L, normal: 28-56 µM/L), prompting the initiation of carnitine supplementation. Urine organic acid analysis demonstrated elevations in lactic acid, ketones, 3-hydroxy isovaleric acid, glutaric acid, and dicarboxylic acids, consistent with secondary mitochondrial dysfunction. Targeted biotin, lipoic acid, and pantothenic acid replacement therapy was initiated and improved her metabolic and neurocognitive function. We present the oldest reported patient with SMVTD at the time of diagnosis. Our work, therefore, expands the clinical spectrum of SMVTD, demonstrating that patients may not develop serious metabolic abnormalities until their teenage years and may experience severe rhabdomyolysis during these episodes. Furthermore, ASD may be part of the neurobehavioral phenotype associated with this condition. Additionally, biochemical evaluations indicate secondary mitochondrial dysfunction. The reduction of analytes in the pantothenate and CoA metabolic pathway in plasma untargeted metabolomics analysis may provide functional validation in variants of uncertain significance in SLC5A6 and potentially be used to monitor treatment efficacy. Our work adds to the limited number of cases of SMVTD and enhances the identification and management of patients with this condition.

Abstract #: 2024PA-00000000039

Presenter: Anne Chiaramello

Intrauterine growth restriction due to idiopathic placental insufficiency causes long-term postnatal mitochondrial pathophysiology and severe neurodevelopmental defects

Uittenbogaard M¹, Brantner CA², Whitehead MT³, Gropman E¹, Gropman AL⁴, Chiaramello A^1*

¹Department of Anatomy and Cell Biology, George Washington University School of Medicine and Health Sciences, Washington, D.C. USA, ²Electron Microscopy Core imaging Facility, University of Maryland Baltimore, School of Dentistry and School of Medicine, Baltimore, M.D. USA, ³ Division of Neuroradiology, Children’s Hospital of Philadelphia, Philadelphia, P.A. USA, ⁴Children’s National Medical Center, Division of Neurogenetics and Developmental Pediatrics, Washington, D.C. USA

*achiaram@gwu.edu (*Corresponding author’s email)

Abstract: The placenta is a nutrient sensor and supplier that plays a pivotal role in providing metabolites required for fetal mitochondrial metabolism to generate high levels of energy, which are essential for normal fetal growth rate and neural development. Placental insufficiency is among the most frequent causes of intrauterine growth restriction (IUGR), resulting in reduced fetal growth rate. However, the question of whether placental insufficiency impacts postnatal mitochondrial physiology and brain development remains unaddressed. Thus, we hypothesized that placental insufficiency mediated by IUGR could provoke dysfunctional fetal mitochondrial metabolism resulting in postnatal mitochondrial pathophysiology and severe neurodevelopmental defects. To test our hypothesis, we focused on a four-year-old male with a history of IUGR, a complex medical history, and a suggested mitochondrial etiology based in lactic acidosis and low levels of citrulline provoked by the administration of Depakote. The proband was born at 36 weeks gestational age due to growth arrest with profound neurodevelopmental and global defects, microcephaly, congenital hypotonia, intractable epilepsy, and the dysmorphic syndrome Pierre-Robin sequence. In the absence of nuclear and/or mitochondrial pathogenic variants by whole exome sequencing of the nuclear genome and deep sequencing of the mitochondrial genome, the genetic causes of his complex phenotypic manifestations remain enigmatic. The family history was noncontributory to IUGR and the proband’s complex phenotypic manifestations. Thus, the most probable cause for IUGR is idiopathic placental insufficiency commonly associated with reduced blood flow through the placenta that curtails levels of oxygen and fuel substrates, resulting in the proband’s grey-matter and white-matter pathologies. Results from live-cell functional mitochondrial OXPHOS assays show a severe deficit in reserve energy, impairing the ability of the proband’s fibroblasts to avert an ATP crisis upon high energy expenditure. Morphometric mitochondrial investigations using transmission electron microscopy confirmed that the proband’s mitochondria display an immature morphology with poorly developed cristae. Thus, we investigated the proband’s metabolic plasticity in favor of glycolysis to compensate for the limited OXPHOS capacity. We found that the proband’s fibroblasts exhibited an increased basal glycolytic response and compensatory glycolytic response, the latter being an indicator of metabolic reprogramming toward glycolysis subsequent to an OXPHOS crisis. Collectively, the immature mitochondrial morphology, the mitochondrial energy deficit and the adaptative metabolic adaptative response toward glycolysis of the proband’s fibroblasts are hallmarks of mitochondria during the early fetal developmental stages characterized by low levels of OXPHOS and oxygen consumption, combined with high glycolysis via passive diffusion of glucose through the placenta. During late fetal stages, mitochondria undergo functional metabolic adaptation toward the OXPHOS metabolism to meet the high energy requirement for cell specification and differentiation in keeping with normal late fetal neurodevelopment. The proband’s impaired mitochondrial metabolic flexibility is congruent with his profound neurodevelopmental defects detected at birth and persisting postnatally. In conclusion, our studies shed light on the long-term postnatal mitochondrial pathophysiology caused by IUGR due to idiopathic placental insufficiency and its negative impact on the energy-demanding development of the fetal and postnatal brain.

Abstract #: 2024PA-0000000043

Presenter: Charles R. Difalco, MD

Untargeted Metabolomics as a Potential Screening Tool for 3-Methylglutaconic Aciduria Syndromes

DiFalco CR^1,3*, Gijavanekar C², Elsea SH², Grace AN^1,4, Odom JD^1,4, Machol K^1,3, Emrick L^1,3, and Scaglia F^1,3,5

¹Department of Molecular and Human Genetics, Baylor College of Medicine, USA, ²Baylor Genetics, USA, ³Texas Children’s Hospital, USA, ⁴CHRISTUS Children’s Hospital, USA, ⁵BCM-CUHK Center of Medical Genetics, Prince of Wales Hospital, Hong Kong SAR

*charles.difalco@bcm.edu

Abstract: 3-methylglutaconic aciduria (3MGA-uria) syndromes comprise a heterogenous group of inborn errors of metabolism defined biochemically by detectable elevation of 3-methylglutaconic acid (3MGC acid) in the urine. In primary 3-methylglutaconic aciduria (or type 1), distal defects in the leucine catabolism pathway directly cause this elevation. Secondary 3-methylglutaconic aciduria syndromes, however, are a wide variety of unique metabolic disorders unrelated to leucine metabolism-specific defects that share a common biochemical phenotype of elevated 3MGC acid. It is currently thought that this accumulation is due to underlying buildup of acetyl CoA in the mitochondria due to impaired oxidation in the TCA cycle with ensuing formation of trans-3MGC CoA and its subsequent byproducts including 3MGC acid. In these disorders, urine 3MGC acid levels are known to be fluctuant and at times undetectable by standard urine organic acid (UOA) analysis, thereby impacting its utility as a means of sensitive biochemical detection of these disorders; this inherent limitation highlights the need for a more sensitive clinical modality. Untargeted metabolomic profiling is a rapidly emerging technology that is being used to detect and characterize biochemical abnormalities in many metabolic disorders. Here, we report on a cohort of seven patients with confirmed inborn errors of metabolism associated with 3MGA-uria who had plasma metabolomics data available for review. In two patients within this cohort, UOA analysis did not show detectable 3-MGC acid levels. Untargeted metabolomic profiling in plasma samples, however, identified significant elevations in 3MGC acid in all individuals. There is promising potential for plasma global metabolomic analysis as a sensitive and comprehensive biochemical screen for metabolic disorders in which there is measurable 3MGC acid elevation.

Abstract #: 2024PA-0000000044

Presenter: Vittoria Rossi, MD

Biallelic variants in POLG2 provide a rare molecular diagnosis in a patient with hepatocerebral syndrome

Vittoria Rossi^1,3, Daniel Brooks^1,3, Hongzheng Dai², Elizabeth Mizerik^1,3, Seema R. Lalani^12,3, Daryl A. Scott^1,3, Fernando Scaglia^1,3,4, Keren Machol^1,3, Mir Reza Bekheirnia^1,3

¹Department of Molecular and Human Genetics, Baylor College of Medicine, Houston TX, USA, ²Baylor Genetics, Houston TX, USA, ³Texas Children’s Hospital, Houston TX, USA, ⁴Joint BCM-CUHK Center of Medical Genetics, Prince of Wales Hospital, Hong Kong SAR, China

*vcrossi@bcm.edu

Abstract: Maintenance of mitochondrial DNA requires the coordination of numerous proteins that contribute to replenishing the mitochondrial nucleotide pool, synthesizing mtDNA, and mediating mitochondrial fusion. Synthesis of mitochondrial DNA (mtDNA) requires the function of polymerase gamma which is a heterotrimer holoenzyme made up of one 140-kDa catalytic subunit (p140) encoded by POLG and a homodimeric processing subunit composed of two p55 accessory proteins encoded by POLG2. While variants in POLG represent the most prevalent single gene cause of mitochondrial disease, there are very few reported cases in the literature of patients with mono-allelic pathogenic variants in POLG2 associated with autosomal dominant progressive external ophthalmoplegia 4 with mtDNA deletions (MIM#610131). Furthermore, only three individuals have been reported with biallelic variants in this gene initially associated with the hepatic type mtDNA depletion syndrome 16 (MIM#618528). In each reported case, the individuals inherited homozygous missense variants with a phenotypic spectrum that varied in both the age of the individuals at presentation- 3 months, 23 years, and 39 years- and their clinical presentation that ranged from fulminant liver failure, optic atrophy with progressive cerebellar ataxia, and focal epilepsy that generalized later in adulthood respectively. Here, we report a fourth case of a patient found to have biallelic variants in POLG2 with a different age at presentation and clinical course when compared to the known reported cases. The 5- week-old female patient presented originally with increased irritability and difficulty feeding that evolved into tonic-clonic seizures and status epilepticus. She was ultimately discharged after her seizures were well controlled on anti-epileptic medications, but she was readmitted for increased lethargy, vomiting, jaundice and was later found to have acute liver failure. Trio whole genome sequencing (WGS) identified a paternally inherited likely-pathogenic variant (c.775C>T, p.R259*) and a maternally inherited variant of uncertain significance (c.445_456del, p.S149_T152del) in POLG2. Follow up lab testing revealed a GDF-15 elevated at 6,000 pg/mL. During her hospital stay, the patient developed worsening coagulopathy and hyperammonemia as sequelae from her worsening liver failure. The patient passed away soon after being released home on hospice care. This case contributes to the phenotypic spectrum associated with POLG2 in addition to highlighting the difficulty in addressing prognosis and goals of care with the family and medical team in the presence of the current limited published literature.

Abstract #: 2024PA-0000000046

Detection and quantification of large scale mtDNA deletions using PacBio long read sequencing

Tanaya Jadhav¹, Avery Zucco¹, Matt Aruta¹, Laura Conlin^1,2, Ramakrishnan Rajagopalan^1,2, Jing Wang^1,2

¹Division of Genomic Diagnostics, Department of Pathology, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA, ²Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

*wangj17@chop.edu

Abstract: Primary mitochondrial diseases (PMDs) are a group of highly heterogenous disorders caused by mutations in either nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). The mtDNA mutation spectrum includes single nucleotide variants (SNVs) as well as single large-scale mtDNA deletion (SLSMD) and multiple mtDNA deletions (MMDs). MMDs are often caused by mutations in nuclear genes responsible for mtDNA biogenesis and maintenance, which is a distinct entity from the syndromes caused by SLSMD. It’s crucial to correctly detect and distinguish MMDs and SLSMD. In addition, accurate quantification of MMDs and SLSMD heteroplasmy is essential for clinical correlation and disease prognosis. Current clinical tests for mtDNA analysis typically involve long-range PCR (LR-PCR) to amplify the full-length mtDNA, followed by short-read Next Generation Sequencing (SR-NGS) to detect SNVs and large-scale mtDNA deletions. If large-scale mtDNA deletion is detected, a separate droplet digital PCR (ddPCR) assay is employed for deletion heteroplasmy quantification. Long-read sequencing (LRS) has shown advantage for large structure variant detection, mostly in nuclear genome. There is limited data of using LRS for mtDNA analysis, especially for MMDs and SLSMD detection and quantification. In this study, we conducted sequencing analysis on 11 samples using PacBio LRS technology, compared with the mtDNA testing results from SR-NGS. The samples include 3 SLSMD positives, 5 MMDs positives and 3 deletion negative controls (N=11). A custom bioinformatics pipeline was developed to identify and cluster large deletions, including SLSMD and MMDs. In addition, mtDNA sequencing variants and heteroplasmy were also evaluated. The LRS is able to sequence the entire 16.5 KB mitogenome in a single read strand. Our results demonstrated that LRS can correctly detect and distinguish SLSMDs and MMDs in sample with deletion heteroplasmy >10%. No large-scale mtDNA deletions were identified in negative control samples using our LRS pipeline. Clustering of the deletion breakpoints revealed that several deletions were recurrent with same breakpoints that were previously thought to be random. Notably, while the heteroplasmy levels calculated from LRS data were different to those determined by ddPCR, their overall trend was found to correlate with the ddPCR results. Furthermore, LRS demonstrated high accuracy in SNV detection and heteroplasmy quantification. The LRS achieved sensitivity of 95.7% and specificity >99.9% compared with SR NGS in these 11 samples. For the SNVs that failed to be detected by LRS, most had heteroplasmy below 10%. In conclusion, our results demonstrated that LRS can detect large scale single and multiple mtDNA deletions. The deletion heteroplasmy calculated using LRS data correlates with that was calculated from ddPCR. The ability to identify precise deletion breakpoints, especially in samples with multiple deletions widens the scope for future studies to investigate the mechanism underlying these deletions such as microhomology mediated end joining vs. blunt end joining. Moreover, LRS has high accuracy for SNV detection and heteroplasmy quantification. It has the potential to be offered as a single clinical test to cover broad mtDNA mutation spectrum.

Abstract #: 2024PA-0000000047

Presenter: Lindsey Miller

Long-Term Elamipretide Treatment in a Patient with Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP) Syndrome: A Case Study

Mary Kay Koenig¹, Lindsey Miller^1*, Maria Poblete¹, S. Nick Russo¹

¹University of Texas McGovern Medical School, Department of Pediatrics, Division of Child & Adolescent Neurology, Center for the Treatment of Pediatric Neurodegenerative Disease, Houston, TX

*Lindsey.B.Miller@uth.tmc.edu

Abstract: Neuropathy, ataxia, and retinitis pigmentosa (NARP) syndrome and maternally inherited Leigh syndrome are part of a continuum of progressive neurodegenerative disorders caused by defects in mitochondrial energy production. Here, we report the long-term use of elamipretide in a patient with a blended phenotype of NARP and Leigh syndromes due to a 91.2% heteroplasmic mutation at m.8993T>G. Prior to elamipretide treatment, the patient presented with primary symptoms of retinitis pigmentosa, sensorineural hearing loss, progressive cerebellar ataxia, cognitive impairment, left eye exotropia, and proximal myopathy. The patient was able to rise from the chair independently, but he was unsteady and braced himself on the chair. He walked with his feet everted with an unsteady gait. At 39 years of age, the patient first received elamipretide therapy at 40mg/day subcutaneously (SC) for ~10 months through the SPIMM-301 trial (Stealth BioTherapeutics). During treatment, the patient experienced an increased ability to work and socialize as well as improvement in exercise ability, balance, and posture. After the trial ended, within ~4 months of elamipretide cessation, the patient’s family reported loss of any improvements made during treatment and noted worsened balance, orientation, mobility, weakness, lethargy, and confusion. At 40 years of age and ~6 months after his last dose of elamipretide through the clinical trial, the patient was enrolled into the SPIES-006 Expanded Access Program (EAP; Stealth BioTherapeutics) for SC elamipretide and reinitiated treatment at 40mg SC. His family reported decreased ataxia, reduced dysarthria, and increased independence after 3 months of treatment. Physical examination revealed improved strength and ambulation, such that the patient stood easily from the chair and ambulated steadily with minimal assistance. After ~1 year on elamipretide through the EAP, the daily dose was increased to 60mg SC to further optimize response. A dramatic improvement in his condition was observed within 6 months of this dose increase and included resolution of previous walking problems, increased strength, return of long-term memory, and improvement in QOL scores. The patient and his family reported increases in his energy, stamina, and walking ability and distance. Ten months after the dose increase, the patient competed in the Special Olympics track event for the first time in over seven years. Eighteen months after the dose increase, the patient reported some return of vision. At 43 years of age and after nearly 5 years of elamipretide treatment, the patient remains fairly stable and active, including continued participation in the Special Olympics. On his most recent exam, the patient did display some intermittent sway when standing and used a cane due to visual impairment only, but there were no signs of ataxia. The patient has developed osteoporosis in his hip for which he has been prescribed vitamin D supplementation. No other changes were noted from previous examination. Overall, the lack of rapid decline in this progressive neurogenerative disease provides support for the clinical benefit of elamipretide treatment in NARP syndrome. Although the patient has experienced injection site reactions, major adverse events have not been reported, demonstrating the safety of long-term elamipretide use.

Abstract #: 2024PA-0000000048

Presenter: Zarazuela Zolkipli-Cunningham

Natural History of Mitochondrial Myopathy objective measures reveals significantly slow, progressive decline

Zolkipli-Cunningham, Z.^1,3*, Flickinger, J.^1,2, Martin, I.¹, Santos, J.D.¹, Rahaman, I.¹, Wellik, A.¹, Ballance, E.^1,2, Liebman, M.¹, Vishnubhatt, S.¹, Peterson, J.¹, Intarachumnum, N.¹, Tindall, A.¹, McCormick, E.¹, Muraresku, C.¹, Bogush, E.¹, MacMullen, L.¹, Stanley, K.¹, Nguyen, S.¹, Sharma, S.^1,3, Goldstein, A.^1,3, Falk, MJ.^1,3, McDermott, M.⁵, Xiao, R.^3,4

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Children’s Hospital of Philadelphia, USA, ²Division of Rehabilitation, Children’s Hospital of Philadelphia, USA, ³Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, USA, ⁴Department of Biostatistics, Epidemiology and Informatics, University of Pennsylvania Perelman School of Medicine, USA, ⁵Department of Biostatistics and Computational Biology, University of Rochester, USA*zolkipliz@chop.edu

Abstract: Introduction: Our prospective Mitochondrial Myopathy Natural History Study (CLARITY) obtains longitudinal objective Mitochondrial Myopathy-Composite Assessment Tool (MM-COAST) measurements¹ in genetically confirmed Mitochondrial Myopathy (MM) subjects, to evaluate disease progression.

Methods: We enrolled 211 pediatric (mean age ± SD, 9.1 ± 4.8 years, 51.2% males) and adult (37.0 ± 14.0 years, 31.7% males) MM subjects harboring either nuclear (n=91, 43.1%) or mtDNA (n=120, 56.9%) genetic etiologies between March 2016 - January 2024 and completed baseline MM-COAST assessments in subjects ⩾ 5 years. Eighty-seven subjects completed longitudinal evaluations up to 63 months from baseline.

Results: Linear mixed-effects models for repeated measures revealed significant slow progression in several MM-COAST domains and the overall Composite Score. In the strength domain, hip flexion changed by -0.26 z-score/year (p=0.002, n=67), ankle dorsiflexion by -0.36/year (p<0.001, n=66), and wrist extension by -0.20/year (p=0.02, n=69). Exercise intolerance measures of 30 second sit-to-stand (30s STS) declined by -0.06/year (p=0.02, n=51) and 6-minute walk (6MWT) by -0.17/year (p<0.001, n=55). In terms of balance, single leg eyes closed changed by -0.12/year (p=0.005, n=75) and tandem stance eyes closed by -0.23/year (p=0.006, n=74). For dexterity, nine-hole peg test (9HPT) declined by -0.80/year (p=0.01, n=72). MM-COAST Composite Score increased by 0.088 points/year (p<0.001, n=75) with a higher value demonstrating greater disease severity.

Our new Mitochondrial Mobility Performance Levels (IMPROVE) classification defines MM subjects by mobility and fatigue levels from non-ambulatory (Level 1) to ambulant without fatigue (Level 6). Most MM subjects in our cohort were IMPROVE Level 3 household ambulators with severe fatigue (n=26, 16%), Level 4 community ambulators with moderate fatigue (n=65, 40%), and Level 5 community ambulators with mild fatigue (n=41, 25%). For Level 3 subjects with severe fatigue, significant change occurred in their 6MWT (-0.28/year, p<0.001, n=12), 9HPT (-1.83/year, p<0.001, n=18), and MM-COAST Composite Score (0.17/year, p<0.001, n=17). By comparison, Level 4 and 5 subjects with moderate and mild fatigue respectively, revealed slower rates of change in their 6MWT (-0.13/year, p<0.001, n=40), 9HPT (-0.19/year, p=0.05, n=47), and MM-COAST Composite Score (0.07/year, p=0.003, n=50), confirming significantly faster decline in Level 3 versus 4 and 5, suggesting that MM phenotype at baseline influences the rate of change over time.

Latent class mixed modeling identified two subclasses of trajectories of the longitudinal MM-COAST Composite Scores, where Class 1 (n=13, 17.3%) showed progressive decline while Class 2 (n=62, 82.7%) had stable or improved trajectory. Class 1 demonstrated significantly higher MM-COAST scores at baseline of 2.07 ± 0.23 (mean ± SEM) representing more severe MM, as compared to Class 2 (0.95 ± 0.10, p=0.0002). The majority of subjects in Class 1 were IMPROVE Levels 3 (n=7/13, 53.8%) and 4 (n=3/13, 23.1%), while Class 2 were mainly IMPROVE Levels 4 (n=28, 45.2%) and 5 (n=18, 29.0%), thus providing additional evidence to confirm that MM phenotype indeed influences MM trajectory.

Conclusion: For the first time, longitudinal objective data demonstrating slow disease progression is available in MM, to better inform clinical trial design and power calculations. Our IMPROVE classification may be transformative for future clinical trial subject selection and cohort longitudinal analyses.

References

 1. Flickinger, J., Fan, J., Wellik, A., Ganetzky, R., Goldstein, A., Muraresku, C. C., Glanzman, A. M., Ballance, E., Leonhardt, K., McCormick, E. M., Soreth, B., Nguyen, S., Gornish, J., George-Sankoh, I., Peterson, J., MacMullen, L. E., Vishnubhatt, S., McBride, M., Haas, R., Falk, M. J., Xiao, R., and Zolkipli-Cunningham, Z. (2021) Development of a Mitochondrial Myopathy-Composite Assessment Tool. JCSM Clinical Reports, 6: 109–127. https://doi.org/10.1002/crt2.41.

Abstract #: 2024PA-0000000049

Presenter: Dr. Kenneth Myers, MD PhD FRCPC

Pyrimidine Deoxynucleoside Treatment for POLG-Related Disorders: Open-Label Human Trial

Myers KA^1,2,3*, Pekeles H², Berrahmoune S¹, Dassi C¹, Cheung ACT⁴, Waters PJ^5,6, Eberhard R², Buhas D^4,7

¹Child Health and Human Development, Research Institute of the McGill University Health Centre, 2155 Guy Street, Suite 500, Montreal, Quebec, H3H 2R9, Canada, ²Division of Neurology, Department of Pediatrics, Montreal Children’s Hospital, McGill University Health Centre, 1001 Décarie Boulevard, Montreal, Quebec, H4A 3J1, Canada, ³Department of Neurology and Neurosurgery, Montreal Children’s Hospital, McGill University Health Centre, 1001 Décarie Boulevard, Montreal, Quebec, H4A 3J1, Canada, ⁴Division of Medical Genetics, Department of Specialized Medicine, McGill University Health Centre, 1001 Décarie Boulevard, Montreal, Quebec, H4A 3J1, Canada, ⁵Medical Genetics Service, Department of Laboratory Medicine, CHUS and Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, J1K 2R1, Canada., ⁶Centre de Recherche du Centre Hospitalier Universitaire de Sherbrooke (CRCHUS), 12e Avenue N Porte 6, Sherbrooke, Quebec, J1H 5N4, Canada., ⁷Department of Human Genetics, McGill University, Montreal, Quebec, Canada

*kenneth.myers@mcgill.ca

Abstract: POLG-related disorders are a group of rare neurodegenerative mitochondrial diseases caused by pathogenic variants in POLG, the gene encoding DNA polymerase gamma. Patients may experience a range of signs and symptoms, including seizures, vision loss, weakness, ataxia, neuropathy, developmental impairment or regression, and liver failure. The diseases follow a progressive, degenerative course, with patients dying a median of 5 months after symptom onset. At present, there are no effective treatments for POLG-related disorders, though some data suggest that deoxynucleoside supplementation could be beneficial. We assessed the safety and efficacy of combination therapy with deoxycytidine and deoxythymidine (dC/dT) in people with POLG-related disorders over a 6-month period as part of a long term open-label, single arm phase 2 trial. dC/dT was given enterally in powder form, dissolved in water. The primary outcome measures included Newcastle Mitochondrial Disease Scale (NMDS) score (sections I-III), serum growth differentiation factor 15 (GDF-15; a biomarker of mitochondrial dysfunction), electroencephalography (EEG), seizure diary, and blood and urine tests to assess end organ and mitochondrial function. Secondary outcome measures included recording of all adverse events to evaluate the safety of the intervention. We present data from the first ten patients enrolled in the trial, six with Alpers-Huttenlocher syndrome, two with ataxia-neuropathy spectrum, and two patients who do not fit into a classical POLG-related phenotype. During the 6 months of treatment, NMDS score improved from a mean of 27.3 at baseline to 20.7 at 6 months (p = 0.0036). The mean serum GDF-15 level decreased from 1031 pg/ml to 729 pg/ml (p = 0.048). Eight of the ten patients had abnormal baseline EEG; of these eight, improvement in EEG was seen in five. There were no significant changes in other blood and urine testing, though trends in decrease of baseline elevated liver function enzymes were observed. Regarding adverse events, two patients experienced diarrhea that spontaneously resolved. There were no other adverse events attributed to the intervention. We conclude that dC/dT is a promising treatment option for people with POLG-related disorders. Further research is needed to assess the long-term safety and efficacy in POLG-related disorders, as well as safety and efficacy in other mitochondrial DNA depletion disorders. This study was primarily funded by the Liam Foundation, with additional funding from the Savoy Foundation, Grand Défi Pierre Lavoie Foundation, and Fonds de Recherche du Québec – Santé.

Abstract #: 2024PA-0000000051

Presenter: Agustin Hidalgo-Gutierrez

Guanylate Kinase 1 Deficiency: A Novel and Potentially Treatable Mitochondrial DNA Depletion/Deletions Disease

Hidalgo-Gutierrez A¹, Shintaku J¹, Ramon J^2,3, Barriocanal-Casado E¹, Pesini A¹, Saneto R⁴, Garrabou G^2,5, Cesar Milisenda J^2,5,7, Mata Garcia A^2,5,7, Gort L^2,6, Ugarteburu O^2,6, Gu Y⁸, Koganti L⁸, Wang T⁹,Tadesse S¹, Meneri M^10,11, Sciacco M¹², Wang S⁹, Tanji K⁸, Horwitz MS¹³, Dorschner MO¹³, Mansukhani M⁸, Pietro Comi G^10,11, Ronchi D^10,11, Marti R^2,3, Ribes A^2,6, Tort F^2,6, Hirano M^1*

¹Department of Neurology, Columbia University Irving Medical Center, New York, NY, USA, ²Biomedical Network Research Centre on Rare Diseases (CIBERER), Instituto de Salud Carlos III, Madrid, Spain, ³Vall d’Hebron Research Institute, Autonomous University of Barcelona, Barcelona, Spain, ⁴Seattle Children’s Hospital, Seattle, WA, USA, ⁵Inherited Metabolic Diseases and Muscle Disorder’s Lab, Cellex – IDIBAPS. Faculty of Medicine and Health Science – University of Barcelona (UB), Barcelona, Spain, ⁶Section of Inborn Errors of Metabolism-IBC. Department of Biochemistry and Molecular Genetics. Hospital Clinic de Barcelona-IDIBAPS, Barcelona, Spain, ⁷Department of Internal Medicine, Hospital Clínic of Barcelona, Barcelona, Spain, ⁸Department of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, NY, USA, ⁹Department of Biostatistics, Mailman School of Public Health, Columbia University, New York, NY, USA, ¹⁰Dino Ferrari Center, Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy, ¹¹IRCCS Fondazione Ca’ Granda Ospedale Maggiore Policlinico, Neurology Unit, Milan, Italy, ¹²IRCCS Fondazione Ca’ Granda Ospedale Maggiore Policlinico, Neuromuscular and Rare Disease Unit, Milan, Italy, ¹³Department of Laboratory Medicine and Pathology, University of Washington, Seattle, USA

*Corresponding Author: Michio Hirano, MD, Columbia University Irving Medical Center, 630 West 168^th Street, P&S 4-423, New York, New York 10032, USA. Phone: 212-305-1048; Email:mh29@columbia.edu

Abstract: Mitochondrial DNA (mtDNA) depletion/deletions syndrome (MDDS) comprises a group of diseases due to primary autosomal defects of mtDNA maintenance^1,2. We have identified a new form MDDS due to biallelic GUK1 pathogenic variants in four probands whose clinical manifestations include: ptosis, ophthalmoparesis, myopathy, hepatopathy, and altered T-lymphocyte profile. GUK1 encodes guanylate kinase (GUK1) an essential enzyme that was originally identified as a cytosolic nucleoside monophosphate kinase (NMPK) that converts deoxyguanosine monophosphate (dGMP) to deoxyguanosine diphosphate (dGDP) and guanosine monophosphate (GMP) to guanosine diphosphate (GDP)^3,4. Long (isoform A) and short (isoform B) isoforms of GUK1 have been identified. Muscle biopsies from probands 1, 2 and 4 showed mtDNA depletion. In proband 3 and 4, mtDNA deletions were also present. All proband showed decreased activities of mitochondrial respiratory chain enzymes. In primary fibroblasts from the probands, we identified decreased guanylate kinase activity causing imbalanced mitochondrial dNTP pools and mtDNA depletion. Pathogenic features present in both replicating and postmitotic fibroblasts indicated that impaired GUK1 affected de novo nucleotide metabolism and the mitochondrial and cytosolic nucleotide salvage pathways⁵. The dual functions are consistent with our observation that the long isoform is intramitochondrial and the short isoform is cytosolic. Importantly, proband fibroblasts treated with deoxyguanosine and/or forodesine, a purine phosphatase inhibitor⁶, ameliorated mtDNA depletion, indicating potential therapeutic interventions that may also be effective for other forms of MDDS with impaired purine deoxynucleotide metabolism. Thus, our data reveal for the first time an impairment of a NMPK, the GUK1, as a new and potentially treatable cause of MDDS with novel pharmacological approaches. Additionally, we demonstrate that the long isoform is the mitochondrial guanylate kinase. Lastly, we have generated a mouse model with variants analogous to those of the most severely affected patients. We are in the process of characterizing this model and plan to test the therapeutic efficacy obtained in vitro on this model.

References

 1. DiMauro, S., Schon, E.A., Carelli, V. & Hirano, M. The clinical maze of mitochondrial neurology. Nat Rev Neurol 9, 429–44 (2013).

 2. Lopez-Gomez, C., Camara, Y., Hirano, M., Marti, R. & nd, E.W.P. 232nd ENMC international workshop: Recommendations for treatment of mitochondrial DNA maintenance disorders. 16 - 18 June 2017, Heemskerk, The Netherlands. Neuromuscul Disord 32, 609–620 (2022).

 3. Khan, N. et al. Solution structure and functional investigation of human guanylate kinase reveals allosteric networking and a crucial role for the enzyme in cancer. J Biol Chem 294, 11920–11933 (2019).

 4. Li, Y., Zhang, Y. & Yan, H. Kinetic and thermodynamic characterizations of yeast guanylate kinase. J Biol Chem 271, 28038–44 (1996).

 5. Carvalho, G., Repoles, B.M., Mendes, I. & Wanrooij, P.H. Mitochondrial DNA Instability in Mammalian Cells. Antioxid Redox Signal 36, 885-905 (2022).

 6. Iino, M., Sato, T., Nakadate, A., Onuki, T. & Takayama, N. Forodesine maintenance therapy for newly diagnosed peripheral T-cell lymphoma: a single-institutional, observational, retrospective analysis. Ann Hematol 101, 2351-2352 (2022).

Abstract #: 2024PA-0000000053

Presenter: Sandra R. Bacman

Reduction of mutant mtDNA by LNP/mRNA delivery of mitoARCUS to skeletal muscle

Bacman SR^1*, Barrera-Paez JD¹, Shoop W², Thompson, R², Moraes CT^1,3

¹Department of Neurology, University of Miami, US, ²Precision BioSciences Inc., ³Department of Cell Biology, University of Miami, US

* sbacman@med.miami.edu

Abstract: Reducing the mutant mitochondrial DNA (mtDNA) load in heteroplasmic cells below the threshold that causes a pathological phenotype is necessary to restore tissue function. We have been using different gene editing tools such as mitoTALEN and mitoARCUS delivered in vivo by viral vectors to eliminate mutant mtDNA in different organs and tissues. However, because changes in mtDNA heteroplasmy do not require long term expression of the gene editing enzyme, we explored transient gene delivery approaches. Significant progress has been achieved with in vivo delivery of mRNA and lipid nanoparticles (LNPs) which contain ionizable lipids. We encapsulated mRNA encoding a mitoARCUS that recognizes and cleaves the mouse mitochondrial tRNA^Ala gene m.5024C>T mutation in LNPs. Mice heteroplasmic for the tRNA^Ala mutation (60-70% of m.5024T) were injected either systemically or intramuscularly with LNP-mitoARCUS or an LNP-GFP control.

Systemic injections caused a rapid and transient expression of mitoARCUS in the liver that induced a robust shift in heteroplasmy (~90%) at 14 days post-injection. A significant decrease in mutant mtDNA (20%) was also observed in the spleen. No change in total mtDNA copy number was observed in these tissues.

Next, we attempted intramuscular injections of these LNP formulations in the Tibialis Anterior (TA) muscle. The right TA (injected with LNP-mitoARCUS) showed a significant decrease (40%) in mutant mtDNA levels compared to the left TA (injected with LNP-GFP). This decrease in mutant mtDNA was observed as early as 7 days after intramuscular injection and was still observed after 120 days. Liver mtDNA heteroplasmy was also decreased after intramuscular injection with LNP-mitoARCUS. There was no change in total mitochondrial copy number between right and left TA at any point after injection. After both systemic and intramuscular injections with LNP-mitoARCUS, the levels of tRNA^Ala, which are destabilized by the m.5024T mutation, were recovered in the liver and muscle, reflecting the increase in wild-type mtDNA.

This study shows that LNPs carrying mitoARCUS mRNA produce a transient expression of mitoARCUS that can markedly reduce mutant mtDNA levels in skeletal muscle and liver and improve molecular phenotypes.

Abstract #: 2024PA-0000000055

Presenters: Malinda Marsh and Annika Knuth

Leber Hereditary Optic Neuropathy (LHON) Scientific Retreat

Marsh, M.^1*, Knuth, A.^2*, Poincenot, L.¹, Barrett, C., PhD³, and Penrod, N., PhD³

¹LHON Collective, USA, ²Patient Advocacy Initiative, University of Notre Dame, USA, ³Science Philanthropy Accelerator for Research and Collaboration, Milken Institute, US

^* Mmarsh@lhon.org

Abstract: Leber hereditary optic neuropathy (LHON) is a rare, inherited mitochondrial disease that primarily affects the optic nerve and can lead to sudden and severe vision loss. LHON is most often caused by disease-specific mutations in mitochondrial DNA (mtDNA), though other genetic variants have also been associated with LHON pathology. There is no current cure for LHON, and there are no FDA-approved therapies. Research in the LHON field is being performed at the foundational biology, translational, and clinical levels, but there is a need for additional research and a funding strategy to accelerate progress toward new therapeutics. LHON Collective partnered with the Milken Institute’s Science Philanthropy Accelerator for Research and Collaboration (SPARC) in 2023 to evaluate the strengths, resources, and needs in the LHON field to identify opportunities where investment in research can transform the therapeutic landscape. We began with a landscape analysis consisting of a series of over 70 interviews with a diverse array of scientific experts. Following this landscape analysis, we convened a select, global group of scientists and clinicians with complementary expertise in LHON and/or aligned diseases or biological mechanisms, model systems, and artificial intelligence and big data, to gather insights to inform priority targets for philanthropic investment at the LHON Scientific Retreat, which took place in February 2024 in Lisbon, Portugal. This retreat introduced new perspectives, allowing experts in aligned, but sometimes disparate, fields to discuss and expand upon current knowledge, and explore mechanisms for advancing research and, ultimately, therapeutic development for LHON. This two-day retreat was a discussion-based forum for cross-disciplinary exchange, featuring short presentations and lightning talks with an emphasis on facilitated group discussions and breakout group activities. Participants’ input was categorized into the following: natural history and patient stratification, model systems to accelerate therapeutic discovery and development, and global research networks and interdisciplinary research ecosystems. Each category was discussed, potential barriers were addressed, and goals and future scientific opportunities were identified. These prioritized areas will lead to better data collection, more reliable models, a better understanding of the genetic causes of disease and mechanisms of pathogenesis, and alignment with other disease areas - all of which are important factors for de-risking translational research and attracting industry partners. Based on the landscape analysis and findings from the LHON Scientific Retreat, we will identify a strategic direction for the most promising opportunities to move scientific research towards a cure for this rare mitochondrial disease. This systematic approach to evaluate LHON and identify its current milestone progress as well as develop expert scientific consensus on future approaches is a model to share with other rare disease communities and the scientific community supporting those rare disease initiatives. It could help focus financial support and scientific approaches to experience the greatest impact towards scientific progress. This approach can provide clarity of scientific short- and long-term goals for philanthropic, bio pharmaceutical and governmental funding to be deployed in a targeted manner with expert-identified consensus on endpoints that show the most promise.

Abstract #: 2024PA-0000000058

Presenter: Laura MacMullen

Elamipretide via Open Label Expanded Access Program in Patients with Genetically-Confirmed Primary Mitochondrial Disorders

MacMullen, LE¹, Stanley, KD¹, Santos, JD¹, Flickinger, J¹, Nguyen, S¹, Muraresku, C¹, Tormey, C¹, Demczko, M^1,2, Zolkipli-Cunningham, Z^1,2, Falk, MJ^1,2, Goldstein, AC^1,2*

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA, USA, ²Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA

*goldsteina@chop.edu

Abstract: While substantial progress has been made in the therapeutic development pipeline for Primary Mitochondrial Disease (PMD), clinical trials remain limited and are often inaccessible to many PMD patients related to eligibility criteria. Expanded Access Programs (EAPs) provide a crucial pathway for providing investigational treatments to PMD patients including those who may fail to meet clinical trial eligibility criteria.

Elamipretide is a small peptide compound delivered via daily subcutaneous injection to target the inner mitochondrial membrane and reversibly bind cardiolipin¹. A total of 28 genetically-confirmed PMD patients have been enrolled into the Elamipretide EAP (Stealth BioTherapeutics) at the Children’s Hospital of Philadelphia (CHOP). 22/28 (79%) patients are actively receiving treatment. 6/28 (21%) individuals passed away from disease progression that was deemed unrelated to treatment. Age at treatment initiation ranged from 0.95 to 66 years. EAP treatment duration to date ranges from 9 to 1,144 days. Patients completed the Primary Mitochondrial Myopathy Symptom Assessment (PMMSA), the 5-level EQ-5D version (EQ-5D-5L), and as standard of care at routine clinic visits completed the Mitochondrial Myopathy-Composite Assessment Tool (MM-COAST)².

Interim analysis of available MM-COAST results in 6/22 (27%) CHOP EAP active study patients revealed that 3/6 (50%) demonstrated improvement, as evidenced by decreased MM-COAST composite score after treatment initiation (a decrease by 1.08 points at 12 months; 0.32 at 12 months, and 0.5 at 20 months in patients 1-3, respectively). By comparison, our longitudinal MM-COAST composite scores of 86 untreated PMD patients demonstrated an increase in composite scores by 0.09 points/year, consistent with incremental progression (Zolkipli-Cunningham et al, unpublished). In the remaining 3/6 patients, two patients showed disease progression (increased 0.77 points at 1 month and 0.7 at 6 months, patients #4 and #5, respectively) and one patient showed disease stabilization (minimal increase of 0.05 at 6 months in patient #6). 6-minute walk test (6MWT) data was available on the 4 ambulatory patients who completed post-treatment MM-COAST assessments. Patients #2 and #3 showed improvement (+38m, +0.5 z-score at 12 months; +97m at 20 months, +0.77 z-score, respectively) while patients #5 and #6 demonstrated decline at 6 months (-72 m, -0.5 z-score; -8m, -0.09 z-score, respectively).

Not all 22 patients were followed with the same outcome assessments, depending on reported symptoms, compliance, and frequency of clinical follow-up visits. Six of 12 (50%) of CHOP EAP patients who completed multiple PMMSA assessments showed improved PMMSA Fatigue 4 summary score (4FS), while 3/12 (25%) showed no change and 3/12 (25%) showed worsening fatigue. Twelve patients (12/18, 67%) reported improvement in their most bothersome symptom, 4/18 (22%) reported no change, and 2/18 (11%) reported worsening. Fifteen patients (15/18, 83%) reported improved mobility on the EQ-5D-5L.

In summary, EAP INDs play an important role in making investigational therapies accessible to PMD patients with pronounced disease burden for whom no effective therapies are available. While interpretation of EAP data is challenging due to real-world data collection and a higher representation of patients in advanced stages of disease, our preliminary descriptive results suggest potential benefits of Elamipretide occurs in some PMD individuals.

References

 1. Szeto HH. First-in-class cardiolipin-protective compound as a therapeutic agent to restore mitochondrial bioenergetics. Br J Pharmacol. 2014; 171: 2029–2050.

 2. Flickinger J, Fan J, Wellik A, et al. Development of a Mitochondrial Myopathy-Composite Assessment Tool. JCSM Clin Rep. 2021; 6(4): 109–127.

Abstract #: 2024PA-0000000059

Presenter: Imon Rahaman

Digital gait and balance sensors characterize fatigue and enhance MM-COAST data in primary mitochondrial disease

Rahaman, I.¹, Flickinger, J.^1,2, Santos, J.D.², Martin, I.², O’Leary, S.^1,2, Fogliano, J.^1,2, Katelynn Stanley¹, Sara Nguyen¹, MacMullen, L¹, Xiao, R.^3,4, Zolkipli-Cunningham, Z.^2,5*

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Children’s Hospital of Philadelphia, USA, ²Division of Rehabilitation, Children’s Hospital of Philadelphia, USA, ³Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, USA, ⁴Department of Biostatistics, Epidemiology and Informatics, University of Pennsylvania Perelman School of Medicine, USA

*zolkipliz@chop.edu

Abstract: Digital and wearable technologies have been increasingly used in Multiple Sclerosis¹ and Parkinson’s disease clinical trials.^2,3,4 While assessment of gait analysis (GaitRite) has been previously studied in Primary Mitochondrial Disease (PMD)^5,6, its clinical validation against a PMD-specific objective outcome measure has not been studied. We performed assessments using the Zeno^TM Walkway⁷ and the mSway^TM inertial sensor⁸ in genetically confirmed PMD and self-reported PMD (SR-PMD) cohorts compared to healthy controls alongside a validated, PMD-specific outcome measure, the Mitochondrial Myopathy Composite Assessment Tool (MM-COAST).⁹ This study was conducted at the inaugural Clinical Research Pavilion at the 2023 United Mitochondrial Disease Foundation (UMDF) Annual Meeting. Subjects completed a six-minute walk test (6MWT) incorporating the Zeno walkway and a 60 second sit-to-stand (60s STS) test with mSway sensors secured to their waist. A total of 19 PMD (mean age ± SD, 23.4 ± 15.1 years, 36.8% female), 20 SR-PMD (39.2 ± 18.39 years, 80% female), and 22 control subjects (45.2 ± 14.7 years, 63.6% female) completed 6MWT assessments, along with MM-COAST assessments. A subgroup of 7 PMD (33.7 ± 17.9 years, 28.57% female), 20 SR-PMD (39.2 ± 18.39 years, 80% female), and 17 control subjects (45.05 ± 16.49 years, 70.58% female) completed 60s STS with mSway. For 6MWT, all gait characteristics including stride width (cm), length (cm), velocity (cm/sec), cadence (steps/min), stance percentage (%), single support percentage (SSP%), and single support center of pressure distance percentage⁸ (SSCOPD%) were found to be significantly different between PMD (n=19) and controls (n=22), p<0.05. The latter three parameters relate to balance. Linear mixed-effects models (LMMs) for repeated measures revealed significant decline from 1 to 6 minutes (slope) in PMD subjects for stride velocity (-1.21 cm/s/min, p=0.03) and trended downward for cadence (-0.94 steps/min², p=0.05) and SSCOPD% (-0.41 %/min, p=0.05), demonstrating increasing fatigue and imbalance over time compared to control subjects with no significant change, p>0.05. In the PMD cohort, we found significant positive and negative correlations between all gait parameters with MM-COAST measures including 6MWT z-scores (positive r ranges, negative r ranges), r₊= [0.62, 0.89], r=[-0.77, -0.74]), composite scores (r₊=[0.49,0.59], r_-=[-0.82, -0.56]), and tandem stance eyes closed balance scores (r₊=[0.46,0.68], r_-= [-0.53, -0.46]), p<0.05 which demonstrates the clinical meaning of the Zeno gait assessment. Preliminary analyses of mSway data for 60s STS included range of vertical acceleration (m/s²) and normalized vertical jerk index⁴ that were significantly slower, with less variability in movements in PMD and SR-PMD subjects compared to controls, p <0.05. LMM analysis revealed a significant decrease over 60s (slope) of range of vertical acceleration for PMD and SR-PMD (-2.18 m/s²/min, p=0.004), demonstrating muscle fatigue, compared to controls who demonstrated an increase over 60 seconds (2.37 m/s²/min, p=0.016). Our preliminary results demonstrate feasibility and clinical meaning of the Zeno walkway and mHealth sensors in the gait and balance assessment of PMD, with ability to enhance the MM-COAST by quantifying a range of deficits not able to be captured by standard clinical assessments. Digital assessments of gait and balance may prove useful for future PMD clinical trials.

References

 1. Hadouiri N, Monnet E, Gouelle A, Sagawa Y, Decavel P. Locomotor strategy to perform 6-minute walk test in people with multiple sclerosis: a prospective observational study. Sensors 2023; 23(7): 3407. Syeda HB, Glover A, Pillai L, Kemp AS, Spencer H, Lotia M, Larson-Prior LJ, Virmani T.

 2. Baudendistel ST, Schmitt AC, Balthaser KC, Wade FE, Hass CJ. The effect of limb selection methods on gait analysis in Parkinson’s disease. Parkinsonism & Related Disorders 2022; published online 12 October 2022.

 3. Sabo A, Gorodetsky C, Fasano A, Iaboni A, Taati B. Concurrent validity of Zeno instrumented walkway and video-based gait features in adults with Parkinson’s disease. IEEE Journal of Translational Engineering in Health and Medicine 2022; 10: 1–11.

 4. Palmerini L, Mellone S, Avanzolini G, Valzania F, Chiari L. Quantification of motor impairment in Parkinson’s disease using an instrumented timed up and go test. IEEE Trans Neural Syst Rehabil Eng. 2013 Jul;21(4):664–73. doi: 10.1109/TNSRE.2012.2236577. Epub 2013 Jan 1. PMID: 23292821.

5. Koene S, Stolwijk NM, Ramakers R, de Vries M, de Boer L, Janssen MCH, de Groot I, Smeitink J. Quantification of gait in children with mitochondrial disease. J Inherit Metab Dis. 2018 Jul; 41(4): 731–740. doi: 10.1007/s10545-018-0148-5. Epub 2018 Mar 12. PMID: 29532198.

6. Galna B, Newman J, Jakovljevic DG, Bates MG, Schaefer AM, McFarland R, Turnbull DM, Trenell MI, Gorman GS, Rochester L. Discrete gait characteristics are associated with m.3243A>G and m.8344A>G variants of mitochondrial disease and its pathological consequences. J Neurol. 2014 Jan; 261(1): 73–82. doi: 10.1007/s00415-013-7129-2. Epub 2013 Oct 23. PMID: 24150688; PMCID: PMC3895207.

 7. Lynall RC, Zukowski LA, Plummer P, Mihalik JP. Reliability and validity of the Protokinetics movement analysis software in measuring center of pressure during walking. Gait Posture. 2017;52:308–311.

 8. Palmerini L, Rocchi L, Mellone S, Valzania F, Chiari L. Feature selection for accelerometer-based posture analysis in Parkinson’s disease. IEEE Trans Inf Technol Biomed. 2011 May;15(3):481-90. doi: 10.1109/TITB.2011.2107916. Epub 2011 Feb 24. PMID: 21349795.

 9. Flickinger, J., Fan, J., Wellik, A., Ganetzky, R., Goldstein, A., Muraresku, C. C., Glanzman, A. M., Ballance, E., Leonhardt, K., McCormick, E. M., Soreth, B., Nguyen, S., Gornish, J., George-Sankoh, I., Peterson, J., MacMullen, L. E., Vishnubhatt, S., McBride, M., Haas, R., Falk, M. J., Xiao, R., and Zolkipli-Cunningham, Z. (2021) Development of a Mitochondrial Myopathy-Composite Assessment Tool. JCSM Clinical Reports, 6: 109–127.

Abstract #: 2024PA-0000000062

Presenter: Allison Hanaford, PhD

Elucidating the role of specific immune populations in CNS and peripheral disease pathogenesis in the Ndufs4(-/-) model of Leigh syndrome

Hanaford AR¹, Khanna A², Liao, R¹, Ching, A¹, Huang S¹, James K¹, Kayser B¹, Truong V⁷, Sedensky M^1,3, Morgan P^1,3, Kalia V^2,4, Sarkar S^2,4, and Johnson SC^5*

¹Center for Integrative Brain Research, Seattle Children’s Research Institute, Seattle, WA, USA, ²Ben Towne Center for Childhood Cancer Research, Seattle Children’s Research Institute, Seattle, WA, USA, ³Department of Anesthesiology and Pain Medicine, University of Washington, Seattle, WA, USA,⁴Department of Pediatrics, University of Washington School of Medicine, Seattle, WA, USA, ⁵Department of Applied Sciences, Translational Bioscience, Northumbria University, Newcastle, UK

*simon.c.johnson@northumbria.ac.uk

Abstract: Subacute necrotizing encephalopathy, or Leigh syndrome (LS), is the most common pediatric presentation of genetic mitochondrial disease. LS is a multi-system disorder with severe neurologic, metabolic, and musculoskeletal symptoms. The presence of progressive, symmetric, necrotizing lesions in the brainstem are a defining feature of the disease, and the major cause of morbidity and mortality, but the mechanisms underlying their pathogenesis have been elusive. Our lab has demonstrated that the immune system plays a key role in the onset and progression and CNS disease in the Ndufs4 knockout (KO) model of LS. Pharmacologic elimination of circulating and peripheral myeloid cells by the CSF1R inhibitor pexidartinib prevents CNS and systemic disease in Ndufs4(KO) mice. Pexidartinib treatment results in depletion of microglia and peripheral macrophage populations, with lower doses considered to target primarily microglia and higher doses targeting circulating macrophage populations in addition to microglia. Response to pexidartinib was dose dependent, implicating the involvement of peripheral immune cells in addition to microglia in LS pathology. To identify the specific immune cell populations contributing to disease pathogenesis in the Ndufs4(KO) mouse, we used genetic and pharmacologic tools. Ndufs4(KO) mice were crossed with genetically engineered mouse models lacking specific immune cell types or signalling components and disease onset and progression monitored. We also used monoclonal antibodies and small molecules to deplete specific cell types in Ndufs4(KO) mice and monitored the impact on disease pathology. These studies revealed that microglia and innate immune cells of peripheral origin both contribute to Ndufs4(KO) disease pathogenesis. We found no evidence for involvement of the adaptive immune system in Ndufs4(KO) pathology. These data support the concept of LS as an innate immune system driven disease and support further investigation into immune modulatory therapeutics. Clinical case reports and other data suggest immune modulatory therapies are efficacious in multiple mitochondrial diseases and that inflammation and the immune system may drive the pathogenesis of multiple mitochondrial diseases, indicating our findings may be relevant to mitochondrial diseases beyond LS. The data presented here have important implications for any future efforts to translate immune-targeting strategies into treatments for genetic mitochondrial disease.

Abstract #: 2024PA-0000000063

Presenter: Lía Mayorga

Manipulating the Nuclear Epigenome: A Novel Approach for Shifting Mitochondrial DNA Heteroplasmy

Pérez MJ^1,2, Colombo RB¹, Branham MT^1,3, Moraes CT⁴, Mayorga L¹

¹Instituto de Histología y Embriología de Mendoza (IHEM, Universidad Nacional de Cuyo, CONICET)- Mendoza, Argentina, ²Facultad de Ciencias de la Nutrición. Universidad Juan Agustín Maza. Mendoza-Argentina, ³Facultad de Ciencias Médicas. Universidad de Mendoza. Mendoza-Argentina, ⁴Department of Neurology, University of Miami Miller School of Medicine, Miami, Florida, USA

*liamayorga@fcm.uncu.edu.ar

Abstract: Mitochondrial disorders (MD) arise from mutations in either nuclear or mitochondrial DNA (mtDNA). The coexistence of disease-causing mutations alongside wild-type mtDNA molecules, known as heteroplasmy, dictates the onset of pathology once it exceeds a certain threshold. Hence, reducing the proportion of mutant to wild-type mtDNA, termed “heteroplasmy shift,” emerges as a promising therapeutic avenue. While gene therapy targeting nuclear genes has made strides in clinical applications across various diseases, editing mtDNA has proven more challenging. Evidence suggests that varying degrees of mitochondrial stress trigger specific modifications in the nuclear epigenome. Notably, we have proposed nuclear DNA methylation patterns as an adaptive response to chronic mitochondrial stress. Given that high heteroplasmy correlates with severe mitochondrial dysfunction, we hypothesized that cells experiencing heightened stress activate this epigenetic mechanism. Our proposal aims to disrupt this epigenetic rearrangement, promoting the survival of cells with lower heteroplasmy, a process we term “epigenetically-mediated cellular heteroplasmy shift.”

We characterized the nuclear DNA methylation of cybrid cells with the m.13513G>A and the m.8344A>G at different heteroplasmy levels using Infinium 850k Methylation EPIC array®. Gene enrichment analysis of the differentially methylated regions (DMR) was performed with Metascape®. Proliferation (Fluorometric Cell Proliferation Assay Kit) and apoptosis (Annexin V) were investigated in response to DNA methylation inhibitors (DMIs) 5-Azacytidine and Decitabine (m.13513G>A and m.8344A>G cybrids) and in response to DNA methyltransferase 1 (DNMT1) inducible down-regulation (m.13513G>A cybrids). Mutation load was measured using RT-qPCR upon treatment with DMIs and DNMT1 down regulation.

The nuclear DNA methylation pattern of the m.13513G>A and m.8344A>G cybrids distinctively separated the samples according to their mutation load and mutation type. Enrichment analysis of the DMR (high versus low heteroplasmy) showed hypermethylation of tissue differentiation pathways and apoptotic genes.

High heteroplasmy cells (m.13513G>A and m.8344A>G) displayed heightened proliferation rates relative to their low heteroplasmy counterparts (p<0.05), situation that was abolished upon treatment with DMIs. In m.13513G>A cybrids, high heteroplasmy cells exhibited reduced apoptosis (p<0.05), which increased upon exposure to DMIs (p<0.05). In a heteroplasmy mosaic scenario involving m.13513G>A cybrids, treatment with Decitabine reduced heteroplasmy ~20%, p<0.01. DNMT1 downregulation cells reduced it ~30% (n=2).

We conclude that there is a distinctive nuclear DNA methylation pattern associated with the level of heteroplasmy in cybrid cells. This epigenetic programming seems to induce an adaptive pro-survival response that is proportional to the heteroplasmy level. When this pattern is disrupted, this adaptive advantage appears to be lost for the high heteroplasmy cells, resulting in a decrease of heteroplasmy.

Abstract #: 2024PA-0000000064

Presenter: Cristina Remes

Preclinical evaluation of dichloroacetate in two C. elegans models of pyruvate dehydrogenase deficiency

Cristina Remes¹, Neal D. Matthew¹, Eiko Nakamaru-Ogiso^1,2, Marni J. Falk^1,2

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA, ²Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA

*remesc@chop.edu

Abstract: Introduction: Pyruvate dehydrogenase complex (PDHc) deficiency is a mitochondrial disorder primarily affecting the brain, liver, and intermediary metabolism. We sought to provide confirmatory, objective, pre-clinical evidence of dose-dependent safety and therapeutic efficacy of a PDHc activator, dichloroacetate (DCA) as a treatment for PDHc deficiency. Two C. elegans RNA interference (RNAi) knockdown strains, pdha-1 and dld-1, were used to model PDHc deficiency and evaluate the efficacy of DCA on animal health.

Methods: Silencing of pdha-1 and dld-1 genes in C. elegans was achieved by feeding RNAi. To titrate the levels of dld-1 knockdown, 3 different concentrations of RNAi bacteria were studied: full dose, 1:20 and 1:100 dilution. Wild-type, dld-1, and pdha-1 knockdown worms were grown from egg stage on solid media NGM plates and treated with DCA ((10 µM, 100 µM, 1 mM, 5 mM, 25 mM) or buffer control seeded with the corresponding RNAi E. coli until Day 1 adult stage. Worm development was quantified by measuring length at stage L4+1. To quantify in vivo mitochondrial unfolded protein response (UPR^mt) stress, green fluorescence was quantified in C. elegans carrying hsp6p::GFP reporter. Activity was analyzed using a thrashing assay to quantify body bends per second in liquid media. C. elegans strains’ lifespans were analyzed in presence of FUDR using an automated image acquisition system (WormScan). DCA treatment effects were evaluated on worm lactate and pyruvate levels.

Results: Knockdown of pdha-1 and dld-1 in C. elegans reduced animal linear growth, decreased worm swimming activity, and reduced mitochondrial health. Increased lactate, pyruvate, and pyruvate:lactate ratio was seen in pdha-1 deficient worms, with decreased lactate levels in dld-1 knockdown worms. While no significant lifespan effect was seen in pdha-1 and dld-1(20x or 100x) worms, full dose dld-1 knockdown increased worm lifespan. DCA treatment had no gross morphologic toxicity, and significantly improved worm linear growth and swim activity. Decreased mitochondrial stress was seen upon DCA treatment at all doses in both pdha-1 and dld-1 (1:100) knockdown strains, without significant rescue for the dld-1 (full dose). DCA treatment at 25 mM in dld-1 and pdha-1 knockdown strains decreased lactate levels with no effect on elevated the pyruvate level in dld-1 worms, and significantly decreased lactate levels in dld-1 (1:20 and 1:100 RNAi).

Conclusions: Preclinical modeling demonstrated that DCA is a promising therapeutic candidate for PDHc deficiency, with significant beneficial effects on C. elegans survival, neuromuscular activity, development, and mitochondrial physiology in dld-1 and pdha-1 deficient models.

Abstract #: 2024PA-0000000065

Presenter: Cristina Remes

Characterization of 19 mitochondrial aminoacyl-tRNA synthetases in C. elegans and the effect of specific amino acid treatment on mt-ARS deficiencies

Cristina Remes¹, Neal D. Matthew¹, Shannon Schrope¹, Eiko Nakamaru-Ogiso^1,2, Marni J. Falk^1,2

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA, ²Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA

*remesc@chop.edu

Abstract: Introduction: Mitochondrial aminoacyl-tRNA synthetases (mt-ARS) are essential components of the mitochondrial translation machinery that catalyze charging of mitochondrial transfer RNAs (tRNAs) with their cognate amino acid. Although mt-ARS has a common biochemical function, patients with mt-ARS mutations develop neurological disorders with a wide range of clinical phenotypes, severity, and age of onset. To systematically characterize the function of each mt-ARS gene, we knocked down the full set of 19 conserved mt-ARS genes using feeding RNA interference (RNAi) in the C. elegans (worm) model organism. In addition, we used these C. elegans models to investigate the effect of specific cognate amino acids as a potential treatment for mt-ARS deficiencies.

Methods: Individual silencing of19 mt-ARS genes in C. elegans was achieved by feeding RNAi specific for each mt-ARS gene for one, two or three generations. We assessed C. elegans knockdown strains development by measuring worm development (length) at larval stage L4. To quantify the degree of in vivo mitochondrial unfolded protein response (UPR^mt) stress induction, we used C. elegans carrying the hsp6p: GFP reporter upon knockdown of each mt-ARS gene. Worm activity was analyzed using a thrashing assay to quantify body bends per second when worms were swimming in liquid media. C. elegans lifespan was analyzed in the presence of FUDR (to prevent progeny development) using an automated image acquisition system (WormScan).

Results: Inhibition of each mt-ARS gene significantly decreased worm length by approximately 20% at larval stage L4, suggesting animal development was delayed in the knockdown strains. Variable levels of UPR^mt stress induction and lifespan effects occurred upon inhibition of each mt-ARS gene. Among the 19 mt-ARS genes, the strongest activation of UPR^mt stress response occurred in mitochondrial aspartyl-tRNA synthetase (DARS2) knockdown worms, which had an approximately 10-fold higher hsp6p::GFP fluorescence induction relative to other mt-ARS knockdown strains screened. In addition, knockdown of hars1, mars2, fars2, kars1 and cars2 resulted in a nearly complete sterile phenotype in C. elegans. Cognate amino acid treatment effects were evaluated on animal neuromuscular activity, development, mitochondrial physiology, and progeny count. Preliminary data showed that multiple mt-ARS knockdown strains significantly improved overall animal health and reduced mitochondrial stress upon treatment with the cognate amino acid, in a dose-dependent fashion.

Conclusions: The comparative impact of inhibiting expression of 19 mt-ARS genes were evaluated on animal development, UPR^mt stress induction, and animal activity in C. elegans. A consistent degree of developmental (growth) delay was seen across all strains, with variable degrees of UPR^mt stress induction and lifespan alteration upon knockdown of individual mt-ARS genes. Importantly, these in vivo treatment data provide specific evidence to demonstrate treatment with their cognate amino acids is a potential therapy for targeting mt-ARS deficiencies.

Abstract #: 2024PA-0000000066

Presenter: Barrera-Paez JD

The mitoDdCBE system as a mitochondrial gene therapy approach

Barrera-Paez JD¹, Bacman SR², Balla T³, Mok BY², Liu DR², Booven DV¹, Griswold AJ¹, Nedialkova D³ and Moraes CT²*

¹Department of Genetics, University of Miami Miller School of Medicine, USA, ²Department of Neurology, University of Miami Miller School of Medicine, USA, ³Max Planck Institute of Biochemistry, Germany, ⁴Broad Institute, Harvard University, and HHMI, USA

*cmoraes@med.miami.edu

Abstract: The mitoDdCBE system was the first base editor developed to alter mtDNA. It is composed of a heterodimer in which each half of a split cytidine deaminase is reconstituted at a target DNA region specified by a pair of TALE DNA binding domains to induce cytosine-to-thymine transitions (C-to-T). We tested the therapeutic potential of this mitochondrial base editor in one of the few existing animal models of heteroplasmic mtDNA mutations, the m.5024T mutant mouse. This mutation falls within the mitochondrial tRNA alanine (mt-tRNA^Ala) gene and destabilizes the tRNA by disrupting a Watson & Crick base pairing in its secondary structure. Given that the m.5024T mutation is a C-to-T transition and cannot be directly corrected by mitoDdCBE, we generated instead a compensatory m.5081C-to-T edit to restore the secondary structure of the molecule.

AAV9-DdCBE viral preparations were administered to young m.5024T mice by a retro-orbital injection. Three months after the injections, Western Blotting and Sanger sequencing showed that different muscle tissues (heart and tibialis anterior) were successfully transduced and edited, whereas no expression nor editing was observed on the liver. Using several AAV9-DdCBE dosages, we confirmed a correlation between the AAV9 dose, and the amount of on-target editing achieved. Moreover, Whole Genome Sequencing results extended this association with the amount of concomitant off-target editing generated. We found that the highest AAV9-DdCBE dosage triggered not only extensive off-target editing in the mtDNA but also a myopathic phenotype with a corresponding lower steady-state levels of COX1 and NDUFB8 proteins, while lower AAV doses were well tolerated in vivo. As expected, there was a linear correlation between the levels of mt-tRNA^Ala and the amount of on-target editing generated in these mice. Further Next Generation Sequencing data generated using a specialized tRNAseq pipeline confirmed an increase in stability in the edited mt-tRNA^Ala and showed that aminoacylation was not affected by the presence of mitoDdCBE-related edits.

In summary, we have successfully edited a cytosine in the mouse mtDNA that acts as a second site suppressor for the pathological m.5024C>T. We are showing, for the first time, that the mitoDdCBE system has therapeutic potential in vivo, but that it is crucial to thoroughly assess off-target editing performance to avoid a negative outcome.

Abstract #: 2024PA-0000000068

Presenter: Ankit Sabharwal

Fishing for Cures: Zebrafish as a Pioneering In Vivo Model to Help Solve Mitochondrial Medicine Odysseys

Sabharwal A^1* Wishman M², Cervera RL², Kar B², Holmberg S², Savage K², Thulung LR², Anderson J³, Farber S³, Ogiso E⁴, Clark K², Ekker SC ^1*

¹Department of Pediatrics, Dell Medical School, The University of Texas at Austin, USA, ²Mayo Clinic, Rochester, Minnesota, USA, ³Johns Hopkins University, Maryland, USA, ⁴Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA

*ankit.sabharwal@auastin.utexas.edu; stephen.ekker@austin.utexas.edu

Abstract: Mitochondria play an important but still largely mysterious role in human physiology, as demonstrated by the enormous biological variation and diverse disorders in patients with mitochondrial disease. Understanding how mitochondria function in normal biology and how human mitochondrial DNA variations contribute to health and disease has been hampered by a few effective approaches to manipulate mtDNA and a lack of existing animal models. Here in this study, we have established disease models caused by nuclear encoded mitochondrial gene and mitochondrial DNA. We present and characterize a new genetic revertible animal model that recapitulates components of Leigh Syndrome French Canadian Type (LSFC), a mitochondrial disorder that includes diagnostic liver dysfunction. lrpprc zebrafish homozygous mutants displayed biochemical and mitochondrial phenotypes similar to clinical manifestations observed in patients, including dysfunction in lipid homeostasis. We were able to rescue these phenotypes in the disease model using a liver-specific genetic model therapy, functionally demonstrating a previously under-recognized critical role for the liver in the pathophysiology of this disease. Understanding the molecular mechanism of the liver-mediated genetic rescue underscores the potential to improve the clinical diagnostic and therapeutic developments for patients suffering from these devastating disorders. Contrary to the wide success of modeling nuclear encoded disorders, editing mtDNA has been a daunting affair due to various challenges posed by the mitochondrial genetics such as random segregation and lack of import of exogenous nucleic acids. However, the advent of mtDNA base editors harnessing the protein import machinery has enabled a series of new cellular and animal models. With advanced methods and effective delivery, near-complete editing efficiency we have demonstrated of introducing over 80% programmed editing (heteroplasmy) in the pioneering animal the zebrafish (Danio rerio). This has enabled us to the establishment of the first designer in vivo models of mtDNA disease. Together, these gene editors and in vivo avatars will enable new approaches for diagnoses and therapies for these terrible diseases. We have extended this work to explore detailed germline transmission analyses with near-complete mutant mtDNA heteroplasmy in F1 animals (over 80%). Zebrafish mtDNA mutants not only show impaired mitochondrial bioenergetics but have an altered transcriptomic signature pertaining to respiration and cholesterol synthesis pathways. Interestingly, zebrafish mutants display differential segregation of pathogenic edits with distinct transcriptomic signatures. The loss of function models for protein coding mtDNA genes enables us to understand the moonlighting roles of these proteins in cellular homeostasis. Using these zebrafish disease models, we are currently exploring the cell specific heterogeneity of segregation of pathogenic mtDNA mutations in disorders such as Leber’s Hereditary Optic Neuropathy (LHON). We have established the first series of zebrafish mitochondrial diseases models, and we are making a series of additional validated zebrafish lines harboring designer mtDNA variants suitable for hypothesis testing as well as discovery science.

Abstract #: 2024PA-0000000069

Presenter: Jean Flickinger

Validation of the Mitochondrial Myopathy Function Scale

Flickinger J^1,2, Santos JD², Martin I², Ballance E^1,2, Intarachumnum N², Xiao R^3,4, Zolkipli-Cunningham Z^1,3*

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Children’s Hospital of Philadelphia, USA, ²Division of Rehabilitation, Children’s Hospital of Philadelphia, USA, ³Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, USA, ⁴Department of Biostatistics, Epidemiology and Informatics, University of Pennsylvania Perelman School of Medicine, USA

*zolkipliz@chop.edu

Abstract: Motor function assessment tools, such as the North Star Ambulatory Assessment (NSAA)¹ for Duchenne Muscular Dystrophy (DMD), have been essential for monitoring disease progression and treatment response. There are currently no existing Mitochondrial Myopathy (MM)-specific motor function measures. The MM-Composite Assessment Tool (MM-COAST) was the first objective outcome measure validated in MM that quantifies impairments in muscle strength, fatigue, exercise intolerance, dexterity, and balance.² However, we are lacking a motor function assessment tool to quantify the consequences of having these MM impairments.

We trialed the NSAA, but observed a ceiling effect as DMD patients are weaker than those with MM. We developed the Mitochondrial Myopathy-Function Scale (MM-Function), to provide a global score of MM motor function. Our initial version consisted of strength-based items. Later iterations included fine motor and endurance-based items to capture the broad functional deficits of MM. The final MM-Function Scale consists of 26 items and yields ordinal data on a scale of 0-3.

We validated MM-Function in 104 genetically confirmed MM subjects including children (n=58, mean age ± SD, 11.73 ± 3.76 years, 48.3% male) and adults (n=46, 38.10 ± 15.01, 26.1% male). Across the cohort, 66 (63.5%) harbored mitochondrial (mt)DNA and 38 (36.5%) nuclear genetic etiologies. Six subjects were non-ambulatory. MM-Function mean score (± SEM) was 55.8 ± 1.9 (71.5%, n=104) of a total score of 78, a higher score indicating better overall motor function. There was no significant difference between child (56.0 ± 2.5, n=58) and adult scores (55.5 ± 3.1, n=46), p=0.91, or between nuclear and mtDNA etiologies (p=0.06). We found a strong positive correlation between MM-Function and NSAA scores (r=0.91, p<0.001, n=48). Notably, we observed a strong negative correlation between MM-Function and MM-COAST Composite Scores, where a high Composite Score indicates greater MM disease severity (r=-0.82, p<0.001, n=101). Specifically, MM-Function scores significantly correlated with each of the MM-COAST individual test z-scores (p<0.001), including dominant strength of elbow flexion (r=0.55, n=96), wrist extension (r=0.47, n=94), hip flexion (r=0.55, n=91), and ankle dorsiflexion (r=0.61, n=92); elbow flexion repetitions for fatigue (r=0.48, n=81); single leg eyes closed (r=0.60, n=100), tandem stance eyes closed (r=0.52, n=100), and tandem stance eyes open (r=0.77, n=54) for balance; nine hole peg test (r=0.78, n=97) and functional dexterity test (r=0.69, n=95); and 30 second sit-to-stand (r=0.67, n=88) and 6-minute walk (r=0.59, n=77). These significant correlations suggest that MM-Function assessments are representative of all MM-COAST domains and thus clinically meaningful. We developed the Mitochondrial Mobility Performance Levels (IMPROVE) classification to define patients by their mobility and fatigue levels. MM-Function mean score for subjects at IMPROVE Level 5 (community ambulators/mild fatigue, 63.0 ± 2.7 (80.8%, n=31) was significantly higher as compared to Level 4 (community ambulators/moderate fatigue, 55.5 ± 2.0 (71.2%, n=32), p=0.002, demonstrating a greater functional impact in MM subjects with moderate fatigue.

These results indicate that our newly validated MM-Function Scale is clinically meaningful and able to quantify motor function in MM individuals. MM-Function Scale may hold utility in characterizing disease trajectory and treatment response in future intervention studies.

References

 1. Scott, E., et al., Development of a functional assessment scale for ambulatory boys with Duchenne muscular dystrophy. Physiother Res Int, 2012. 17(2): p. 101–9.

 2. Flickinger, J., Fan, J., Wellik, A., Ganetzky, R., Goldstein, A., Muraresku, C. C., Glanzman, A. M., Ballance, E., Leonhardt, K., McCormick, E. M., Soreth, B., Nguyen, S., Gornish, J., George-Sankoh, I., Peterson, J., MacMullen, L. E., Vishnubhatt, S., McBride, M., Haas, R., Falk, M. J., Xiao, R., and Zolkipli-Cunningham, Z. (2021) Development of a Mitochondrial Myopathy-Composite Assessment Tool. JCSM Clinical Reports, 6: 109–127. https://doi.org/10.1002/crt2.41.

Abstract #: 2024PA-0000000071

Presenter: Ibrahim George-Sankoh

“MMFP-Tableau: Data warehouse usage and optimization to guide therapeutic development and clinical research in mitochondrial disease”

Ibrahim George-Sankoh^1,3*, Laura MacMullen¹, Katelynn Stanley¹, Zarazuela Zolkipli-Cunningham^1,2, Elizabeth McCormick¹, Asif Chinwalla³, Deanne Taylor^2,3, Marni J. Falk^1,2

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Children’s Hospital of Philadelphia, Philadelphia PA, ²Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, ³Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, PA

*georgesani@chop.edu

Abstract: Translational and clinical research in healthcare relies on secure databases for data extraction, interpretation, and subsequent modeling of datasets to aid in therapeutic development and inform clinical care. Precision medicine requires adequate and relevant data to facilitate predictive diagnostics and empower data-driven treatment decisions in clinical practice. Challenges to this effort include integration of unharmonized data silos that have varying levels of validity and utility, unstructured data in disparate data sources, and a lack of relevant, clear clinical information for cohort descriptions. The Mitochondrial Medicine Frontier Program (MMFP)-Tableau Data Warehouse utilizes a novel data management approach to collate, integrate, and validate all clinical data sources that can be readily queried by clinicians and researchers to facilitate their effective utilization for clinical care, genomics, research studies, retrospective cohort analyses, and drug development. The collection and usage of quality datasets in our data warehouse is key to developing new therapies and developing and refining diagnoses that enable collaborations and research studies. Clinical data models are built to help the deployment of specific regulations relevant to pharmaceutical companies to narrow the gap between drug trial design and data-based research. When employed effectively, our robust data warehouse and tools extract and unify all siloed sources into a single database server that hosts high-quality datasets of genomic, clinical, and research data, each embedded with regularly scheduled streaming updates on all sources and workflows developed. The ability to continually update and develop workflows facilitates model changes and new outcomes that can be visualized to improve drug development efforts. Specifically, Alteryx is a high-end analytics platform that serves as a data stage for complex analytics by a central data integration analyst (IGS) who creates supervised and unsupervised data models, data queries, and customized analysis of viable, highly dimensional data. Outcomes and data deliverables are then output to Tableau, a commercial software hosted on a virtual machine. Tableau allows for interactive dashboards which give end-users easily accessed, rapid insights. Programmatic leveraging of these tools empowers data-driven decisions, including enabling analysis of subject and cohort data for phenotyping, evaluating clinical outcomes, and developing targeted therapies. Ontology-based phenotyping leverages high-quality datasets and data models to inform clinical research outcomes. Ultimately, data-centric machine learning approaches could help to predict mitochondrial therapeutic responses. This resource is readily deployed for both internal clinical research programs as well as to facilitate biopharma sponsored research across a diverse array of mitochondrial disease queries. Indeed, the Mitochondrial Medicine Frontier Program Data Warehouse model provides an institutional framework to partner with pharmaceutical companies to inform patient data-driven strategies for therapeutic development. Database optimization is constantly ongoing as this system is deployed as the central hub for integration of both subject-level and cohort-level data by defining explicit clinical potentials in the context of efficacy for therapeutic development and downstream collaborations.

Reference

https://www.medrxiv.org/content/10.1101/2024.01.03.24300791v1

Abstract #: 2024PA-0000000072

Presenter: Mizuki Kobayashi

Apomorphine and derived synthetic aporphine alkaloids are potential therapeutics for mitochondrial diseases with anti- ferroptotic effect

Kobayashi M¹, Miyauchi A¹, Jimbo EF¹, Aoki S¹, Watanabe M¹, Yoshikawa Y², Akiyama Y², Yamagata T¹, Osaka H¹*

¹Department of Pediatrics, Jichi Medical University, Japan, ²Department of Computer Science, School of Computing, Tokyo Institute of Technology, Japan

*hosaka@jichi.ac.jp

Abstract: Apomorphine is a dopamine agonist that originally had broad affinity for dopamine D1 to D5 receptors. We previously discovered apomorphine from a central nervous system (CNS) agonist library for compounds with anti-cell death activity against skin fibroblasts derived from patients with mitochondrial disease and reported that apomorphine has potential as a therapeutic agent for mitochondrial disease¹. Furthermore, we showed that apomorphine inhibited ferroptosis in patients-derived skin fibroblasts from those with various types of mitochondrial diseases and normal control cells, independent of dopamine receptors². While apomorphine has anti-ferroptotic effects on mitochondrial disease patients-derived skin fibroblasts, its strong dopamine receptor agonist effects produce emetic side effects in vivo. In addition, the in vivo half-life of the compounds is extremely short due to their scaffolds. Therefore, we attempted to synthesize as many apomorphine-like compounds and derivatives as possible, and to find compounds that retain anti-cell death activity against mitochondrial disease-derived skin fibroblasts as apomorphine, but without the dopamine receptor agonist effect. The 26 apomorphine analogues were obtained and 20 apomorphine derivatives were synthesized. Their anti-cell death and dopamine agonist activities were examined. As a result, three compounds with a hydroxy group at the 11th carbon of the aporphine framework showed high anti-cell death activity against LS patient-derived skin fibroblasts without dopamine agonist activity. These synthetic aporphine alkaloids have the potential to be therapeutic agents for mitochondrial disease without the side effect of emesis and may overcome the low bioavailability of apomorphine. In addition, their high anti-ferroptotic activity may make them a potential therapeutic agent for ferroptosis-related diseases.

References

 1. Miyauchi, A. et al. Apomorphine rescues reactive oxygen species-induced apoptosis of fibroblasts with mitochondrial disease. Mitochondrion 49, 111–120, doi:10.1016/j.mito.2019.07.006 (2019).

 2. Miyauchi, A. et al. Apomorphine is a potent inhibitor of ferroptosis independent of dopaminergic receptors. Sci Rep 14, 4820, doi:10.1038/s41598-024-55293-1 (2024).

Abstract #: 2024PA-0000000073

Presenter: *Tiago Branco

The mitochondrial fission protein Fis1 safeguards from sterile inflammation in vivo

Branco T^1,2, Bartolai F^1,2, Wang Z^1,2, Barbieri E^1,2, Bean C^1,2, Ceruti R², Herkenne S³, Serneels L⁴, Samardzic D², Kasahara A², Martinvalet D², De Strooper B⁴, Viscomi C², Scorrano L^1,2*

¹Veneto Institute of Molecular Medicine, Padua, Italy, ²Department of Biology, University of Padua, Italy, ³Laboratory of Molecular Angiogenesis, University of Liège, Belgium, ⁴VIB, Leuven, Belgium

*luca.scorrano@unipd.it

Abstract: Introduction: Fission protein 1 (Fis1) is the sole outer mitochondrial membrane receptor of Drp1 in yeasts, but its role in mammalian mitochondrial fission is unclear. Here we set out to elucidate the physiological roles of Fis1 in vivo.

Methods: We generated a homozygous hypomorphic Fis1 mouse (Fis1^h/h) and a conditional inducible ubiquitous Fis1 knockout mouse by crossing a Fis1^flx/flx mouse with a tamoxifen-inducible Actin-CRE that was fed with a tamoxifen containing chow for 1 month, followed by recovery. We crossed the Fis1^h/h mice with the in vivo reporter of mitophagy mitoQC. We analyzed mice survival, tissue histopathology, serum levels of Fgf21, inflammation markers and tissue ultrastructure by electron microscopy. We isolated mitochondria by standard differential centrifugation and measured mitochondrial oxygen consumption by Oroboros respirometry and recruitment of proteins to mitochondrial membranes by immunoblotting.

Results: During their first week of life, Fis1^h/h pups are indistinguishable from their control littermates, but they soon become progressively weaker and kyphotic, not surviving past 3 weeks. Remarkably, Fis1^hh pups present a spongiform vacuolization with striking astrogliosis and microgliosis that affects mostly the brainstem and spinal cord. Mechanistically, Fis1^h/h mitochondrial respiration and ultrastructure are deranged in several tissues already at the asymptomatic stage. Indeed, damaged mitochondria accumulate in the Fis1^hh animals as evident by the use of the mito-QC reporter and by the serum accumulation of Fgf21 and the higher expression of inflammatory genes, including cGAS-STING. Notably, these findings were recapitulated in adult life upon tamoxifen-induced Fis1 deletion. Indeed, adult Fis1 KO adults lose weight, become gradually weaker and kyphotic and they present similar brain lesions such as those observed in Fis1^hh pups. Acute Fis1 loss reduces recruitment of the master fission regulator Drp1 and of the lysosomal adaptor TBC1D15 to mitochondria.

Conclusion: Fis1 is an essential mammalian protein that prevents the buildup of mitochondrial stress and sterile inflammation.

Key words: Fis1, Encephalopathy, Mitochondrial fission, and inflammation.

Abstract #: 2024PA-0000000074

Presenter: Caterina Garone

Deoxyguanosine kinase deficiency: natural history and liver transplant outcome

Manzoni E^1,2†, Carli S^1†, Gaignard P³, Schlieben LD^4,5, Hirano M⁶, Ronchi D⁷, Gonzales E⁸, Shimura M⁹, Murayama K^9,10, Okazaki Y¹⁰, Baric Y¹¹, Ramadza DP¹¹, Karall D¹², Mayr J¹³, Martinelli D¹⁴, La Morgia C^15,16, Primiano G^17,18, Santer R¹⁹, Servidei S^17,18, Céline Bris²⁰, Aline Cano²¹, Furlan F²², Gasperini S²³, Laborde N²⁴, Lamperti C²⁵, Lenz D²⁶, Mancuso M²⁷, Vincenzo Montano²⁷, Menni F²², Musumeci O²⁸, Nesbitt V²⁹, Procopio E³⁰, Rouzier C³¹, Staufner C²⁶, Taanman JW³², Tal G^33,34, Ticci C³⁰, Cordelli DM^1,2, Carelli V^15,16, Procaccio V²⁰, Prokisch H^4,5, Garone C^1,2

¹Department of Medical and Surgical Sciences, Alma Mater Studiorum University of Bologna, 40138 Italy, ²IRCCS Istituto delle Scienze Neurologiche, UO Neuropsichiatria dell’età pediatrica di Bologna, Bologna, 40124, Italy, ³Department of Biochemistry, Bicêtre Hospital, Reference Center for Mitochondrial Disease, University of Paris-Saclay, Assistance Publique-Hôpitaux de Paris, 94275, France, ⁴School of Medicine, Institute of Human Genetics, Technical University of Munich, 80333, Germany, ⁵Institute of Neurogenomics, Computational Health Center, Helmholtz Zentrum München, Neuherberg, 80333, Germany, ⁶H. Houston Merritt Neuromuscular Research Center, Department of Neurology, Columbia University Irving Medical Center, New York, 10033, NY, USA, ⁷Dino Ferrari Center, Department of Pathophysiology and Transplantation, University of Milan, Milan, 20122, Italy, ⁸Pediatric Hepatology and Pediatric Liver Transplantation Unit, Bicêtre Hospital, Reference Center for Mitochondrial Disease, University of Paris-Saclay, Assistance Publique-Hôpitaux de Paris, Paris, 94270, France, ⁹Center for Medical Genetics, Department of Metabolism, Chiba Children’s Hospital, Chiba, 260-0842, Japan, ¹⁰Diagnostics and Therapeutic of Intractable Diseases, Intractable Disease Research Center, Graduate School of Medicine, Juntendo University, Tokyo, 113-8421, Japan, ¹¹Department of Pediatrics, University Hospital Centre Zagreb and University of Zagreb, School of Medicine, Zagreb, 10000, Croatia, ¹²Clinic for Pediatrics, Division of Inherited Metabolic Disorders, Medical University of Innsbruck, 6020 Innsbruck, Austria, ¹³University Children’s Hospital, Paracelsus Medical University (PMU), 5020 Salzburg, Austria, ¹⁴Division of Metabolism, Bambino Gesù Children’s Hospital IRCCS, Rome, 00165, Italy, ¹⁵Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, 40123, Italy, ¹⁶IRCCS Istituto di Scienze Neurologiche di Bologna, Programma di Neurogenetica, Bologna, 40124, Italy, ¹⁷Dipartimento di Neuroscienze, Organi di Senso e Torace -Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Rome, 00136, Italy, ¹⁸Dipartimento Di Neuroscienze, Università Cattolica del Sacro Cuore, Rome, 00168, Italy, ¹⁹Department of Pediatrics, University Medical Center Eppendorf, Hamburg, 20246, Germany, ²⁰Univ Angers, Angers Hospital, Inserm, CNRS, MITOVASC, SFR ICAT, F-49000 Angers, France, ²¹Centre de référence des maladies héréditaires du métabolisme, CHU la Timone Enfants, Marseille, 13005, France, ²²Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Regional Clinical Center for expanded newborn screening, Milan, 20122, Italy, ²³Department of Pediatrics, Fondazione IRCCS San Gerardo dei Tintori, 20900 Monza, Italy, ²⁴Unité de Gastroentérologie, Hépatologie, Nutrition et Maladies Héréditaires du Métabolisme, Hôpital des Enfants, CHU de Toulouse, Toulouse, 31300, France, ²⁵Division of Medical Genetics and Neurogenetics, Fondazione IRCCS Neurological Institute "C. Besta", Milan, 20133, Italy, ²⁶Division of Neuropaediatrics and Paediatric Metabolic Medicine, Center for Paediatric and Adolescent Medicine, University Hospital Heidelberg, Heidelberg, 69120, Germany, ²⁷Department of Clinical and Experimental Medicine, Neurological Institute, University of Pisa & AOUP, 56126, Italy, ²⁸Unit of Neurology and Neuromuscular Disorders, Department of Clinical and experimental Medicine, University of Messina, 98125, Italy, ²⁹Department of Paediatrics, Medical Sciences Division, Oxford University, Oxford OX3 9DU, UK, ³⁰ Metabolic Unit, Meyer Children’s Hospital IRCCS, Florence, 50139, Italy, ³¹Centre de référence des Maladies Mitochondriales, Service de Génétique Médicale, CHU de Nice, Université Côte d’Azur, CNRS, INSERM, IRCAN, 06000, France, ³²Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, University College London, London, WC1N 3BG, UK, ³³Metabolic Clinic, Ruth Rappaport Children’s Hospital, Rambam Health Care Campus, Haifa, 3109601, Israel, ³⁴ The Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, 3109601, Israel

*caterina.garone@unibo.it

Abstract: Deoxyguanosine kinase (dGK) deficiency is an autosomal recessive disorder presenting with early onset of liver failure with/without neurological involvement and a clinical course rapidly progressive to death. The lack of enzyme activity causes mitochondrial deoxynucleotides (dNTPs) pool unbalance leading to quantitative (depletion) and qualitative (multiple deletions and point mutations) impairment of mtDNA synthesis and multiple OXPHOS defect. Understanding the natural history of the disease is critical for translating experimental therapies from pre-clinical studies into clinical trials with nucleosides replacement therapies and/or gene therapy. Here we present an international, multicentre retrospective natural history study aiming to define clinical, biochemical, and molecular genetic characteristics of dGK deficiency. A systematic literature review from January 2001 to June 2023 was conducted. Physicians of research centers or clinicians all around the world caring for previously reported patients were contacted to provide follow-up information or additional clinical, biochemical, histological/histochemical, and molecular genetics data for unreported cases with a confirmed molecular diagnosis of dGk deficiency. A cohort of 202 genetically confirmed patients, 36 unreported, and 166 from the systematic literature review, were analyzed. Patients had a neonatal onset (⩽ 1 month) in 55.7% of cases, infantile (>1 month and ⩽ 1 year) in 32.3%, pediatric (>1 year and ⩽18 years) in 2.5%, and adult (>18 years) in 9.5%. Kaplan-Meier analysis showed a statistically significant difference in the survival rate among the age-at-onset subgroups (p <0.0001) with the highest mortality for the neonatal group. Median survival was 0.5 years for the neonatal onset, 1.17 years for the infantile-onset, and 67 years for the adult-onset group. Median survival for the pediatric onset was not directly estimable because of the low number of censored patients. Based on the clinical phenotype, we defined four different clinical subtypes: hepatocerebral (58.8%), isolated hepatopathy (21.9%), hepatomyoencephalopathy (9.6%), and isolated myopathy (9.6%). Muscle involvement was predominant in adult-onset cases whereas liver dysfunction causes morbidity and mortality in early-onset patients with a median survival of less than 1 year. No genotype-phenotype correlation was identified. Liver transplant (LTx) significantly modified the survival rate in 26 treated patients when compared with untreated. Only six patients had additional mild neurological signs after LTx. In conclusion, dGk deficiency is a disease spectrum with a prevalent liver and brain tissue-specificity in neonatal and infantile-onset patients and muscle tissue-specificity in adult-onset cases. Our data will be essential for clinical trial planning and immediate intervention with LTx and/or nucleosides supplementation.

Abstract #: 2024PA-0000000075

Presenter: Caterina Garone

Developing in vivo models for RRM2B mitochondrial encephalomyopathy

Carli S^1,2, Sabeni S^1,2, Garone C^1,2*

¹Department of Medical and Surgical Sciences, Alma Mater Studiorum University of Bologna, ²Center for Applied Biomedical Research, Alma Mater Studiorum University of Bologna

*caterina.garone@unibo.it (corresponding author)

Abstract: Encoded by the nuclear RRM2B gene, the human ribonucleotide reductase plays a role in the mitochondrial nucleotide pool salvage pathway. Pathogenic variants in RRM2B have been associated with a disease spectrum presenting with a) autosomal recessive infantile encephalomyopathy with renal tubular acidosis, multi-system involvement, and mtDNA depletion; b) autosomal recessive childhood/adult-onset Kearns-Sayre like syndrome with mtDNA multiple deletions; c) autosomal recessive adult-onset mitochondrial neuro gastrointestinal encephalopathy like syndrome with mtDNA depletion; d) autosomal dominant or recessive adult-onset chronic external ophthalmoplegia with variable degree of multi-system involvement and multiple mtDNA deletions. A dosage mechanism and impairment of the complex assembly have been proposed but never demonstrated. Similarly, nucleoside supplementation therapy has been tested in vitro but no data are available in vivo models. Our project aimed to develop a rrm2b knockout model for studying the disease mechanism and testing novel experimental pharmacological and gene therapies. We have applied CRISPR-Cas9 technology by using two guides RNA and Cas9 protein to excise the exon 4 containing the catalytic site of the gene in mouse embryonal stem cell line. Genetic screening of 200 clonal cell lines demonstrated the efficiency of our strategy with three cell lines carrying a homozygous deletion of exon 4 in the rrm2b gene. Results were confirmed by direct Sanger sequencing. Rrm2b gene expression was reduced in all the selected clones. Western-blot analyses demonstrated a reduction in the steady-state level of rrm2b protein in the selected clones except for one in which the protein was almost absent. Further studies are currently ongoing to demonstrate the functional effect of the deletion in mitochondrial DNA replication. In conclusion, we were successful in generating knockout rrm2b cell lines by removing the catalytic site. Those cell lines will be fundamental for preliminary in vitro studies on the efficacy and safety of experimental therapy and for generating the in vivo model.

Abstract #: 2024PA-0000000077

Presenter: Lindsey Miller

Making A Comeback: A Case Study of an Adolescent with TK2d Treated with Doexecitine and Doxribtimine

Miller L^1*, Clearman A¹, Koenig MK¹, Russo S N1

University of Texas McGovern Medical School, Department of Pediatrics, Center for the Treatment of Pediatric Neurodegenerative Disease, Houston, Texas

*lindsey.b.miller@uth.tmc.edu

Abstract: We report the case of a 17-year-old female with thymidine kinase 2 (TK2) deficiency. At 14 years of age the patient began to notice difficulty climbing stairs and brushing her hair leading to genetic testing neurological evaluation. Genetic testing identified compound heterozygous pathogenic variants at c.547C>T and c.248T>C in the TK2 gene. Muscle biopsy confirmed the diagnosed with mtDNA quantification at 11% of control. Shortly after diagnosis the patient began to rapidly decline, becoming wheelchair bound two years after symptom onset. She developed dysphagia, rapid weight loss, and worsening pulmonary function. Due to her rapid decline and worsening pulmonary function, in December 2023 she started compassionate use combination therapy of nucleosides doexecitine and doxribtimine. She weighed 55.9 kg (BMI 19.3%) and was started on 130 mg/kg/day (49 ml TID). Target dose was 400 mg/kg/ day however titration was halted at 260 mg/kg/day due to GI side effects, namely diarrhea. At her 3-month follow up she weighed 60.5 kg (BMI 20.8%), and her dose was weight adjusted. Since initiating therapy, she has experienced improvement in her ability to stand for longer periods of time, walk unassisted for short distances, and has had increased food intake. Her dyspnea has improved. She uses nighttime BiPAP but has not required the use of any inhaler, cough assistance, or any other form of respiratory support. At baseline she spoke in soft tones and short sentences, at her 3-month visit she was able to speak in full sentences with greatly improved dysarthria. Also, at baseline she had to “crawl” her hands up her face to reach the top of her head, three months later she can reach her face and perform regular activities of daily living like washing her face and performing skin and hair care. At her initial visit she was unable to complete a 6-minute walk test and required a substantial amount of assistance for transfers. Three months later she completed the six-minute walk test, walking 100 meters unassisted and the last 31.7 m w/ upper extremity support. Overall doexecitine and doxribtimine therapy has improved this patient’s quality of life as evidenced by her ability to perform her ADLs and walk independently for short distances. The authors would like to acknowledge UCB for providing the investigational product doexecitine and doxribtimine under IND # 169735 and supporting the EAP under IRB # HSC-MS-23-0999 UT Health Houston.

Abstract #: 2024PA-0000000078

Presenter: Laura Jameson

Environmental contaminant 6PPD-quinone has metabolic effects consistent with redox cycling activity in C. elegans

Jameson L E¹, Meyer, J N^1*

¹Nicholas School of the Environment, Joel N Meyer, Duke University, USA

*Joel.Meyer@duke.edu

Abstract: An emerging environmental contaminant of concern is the tire rubber leachate (N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine) 6PPD and its metabolite 6PPD quinone (6PPDQ) in roadway water runoff and streams. This toxicant first gained attention for causing mass toxicity of Coho salmon, and emerging evidence suggests it poses a human exposure risk. Research in various environmental matrices has shown 6PPD and 6PPDQ to be present in human relevant media including roadway water, urban air pollution and dust, rubber-containing cleaning and beauty products, and household water. Recent work has found 6PPDQ in 100% of adults via urinalysis in sex and age dependent concentrations, and another study found 6PPDQ in 72% of adults’ blood serum. Due to its frequent human exposure and potential for bioaccumulation which has been suggested by adults having notably higher urine concentrations than children, understanding 6PPDQ’s mechanism of toxicity is critical. There is considerable inter-species variability in susceptibility to 6PPDQ toxicity, and several mechanisms of toxicity have been proposed including hepatotoxicity, lipid oxidation, and mitochondrial dysfunction. Some reports suggest that 6PPDQ may uncouple mitochondria. Investigation of the mechanisms of action is necessary to accurately model risk and potential adverse health outcomes. We are utilizing the model organism Caenorhabditis elegans to identify specific metabolic processes impacted by developmental and acute 6PPDQ exposure in whole body and in the context of dopaminergic neurons in vivo. We exposed the C. elegans to 6PPDQ in a wide range of concentrations from 0.1 nM to 33 µM throughout development, from egg until early adulthood, and measured growth and dendritic degeneration in a fluorescent CEP neuron strain. We observed a slight inhibition of body volume and length and mild dendritic degeneration. Whole body ATP, ratio of ATP:ADP, and oxidized: reduced glutathione was measured utilizing a GFP-normalized luciferase transgene, a dopaminergic neuron-localized Perceval strain, and a mitochondrial roGFP strain respectively. We found that 6PPDQ has sub-lethal metabolic and toxic effects in C. elegans effects consistent with redox cycling. Our results indicate that exposure to 6PPDQ may result in a more oxidized cellular state. Further testing will examine whether this oxidized redox environment induces dopaminergic neurodegeneration. Because dopaminergic neurodegeneration is associated with Parkinson’s Disease (PD), and PD is associated with exposure to mitotoxicants, we will also investigate mitotoxins with alternate mechanisms of action, including uncouplers.

Abstract #: 2024PA-0000000079

Presenter: Shahreen Tina Amin

Relevance of Mitochondrial Biogenesis and mitochondria-Lysosome crosstalk in Neurodevelopmental and neurodegenerative disorders

Shahreen Tina Amin* and John Wolfe

Children’s Hospital of Philadelphia, Philadelphia, PA, USA Department of Neurology

*aminst@chop.edu

Abstract: Mitochondria-lysosome inter-organelle contact modulates mitochondrial dynamics and aberrant mitochondria-lysosome crosstalk pathways impair mitochondrial turnover. Reduced autophagic flux resulting in persistence of dysfunctional mitochondria has been observed in lysosomal storage diseases like Gaucher’s disease, MPS IIIC and MPS VI. Impaired mitochondrial turnover or mitophagy has been implicated in neurodevelopmental and neurodegenerative disorders, like Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease. Mitophagy involves intrinsic lysosomal acid hydrolases, recruitment of cytosolic protein to mitochondria and alteration of intrinsic mitochondrial proteins namely those in cellular bio-energetic processes and apoptosis. Mitochondria, as powerhouse is trafficked to axon terminals thereby demanding decent trafficking, fission, fusion and deliver energy generated by OXPHOS to axon terminals and synapse. Mucopolysaccharidosis VII (MPS VII) or Sly disease associated deposition of glycosaminoglycans leads to reversible neurological and skeletal anomalies due to deficient or mutated GUSB. Whereas ARSACS (Autosomal recessive spastic ataxia of Charlevoix-Saguenay) is a neuro-developmental disease due to mutations in molecular Chaperone Sacsin. Proteomic and genomic analysis of MPS VII mouse brain hippocampus has revealed altered expression in both nuclear and mitochondrial DNA encoded mitochondrial proteins thereby signifying mitochondria-lysosome crosstalk. Proteomic analysis of ARSACs interactome has revealed altered expression of proteins involved in mitochondrial biogenesis thereby also signifying mitochondria-lysosome crosstalk. I identified altered expression of intrinsic lysosomal proteins involved in mitophagy (pathway involving both mitochondria and lysosome), proteins dually expressed in lysosome and mitochondria, and notably intrinsic mitochondrial proteins (proteins involved in mitochondrial energy metabolism pathways, intrinsic apoptotic pathway, and mitochondrial translation machinery) in Sly disease and ARSACs. The next step in understanding this crosstalk therefore would include detecting the interplay of functional mitochondrial and lysosomal protein complex assemblies disintegrated in MPS VII and ARSACs disease process and include identifying GUSB and SACSIN specific interactome. The impetus is to understand the disintegration of complexes associated with disease specific neuronal phenotype, which can be reinstated by gene therapy or isogenic repair.

Abstract #: 2024PA-0000000080

Presenter: Jillian Jetmore

Aberrant glycosylation in mitochondrial disease enhances influenza A virus pathogenesis

Jetmore JW¹, Fuchs AL¹, Marin Franco J¹, Singh B¹, Tarasenko T¹, McGuire PJ^1*

¹Metabolism, Infection, and Immunity Section, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD

*peter.mcguire@nih.gov

Abstract: High risk populations, such as children with mitochondrial diseases (MtD), are prone to develop severe morbidity and mortality from viral infections. Children with Leigh syndrome (LS), a severe MtD phenotype, represent a particularly medically vulnerable group. Respiratory infections like influenza A virus (IAV) can trigger life-threatening neurodegenerative events in LS, necessitating investigation into host-viral pathogen interactions in this disorder. At the cellular level, cells affected by LS adopt Warburg-like metabolism, which is marked by increased glycolysis, pentose phosphate pathway flux, and fatty acid synthesis. These metabolic characteristics are similar to metabolic reprogramming induced during IAV infection. We hypothesized that respiratory epithelial cells affected by LS would facilitate viral pathogenesis due to their baseline metabolism mirroring the alterations induced in IAV-infected cells. To test this hypothesis, we began by examining CRISPR-edited Ndufs4 knock out (KO) murine lung epithelial type I (LET1) cells, a model of LS due to complex I deficiency. Wild type (WT) and Ndufs4 KO cells were infected with a mouse adapted H3N2 subtype of IAV, which causes mild illness in mice. At 24 hours post-infection, Ndufs4 KO cells displayed markedly increased viral load by RT-PCR for several IAV gene segments, including NS1, NP, PB2, M1, M2, and PA. In addition, at 10 and 20 minutes post-infection, viral attachment was significantly increased in Ndufs4 KO cells relative to WT. Host glycosylation patterns are known to be a crucial determinant factor for viral attachment; therefore, we examined surface glycans in Ndufs4 KO cells using fluorescently tagged lectins. Ndufs4 KO cells exhibited elevated glycoprotein/glycolipid sialylation, the primary receptors for IAV. Increased sialylation may be attributed to metabolic reprogramming in LS cells marked by increased utilization of the hexosamine and sialic acid biosynthetic pathways. To better understand this enhanced viral pathogenesis in vivo, we infected Ndufs4 KO mice with nebulized IAV to induce viral pneumonia. Following infection, Ndufs4 KO mice demonstrated elevated sialic acid in the lungs by fluorescence microscopy for lectin binding. Lung viral load was also increased, and the mice displayed worse clinical severity scores and amplified weight loss relative to WT. Moreover, we observed systemic cytokine storm in IAV-infected Ndufs4 KO mice, with elevated levels of inflammatory cytokines, including IFN-γ, IP-10, IL-6, and TNF-α. Altogether, our results suggest that aberrant glycosylation leads to increased IAV attachment, producing higher viral loads, which provokes host cytokine storm and heightened illness in LS.

Abstract #: 2024PA-0000000082

Presenter: Brittni Walker

Restoration of defective Oxidative Phosphorylation in a subset of neurons is sufficient to prevent the development of mitochondrial encephalopathy in mice

Walker B^1*, Theard LM², Pinto M², Rodriguez-Jozic M², Bacman SR² and Moraes CT^2,3,4

¹Neuroscience Graduate Program, University of Miami Miller School of Medicine, ²Dept. of Neurology, University of Miami Miller School of Medicine, ³Dept. of Ophthalmology, University of Miami Miller School of Medicine, ⁴Dept. of Cell Biology, University of Miami Miller School of Medicine

*Corresponding author (cmoraes@med.miami.edu)

Abstract: Oxidative Phosphorylation (OXPHOS) defects can cause severe encephalopathies and no effective treatment exists for these disorders. To assess the ability of gene replacement to prevent the progression of the disease, we subjected two different CNS-deficient mouse models (Ndufs3/complex I or Cox10/complex IV conditional knockouts) to a gene therapy approach. We designed recombinant AAV-PHP.eB viruses carrying the missing gene driven by the human synapsin promoter for neuronal expression. Juvenile, presymptomatic mice were injected retro-orbitally (i.v.) and their behavior analyzed weekly. In both models, we observed no progression of the encephalopathy and survival extended from 5-7 months, when the mice are severely debilitated, to more than 15 months, with no detectable disease phenotypes. Molecular analyses showed recovery of protein levels and improved enzymatic activity of OXPHOS complexes. Confocal imaging supported reintroduction of the protein of interest and reduced neuroinflammation. Surprisingly, these remarkable phenotypic improvements were achieved with only approximately 30% of neurons expressing the transgene from the rAAV-PHP.eB vector in the conditions used. These findings suggest that neurons lacking OXPHOS are protected by the surrounding neuronal environment and that partial compensation for neuronal OXPHOS loss can have disproportionately positive effects. This study offers a potential therapeutic approach to mitochondrial disease by early gene replacement therapy. Because of the species-specific limitations of AAV-PHP.eB, delivery to the human CNS remains a challenge.

Abstract #: 2024PA-0000000083

Presenter: Javier Huayta

Dopaminergic Neurodegeneration is Induced by Inhibition of Mitochondrial Complex III in Caenorhabditis elegans

Huayta J¹ and Meyer JN^1*

¹Nicholas School of the Environment, Duke University, USA

joel.meyer@duke.edu (^*Corresponding author’s email)

Abstract: Environmental factors are important contributors to Parkinson’s Disease (PD). Laboratory, clinical, and epidemiological studies have demonstrated a role for several chemical exposures. All these chemicals affect mitochondria. However, there is strong evidence for association with PD for only a few chemicals, and because relatively few people are exposed to significant amounts of those chemicals, they collectively likely explain only a small fraction of PD. It is not feasible to test all the chemicals that induce mitochondrial dysfunction. Our approach is aimed to assess the mechanisms of toxicity for these chemicals. These include inhibition of all four electron chain complexes, ATP synthase, and Krebs cycle enzymes; redox cycling; mtDNA damage; and uncoupling of ATP production from oxygen consumption. We are working to define which forms of mitochondrial dysfunction result in dopaminergic neurodegeneration, as well as whether oxidative stress, and ATP depletion, are required for dopaminergic neurodegeneration. Thus, we can narrow the focus of efforts to identify chemicals that could contribute to PD. We are using the model organism C. elegans to evaluate the in vivo effects of toxic mitochondrial exposures on dopaminergic neurodegeneration (based on morphological and behavioral changes), ATP levels, and redox state. We exposed C. elegans embryos to chemicals toxic to mitochondrial until the L4 larval stage. We acquired images of the animals’ heads using fluorescence microscopy, capturing the 4 cephalic dopaminergic neurons. Neurodegeneration was quantified by assigning a score of 0 to 6 to each neuron, dependent on the type of damage. Complex III inhibitors Antimicyn A and Pyraclostrobin -which are environmentally relevant pesticides- caused dopaminergic neurodegeneration in a dose dependent manner, and significant dopaminergic neurodegeneration was observed for concentrations of 500 nM and 50 µM respectively. Complex III inhibitors may also induce neurodegeneration in glutamatergic and cholinergic neurons, and in neuronal sheath cells, but not in serotonergic or GABAergic neurons. Rescue experiments with antioxidants and signaling modifiers suggest that neurodegeneration linked to Complex III inhibitors exposure is caused by an increased production of mitochondrial reactive oxygen species (ROS), but additional experiments will be performed to determine if dopaminergic neurodegeneration is caused by ROS damage to neurons or changes in signaling pathways in mitochondria. These results will serve to elucidate the mechanistic aspects of chemicals exposures leading to PD, steering future research in vertebrate models.

Abstract #: 2024PA-0000000084

Presenter: David Thorburn

What determines diagnostic yield? Is 70% now achievable for mito disease?

Thorburn DR^1,2,3*, Rius R^2,4, Amarasekera SSC^1,2, Lake NJ^1,2,5, Frajman LE^1,2, Thirukeswaran S^1,2,3, Mountford HS^1,2,6, Frazier AE^1,2, Welch AME^1,7,8, Hock DH^1,3,9, Semcesen L⁹, Stroud DA^1,3,9, Calvo SE^10,11,12, Mootha VK^10,11,12, Christodoulou J^1,2,3and Compton AG^1,2,3

¹Genomic Medicine Theme, Murdoch Children’s Research Institute, Melbourne, Australia, ²Department of Paediatrics, University of Melbourne, Melbourne, Australia, ³Victorian Clinical Genetics Services, Melbourne, Australia, ⁴Centre for Population Genomics, Murdoch Children’s Research Institute, Melbourne, Australia, ⁵Department of Genetics, Yale School of Medicine, New Haven, CT, USA, ⁶School of Philosophy, Psychology and Language Sciences, University of Edinburgh, Edinburgh, UK, ⁷Blood Cells and Blood Cancer Division, Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia, ⁸Department of Medical Biology, University of Melbourne, Melbourne, Australia, ⁹Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, Australia, ¹⁰Howard Hughes Medical Institute and Department of Molecular Biology, Massachusetts General Hospital, Boston, USA, ¹¹Broad Institute of MIT and Harvard, Cambridge, USA, ¹²Department of Systems Biology, Harvard Medical School, Boston, USA

*Corresponding author’s email: david.thorburn@mcri.edu.au

Abstract: In patients suspected of mitochondrial disease (MD), a “genomics first” approach has largely replaced traditional enzyme and histochemical testing, sparing the need for invasive biopsies on many patients. Strikingly though, publications on genome sequencing (GS) of patient cohorts report diagnostic yields ranging from <30% to >70%. This variability is due to both numerator (i.e., diagnoses) and denominator (i.e., cohort) issues. The former include the genomic approach and availability of samples, budget, and expertise for functional genomic follow-up. The latter include entry criteria for the cohort, e.g., prospective versus retrospective, age, referral sources and degree of suspicion. Our aim was to identify the major determinants of diagnostic yield by comparing one published and two unpublished cohorts with contrasting results.

The UK 100,000 genomes study performed GS on 345 pediatric- or adult-onset suspected MD patients (from 319 families) and found a probable or definite genetic diagnosis in 31% of families¹. Surprisingly, 63% of diagnoses were in non-MD genes (so called phenocopy genes or “mitochondrial mimics”) rather than MD genes, emphasizing how the wide phenotypic spectrum of MD can overlap with many non-MDs.

The Australian Genomics Mitochondrial Flagship studied 140 probands with pediatric- or adult-onset MD who were randomized to have either GS or exome plus mtDNA sequencing. Initial sequencing used blood DNA and proteomic or other follow-up analyses were performed when feasible on available samples. In both studies, recruitment was based on modified Nijmegen mitochondrial criteria (MNC) scores >4 (5-7 = probable MD, 8-12 = definite MD). The HPO terms used in MNC scoring varied and were applied retrospectively in the UK study and prospectively in the Australian study. Overall diagnostic yield in the Australian study was 55%. It was higher in pediatric-onset (71%) than adult-onset (31%) cases, and comparable in children with non-European versus European ancestry. 29% of diagnoses were in non-MD genes but in patients with MNC scores of 8-12, all diagnoses were in MD genes.

The third study comprises 3 overlapping retrospective cohorts (Leigh syndrome spectrum, complex I deficiency and population-based) totaling 264 pediatric-onset MD cases with stringent inclusion criteria, equivalent to definite MD diagnoses. 60 of the 264 cases who remained unsolved following genetic and exome studies underwent multi-omic analyses, including GS (with periodic re-analysis), RNAseq, proteomics or targeted studies. Each sub-cohort and the overall cohort had a diagnostic yield of 90% with only 11% of total diagnoses being in non-MD genes. Cryptic diagnoses in the exome-unsolved cases were enriched for complex structural rearrangements, de novo dominant variants, deep intronic variants and splice-site altering synonymous variants, which can be difficult to prioritize in singleton clinical GS.

Clinical diagnostic yields of GS typically remain in the 30-50% range depending on referral patterns and whether gene lists include non-MD genes but strategies such as periodic GS re-analysis, RNAseq and proteomics can each increase diagnostic yield by 10% or more.

This research was supported by the US Department of Defense Congressionally Directed Medical Research Programs PR170396 and by the Australian NHMRC, MRFF and Mito Foundation.

References

 1 Schon, K et al., BMJ 2021;375:e066288

Abstract #: 2024PA-0000000085

Presenter: Elizabeth M. McCormick, MS, LCGC

Standardized assessment of the relationship between 37 mitochondrial DNA genes and Primary Mitochondrial Disease using the ClinGen Clinical Validity Framework

McCormick EM¹*, Peterson JT¹, Taylor JP², Bluske K², Clause AR², Chandrasekhar A², Lowry J², Coffey AJ², Gai X^3,4, Falk MJ^1,5, Zolkipli-Cunningham Z^1,5, Rahman S⁶

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children’s Hospital of Philadelphia, USA, ²Illumina Laboratory Services, Illumina Inc., USA, ³Center for Personalized Medicine, Department of Pathology & Laboratory Medicine, Children’s Hospital Los Angeles, USA, ⁴Keck School of Medicine, University of Southern California, USA, ⁵Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, USA, ⁶Genetics and Genomic Medicine, UCL Great Ormond Street Institute of Child Health, UK

*mccormicke@chop.edu

Abstract: Primary mitochondrial diseases (PMD) are a phenotypically heterogenous group of disorders caused by impaired mitochondrial energy metabolism. PMD is caused by pathogenic variants in either nuclear or mitochondrial DNA (mtDNA), including in each of the 37 mtDNA genes that has been reported to be associated with PMD. The ClinGen Mitochondrial Disease Gene Curation Expert Panel (GCEP, https://clinicalgenome.org/affiliation/40027/), funded since 2017 through the National Institutes of Health (NIH) National Institute of Child Health and Human Development (NICHD) U24 grant program and re-funded in 2021 by NICHD together with the National Institute of Neurological Disorders and Stroke (NINDS), has been a highly productive effort engaging more than 50 international PMD experts. The first three-year project period focused on systematic expert panel evaluation of the strength of evidence between select nuclear and mtDNA genes and Leigh syndrome spectrum (LSS) and the current project period has expanded to perform rigorous expert panel curation of all genes associated with PMD. Gene curation, according to the ClinGen Gene-Disease Validity Curation Process, of the 37 mtDNA genes in association with PMD was prioritized and completed. The ClinGen Gene-Disease Validity Curation Process was developed for curation of nuclear-encoded genes and was therefore not directly applicable for curation of genes encoded by mtDNA. Unique features of mtDNA were considered and an approach for mtDNA gene curation was developed including defining baseline inclusion criteria for scoring variants in reported cases and approaches to scoring variants with varying levels of functional validation, including cybrid cell lines and single fiber studies. This new standardized framework allowed for systematic and rigorous review of published literature to reach consensus on the strength of the relationship between each mtDNA gene and PMD. The Mitochondrial Disease GCEP has completed curation of all 37 mtDNA genes in association with PMD. Among these curations, 26/37 (70%) reached a “Definitive” classification, 6/37 (16%) reached a “Moderate” classification, and 5/37 (14%) reached a “Limited” classification. This work has established the varied strength of relationship of mtDNA genes to PMD based on published literature evidence. This collaborative international effort remains actively underway to rigorously review gene-disease relationships for PMD, which is critical for accurate variant interpretation and confirmation of genetic diagnoses necessary to optimize medication management, tailored multi-system organ screening, accurate recurrence risk counseling and prevention, and clinical trial inclusion.

Abstract #: 2024PA-0000000086

Presenter: Elizabeth M. McCormick, MS, LCGC

Expert panel curation of the MITOMAP-Confirmed disease-associated variants according to the mitochondrial DNA variant interpretation specification guidelines: Outcomes, strengths, limitations, and plans for optimization

McCormick EM¹*, Lott MT², Muraresku CC¹, Sheta L³, Wong S⁴, Procaccio V⁵, Wallace DC^2,6, Gai X^7,8, Falk MJ^1,6

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Children’s Hospital of Philadelphia, USA, ²Center for Mitochondrial and Epigenomic Medicine, Children’s Hospital of Philadelphia, USA, ³Variantyx, Inc, Framingham, Massachusetts, USA, ⁴Ambry Genetics, USA, ⁵Genetics Department, MitoVasc Institute, Angers Hospital University, France, ⁶Perelman School of Medicine, University of Pennsylvania, USA, ⁷Center for Personalized Medicine, Department of Pathology & Laboratory Medicine, Children’s Hospital Los Angeles, USA, ⁸Keck School of Medicine, University of Southern California, USA

*mccormicke@chop.edu

Abstract: The unique features of the mitochondrial DNA (mtDNA) genome require special consideration when classifying mtDNA variants including variant heteroplasmy, threshold effect, absence of splicing, presence of haplogroups, and maternal inheritance. The Clinical Genome Resource (ClinGen) Mitochondrial Disease Variant Curation Expert Panel (Mito VCEP), funded by ClinGen in 2016, subsequently by the National Institutes of Health (NIH) National Institute of Child Health and Human Development (NICHD) U24 grant program in 2017, and re-funded in 2021 by the NICHD and National Institute of Neurological Disorders and Stroke (NINDS), has engaged over 50 international experts for a highly productive effort over 8 years (https://clinicalgenome.org/affiliation/50027). After critically reviewing the 2015 American College of Medical Genetics (ACMG) and Association of Molecular Pathology (AMP) standards and guidelines widely used for clinical interpretation of DNA sequence variants, further specifications, and additional guidance for mtDNA variant classification were developed and published (Richards et al., 2015, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4544753; McCormick et al., 2020, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7717623). Issues addressed included mtDNA genome composition and structure, haplogroups and phylogeny, mtDNA genomic databases and computational algorithms, maternal inheritance, heteroplasmy, and functional analyses unique to mtDNA. The Mito VCEP now actively curates and classifies mtDNA variants according to the specifications published by this group. Curation of the 97 mtDNA variants listed as “Confirmed” in MITOMAP as of 2022 was prioritized as these variants are widely considered to be the “gold standard” variants associated with primary mitochondrial disease by clinicians and researchers. MITOMAP variant curation differs from the ACMG/AMP classification system in several aspects, including that MITOMAP curation has largely been performed by a single manual curator (MTL) reviewing all published literature, followed by review from the heads of the MITOMAP clinical team (VP, DCW). Curation of all 97 MITOMAP “Confirmed” mtDNA variants has been completed. Thirty-four variants reached a classification of uncertain significance, however the Mito VCEP elected to upgrade the classification of 11 to likely pathogenic as they agreed this was the most appropriate classification. Forty-seven variants reached a classification of likely pathogenic though the Mito VCEP elected to upgrade the classification of five to pathogenic. An additional 16 reached a classification of pathogenic. The 16/97 variants that did not reach the appropriate classification, as agreed upon by the Mito VCEP, highlight the limitations to the current ACMG/AMP classification system specified for mtDNA variants. To address current limitations, the Mito VCEP is developing a second specification version, bolstering guidelines around functional studies, in silico predictors, proband counting, and beyond, to further optimize mtDNA variant classification and interpretation. Of note, MITOMAP now includes this VCEP’s assessed pathogenic variants as “Confirmed,” with the total number of variants with this categorization now being 115.

Abstract #: 2024PA-0000000087

Presenter: Piotr Kopinski, MD, PhD

Transcriptional analysis and metabolic characterization of uveal melanoma suggest an altered epigenome

Kopinski, PK, Ligezka AN, Miley D, Erickson S, Morava E, Kozicz T, Dalvin, LA*

Mayo Clinic, Rochester, MN, USA

*dalvin.lauren@mayo.edu

Abstract: Uveal melanoma is a deadly, highly metastatic cancer for which there is no molecularly targeted therapy. Transcriptional analysis of uveal melanoma samples showed alterations in transcription of several epigenetic and metabolic genes. Given the known association between mitochondrial function, the epigenome, and oncogenesis, we isolated primary uveal melanocytes from human donors and compared their mitochondrial metabolism to that of uveal melanoma cell lines. We analyzed the expression patterns of metabolic and epigenetic enzymes of primary uveal melanoma samples using the TCGA database. We successfully isolated and propagated six new primary human uveal melanocyte cell lines. We sequenced the cell lines to ensure no uveal melanoma-associated mutations were present and characterized their growth and morphology compared to established human uveal melanoma cell lines. Using Seahorse, we quantified oxygen consumption rate in basal and proton leak conditions, as well as determined their maximal respiratory capacity and media acidification rate. Uveal melanoma transcriptional analysis showed alterations in citrate synthase (CS), ATP-dependent citrate lyase (ACLY), and multiple epigenetic enzymes, including NAD-dependent deacetylases (SIRT1 and SIRT6), histone demethylases (KDM3A/B, JMJD1C), and DNA methyltransferases (DNMT3A/B). Primary uveal melanocytes exhibited highly regular morphology, intense pigmentation, and were positive for melanocyte markers Melan-A and SOX10 and negative for the RPE marker Bestrophin-1. Compared to uveal melanoma, primary uveal melanocytes showed slower growth and altered metabolism. Specifically, primary uveal melanocytes had higher basal oxygen consumption and showed tight mitochondrial coupling. Moreover, their media acidification rate was lower compared with uveal melanoma samples, which showed a more glycolytic profile. Transcriptional analysis of uveal melanoma suggests alterations in mitochondrial metabolism and the epigenome. We have shown that primary uveal melanocytes show a distinct metabolic profile from uveal melanoma cell lines, which are highly glycolytic. Taken together, this suggests that uveal melanoma may have an altered epigenome whose further study might uncover new therapeutic targets.

Abstract #: 2024PA- 0000000088

Presenter: Stephanie Lucas*, MD/PhD

Pearson Syndrome Natural History Study

Radhakrishnan K¹, Aziz P², and Parikh S³

¹Department of Pediatric Gastroenterology, CCF, USA, ²Department of Pediatric Cardiology, CCF, USA, ³Department of Pediatric Neurology, CCF, USA

*lucass4@ccf.org

Abstract: Pearson Syndrome is the infantile onset form of single, large-scale mitochondrial DNA deletion syndromes. Individuals with this type of disorders are classically divided into three different syndromes based on presentation: 1) Kearns-Sayre Syndrome (KSS) – a multisystem childhood/young adult syndrome characterized by sensorineural hearing loss, heart block, failure to thrive, ophthalmoplegia, and multiple other symptoms; 2) chronic progressive external ophthalmoplegia – the most mild presentation, characterized by extraocular muscle weakness and ptosis +/- more widespread muscle weakness; and Pearson Syndrome – the most severe presentation characterized by infantile onset transfusion dependent sideroblastic anemia and exocrine pancreatic dysfunction. Pearson Syndrome is a universally fatal disease with some patients dying in infancy, while others will go on to develop signs of KSS in their childhood/teenage years before succumbing to the disease. Studies have been done to better characterize onset and progression of patients diagnosed with single, large-scale mitochondrial deletions as a whole. In this study, a retrospective chart review of 17 patients diagnosed with Pearson Syndrome and seen at CCF for at least one visit was done to determine onset of various symptoms associated with Pearson/KSS with the hope to better understand the progression of this disease. On review, most patients developed sideroblastic anemia prior to 1 year with resolution of anemia between 1 year to 6 years. The onset of pancreatic insufficiency was much more variable with patients developing between <1y to 9y, while others never developed it. Three patients died young (< 4y), prior to onset of KSS. Of the remaining patients who were followed long enough to develop KSS, the earliest noted symptoms were generally ophthalmologic followed closely by hearing loss then heart block with all symptoms developing within a 1 to 5-year timeframe from onset of initial symptom of KSS. Similarly, in those patients who did not receive a preventative pacemaker at first sign of conduction abnormality, went on to develop 3^rd degree heart block within 1-1.5 yrs. from first presentation of dysfunction and required emergent pacing. Development of other symptoms including chronic kidney disease, renal tubular acidosis, diabetes, and other endocrine changes was much more variable. This retrospective analysis lays out the progressive development of various symptoms related to Pearson/KSS. By better understanding the timeline from onset of Pearson syndrome to transition to KSS and death, doctors may better counsel patients on the natural progression of this disorder. Similarly, to determine any benefit from therapeutic interventions, the natural course of the disease must be well established.

Abstract #: 2024PA-0000000089

Presenter: Eliza Gordon-Lipkin

Immunoglobulin Therapy in the Mitochondrial Disease Community

Gordon-Lipkin, EM¹*, Kruk, S¹, McGuire, PJ¹

¹Metabolism, Infection and Immunity Section, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA

*eliza.gordon-lipkin@nih.gov

Abstract: Background: Children with mitochondrial disease (MtD) often receive immunoglobulin replacement therapy (IRT) as part of their clinical care regimen. Anecdotal reports from caregivers with a child with MtD indicate reduced hospitalizations as well as improvements in symptomatology after infusion, especially neurologic symptoms such as seizures and movement disorders. Symptoms often return just prior to the next infusion (i.e., 1 month later). This cycling of symptoms appears to coincide with the kinetics (i.e., peak and trough) of intravenous IRT. However, because this therapy is not officially approved for mitochondrial disease by the Food and Drug administration, the route, dosing, intervals, and indications for which patients are receiving this medication via their home insurance are widely variable. Methods: An online questionnaire was distributed through MtD advocacy groups from March 2024 to June 2024. The target population will be adult caregivers of children with mitochondrial disease. The invitation to participate will be distributed via an internet link. Respondents must be at least 18 years of age to participate. No personal identifying information is collected. The questionnaire was designed for this study by an expert panel of mitochondrial disease experts at NHGRI. The survey contains questions about demographics (age, gender, race, ethnicity), phenotype and severity of mitochondrial disease (hospitalization rate, IPMDS), an immunodeficiency screening questionnaire, use of IRT (indications, administration, dosing, frequency), and the efficacy and adverse effects of IRT. Results: Pilot data is currently available. Full, expanded data after distribution via the UMDF listserv of >1200 families will be presented at the UMDF Symposium. Based on prior surveys launched in partnership with UMDF, we anticipate a response rate of >100 families. Preliminary responses include 16 families, 3 of which have received IRT and 1 of which was recommended but has not received. We will also compare patients who have and have not received IRT to identify if specific MtD factors are associated with IRT use or response. Conclusion: This study will allow us to measure and characterize the incidence of IRT use in individuals with mitochondrial disease. These data will be the starting point of an analysis to determine the uptake and acceptance of IRT in the MD community.

Abstract #: 2024PA-0000000090

Presenter: Allison Hanaford, PhD

Interferon-gamma contributes to disease progression in the Ndufs4(-/-) model of Leigh syndrome

Allison R Hanaford^1*, Asheema Khanna², Katerina James¹, Vivian Truong¹, Ryan Liao¹, Yihan Chen¹, Michael Mulholland¹, Ernst-Bernhard Kayser¹, Kino Watanabe¹, Erin Shien Hsieh¹, Margaret Sedensky^1,5, Phil Morgan^1,5, Vandana Kalia^2,6, Surojit Sarkar^2,6, Simon C Johnson^1,3,4,5,7*

¹Center for Integrative Brain Research, Seattle Children’s Research Institute, Seattle, WA, USA, ²Ben Towne Center for Childhood Cancer Research, Seattle Children’s Research Institute, Seattle, WA, USA, ³Department of Laboratory Medicine and Pathology, University of Washington. Seattle, WA, USA, ⁴Department of Neurology, University of Washington, Seattle, WA, USA, ⁵Department of Anaesthesiology and Pain Medicine, University of Washington, Seattle, WA, USA, ⁶Department of Paediatrics, University of Washington School of Medicine, Seattle, WA, USA, ⁷Department of Applied Sciences, Translational Bioscience, Northumbria University, Newcastle, UK

*allison.hanaford@seattlechildrens.org

Abstract: Leigh syndrome (LS), the most common pediatric presentation of genetic mitochondrial dysfunction, is a multi-system disorder characterized by severe neurologic and metabolic abnormalities. Symmetric, bilateral, progressive necrotizing lesions in the brainstem are defining features of the disease. Patients are often symptom-free in early life but typically develop symptoms by about 2 years of age. The mechanisms underlying disease onset and progression in LS remain obscure. Recent studies have shown that the immune system causally drives disease in the Ndufs4(-/-) mouse model of LS: treatment of Ndufs4(-/-) mice with the macrophage-depleting Csf1r inhibitor pexidartinib prevents disease. While the precise mechanisms leading to immune activation and immune factors involved in disease progression have not yet been determined, interferon-gamma (IFNγ) and interferon gamma-induced protein 10 (IP10) were found to be significantly elevated in Ndufs4(-/-) brainstem, implicating these factors in disease. To establish the role of IFNγ and IP10 in LS, we generated IFNγ and IP10 deficient Ndufs4(-/-)/Ifng(-/-) and Ndufs4(-/-)/IP10(-/-) double knockout animals, as well as IFNγ and IP10 heterozygous, Ndufs4(-/-)/Ifng(+/-) and Ndufs4(-/-)/IP10(+/-), animals. We monitored disease onset and progression to define the impact of heterozygous or homozygous loss of IFNγ and IP10 in LS. Loss of IP10 does not significantly impact the onset or progression of disease in the Ndufs4(-/-) model. IFNγ loss significantly extends survival and delays disease progression in a gene dosage-dependent manner, though the benefits are modest compared to Csf1r inhibition. IFNγ contributes to disease onset and progression in LS. Our findings suggest that IFNγ targeting therapies may provide some benefits in genetic mitochondrial disease but targeting IFNγ alone would likely yield only modest benefits in LS.

Abstract #: 2024PA-0000000091

Presenter: Marni J. Falk, MD

Glycosylation serves as a physiologic brake to reduce rapid Mg²⁺ influx capacity into mitochondria and dynamically communicate relative cellular nutrient status and bioenergetic capacity

Min Peng¹, Vernon E. Anderson¹, Marni J. Falk,^1,2* and Eiko Nakamaru-Ogiso^1,2*

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA, ²Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, *Equal Contribution

*falkm@chop.edu and ogisoe@chop.edu

Abstract: N-linked glycoproteins function in numerous biological processes, modulating enzyme activities as well as protein folding, stability, oligomerization, and trafficking. While N-glycosylation of mitochondrial proteins has been detected by untargeted MS-analyses, the physiological existence and roles of mitochondrial protein N-linked glycosylation remain under debate. Here, we report that MRS2, a mitochondrial inner membrane protein that functions as the high flux magnesium transporter, is N-glycosylated to various extents depending on cellular bioenergetic status. Both N-glycosylated and unglycosylated isoforms were consistently detected in mitochondria isolated from mouse liver, rat, and mouse liver fibroblast cells (BRL 3A and AFT024, respectively) as well as human skin fibroblast cells. Immunoblotting of MRS2 showed it was bound to, and required stringent elution conditions to remove from, lectin affinity columns with covalently bound concanavalin A or Lens culinaris agglutinin. Following peptide: N-glycosidase F (PNGase F) digestion of the stringently eluted proteins, the higher M_r MRS2 bands gel-shifted to lower M_r and loss of lectin affinity was seen. BRL 3A cells treated with two different N-linked glycosylation inhibitors, tunicamycin or 6-diazo-5-oxo-l-norleucine, resulted in decreased intensity or loss of the higher M_r MRS2 isoform. These data demonstrate that MRS2 is an N-glycosylated protein. To investigate the possible functional role of MRS2 N- glycosylation, we measured rapid Mg²⁺ influx capacity in intact mitochondria isolated from BRL 3A cells in control media or following treatment with tunicamycin or 6-diazo-5-oxo-l-norleucine. Interestingly, rapid Mg²⁺ influx capacity increased in mitochondria isolated from BRL 3A cells treated with either N-glycosylation inhibitor. Forcing reliance on mitochondrial respiration by treatment with either galactose media (24 h) or the glycolytic inhibitor 2-deoxyglucose similarly reduced the N-glycosylated isoform of MRS2, with a correlated concomitant increase in rapid Mg²⁺ influx capacity. Conversely, inhibiting mitochondrial energy production in BRL 3A cells with either rotenone or oligomycin resulted in an increased fraction of N-glycosylated MRS2, with decreased rapid Mg²⁺ influx capacity. Collectively, these data provide strong evidence demonstrating that MRS2 N-glycosylation occurs and is directly involved in the regulation of mitochondrial matrix Mg²⁺. MRS2 N-glycosylation serves as a physiologic brake on the influx of mitochondrial matrix Mg²⁺ under conditions of glucose excess (which stimulates glycolysis) or mitochondrial bioenergetic impairment. Functionally, this post-translational modification serves as a biological switch to dynamically communicate relative cellular nutrient status and bioenergetic capacity.

Abstract #: 2024PA-0000000092

Presenter: Mariana Zarate Mendez

Deoxyribonucleoside supplementation rescues mtDNA depletion in neuronal models of POLG mutations

Zarate Mendez M¹*, Hathazi D¹, Munro B¹, Horvath R¹

¹Department of Clinical Neurosciences, University of Cambridge, UK

*mz487@cam.ac.uk

Abstract: DNA polymerase gamma (POLγ) is the main protein responsible for the replication and maintenance of the mitochondrial DNA (mtDNA). Mutations in POLG, the gene encoding the catalytic subunit of POLγ, are among the most common single-gene causes of inherited mitochondrial diseases. While POLG-related disorders constitute a wide and heterogenous spectrum of phenotypes, they consistently lead to mtDNA depletion and the accumulation of mtDNA deletions or point mutations. Previous studies have suggested that the mtDNA replication rate is limited by the physiologic deoxyribonucleoside triphosphates (dNTPs) concentration, and thus increasing dNTP availability might have a therapeutic effect through any residual POLG activity in patients with POLG mutations. Indeed, it has been shown that supplementing patient-derived cells with dNTP precursors, deoxyribonucleosides (dNs), significantly increased mtDNA repopulation rates after EtBr-induced mtDNA depletion. However, dN supplementation studies have been restricted to mitotic tissue that requires EtBr treatment to achieve the mtDNA depletion observed in patients. To test whether dN supplementation would be effective in treating mtDNA depletion induced by POLG mutations in post-mitotic tissue, we reprogrammed patient fibroblasts into iPSCs and differentiated them into cortical neurons. We found that while there was no significant difference in mtDNA copy number between patient and control lines at the neuronal stem cell (NSC) stage, the NSCs with POLG mutations failed to increase their mtDNA copy number during neuronal differentiation at the same rate as controls. Of note, supplementing the cortical neurons with all four dNs plus an inhibitor of deoxyadenosine degradation significantly increased the mtDNA copy number in cells with POLG mutations after three weeks in culture. Interestingly, while nucleoside supplementation had a positive effect in all patient cell lines, the extent of the mtDNA increase varied between cells with different POLG mutations. We are currently investigating mtDNA deletions and point mutations in individual patient cell lines before and after supplementation. To further investigate the effect of dN supplementation, we developed a brain organoid model carrying compound heterozygous POLG mutations. In POLG mutant organoids we detected mtDNA depletion, and we are in the process of treating them with nucleosides. In our models, mutations in POLG resulted in defective mtDNA maintenance leading to mtDNA depletion. In neurons, the mtDNA depletion was underlain by an inability to increase mtDNA copy number during neuronal differentiation, in turn preventing the NSCs from undergoing the necessary metabolic shift from glycolysis towards oxidative phosphorylation. Therefore, rescuing the mtDNA depletion through dN supplementation might help to restore the OXPHOS deficiencies that underlie the clinical phenotypes observed in patients with POLG mutations. We are further investigating the effect of dN supplementation to treat POLG related disorders using zebrafish models, and if results are positive, we will begin to develop a clinical trial.

Abstract #: 2024PA-0000000094

Presenter: Kelsey Keith

Modulating Mitochondrial Function through Nutrition as a Therapeutic Strategy in Pediatric Osteosarcoma

Peng M^1*, Keith K^2*#, Dalwadi S¹, Anderson VE¹, Ogiso-Nakamaru E^1,3, Resnick A⁴, Falk MJ^1,3

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA, ²Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, PA, ³Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, ⁴Center for Data Driven Discovery (D3B), Department of Surgery, Children’s Hospital of Philadelphia and University of Pennsylvania Perelman School of Medicine, Philadelphia, PA

*Corresponding author falkm@chop.edu

^#These authors contributed equally to the work.

Abstract: Pediatric osteosarcoma is the most prevalent pediatric malignant bone tumor but has limited treatment efficacy, particularly in metastatic osteosarcoma where the adjusted 5-year survival rate is 20%. There are no recent therapies, with treatment currently relegated to surgical resection and standard DNA-damaging chemotherapies like methotrexate, doxorubicin, and cisplatin. Given the lack of effective treatments available, interest is growing in how modulating tumor metabolism through nutrient availability could be harnessed as a therapeutic. As illustrated by the well-known Warburg effect, which is characterized by increased glycolysis even in the presence of oxygen, mitochondrial metabolism is imperative for tumor growth, progression, and metastasis, representing an essential pathway to target.

To investigate the relationship between mitochondrial function and cancer growth, osteosarcoma cell viability was evaluated in 7 different cell lines grown in moderate glucose, low glucose, or galactose, where the absence of glucose forces cells to switch from glycolysis to oxidative phosphorylation. Building on nutrient modulation, combinations of potentially novel metabolic therapies for osteosarcoma were studied including metformin (indirect respiratory chain inhibitor), cycloheximide (cytosolic translation inhibitor that upregulates mitochondrial translation), as well as the imipridones ONC201 and ONC206 (a fluorinated derivative of ONC201) that act as agonists of caseinolytic protease P (ClpP) in the mitochondrial unfolded protein response (UPR^mt) and have shown promising results in clinical trials as a therapy for gliomas and glioblastomas.

Nutrient modulation had significant effects on osteosarcoma cells, where lower glucose concentrations slowed cellular growth. Each of the drug treatments in low glucose media selectively suppressed 143B osteosarcoma cells’ growth, without affecting normal osteoblast controls, suggesting these therapies may have therapeutic potential with low toxicity to healthy cells. Each drug tested also showed efficacy in at least one other, but not all cell lines in low glucose media, highlighting the need for preclinical modeling and personalized treatments of individual tumors. Further, drug effects were reduced at physiological glucose levels, demonstrating the power of nutrient modulation to modulate drug therapy for osteosarcoma. RNA-seq was performed to further dissect therapeutic effects, showing that each individual tumor line had individual responses to treatment, reinforcing the need for personalized therapies.

Osteosarcoma cell lines derived from different patients’ tumors have highly variable response to metabolic drug therapies but had consistently decreased cell viability under conditions that force cellular reliance on oxidative phosphorylation. This work further highlights the opportunity to develop tumor selective therapies that target metabolic -specific tumor function.

Abstract #: 2024PA-0000000095

Presenter: Kelsey Keith

A Disease-Agnostic High-Throughput Screening Platform for PMD Candidate Drug Therapies and Mitotoxicants

Keith K^1#, Matthew N^2,3#, Schrope S², Mendel R², Remes C², Nakamaru-Ogiso E^2,3, Haroon S^2,3, Falk MJ^2,3*

¹Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, PA, ²Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA, ³Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA

*Corresponding author falkm@chop.edu

^#Equal contribution

Abstract: Primary mitochondrial disease (PMD) is a highly heterogenous disease for which there are no FDA-approved therapies. Treatment relies mainly on addressing symptoms, with minimal interventions available to directly target disease causes or consequences. Furthermore, mitochondrial disease patients are more vulnerable to both environmental toxicants and drugs that may otherwise appear to be tolerated in the general population. Here, we report the development of a high-throughput screening (HTS) platform using C. elegans that is broadly applicable to any etiology of PMD to screen for compounds that are potentially therapeutic or toxic in the setting of PMD.

The advantage of performing HTS in C. elegans is to allow for assessing integrated physiologic effects or toxicity at a whole organism level that has multiple cell types. The assay utilizes a C. elegans fluorescent strain harboring two markers: myo-2p::mCherry marks the head of each animal to enable assay normalization and hsp-6p::gfp enables quantitation of mitochondrial stress induction at the level of the mitochondrial unfolded protein response (UPR^MT). This strain is then crossed to PMD gene mutants and/or exposed to feeding RNA interference (RNAi) to modulate target gene expression. Mutant and control animals are plated on a 384-well plate on which 80 compounds are tested in 4 replicate wells per drug for 24 hours before plate scanning, first to assess animal activity as a readout of toxicity (WormScan) and then on a high content imager (CX5, Thermo Fisher) to quantify the degree of mitochondrial stress induction. A semi-automated data analysis and reporting pipeline has been established to automatically perform data quality control of animal number, fluorescence, missingness, and plate dynamic range. Data are then normalized as percent reduction in fluorescence, followed by candidate hit identification. Candidate beneficial or negative hits, respectively, display a high or low percent reduction in green fluorescence intensity. Worm activity is used as a negative filter to remove candidate beneficial hits with low activity and to identify potentially toxic hits.

To date, we have completed HTS of a 2,560-compound library of FDA-approved drugs and natural compounds in 3 distinct PMD genetic models, including in missense mutant lines for NDUFS2 gas-1(fc21) and OPA1 opa1(R289Q), as well as in a heteroplasmic single large-scale mitochondrial deletion model (SLSMD, uaDf5). N2 Bristol worms are used as a wild-type control. These HTS have led to identification of reproducible potentially therapeutic hits to take forward toward dose-range and multi-phenotype validation in each of these strains, including 8 in gas-1(fc21), 16 in opa1(R289Q), and 10 in uaDf5. In addition, this work identified 200 compounds having potential toxicity in PMD based on the gas-1(fc21) HTS library screen.

In conclusion, we have developed a robust and flexible preclinical analysis platform to facilitate the expeditious and unbiased HTS analysis of candidate therapies and toxic compounds that can be readily applied to any cause of PMD.

Abstract #: 2024PA-0000000096

Presenter: Colin Kremitzki

Phenotypic Analysis of Mitochondrial Perturbation in Human Cells, a Path to Uncovering Differential Susceptibilities in Neurodegenerative Disease

Colin Kremitzki^1*, Jason Waligorski¹, Lina Ali¹, Diana Grigore¹, Graham Bachman¹, Serena Elia¹, Manny Gerbi¹, Mallory Wright¹, Uma Kaushik¹, Saul Weiss¹, Nico Zaharia¹, Jimin Lee¹, Purva Patel¹, Josh Langmade¹, Waleed Minzal¹, Aldrin Yim¹, Josh Milbrandt¹, Paul Hime¹, Rob Mitra¹, Jeff Milbrandt¹, William Buchser¹

¹Washington University School of Medicine – Dept. of Genetics

*ckremitz@wustl.edu

Abstract: Mutations in mitochondrial-related genes are the underlying cause in most neurodegenerative diseases, yet the significance of most variants remains uncertain. Several thousand genes have been shown to regulate mitochondria in eukaryotic cells, but which of these genes are necessary for proper mitochondrial function and dynamics? We investigated the degree of morphological disruptions in mitochondrial gene-silenced cells to understand the magnitude of genetic contribution to properly functioning mitochondria in human U2OS cells. We analyzed over five thousand gRNAs in a high dimensional phenotypic dataset produced by the image-based pooled analysis platform Raft-Seq. Using an MFN2-mutant phenotype, we uncovered TMEM11, TIMM8A, and three NADH Ubiquinone proteins, as crucial for normal mitochondrial morphology in human cells. Reanalysis with anomaly detection revealed other critical genes, including APOOL, MCEE, NIT, PHB, and SLC16A7, which perturb mitochondrial network morphology in a manner divergent from MFN2. These studies offer insights into the molecular basis for mitochondrial dysfunction and reveal potential targets for further investigation into neurodegenerative disease pathways.

Abstract #: 2024PA-0000000097

Presenter: Nicole Wilson*

Curating Patient-Provided Genetic Diagnostic Lab Reports in a Mitochondrial Disease Registry

Wilson N.¹*, McCormick E., MS, LCGC²

¹United Mitochondrial Disease Foundation (UMDF), Pittsburgh, USA, ²Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA

*nicole@umdf.org

Abstract: mitoSHARE is a UMDF worldwide patient-populated registry with the goal of advancing scientific research using data gathered from patients and families affected by mitochondrial disease. In the present work we aim to 1) improve the accuracy of patient-provided genetic testing data utilizing a novel on-platform curation process and 2) return to the patient participants a lay summary of the curation report to improve understanding of genetic testing results. Patient registries are a critical component of therapeutic development, enabling the identification and characterization of a disease patient community from the patient perspective. Self-reported genetic testing data may be inaccurate due to lack of understanding of complex diagnostic lab reports typically written for clinician consumption. 396 patient participants in mitoSHARE self-report as having had genetic testing performed. These participants are encouraged to upload copies of their genetic testing report into mitoSHARE which are then curated by a genetic counselor to extract data into a standardized form. Each variant in a primary mitochondrial disease gene listed on a report is reviewed to ensure the most updated information is provided, and this includes review of case reports with the variant(s) under review, population frequency, and any functional validation that has been performed. Furthermore, any phasing information is also considered. In return, the patients receive a lay summary report of the curation, aiding in their understanding of the outcome of the genetic testing while also ensuring high fidelity research data are collected. Over 60 different types of clinical and genetic diagnoses are represented in the registry, although nearly 29% of patient participants self-report as “suspected mitochondrial disease”, reinforcing the challenging diagnostic journey many members of the mitochondrial disease community endure. 54% of the patients reported that they are genetically diagnosed. 132 genetic testing reports from 92 individuals have been curated and 82 have had positive results. Some documents uploaded by registry participants as genetic testing results are removed from the genetic testing review queue as they are not diagnostic laboratory testing reports. Since its creation, mitoSHARE has established itself as an effective tool for identifying and characterizing mitochondrial disease patients and caregivers around the world. Genetic testing is the accepted gold standard for diagnosing patients, and therefore providing patients with a lay summary report based on curation by a certified professional is a crucial addition to their diagnostic journey while in parallel providing rigorous registry data for use in research.

Abstract #: 2024PA-0000000099

Presenter: Daniel E. McGinn

Next-generation sequencing, genomic data analysis, utility, and diagnostic rate of a patient advocacy group-led no-cost genetic testing program for primary mitochondrial diseases

McGinn DE¹*, McCormick EM¹, Shen L², Falk MJ^1,3, Yeske P⁴

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA, USA, ²Center for Personalized Medicine, Department of Pathology & Laboratory Medicine, Children’s Hospital Los Angeles, Los Angeles, CA, USA, ³Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA, ⁴United Mitochondrial Disease Foundation, Pittsburgh, PA, USA

*mcginnde@chop.edu

Abstract: Primary mitochondrial diseases (PMD) are recognized to be caused by pathogenic variants in more than 400 genes across both the nuclear and mitochondrial genomes. However, many individuals with medical concerns for PMD lack a confirmed genetic etiology. In some individuals, this is due to socioeconomic barriers preventing access to clinical diagnostic genetic testing. To address this inequity, the United Mitochondrial Disease Foundation (UMDF) in collaboration with Medical Neurogenetics (MNG) clinical diagnostic laboratory, launched in 2022 a no-cost pilot genetic testing program. The aim of the program was to aid clinicians in obtaining genetic diagnoses for patients with suspected PMD. Within the program, a participating clinician could order next-generation sequencing (NGS)-based test of a large panel of known PMD genes (320 nDNA genes) as well as full mtDNA genome sequencing (Comprehensive Cellular Energetics Defects, NGS301). NGS sequencing, raw genomic sequencing data quality control (QC), and variant calling were conducted by the MNG lab. As MNG Laboratories sent findings directly to participating clinicians, the diagnostic rate and overall utility of the no-cost pilot genetic testing program is unknown. To address this knowledge gap, de-identified Variant Call Format (VCF) files were securely transferred from MNG lab to UMDF for 271 participating individuals. De-identified VCF files were then transferred to the Children’s Hospital of Philadelphia (CHOP) Mitochondrial Medicine research group for systematic reanalysis using the Mitochondrial Disease Sequence Data Resource (MSeqDR) Quick-Mitome Web resource¹ followed by Genetic Counselor (D.E.M.) expert review. Interim analysis has been completed on 22 individuals with variants identified in 18 of whom, including: 41 Pathogenic or Likely pathogenic (P/LP) variants (per ClinVar) including 4 variants in mtDNA, 23 nDNA variants inherited in an Autosomal Dominant (AD) manner, 11 nDNA variants inherited in an Autosomal Recessive (AR) manner, and 3 nDNA variants inherited in an X-linked manner. While 10 of these P/LP variants had overlap with subject symptoms, further variant curation is underway. Analysis and curation of the variants in the remaining study participants remains ongoing and will be shared with the mitochondrial disease community along with overall diagnostic success rate of this novel UMDF pilot genetic testing program. Overall, significant socioeconomic barriers exist for PMD patients to access genetic testing, which has become indispensable to confirm the underlying genetic etiology, achieve a specific molecular diagnosis, and end the diagnostic journey for previously undiagnosed individuals with suspected mitochondrial disease. Obtaining genetic confirmation for PMD patients is critical to optimize medication management, tailor multi-system organ screening, provide accurate recurrence risk counseling and prevention, and enable clinical trial inclusion. Data from this pilot testing program will help inform future strategies for sustaining equitable access to genetic testing programs supported by PMD advocacy groups including the UMDF.

Reference

 1 Shen L, Falk MJ, Gai X. MSeqDR Quick-Mitome (QM): Combining Phenotype-Guided Variant Interpretation and Machine Learning Classifiers to Aid Primary Mitochondrial Disease Genetic Diagnosis. Curr Protoc. 2024 Jan; 4(1): e955. doi: 10.1002/cpz1.955. PMID: 38284225.

Abstract #: 2024PA-0000000100

Presenter: Amel Karaa

SPIMD-301 (NuPower) Target Patient Population and Demographics

Amel Karaa¹*, Alana Sullivan², Anthony Abbruscato², Michelangelo Mancuso³

¹Division of Genetics, Massachusetts Hospital, Harvard Medical School, Boston, MA, USA, ²Stealth BioTherapeutics, Needham, MA, USA, ³Department of Clinical and Experimental Medicine Neurological Institute University of Pisa & AOUP, Italy

*akaraa@mgh.harvard.edu (Corresponding author’s email)

Abstract: SPIMD-301 (NuPower), is a fully enrolled, ongoing Phase 3 randomized, double-blind, parallel-group, placebo-controlled trial designed to evaluate the efficacy and tolerability of daily subcutaneous injections of elamipretide 60 mg in subjects with primary mitochondrial disease (PMD) resulting from pathogenic nuclear DNA (nDNA) mutations. SPIMD-301 was designed based on findings from the SPIMM-301 (MMPOWER-3) Phase 3 clinical trial which evaluated the use of elamipretide for treating subjects with primary mitochondrial myopathy (PMM). Enrolled subjects in SPIMM-301 had a variety of pathogenic variants in nDNA or mitochondrial DNA (mtDNA) myopathy-causing genes. Although the study did not meet the primary endpoint in the highly heterogeneous study cohort, the pre-specified, post-hoc genetic subgroup analyses demonstrated that patients with pathogenic nDNA variants experienced an improvement in six-minute walk test (6MWT) versus placebo. This benefit was especially prominent in patients having mtDNA replisome-related mutations and chronic progressive external ophthalmoplegia (CPEO). Therefore, nuclear encoded control of mtDNA maintenance and replication were identified as potential therapeutic targets for functional improvements. The 6MWT is an important tool for determining the level of physical health, and therapeutic efficacy in PMD studies, and is frequently chosen as the primary outcome in clinical trials. However, a clear understanding of age-matched normative 6MWT values relative to those observed in PMD patients may not be fully appreciated. A post hoc analysis of MMPOWER-3 informed the design of SPIMD-301 (NCT05162768). The trial design consists of a screening (⩽28 days), treatment (48 weeks), and follow-up period (4 weeks). The primary efficacy endpoint will be change in distance walked (meters) on the 6MWT at week 48. Biomarker and pharmacokinetic evaluations will also be explored. A total of 102 subjects have been enrolled and randomized in a 1:1 ratio to receive either elamipretide or matching placebo. Inclusion criteria consisted of being aged ⩾18 years and ⩽70 years with clinical manifestations of PMD (exercise intolerance and/or skeletal muscle weakness) associated with a genetically confirmed pathogenic replisome-related nDNA variant(s), and co-morbidity of CPEO at screening. Subjects who received elamipretide in the past year or walked <150 meters or >450 meters during the 6MWT during screening were excluded. Of enrolled subjects, demographics were mean age 49.8 years and 33.5 years at symptom onset, 65% female, 94% White, and mean BMI 25.7kg/m². Replisome-related genotypes of subjects included POLG, TWNK, DGUOK, TK2, TYMP, RRM2B, DNA2, ANT1. Baseline functional-assessment data consisted of the following means: 348.8 meters walked on the 6MWT, 19.8 seconds on the 5XSST, and 37.7 seconds 3TUG test. Baseline functional assessment data was similar to that in subjects from the nDNA subgroups of SPIMM-301 (mean 6MWT of 322.1 meters in elamipretide-treated subjects) and SPIMM-300 (RePOWER; mean 6MWT of 339.1 meters), an observational study in subjects with PMD. Compared to the reported normative findings in adults of 638±44 meters in males and 593±57 meters in females, the baseline findings in these trials highlight the level of impairment present in patients with PMD and PMM prior to treatment.

Abstract #: 2024PA-0000000101

Presenter: Dana V. Mitchell

Using genome-scale metabolic modeling to prioritize genetic variants in patients with rare mitochondrial disorders

Mitchell DV^1*, Zhang Y¹, Keith K¹, McCormick EM^1,2, George-Sankoh I¹, Kim MS³, Yoon H², Cheng Y², Falk MJ^2,4, Taylor D^1,4

¹Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, PA, ²Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA, ³School of Medicine, Kyung Hee University, Dongdaemun-gu, Seoul, South Korea, ⁴Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA

*mitchelldv@chop.edu

Abstract: Primary mitochondrial diseases (PMD) manifest with a broadly heterogenous set of multi-system symptoms resulting from bioenergetic dysfunction, which may progressively include muscle weakness, exercise intolerance, fatigue, neurodevelopmental disabilities, cardiomyopathy, arrhythmias, hearing and/or vision loss, renal failure, gastrointestinal symptoms, and early death. These symptoms vary across patients and may differ even between patients with shared genetic etiologies. No FDA approved therapies exist for PMD. More than 400 genes have been associated with causing PMD to date, while approximately 35% of suspected cases remain without a definitive pathogenic etiology. Here, we report a novel systems biology based metabolic modeling approach to identify genetic etiologies using genome-scale metabolic models generated with RNA-Seq transcriptome data. The data is from fibroblast cell lines grown in galactose media, which unmasks mitochondrial dysfunction by forcing the cells to rely on their defective oxidative phosphorylation machinery and consists of 36 suspected mitochondrial disease patients with uncertain genetic etiologies, 12 healthy controls, and 45 definite mitochondrial disease patients. Metabolic models were reconstructed of biochemical reactions comprising cellular processes of interest, which, when constrained by transcriptome data, simulate metabolic capacity and potential metabolic flux. Regulatory changes in undiagnosed mitochondrial disease patient cells were identified when compared with healthy controls and patients with molecularly confirmed genetic etiologies, after validation in patients with confirmed diagnoses. A previously unrecognized fatty acid synthesis disorder was suspected in one undiagnosed patient, as demonstrated by reduction in the flux potential of Long Chain Fatty-Acid Coenzyme A Ligases reactions; decreased expression was also detected of Acyl-CoA Synthetase Long Chain Family Member 5 (ACSL5), a gene essential to this reaction. In a second undiagnosed individual, severe differences in the flux of the GalNAc transferase reaction were suggestive of a GALNT2-congenital disorder of glycosylation. Pathogenic variant analyses and functional validation studies to confirm causal genetic etiologies in these subjects remain underway. Adding transcriptome profiling modeling to diagnostic searches, including metabolic flux capacity modeling, may highlight previously unrecognized gene candidates in undiagnosed mitochondrial disease patients.

Abstract #: 2024PA-0000000102

Presenter: Chad Glasser PharmD

PROMIS® for MELAS: Measuring What’s Important from the Patient and Clinician Perspectives

Medrano P¹, Banderas B¹, Walker M¹, Settel L¹, Berger S¹, Shields A¹, Webster M², Chickering J², Glasser C², Gwaltney C³, Wilson P^2*

¹Adelphi Values; ²Tisento Therapeutics; ³Gwaltney Consulting

*pwilson@tisentotx.com (Corresponding author’s email)

Abstract: MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) is a rare, progressive genetic disorder associated with shortened life expectancy and characterized by a wide and heterogeneous range of symptoms. There are currently no approved treatments or validated outcome measures for MELAS. Zagociguat, an oral, once-daily, investigational drug, is being evaluated for efficacy and safety in the Phase 2b PRIZM study in participants with MELAS. Qualitative research activities were conducted to support the development of a measurement strategy for the Phase 2b study. The goals of this qualitative study were to 1) identify the key signs, symptoms, and impacts of MELAS from patient and therapeutic-area clinical expert perspectives; 2) evaluate the Patient-Reported Outcomes Measurement Information System (PROMIS®) Fatigue Mitochondrial Disease Short Form and the draft PROMIS Cognitive Function MELAS Short Form for use in adult MELAS patients; and 3) compare the content assessed by the PROMIS questionnaires and performance outcomes measures (PerfOs; 30 Second Sit-to-Stand and tests of cognitive functioning) to clinician- and patient-reported signs, symptoms, and impacts. Interviews were conducted with therapeutic-area clinical experts and adult MELAS patients. Sixty-minute clinician interviews (n=5) elicited information about signs, symptoms, and impacts of MELAS and patients’ ability to report their symptoms using a PRO measure. Patient interviews (n=16) were conducted in two 45-minute sessions: 1) concept elicitation (CE) and 2) cognitive debriefing (CD) interviews. Interviews used qualitative methods to understand the patient experience of MELAS and examine the ability of the PROMIS questionnaires to capture that experience. Clinician- and patient-reported signs, symptoms, and impacts were then mapped to concepts assessed by the PROs and the PerfOs. All clinicians (n=5) reported MELAS patients experienced physical fatigue, mental fatigue, exercise intolerance, memory loss, impaired executive function, seizures, and stroke-like episodes as key symptoms associated with MELAS. During CE interviews, patients’ most frequently reported symptoms included physical fatigue (n=15/16), hearing loss (n=13/16), mental fatigue (n=12/16), exercise intolerance (n=11/16), and memory loss (n=11/16). Clinicians and patients reported a wide range of impacts, with clinicians most frequently reporting patients requiring help from others (n=5/5), decreased performance in school/work (n=5/5), and impacted sleep (n=4/5), and patients most frequently reporting the need for hearing aids/cochlear implants (n=10/16), inability to work (n=8/16), and impacted family relationships (n=7/16). During CD interviews, at least 12/16 patients interpreted instructions, recall period, items, and response options for the PROMIS PROs as intended and considered the questionnaires relevant to their MELAS experience. Concept mapping demonstrated that the PROMIS questionnaire items and the PerfOs corresponded with a clinician– or patient-reported symptom or impact. Interview results suggested mental fatigue is an important and relevant component of the MELAS experience; therefore, two items assessing mental fatigue were added to the PROMIS Fatigue Mitochondrial Disease Short Form to form the PROMIS Fatigue MELAS Short Form. Combined, these qualitative interviews establish the key signs, symptoms, and impacts of MELAS from patient and clinician perspectives, highlight the heterogeneity of this complex disease, and support the appropriateness of the PROMIS questionnaires and PerfOs to assess the symptoms and impacts of MELAS in adults.

Abstract #: 2024PA-0000000103

Presenter: Kodi Harris

Barth Syndrome and the brain: A novel bioenergetic phenotype in TAFAZZIN deficient neural progenitor cells

Harris, K.¹*, Carney, O.¹, Anzmann, A.¹, Lee, K.¹, Vernon, H.¹

¹Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

For correspondence: Kodi Harris *kharr149@jhmi.edu

Abstract: Barth Syndrome (BTHS) is a rare, X-linked mitochondrial disease caused by pathogenic variants in the gene TAFAZZIN (TAZ), which encodes for the transacylase responsible for remodeling monolysocardiolipin (MLCL) into mature cardiolipin (CL), the final step in CL synthesis. BTHS is characterized by cardiomyopathy, neutropenia, and skeletal myopathy. The neurodevelopmental phenotype in BTHS is comparatively milder and features may include learning difficulties, cognitive fatigue, and attention deficits. The impact of TAZ-deficiency in the brain has been relatively understudied compared to impacts on other organ systems. To model the impact of TAZ-deficiency in the brain, we generated CRIPSR edited TAFAZZIN-KO (TAZ-KO) human induced pluripotent stem cells (iPSCs) and differentiated them into neural progenitor cells (NPCs) and mature cortical neurons. TAZ-deficient NPCs had the characteristic biochemical phenotype seen in BTHS, including elevated MLCL and decreased CL. The NPCs also had subtle abnormalities in mitochondrial morphology including decreased mitochondrial count and mitochondrial area. Interestingly, we discovered alterations in OXPHOS assembly and function in the TAZ-deficient NPCs, including a significant decrease in the assembly of the CIV portion of the CIII-CIV intermediate complex. Importantly, this intermediate complex is facilitated through CL binding interactions and is known to be elevated in glycolytic cell types, such as NPCs. Additionally, we discovered that TAZ-deficient NPCs have reduced oxygen consumption compared to WT counterparts. These findings highlight the critical role of TAZ in the neuronal mitochondrial function and suggest a bioenergetic deficit. Currently, we are using small molecule approaches to rescue the NPC mitochondrial phenotype including targeting CL content and pyruvate dehydrogenase function (PDH). Targeting PDH is of particular interest in the NPCs, because PDH has been shown to be involved in assembly of CIII-CIV complexes.

Abstract #: 2024PA-0000000104

Presenter: Katelynn Stanley

The Champ Foundation Registry (CFR): Single Large-Scale Mitochondrial Deletion Syndrome Natural History Study (SLSMDS-NHS)

Stanley, KD¹, Reynolds, E², MacMullen, LE¹, Qunell, E¹, Weis, M¹, Tormey, C¹, Demczko, M^1,3, Goldstein, AC^1,3, Falk, MJ¹, Parikh, S⁴, Ganetzky, R¹*

¹Division of Human Genetics, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA, ²The Champ Foundation, Durham, NC, USA, ³Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA, ⁴Mitochondrial Medicine Center, Center for Pediatric Neurosciences, Cleveland Clinic, Cleveland, OH, USA

*ganetzkyr@chop.edu

Abstract: Single Large-Scale Mitochondrial DNA Deletion Syndromes (SLSMDS) are rare primary mitochondrial diseases (PMD) that include 3 described phenotypes: Pearson Syndrome (PS), Kearns-Sayre Syndrome (KSS), and Chronic Progressive External Ophthalmoplegia (CPEO). PS presents in the first year of life with transfusion dependent sideroblastic anemia and variable exocrine pancreatic failure, with poor prognosis due to increased risk2 of sepsis (due to neutropenia) and lactic acidosis. Patients who survive PS will often have a resolution of their PS symptoms in early childhood and then develop symptoms of KSS, characterized by cardiac conduction abnormalities, pigmentary retinopathy, progressive external ophthalmoplegia, ataxia, renal involvement, and endocrinopathies (diabetes mellitus, hypoparathyroidism) with symptoms generally presenting before the age of 20. Patients who have signs of progressive external ophthalmoplegia without pigmentary retinopathy or cardiac conduction defects are classified as CPEO. These patients may have additional findings, such as skeletal myopathy (CPEO+). In addition to these three named phenotypes, many patients exist who do not fit the historically defined syndromes (SLSDMS not otherwise specified). The natural history of SLSMDS has not been fully characterized, in particular regarding the timing of disease progression and symptom onset. The Champ Foundation, a family-run patient advocacy group dedicated to supporting research toward better treatments and a cure for SLSMDS, established two SLSMDS Centers of Excellence at the Children’s Hospital of Philadelphia and the Cleveland Clinic in an effort to standardize clinical care for patients with SLSMDS and improve understanding of the natural course of SLSMDS through an observational, prospective, multi-site natural history study. Outcome measures and data collection are conducted at patients’ regularly scheduled clinic visits that typically occur every 3 to 12 months. Outcome measures include objective clinician assessments of mitochondrial disease burden, general and neurologic examination, and ataxia rating scale. Preliminary SLSMD cohort data will be presented including demographics, symptom development and progression, laboratory results, physical characteristics, and survivability. Natural history study data will deepen our understanding of the differences between individual clinical phenotypic groups and identify any unifying trends across all subjects with SLSMD disorders. Furthering understanding of SLSMDS natural history, of disease progression and burden over time will facilitate development of outcome measures for future clinical trials.

Abstract #: 2024PA-0000000105

Presenter: Dana V. Mitchell

Modeling the metabolic flux capacity of mitochondrial disease patient cell using transcriptomics data in galactose growth media

Mitchell DV^1*, Zhang Y¹, Keith K¹, McCormick EM^1,2, George-Sankoh I¹, Kim MS³, Falk MJ^2,4, Taylor D^1,4

¹Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, PA, ²Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA, ³School of Medicine, Kyung Hee University, Dongdaemun-gu, Seoul, South Korea, ⁴Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA

*mitchelldv@chop.edu

Abstract: Primary mitochondrial disease (PMD) cellular and animal models with impaired aerobic energy production have improved health and viability when given glucose, exploiting their upregulated glycolytic anaerobic energy production capacity. However, our previous preclinical modeling studies showed that glucose administration masks mechanistic investigation into metabolic stress adaptations that occur in PMD cells. We hypothesized that culturing PMD patient fibroblast cells in galactose medium would unmask underlying cellular adaptations, enabling evaluation of mitochondrial disorders on intermediary metabolic processes and functions. 12 human fibroblast cell lines from PMD patients with confirmed (n=2) or suspected (n=10) mitochondrial disease as well as healthy controls (n=2), were cultured at ~80% confluence in glucose (10 mM) or galactose (10 mM) media for 24 hours, then RNA-Seq based transcriptome profiling was performed. Using the RNA-seq for a novel metabolic modeling approach to discern variations in metabolic potential between healthy and diseased cells, this systems biology focused method uses genome-scale metabolic models that are reconstructed from biochemical reactions comprising cellular processes of interest, which, when constrained by transcriptome data, simulate metabolic capacity and potential metabolic flux. Using these methodologies, significant differences were identified in gene expression, pathway enrichment, and biochemical regulation between PMD cells grown in galactose versus glucose media using our methodologies. By contrast, few differences were identified in glucose vs galactose exposed healthy control cells. Having established the utility of galactose media to elucidate cellular metabolic adaptations, our methodology and analysis was validated on a larger cohort of 92 additional samples comprising 12 healthy controls, 36 suspected mitochondrial disease patients with uncertain genetic etiologies, and 45 definite PMD patients cohorted by molecularly-related etiologies (6 complex I deficiency, 3 complex III/V deficiency, 4 complex IV deficiency, 2 fission/fusion defects, 9 mtDNA maintenance disorders, 1 single large-scale mtDNA Deletion (SLSMD), 2 nDNA Gene Disorders, 5 pyruvate dehydrogenase disorders, and 13 mitochondrial translation disorders). This expanded investigation aimed to elucidate discrepancies in the potential activity of biochemical reactions between healthy controls and PMD patients with varying genetic etiologies. Our findings demonstrated that significant differences occurred in potential flux across numerous metabolic reactions between healthy controls and PMD patients with mtDNA maintenance disorders, complex I deficiency, nuclear DNA gene aberrations, and other conditions. This study underscores the critical importance of carefully controlling for cellular growth conditions when studying PMD and utilizing transcriptome profiling to investigate cellular mechanisms underlying complex metabolic disorders. Further, metabolic flux modeling is a novel approach to unveil previously unrecognized systemic biochemical disparities that occur among different classes of mitochondrial pathology.

Abstract #: 2024PA-0000000106

Presenter: Elizabeth M. McCormick, MS, LCGC

Harnessing resilience in adults with primary mitochondrial disease: A pilot study investigating the feasibility of the Promoting Resilience in Stress Management (PRISM) and clinical-focused narrative (CFN) interventions

Qunell E¹, Muraresku CC¹, Falk MJ^1,2, Lazariu V³, McCormick EM¹*

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children’s Hospital of Philadelphia, USA, ²Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, USA, ³Biostatistics and Data Management Core, Children’s Hospital of Philadelphia, USA

*mccormicke@chop.edu

Abstract: Individuals with primary mitochondrial disease (PMD) experience an average of 16 clinical symptoms that not only affect their physical health but significantly impact their daily life, leading to significant psychosocial burden. Resilience is defined as the ability to ‘bounce back’ and/or maintain well-being during life stressors. Increased resilience has been associated with improved health outcomes in those living with chronic illness. Given its potential benefit not only in psychosocial well-being but on physical metrics of health, increasing resilience in those with PMD is an important goal. Promoting Resilience in Stress Management (PRISM) is a psychosocial intervention with origins in cognitive behavioral therapy methods that teaches individuals skills associated with improved resilience. Similarities and differences exist between the chronic diseases included in previous pilots of PRISM and PMD, such that the ways in which adults with PMD would respond to the PRISM intervention is unknown. Separately, the clinical-focused narrative (CFN) intervention is rooted in narrative medicine and information collected at mitochondrial medicine center visits, where through discussing their medical, developmental, social, and family history information, affected individuals share their stories. Eighty-seven adults with a confirmed genetic etiology of PMD were recruited from Children’s Hospital of Philadelphia (CHOP) Institutional Review Board (IRB) study #6177 and invited to participate in this combined phase 1 and phase 2a randomized control pilot study evaluating PRISM (active intervention) and CFN (standard intervention). After completing the informed consent process but prior to the first intervention session, participants were emailed a Web link to complete surveys that include validated assessments of coping, resilience, and quality of life, as well as demographic and confounding variables. Surveys assessing coping, resilience, and quality of life were again sent to the participants after completing the study intervention. Web-based discussion groups were held after participants completed the intervention and were recorded and transcribed to facilitate thematic analysis. Enrollment into this pilot study is complete as sixteen participants (aged 25 to 67 years; 15 female, 1 male; 5 with nuclear DNA etiologies, 11 with mitochondrial DNA etiologies) enrolled and were randomized (1:1) to the PRISM or CFN intervention. All 16 participants completed the intervention, participated in discussion groups, and completed all scales. Statistical analyses of demographics, confounding variables, and validated scales remain underway. Participants receiving both interventions had generally positive feedback on the timing, format, duration, and content of the interventions and provided suggestions on when during the PMD journey might be the best time to participate in such an intervention. While additional analyses are ongoing, these interventions have already been shown to be feasible in, and accepted by, adults with PMD. The basic statistical pilot information generated by this North American Mitochondrial Disease Consortium (NAMDC)-funded pilot project program will be used to develop a first efficacy study of these behavioral interventions.

Abstract #: 2024PA-0000000107

Presenter: Dana V. Mitchell

Harmonization of Phenotype Terminology: Identifying Common Human Phenotype Ontology (HPO) Terms for Primary Mitochondrial Disease Across Six Mitochondrial Disease Centers

Dana V. Mitchell^1*, Elizabeth M. McCormick², Boriana Büchner³, Michio Hirano⁴, David Thorburn^5,6, John Christodoulou^5,6,7, Carolyn Sue^8,9,10, Robert McFarland^11,12, Yi Ng^11,12,13,14, Enrico Bertini¹⁵, Daria Diodato¹⁵, Michelangelo Mancuso¹⁶, Costanza Lamperti¹⁷, Shamima Rahman^18,19, Lishuang Shen²⁰, Xiaowu Gai^20,21, Holger Prokisch^22,23, Deanne Taylor^1,24, Marni J. Falk^2,24, Zarazuela Zolkipli-Cunningham^2,24

¹Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, PA, ²Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA, ³Friedrich-Baur-Institute at the Department of Neurology, LMU University Hospital, LMU Munich, 80336 Munich, Germany, ⁴Department of Neurology, H. Houston Merritt Center, Columbia Translational Neuroscience Initiative, Columbia University Irving Medical Center, New York, NY, USA, ⁵Brain and Mitochondrial Research Group, Murdoch Children’s Research Institute, Melbourne, Victoria, Australia, ⁶Department of Paediatrics, University of Melbourne, Melbourne, Victoria, Australia, ⁷Faculty of Medicine and Health, Discipline of Child and Adolescent Health, The University of Sydney, Sydney, New South Wales, Australia, ⁸Neuroscience Research Australia, Sydney, NSW, Australia, ⁹Kolling Institute for Medical Research, University of Sydney, NSW, Australia, ¹⁰University of New South Wales, Sydney, NSW, Australia, ¹¹Wellcome Centre for Mitochondrial Research, Faculty of Medical Sciences Newcastle University Newcastle upon Tyne UK, ¹²Translational and Clinical Research Institute, Faculty of Medical Sciences Newcastle University Newcastle upon Tyne UK, ¹³NHS Highly Specialised Service for Rare Mitochondrial Disorders, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne NE2 4HH, UK, ¹⁴NIHR Newcastle Biomedical Research Centre, Biomedical Research Building, Campus for Ageing and Vitality, Newcastle upon Tyne NE4 5PL, UK, ¹⁵Unit of Muscular and Neurodegenerative Diseases, Bambino Gesu’ Children’s Hospital, IRCCS, Rome, Italy, ¹⁶Department of Experimental and Clinical Medicine, Neurological Institute, University of Pisa, Pisa, Italy, ¹⁷Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS (Istituto di Ricovero e Cura a Carattere Scientifico) Istituto Neurologico Carlo Besta, Milan, Italy, ¹⁸Mitochondrial Research Group, UCL Great Ormond Street Institute of Child Health, London, UK, ¹⁹Metabolic Unit, Great Ormond Street Hospital NHS Foundation Trust, London, UK, ²⁰Center for Personalized Medicine, Department of Pathology and Laboratory Medicine, Children’s Hospital Los Angeles, Los Angeles, CA, USA,²¹Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA, ²²Institute of Human Genetics, School of Medicine, Technical University of Munich, Munich, Germany, ²³Computational Health Center, Helmholtz Center Munich, Neuherberg, Germany, ²⁴Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA

*mitchelldv@chop.edu

Abstract: As large patient databases were created, no standardized terminology for the highly diverse class of Primary Mitochondrial Diseases (PMD) had historically been established across the global PMD community. Rather, use of clinical phenotype terms has extensively varied related to the broad phenotypic spectrum of PMD. More recently, adoption of Human Phenotype Ontology (HPO) standardized term utilization has been transformative to facilitate effective communication and collaboration. However, the large ontological database of descriptors that vary in level of detail continues to foster widespread differences in phenotype terms used in PMD patient registries. Here, we collected comprehensive lists of PMD phenotypic terms from six international Mitochondrial centers and/or national registries in the United States (North American Mitochondrial Disease Consortium (NAMDC) registry), Italy (Mitocon registry), Germany/Austria (mitoNET registry), Australia, Newcastle, England, and London, England. In the first-tier analysis, phenotype terms from each center were manually mapped to HPO terms with preservation of the original ontological level by two independent investigators (EM, ZZC). Mapped HPO terms were reviewed and approved by the respective centers. Statistical analysis was used to determine the overlap and overall differences in the levels of the HPO tree utilized by each center (cosine similarity metrics). In the second-tier analysis, a novel ‘going up the tree’ analysis was then applied to standardize resolution of HPO terms from each center by using the branching structure of the HPO database to add the corresponding HPO parent term. Dramatic discordance was evident on first-tier analyses of all HPO terms, where only 7 common HPO terms existed across the 6 centers/registries. Detailed examination of the discordance between centers/registries revealed differences in the level of specificity of their HPO terms. For example, all 6 centers used the HPO term “Seizure [HP:0001250]”, yet mapped terms from the NADMC registry used more specific terms (e.g. Febrile seizure [HP:0002373]). Our second tier, ‘going up the tree’ analyses accounting for HPO ontology level revealed a greater overlap, consisting of 75 common HPO terms across all 6 centers/registries. We also observed greater overlap between pairs of centers (e.g. Germany and Italy). Despite increased HPO term overlap, statistical similarity metrics remained low across the cohort. A total of 602 HPO terms were unique to a single center, demonstrating the wide breadth of heterogeneity in phenotype terms. Contributory factors included variable patient cohort characteristics including disease category (neurology vs. genetics focus), age (adult vs. pediatric), and the source of each center’s list (clinical intake vs. existing registry). Altogether, a core set of 1,076 HPO terms were identified that represented the most common phenotypes at each of the 6 centers/registries. We identified a 78% overlap between our core set of HPO terms and the published Mitophen HPO database of mitochondrial (mt)DNA disorders¹. Our results highlight the critical need to foster international collaboration to implement harmonization of PMD patient registries. Based on the comparison of HPO terms across six international Mitochondrial centers and/or existing national registries, the GENOMIT initiative developed a global platform to collect and analyze data from PMD cohorts.

References

 1. Thiloka E Ratnaike, Daniel Greene, Wei Wei, Alba Sanchis-Juan, Katherine R Schon, Jelle van den Ameele, Lucy Raymond, Rita Horvath, Ernest Turro, Patrick F Chinnery, MitoPhen database: a human phenotype ontology-based approach to identify mitochondrial DNA diseases, Nucleic Acids Research, Volume 49, Issue 17, 27 September 2021, Pages 9686–9695, https://doi.org/10.1093/nar/gkab726.

Abstract #: 2024PA-0000000109

Presenter: Leonard Burg

Interrogating mechanisms of brain cell death induction upon acute complex IV inhibition in a surf1^-/- zebrafish model of Leigh Syndrome

Burg L¹*, Falk M.J.^1,2

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, ²Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104

*Burgl@CHOP.edu

Abstract: Leigh Syndrome (LS) is a progressive neurodegenerative disorder caused by impaired mitochondrial energy production from pathogenic variants in one of over 111 nuclear or mitochondrial genes, the most common of which is SURF1 that encodes a complex IV assembly factor. Our CHOP research lab has generated and characterized two surf1^-/- zebrafish knockout lines that exhibit 80-95% reduction in complex IV activity that is further reduced and elicits pronounced neuromuscular dysfunction and stroke-like brain cell death upon acute challenge with low-dose azide. While stroke upon stress exposure commonly occurs with complex IV inhibition, the cell death mechanism(s) remain unknown. We hypothesized that identifying the precise cellular death mechanisms following stressor exposure in surf1^-/- zebrafish will better inform and identify novel therapeutic approaches for the unique neurodegenerative phenotype of LS. Here we evaluated five potential cell death mechanisms (necrosis, apoptosis, autophagy, ferroptosis, pyroptosis) to determine which are activated in surf1^-/- zebrafish brains following low-dose azide stress. To identify the predominant cell death mechanism(s), we will utilize available pharmacologic cell death inhibitors to determine which may prevent brain cell death upon azide exposure in surf1^-/- larvae, as well as evaluating morphologic criteria, immunohistochemical, immunoblot, biochemical, and fluorescence read-outs of specific cell death pathway activation at various timepoints following azide exposure in surf1^-/- larvae. Efforts are underway to characterize the specific cell death pathway(s) exhibited following azide stressor exposure in the surf1^-/- zebrafish model, and here we will present data from morphological evaluation, cell staining and fluorescent labeling, and pharmacologic inhibition for each of the five potential pathways. This information will be useful in future studies of LS models, as knowledge of the specific cell death mechanism may provide therapeutic targets and novel strategies for inhibiting their brain cell death. Indeed, this work holds strong human disease relevance as stressor-induced acute neuro-regression and decompensation, including metabolic strokes, is a major cause of morbidity and mortality in human LS. The long-term goal of this work is to improve mechanistic understanding of neurologic dysfunction in SURF1 disease and identify prioritized therapies for the prevention of neurodevelopmental regression and death in pediatric LS.

Abstract #: 2024PA-0000000110

Presenter: Bogush E.

Facilitating community-based genomic data analysis in primary mitochondrial disease: mitoSHARE patient registry and Mitochondrial Disease Sequence Data Resource (MSeqDR) collaboration to accelerate genomic data discoveries

Bogush E.¹*, Wilson N.², McCormick E.M.¹, Qunell E.¹, Gai X.^3,4, Yeske P.², Falk M.J.^1,5

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA, ²United Mitochondrial Disease Foundation (UMDF), Pittsburgh, PA, ³Center for Personalized Medicine, Department of Pathology & Laboratory Medicine, Children’s Hospital Los Angeles, Los Angeles, CA, ⁴Keck School of Medicine, University of Southern California, Los Angeles, CA, ⁵Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA

*bogushe@chop.edu

Abstract: While more than 400 genes across both nuclear and mitochondrial genomes have been associated with primary mitochondrial disease (PMD), many individuals with medical concerns highly concerning for PMD lack a confirmed genetic etiology. Clinical diagnostic genomic testing for PMD has become widely utilized, yet analysis of genomic data often remains limited to clinical diagnostic laboratories that typically report variants only in known disease genes. Opportunity exists for novel gene discovery and/or more complex genomic analyses of existing genomic data from PMD subjects. We describe here a community-wide mechanism to facilitate such analyses through a collaboration between the United Mitochondrial Disease Foundation (UMDF) worldwide patient-populated registry, mitoSHARE, and the Mitochondrial Disease Sequence Data Resource (MSeqDR) Consortium. Participants enrolled in mitoSHARE are encouraged to share their diagnostic state during intake, allowing for identification of participants who have had genetic testing performed. If a participant indicates interest in learning more about sharing their existing genomic data, they are provided with contact information to learn more about the Children’s Hospital of Philadelphia (CHOP) Institutional Review Board (IRB)-approved MSeqDR genomic data repository study. Once consented and enrolled, participant genomic data is transferred from clinical diagnostic testing laboratories directly to a secure Cloud-based Web server. Participant eligible data includes exome, genome, and/or RNA-Seq transcriptome data to allow for more robust data analysis. Participant data is coded with a global universal identifier (GUID) that is provided back to the participant for them to directly share with a medical and/or scientific professional of their choosing, deemed as their proxy. Genomic data is then made available to the patient-selected proxy within a secure, Web-based, genomic data analysis environment through a user-friendly data query platform, Genesis, allowing for the proxy’s direct analysis of the full data set without need for bioinformatics expertise. 396 mitoSHARE participants indicated having had previous clinical diagnostic genetic testing performed. Following low numbers of participants reaching out to the study team after initially having indicated interest, personalized email correspondence from the mitoSHARE coordinator that informed participants of their MSeqDR study eligibility led to increased participant contact with the study team. To date, 58 participants have completed the informed consent process and 11 genomic data sets have been transferred from testing laboratories. GUID-encoded genomic data sets are being made available to proxies within the genomic data query platform Genesis. Video tutorials created by the MSeqDR study team will be made available to proxies to learn how to self-navigate Genesis’ platform. Participant enrollment and genomic data requests remain ongoing to facilitate research analyses and is open both to those with solved and unknown genetic etiologies. Through this novel, patient-academic partnership established between mitoSHARE and MSeqDR, individuals with PMD learn about an opportunity to participate in clinical research, leading to a more robust genomic data analysis pipeline for mitochondrial disease research. Importantly, this community research project enables individuals and families with either definitive or suspected PMD to choose specific medical professionals to access and meaningfully evaluate their complex genomic data.

Abstract #: 2024PA-0000000111

Presenter: Andrea Cortes Fernandez

Clinical Utility in Hospital-Wide Use of GDF15 as a Biomarker for Mitochondrial DNA Encoded Primary Mitochondrial Disorders

Cortes Fernandez A¹, Estrella J^1,2, Oglesbee D³, Larson A¹, Van Hove J¹

¹Department of Pediatrics, Section of Clinical Genetics and Metabolism, University of Colorado, Aurora, Colorado, USA, ² Department of Diagnostic Genomics, PathWest Laboratory Medicine WA, Nedlands, Western Australia, Australia, ³ Department of Laboratory Medicine and Pathology, Mayo Clinic School of Medicine, Rochester, Minnesota, USA

*andreacortesfernandez@gmail.com

Abstract: Clinical recognition of primary mitochondrial disorders (PMD) is difficult due to the extreme clinical and genetic heterogeneity. Traditional biomarkers such as lactate and pyruvate have low sensitivity and specificity. New protein biomarkers growth differentiation factor 15 (GDF15) and fibroblast growth factor 21 (FGF21) have shown promise in structured clinical studies but have been questioned for their ability to use in a hospital-wide uncontrolled setting. GDF15 is known to be elevated more frequently in mitochondrial DNA (mtDNA) encoded mitochondrial disorders (deletions, tRNA mutations), and to be impacted by liver diseases and systemic inflammatory responses.

We aim to retrospectively review the clinical utility of GDF15 when used hospital-wide by clinicians in the recognition of mtDNA-encoded PMD.

In a tertiary care hospital setting, the medical records were reviewed of all patients in whom GDF15 had been measured on a clinical basis in both inpatient and outpatient setting and classified into patients with PMD due to mtDNA defects, PMD due to nuclear defects, definite or unlikely non-mitochondrial disease, whereas patients with liver disease (>2.5x ULN) or systemic inflammatory illness were separately identified. GDF15 was clinically assayed in a CLIA laboratory (Mayo), which listed as a cutoff 750 pg/mL.Of 418 identified patients (213 male, 205 female) that had GDF15 clinically measured, there were 38 mtDNA encoded PMD (GDF15 >750 pg/mL in 76%), 37 nuclear DNA encoded PMD (66% elevated GDF15), 97 definite non-mitochondrial disorders (20% elevated GDF15), 213 unlikely mitochondrial disorders (10% elevated GDF15). Patients with liver pathology (N=20) or systemic disease in ICU setting (n=16) were excluded. The clinical utility was compared at the lab reported cutoff value of 750 pg/mL and our previously reported cutoff for GDF15 of 874 pg/mL in order to detect mtDNA encoded PMD compared to all other classes. At 874 pg/mL, sensitivity was 71.1%, and specificity was 90%, resulting in an AUC of 0.893 on the receiver operating curve analysis, with a PPV of 42% and a NPV of 97%. At 750 pg/mL, sensitivity was 76.3%, and specificity was 85%, with a PPV of 35% and a NPV of 97%. Based on the highest Youden J-index, the optimal cut-off value was 815 pg/mL. At this cutoff, the sensitivity was 76.3%, the specificity was 87.8%, with a positive predictive value (PPV) of 40% and a negative predictive value (NPV) of 97%. At this optimized cutoff level, mtDNA encoded PMD patients had elevated GDF15 in 76%, nuclear DNA encoded PMD in 26%, and non-mitochondrial disorders in 11% of patients. When used in the hospital-wide clinical setting, after excluding abnormal liver function and critical illness, GDF15 had good clinical utility in predicting mtDNA-encoded PMD, but less nuclear encoded PMDs. With a 40% PPV, an elevated GDF15 level would be a good indicator to include next-gen sequencing analysis of mtDNA in the genetic work-up. To our knowledge, this is the first study that shows the utility of GDF-15 in a real-life setting outside structured studies.

Abstract #: 2024PA-0000000112

Presenter: Cassandra Tormey, CRNP

Avoidant/Restrictive Food Intake Disorder (ARFID) in Mitochondrial Disease – a Dangerous Diagnosis

Tormey, C.¹*, Bogush, E.¹, Goldstein, A.¹, Demczko, M.¹, Falk, M.J.^1,2

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA, ²Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA

*tormeyc@chop.edu

Abstract: Gastrointestinal features of Primary Mitochondrial Disease (PMD) are well described throughout the phenotypes of both nuclear and mitochondrial DNA (mtDNA) disease, which can manifest as unintentional weight loss and pose challenges for managing providers in promoting adequate growth in PMD patients. Here, we discuss another implication of unintentional weight loss in both diagnosed and undiagnosed patients by presenting three cases in which GI manifestations were incorrectly labeled as disordered eating. Broader awareness of these PMD patient experiences may allow providers to better elicit a medical history and advocate for patients whose medical specialists may be unfamiliar with the severity of GI manifestations in PMD. Patient 1 is a 17-year-old female with a remote history of absence seizures and asthma who presented to medical attention due to myalgia preventing ambulation, emesis and 23-pound weight loss over the preceding 6-months. Upon arrival to the emergency department, hyperlactatemia was presumed to be secondary to restrictive eating. While awaiting admission for nutritional rehabilitation, she was found unresponsive with upper extremity myoclonus. This decompensation led to intubation requiring inotrope support with persistent lactic acidosis and scattered lesions on brain MRI. Comprehensive mtDNA analysis identified a pathogenic MT-TK variant, m.8363G>A at 99% heteroplasmy in blood that was causal of her MERRF/Leigh phenotype. Patient 2 was a 17-year-old female with a history of muscle fatigue, and exercise intolerance with bilateral pes cavus. Her history was most notable for poor weight gain. Presenting with failure to thrive at 5 years old, she was later diagnosed with anorexia and avoidant/ restrictive food intake disorder with multiple hospitalizations for nutritional rehabilitation. At 11 years, the Division of Child Protective Services was consulted for neglect due to patient’s weight. Despite enteral nutrition, she became total parental nutrition (TPN) dependent in the months leading to an acute decompensation secondary to ischemic bowel with multiple perforations requiring resections complicated by septic shock. Whole genome sequencing identified two pathogenic, compound heterozygous variants in POLG (c.1943 C>G, p.(P648R) and c.679C>T, p.(R227W)). Due to the development of worsening shock, care was compassionately withdrawn. Patient 3 is a 23-year-old female who was found to harbor a familial, pathogenic MT-TL1 m.3243 A>G variant at 52% and 83% heteroplasmy levels in buccal and urine, respectively. At 22 years old, she experienced a 15-pound weight loss over a year and a half period, with resulting weight of 75 pounds. An admission to an inpatient treatment center for nutritional rehabilitation was coordinated. Physicians at the Mitochondrial Medicine Frontier Program at the Children’s Hospital of Philadelphia were able to collaborate with family and medical team to prevent an inappropriate admission and advocate for an expedited GI work up to promote weight gain. While the exact prevalence is unknown, it is likely that these cases are harbingers of broader misdiagnoses of PMD as nutritional disorders, rather than isolated events. Unintentional weight loss, particularly in teenage girls, can be misdiagnosed as restrictive or disordered eating and mask a broader underlying mitochondrial disease phenotype. As determining that distinction may be complex, awareness is needed by medical specialists to advocate for appropriate patient care and prevent misdiagnoses.

Abstract #: 2024PA-0000000113

Presenter: Melis Kose

Cockayne syndrome cells display pronounced mitochondrial function and acute stress sensitivity that is rescued by N-acetylcysteine

Kose M^1*, Haroon S^1,2, McCormick E¹, Nakamaru-Ogiso E^1,2, Falk MJ^1,2

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, ²Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104

*Corresponding Author Email: kosem@chop.edu

Abstract: Cockayne syndrome (CS) is a rare autosomal recessive disorder caused by pathogenic variants in nuclear genes encoding Cockayne syndrome A (CSA), Cockayne syndrome B (CSB), and ultraviolet stimulated scaffold protein A (UVSSA) proteins that are involved in transcription coupled repair (TCR). Hallmark symptoms of CS include premature aging, cachexia, and neurodegeneration, which overlap with symptoms seen in primary mitochondrial disease (PMD). Published studies have suggested mitochondrial dysfunction occurs in CS¹, although no mitochondrial-targeted therapies are available. Here, preclinical modeling of CSB^-/- patient fibroblast cell lines was performed to characterize diverse aspects of their mitochondrial physiology and evaluate the potential efficacy of mitochondrial focused therapies.

CS human patient primary fibroblast cell lines from two siblings with ERCC6 (CSB) pathogenic variants were characterized under metabolic stress conditions to assess baseline mitochondrial DNA (mtDNA) content (qPCR), and mitochondrial content (western immunoblot analysis). Cell survival analysis (CellGlo) was also performed in media containing galactose (10 mM), glutamine (0.5 mM) and L-butionine (S, R)-sulfoximine (BSO, (50 µM)) to acutely induce oxidative stress by depleting glutathione. Therapeutic modeling was performed of candidate therapies effects in human fibroblasts, including N-acetylcysteine (NAC), taurine, and several novel mitochondrial disease therapeutic candidates. In addition, CS patient platelets were isolated for analysis of electron transport chain and citrate synthase enzymatic activities.

Consistent with previous reports showing increased mtDNA mutation and deletion frequency in CSB deficient cells², mtDNA content was 47% reduced in CS cells relative to controls (p<0.05). Interestingly, CS cells had mitochondrial proliferation, with significantly increased protein levels of citrate synthase (37%) and TOM20 (45%) (p<0.01). Platelet analyses revealed increased citrate synthase activity in one CS patient and reduced levels of complex I activity in both CS patients relative to controls, with reduced complex I protein expression by 26% and 56% relative to controls. While control fibroblasts maintained full viability under BSO oxidative stress, CS fibroblasts displayed marked acute oxidative stress sensitivity evidenced by reduced cell survival in BSO (10-15% of controls at 72 h). Remarkably, cell survival upon co-exposure for 72 to BSO with NAC (5 mM) rescued cell CS cell survival to 80-85% of control levels. Upon investigation of whether antioxidant therapy could reverse acute oxidative stress effects, CS cells exposed first to BSO for 72 h followed by 7 days of NAC (5 mM) therapy, with complete morphologic rescue observed by microscopic analysis. Additional investigations of mitochondrial bioenergetics and oxidative stress at baseline and with additional mitochondrial disease therapies are ongoing in CS cell and C. elegans models.

Overall, CS cells have disrupted mitochondrial physiology evidenced by mtDNA depletion, mitochondrial proliferation, pronounced oxidative stress sensitivity, and reduced complex I activity. CS cell survival was significantly increased with antioxidant treatment, both concurrent and subsequent to acute oxidative stress exposure, suggesting NAC may hold therapeutic potential for CS and should be pursued in clinical trials.

References

 1 Scheibye-Knudsen M, Croteau DL, Bohr VA. Mitochondrial deficiency in Cockayne syndrome. Mech Ageing Dev. 2013 May-June; 134(5-6): 275–83. doi: 10.1016/j.mad.2013.02.007. Epub 2013 Feb 19. PMID: 23435289; PMCID: PMC3663877.

 2 Aamann MD, Sorensen MM, Hvitby C, Berquist BR, Muftuoglu M, Tian J, de Souza-Pinto NC, Scheibye-Knudsen M, Wilson DM 3rd, Stevnsner T, Bohr VA. Cockayne syndrome group B protein promotes mitochondrial DNA stability by supporting the DNA repair association with the mitochondrial membrane. FASEB J. 2010 Jul; 24(7): 2334–46. doi: 10.1096/fj.09-147991. Epub 2010 Feb 24. PMID: 20181933; PMCID: PMC2887265

Abstract #: 2024PA-0000000115

Presenter: Shilpa Iyer

Leigh Syndrome Induced pluripotent stem cells exhibit defects in neural and cardiac differentiation

Fibi Meshrkey ^1,2,3, Christopher L. Littlejohn^4,5, Kelly M. Scheulin^4,5, Indrachapa Ekanayaka^1,2, Joshua Stabach,¹, Edward J. Lesnefsky⁶, Raj R. Rao⁷, Franklin D. West^4,5, and Shilpa Iyer^1,2

¹Department of Biological Sciences, Fulbright College of Arts and Sciences, University of Arkansas, Fayetteville, AR, USA, ²Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR, USA, ³Department of Chemical Engineering, College of Engineering, University of Arkansas, Fayetteville, AR, USA, ⁴Regenerative Bioscience Center, University of Georgia, Athens, GA, USA, ⁵Department of Animal & Dairy Science, University of Georgia, Athens, GA, USA, ⁶Cardiology Section Medical Service, McGuire Veterans Affairs Medical Center, Richmond, VA, USA, ⁷Department of Biomedical Engineering, University of Arkansas, Fayetteville, AR, USA

*si012@uark.edu

Abstract: Introduction: Leigh syndrome (LS) is a fatal pediatric neurodegenerative disorder caused by mutations in the mitochondrial DNA (mtDNA). Symptoms associated with LS widely ranged from mild fatigue to lactic acidosis, and abnormal neural and followed by cardiac developmental defects. Due to the disease heterogenicity, there are neither effective treatment options nor adequate human models to explain LS-associated neural and cardiac defects in infancy, especially during embryonic development. Human induced pluripotent stem cells (hiPSC) serve as adequate early developmental models for studying LS. Thus, this study focuses on analyzing the neural and cardiac differentiation potential of LS-hiPSC cell lines.

Results: Three LS- hiPSCs (carrying mtDNA mutations 8993T>G, 9185T>C, 12706T>C) were differentiated into neurons and cardiomyocytes based on protocols established in our laboratory. Our results indicated that after 10 days of differentiation using defined medium (E6 + BMPi,) rosettes were generated and were abundant by day 15, thus addition of BMPi was necessary and endorsed neural differentiation in diseased LS-hiPSCs. Next, we differentiated LS-hiPSCs into cardiomyocytes using established protocols in our laboratory. Immunocytochemical analysis demonstrated cardiac marker expression such as cardio-troponin T and α-sarcomeric actinin in both control BJ-hiPSC-CMs and LS-hiPSC-CMs. At day 30, unpurified population of one LS-hiPSC-CMs shows significantly faster beating frequency than control hiPSC-CMs (p < 0.0001); while another LS-hiPSC-CM demonstrated a reduced beating frequency than control hiPSC-CMs (p<0.0001), indicative of overall aberrant cardiac differentiation outcomes.

Conclusion: Leigh syndrome is mostly characterized by defects in early development of central nervous system, followed by high occurrence of cardiac defects. Our results indicate that neural and cardiac symptoms manifest simultaneously early on in embryonic development in LS-hiPSCs carrying mtDNA mutations within genes encoding for complex I and mitochondrial ATP synthase.

Abstract #: 2024PA-0000000117

Presenter: Amber J. Dinchman

Toward targeted therapies: the diverse pharmaceutical and nutritional responses of Leigh syndrome patient derived induced pluripotent stem cell-cardiomyocytes

Dinchman AJ^1*, Yamamoto TH¹, Huang CC¹, Cutright AK¹, Dinchman KC¹, Kang SJ², Hord C¹, Edmiston PL³, Piontkivska H⁴, Cohen BH^1,5, Chilian WM¹, and Kang PT^1*

¹Department of Integrative Medical Sciences, Northeast Ohio Medical University, OH, ²Northwestern University, IL, ³The College of Wooster, OH, ⁴Kent State University, OH, ⁵Akron Children’s Hospital Rebecca D. Considine Research Institute, OH

*(Corresponding author’s email pkang1@neomed.edu)

Abstract: Leigh syndrome (LS) is a rare inherited neurometabolic disorder that affects the central nervous system with severe adverse consequences. Being a mitochondrial disease, LS presents defective mitochondrial energy metabolism. A conundrum in finding adequate therapy for LS may be in part the tremendous genetic variation resulting in diverse disease burden and progression. One drug may work on a subset of patients, but this beneficial effect may not apply to the entire cohort. Approximately 80% of the LS patients had deficiencies of the respiratory chain enzyme complex, and mutations on pyruvate dehydrogenase complex are also believed to cause LS. Mutations of the MT-NDs and MT-ATP6 genes are the most common mtDNA mutation genotypes. We propose that patient-derived induced pluripotent stem cells (iPSCs) can serve as surrogates for studying mitochondrial diseases, provided a standardized protocol is used to mitigate effects of mtDNA heteroplasmy. Accordingly, we reprogrammed peripheral blood mononuclear cells (CytoTune-iPS 2.0 Sendai Reprogramming) from healthy donors and LS patients with epilepsy enrolled in clinical trials. Informed consent for this study was obtained and approved by the institutional IRB. Subject C01 was the unaffected parent of subject LS01, and subject C02 was the unaffected parent of subject LS02. LS01 carries mutations on NDUFAF3 gene (NADH dehydrogenase (ubiquinone) complex I, assembly factor 3) with mutation 1: G164SfsX29 c.489_490delTG and mutation 2: Y11DF c.31T>C. LS02 carries mutations on SUCLG1 with mutations c.713A>G (p.D238G) in trans with c.787G>A (p.E263K) causing deficiency in Succinyl-CoA ligase [GDP-forming] subunit alpha, mitochondrial; also a c.472_474delAAA (p.K158del) in the RARS2 gene (hetero). Subject LS03 carries m.8993T>G mutation. Subject LS04 carries mutations on SURF1 gene with mutation c.312_321del10iinsAT (p.Leu105X), homozygous. Subject C03 and C04 were healthy donors. After culturing the reprogrammed cells, three to six iPSC cell colonies were selected (Alkaline Phosphatase Live Stain) from each donor, pooled, and sorted by SSEA4+ marker at passage 6. Pluripotency was confirmed using OCT4 immuno-staining and RT-PCR for OCT4, NANOG, and SOX2 at passage 10. The transcriptome profiling (Genechip Probe Array) of iPSCs revealed decreased expression of genes in neuronal-developmental (EGR1, CXCL5, SEPT6, BCHE, CA3), decreased antioxidant (GSTM3) and compensatory increased expression of substrate utilization genes (GPAT2, PCK2) in LS-iPSCs. We further differentiated (STEMdiff Ventricular Cardiomyocyte Differentiation) iPSCs into cardiomyocytes (iCMs) to investigate the mitochondrial function by Seahorse extracellular flux assay. LS01-iCMs exhibited improved basal oxygen consumption rate (OCR) after drug PTC743 treatment, and both PTC743 and NV118 enhanced the LS01-iCMs respiratory reserve capacity. However, the basal OCR of LS04-iCMs were not responding to PTC743 and NV118 of the same dosage. We then screened the substrate utilization preferences of the iCMs using Phenotype MicroArray Mammalian assays (Biolog). LS03-iCMs thrived in the presence of acetoacetic acid and α-keto-butyric acid with reduced oxidative stress (AmplexRed). LS03-iCMs failed to survive in the presence of monomethyl-succinate, but the same substrate exhibited beneficial effect on LS01-iCMs. Our findings suggest that diverse outcomes to pharmaceutical and nutritional interventions must be tailored to each patient. The mechanism studies on LS-iPSC-derived disease model system can contribute to the “Treatabolome” for targeted therapies.

Abstract #: 2024PA-0000000118

Presenter: Jeffrey A Haltom*

SARS-COV-2 ORF10 INDUCES MITOCHONDRIAL DYSFUNCTION

Haltom J.A^1,2,3,5*, Trovao N.S^4,5, Guarnieri J.W^3,5, Pan V⁴, Singh U^1,2,5, Tsoy S⁶, O’Leary C.A^7,8, Bram Y⁶, Widjaja G.A³, Cen Z³, Meller R⁹, Baylin S.B^10,11, Moss W.N^1,7, Nikolau B.J^1,7, Enguita F.J¹², Beheshti A^5,14,15, Wallace D.C^3,5,13, Schwartz R.E^6,16,17*, and Wurtele E.S^1,2,5*

¹Bioinformatics and Computational Biology Program, Genetics Program, Iowa State University, Ames, IA 50011, USA, ²Department of Genetics Development and Cell Biology, Iowa State University, Ames, IA 50011, USA, ³Center for Mitochondrial and Epigenomic Medicine, Division of Human Genetics, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA, ⁴Division of International Epidemiology and Population Studies, Fogarty International Center, National Institutes of Health, Bethesda, Maryland, 20892, USA, ⁵COVID-19 International Research Team, Medford, MA 02155, USA, ⁶Division of Gastroenterology and Hepatology, Department of Medicine, Weill Cornell Medicine, New York, NY, USA, ⁷Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA, ⁸Department of Biology and Chemistry, Cornell College, Mount Vernon, IA 52314, USA, ⁹Morehouse School of Medicine, Atlanta, GA, 30310-1495, USA, ¹⁰Department of Oncology, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD 21231, ¹¹Van Andel Research Institute, Grand Rapids, MI 49503, ¹²Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal, ¹³Department of Pediatrics, Division of Human Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104 USA, ¹⁴Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA, ¹⁵Blue Marble Space Institute of Science, Seattle, WA, 98104 USA, ¹⁶Department of Physiology, Biophysics and Systems Biology, Weill Cornell Medicine, New York, NY, USA, ¹⁷Department of Biomedical Engineering, Cornell University, Ithaca, NY, USA

*Correspondence: mash@iastate.edu, res2025@med.cornell.edu

Abstract: Expression of SARS-CoV-2 ORF10 has been shown to induce mitophagy resulting in the degradation of the mitochondrial antiviral signaling (MAVS), antagonizing an innate immune response.¹ However, nobody has investigated the broader effect of ORF10 expression on the host transcriptome. We analyzed relative expression levels of host genes in A549 and 293T-cells, which expressed either ORF10 or GFP. In A549-cells, expression of ORF10 decreases expression of 12S-MT-RNR2 and 16S-MT-RNR1 and nine genes required for the electron transport chain, including complex I subunits MT-ND1, MT-ND4, MT-ND4L, MT-ND5, complex III subunit MT-CYB, complex IV subunits MT-CO1, MT-CO2, MT-CO3, and complex V subunit MT-ATP6. In 293T-cells, expression of ORF10 results in a robust decrease in the expression of the nuclear-encoded mitochondrial ribosomal proteins MRPL4, MRPS34, MRPL41 and complex I subunits (NDUB7, NDUFS7). This alteration of mitochondrial transcripts would disrupt mitochondrial function by decreasing oxidative-phosphorylation and mitochondrial membrane potential, leading to elevated mitochondrial reactive species production. ORF10 is unlikely to directly inhibit transcription of either mtDNA or nDNA genes since the transcriptional response of the two cells is opposite. Yet, ORF10 must be inhibiting some common feature of mitochondrial function to elicit the strong transcriptional responses observed. A study of ORF10 protein interactions revealed that it binds to TIMM8B, THTPA, PPT1, MAP7D1, and the CUL2 complex.² All identified ORF10 protein interactions would directly or indirectly inhibit mitochondrial function and through HIF-1α push metabolism toward glycolysis to enhance SARS-CoV-2 biogenesis.³ Furthermore, we computationally predicted the 3D protein structure of ORF10 and found that it forms an amphiphilic α-helix with positively charged arginines on one face and hydrophobic amino acids on the other face. This is the classical structure for a mitochondrial import sequence. Moreover, we found that a less pathogenic ORF10 mutation rearranges the positioning of the critical clustered arginines. We have reported, SARS-CoV-2 infection targets the mitochondria and induces mitochondrial suppression in many organs, which could cause long lasting systemic impact associated with the symptoms that appear during post-acute sequelae of COVID-19 (PASC).³ Interestingly, ORF10 displays prolonged elevation in specific tissues, which could also contribute to long COVID or PASC. Together, this data indicates that the ORF10 protein causes mitochondrial dysfunction by targeting mitochondrion proteins of the TCA cycle and OXPHOS, which ultimately triggers mitophagy and the degradation of MAVS to abate the immune response. Consistent with this, we observed a downward trend of expression of innate immune genes in ORF10 expressing A549-cells and 293T cells.

Reference

 1. Xingyu Li, et al. Sars-cov-2 orf10 suppresses the antiviral innate immune response by degrading mavs through mitophagy. Cellular & molecular immunology, 19(1): 67–78, (2022).

 2. Gordon, D. E, et al. A SARS-CoV-2 protein interaction map reveals targets for drug repurposing. Nature 583, 459–468, doi:10.1038/s41586-020-2286-9 (2020).

 3. Guarnieri, J. W. et al. Core mitochondrial genes are down-regulated during SARS-CoV-2 infection of rodent and human hosts. Sci. Transl. Med. 15, eabq1533, doi:10.1126/scitranslmed.abq1533 (2023).

Abstract Number #: 2024PA-0000000120

Presenter: Ryan M. Mendel

Identification of FDA-approved compounds that rescue mitochondrial stress in a heteroplasmic single large-scale mtDNA deletion (SLSMD) C. elegans animal model

Mendel RM^1*, Cheng Y¹, Tara Z¹, Schrope S¹, Keith K³, Matsuno S¹, Mathew N¹, Ogiso E¹, Falk MJ^1,2, and Haroon S^1,2

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA.,²Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA.,³Department of Biomedical and Health Informatics (DBHi), Children’s Hospital of Philadelphia, Philadelphia, PA

*mendelr@chop.edu

Abstract: Background: Kearns-Sayre syndrome (KSS) and Pearson Syndrome (PS) are primary mitochondrial diseases caused by single large-scale mitochondrial DNA (mtDNA) deletions (SLSMD). SLSMD syndromes, as with most mitochondrial diseases, have no effective treatments or FDA-approved therapies. One major roadblock to therapeutic development is the lack of tools to genetically engineer models harboring mtDNA deletions that are stable and transgenerational. As such, studying naturally occurring animal models helps to advance understanding of disease progression and enable therapeutic development.

Methods: The uaDf5 C. elegans model harbors a heteroplasmic 3.1 kilobase mtDNA deletion of 11 genes that encode 7 MT-tRNAs and 4 mitochondrial proteins. Here, we use a mitochondrial stress phenotype in the uaDf5 animals to screen for potential therapeutic leads from two libraries, consisting of 62 mitophagy modulators and 2,560 FDA-approved and natural compounds. To conduct the screen, uaDf5 animals were crossed with the myo2::mcherry reporter, a red fluorescent pharyngeal bulb marker for automated animal count, and an hsp6p::GFP reporter that fluoresces green upon mitochondrial stress induction. These triple transgenic animals were used to screen potential therapies using the CX5 high content imager (Thermo Fisher).

Results: 3 drugs empirically identified compounds previously found to rescue survival in a C. elegans gas-1(fc21) complex I disease model were identified as positive controls for the library screen based on their ability to rescue mitochondrial stress in uaDf5 animals. Upon screening the mitophagy modulator library, 5 additional compounds were identified that significantly rescued mitochondrial stress in uaDf5 mutant animals. Testing these 5 compounds at 4 different concentrations (ranging from 12.5 µM to 100 µM) revealed only 2 that reproducibly rescued mitochondrial stress in a dose-dependent manner across multiple biological replicate experiments. Validation studies of these two modulators effects on reducing SLSMD heteroplasmy levels in uaDf5 worms are underway, as well as combination treatments of positive control hit compounds with mitophagy modulators. Furthermore, a high-throughput screen of a 2,560 FDA-approved drug and natural product library has been completed, with validation of 10 hits across a 1-80 µM dose curve. Importantly, a uaDf5 line harboring a higher SLSMD heteroplasmy level (60%, as compared to drug screen done in animals with ~30% SLSMD heteroplasmy) has now been established, which displays delayed development, reduced fecundity and neuromuscular defect. Lead compounds identified in the HTS will now be evaluated to study their ability to rescue these SLSMD worm phenotypes.

Conclusions: In conclusion, uaDf5 worm models of SLSMDs enable mechanistic and therapeutic modeling insights, as well as high throughput screening of drug libraries to identify novel therapeutic leads for SLSMD diseases.

Abstract #: 2024PA-0000000123

Presenter: Arguello Tania

Absence of MgmeI results in an increase rate of mtDNA deletion formation in dopaminergic neurons in vivo but does not contribute to accumulation of deletions

Arguella T*¹, Nissanka N ¹, Moraes C¹

¹Department of Neurology, University of Miami Miller School of Medicine, Miami, FL 33136, USA*cmoraes@med.miami.edu

Abstract: Mitochondria DNA (mtDNA) damages have been associated with primary mitochondrial diseases and neurodegeneration; however, the mechanism of generation, accumulation, and susceptibility of different neuronal types to mtDNA damage is still not understood. To better understand the mechanism of mtDNA deletion formation and accumulation in specific neuronal subtypes we used a mouse model of Parkinson disease, characterized by loss of dopaminergic neurons preferentially in the substantia nigra. This mouse model has an inducible mitochondrial-targeted restriction endonuclease PstI (mitoPstI-DATtTA (+/-)) in dopaminergic neurons. We have previously shown that mitoPstI expression leads to the formation of mtDNA deletions. We induced mitoPstI-generated mtDNA deletions during the first 2 months after birth followed by silencing of mitoPstI expression by administration of DOX for 4 additional months. In addition, we also analyzed the same mouse model lacking the mitochondrial Genomic Maintenance Exonuclease MGME1 gene, which has been implicated in mtDNA replication and degradation of fragmented mtDNA. Using laser microdissection followed by digital PCR in DNA extracted from 2 month and 6-month-old mice, we found no preferential accumulation of mito-PstI induced deletions between substantia nigra (SNc) and ventral tegmental area (VTA) regions. We observed a significant increase of mito-PstI induced deletions from 2 months to 6-month-old mice in both SNc and VTA in the (mitoPstI- DATtTA (+/-); However, mtDNA depletion was detected only in the SNc region. On the other hand, the levels of mtDNA deletions at 2 months were higher in the mitoPstI-DATtTA (+/-); Mgme1(-/-) SNC and VTA when compared to mitoPstI-DAT (+/-) alone but they were maintained at the same rate from the generation during the first 2 months until 6-month-old. These results suggest that absence of MgmeI results in an increase rate of mtDNA deletion formation but does not contribute to the increased accumulation of deletions through time in both SNc and VTA neuronal regions. However, we found higher susceptibility to mtDNA depletion in the SNc, likely as a result of poor compensation for the mito-PstI induced double-strand breaks, which could lead to preferential neuronal loss in this area.

Abstract #: 2024PA-0000000124

Presenter: Laura MacMullen

Leigh Syndrome Roadmap Project: A Natural History Study

MacMullen, LE¹, Stanley, KD¹, Yeske, P², Wilson, N², Koenig, MK³, Clearman, A³, Russo, N³, Poblete, M³, Guerra, WH³, Patel, N³, Cohen, BH⁴, Rossman, I⁴, Ginsberg, M⁴, Steiner, S⁴, Coppenger, J⁴, Tonni, H⁴, Erdesky, A⁴, Haas, R⁵, Yang, J⁵, Ruiz, C⁵, Reiner, G⁵, Murray, S⁶, Maguire, A⁷, Massucci, S⁸, Martin, L⁹, Christodoulou, J¹⁰, Thorburn, D¹⁰, Rahman,S¹¹, McFarland, R¹², Bertini, E¹³, Elsharkawi, I¹, George-Sankoh, I¹, Tormey, C¹, Demczko, M^1,14, Zolkipli-Cunningham, Z^1,14, Goldstein, AC^1,14, Falk, MJ^1,14*

¹Division of Human Genetics, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA, ²United Mitochondrial Disease Foundation, Pittsburgh, PA, USA, ³The University of Texas McGovern Medical School, Department of Pediatrics, Division of Child and Adolescent Neurology, Houston, TX, USA, ⁴Department of Pediatrics, Rebecca D. Considine Research Institute, Akron Children’s Hospital, Akron, OH, USA, ⁵Rady Children’s Hospital, UC San Diego School of Medicine, La Jolla, CA, USA, ⁶Mito Foundation, Sydney, Australia, ⁷Lily Foundation, Warlingham, England, ⁸Mitocon, Rome, Italy, ⁹People Against Leigh Syndrome, Houston, PA, USA, ¹⁰Murdoch Children’s Research Institute and Department of Paediatrics, University of Melbourne, Melbourne, Australia, ¹¹UCL Great Ormond Street Institute of Child Health and Great Ormond, Street Hospital for Children NHS Foundation Trust, London, UK, ¹²WCMR Newcastle University, Newcastle NE1 7RU, UK, ¹³Genetics and Rare Diseases Research Division, Unit of Neuromuscular and Neurodegenerative Disorders, Bambino Gesù Children’s Hospital, Rome, Italy. ¹⁴Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA

*falkm@chop.edu

Abstract: Leigh syndrome spectrum (LSS) is a rare, progressive neurodegenerative condition caused by pathogenic variants in more than 113 mitochondrial or nuclear DNA genes. The natural history of LSS is poorly understood and has not been rigorously studied. Through an international collaborative effort governed and financially supported by several international mitochondrial disease patient advocacy groups, we are working to define the natural history of LSS with defined molecular etiologies through objective and subjective assessments of symptom involvement over time including objective clinician assessments, subjective patient or parent-reported outcomes, and patient medical history. Overall, this observational prospective cohort study to evaluate the natural history of LSS involves outcome measure administration in disease-relevant domains at 3 to 6 months intervals. Retrospective medical history-based objective outcomes are also being evaluated. Primary outcome measurements include 1) objective clinician assessments of movement (Movement Disorder Childhood Rating Scale, MD-CRS), dystonia (Barry Albright Dystonia Scale, BADS), ataxia (Scale for the Assessment and Rating of Ataxia, SARA) and mitochondrial disease burden (Newcastle Pediatric Mitochondrial Disease Scale, NPMDS, and Newcastle Mitochondrial Disease Scale for Adults, NMDAS) 2) subjective measures of fatigue (Modified Fatigue Impact Scale, MFIS), quality of life (Pediatric Quality of Life Inventory, PedsQL), daily signs and symptoms (Observer Reported Outcome, ObsRO) and caregiver burden (Zarit Burden Interview, ZBI). Assessments can be done in person or by telehealth. Since study initiation in February 2022, 68 genetically confirmed LSS subjects have been enrolled to date across four active US sites, with international site activation in three additional countries underway. Deidentified data is sent from participating sites to a centralized data coordinating center at Children’s Hospital of Philadelphia (CHOP), where it is integrated and modeled to create user-friendly visualizations in the CHOP MMFP-Tableau data query tool and shared with investigators and project stakeholders at quarterly Web meetings. Interim longitudinal data will be presented, including cohort demographics and genetic distribution, common symptoms, and progression of symptoms over time, infection history, and results from clinical and developmental assessments. Longitudinal natural history study data will help to establish baseline outcome measures to prioritize for use in future clinical trials to test candidate therapies for LSS.

Abstract #: 2024PA-0000000125

Presenter: Ngoc Hoang

MitoTEMPO improves colonic mitochondrial dysfunction in the hyperandrogenemic rat model of PCOS

Hoang, N.H., Rezq, S., Shawky, N.M., Brooks, K., Quin, R.M., Yanes Cardozo, L.L., Romero, D.G., and Edwards*, K.

Department of Pharmacology and Toxicology, The Women’s Health Research Center, Mississippi Center of Excellence in Perinatal Research, University of Mississippi Medical Center Jackson, MS, USA

*Corresponding author’s email: kedwards1@umc.edu

Abstract: Polycystic ovarian syndrome (PCOS) is the most common endocrine disorder in women with approximately 80% of PCOS women suffer from hyperandrogenemia. PCOS women often exhibited symptoms of irritable bowel syndrome (IBS) that is associated sub-acute gastrointestinal (GI) inflammation. Mitochondrial dysfunction is associated with the development of inflammation and hyperandrogenemia can alter mitochondrial function. We have shown increased inflammation and mitochondrial dysfunction are present in the colons in the hyperandrogenemic female (HAF) rats, which exhibit similar characteristics to PCOS women. Targeting mitochondria is a feasible treatment option to improve mitochondrial function and inflammation in the colon. This study aims to investigate if the mitochondrial-targeted therapy, mitoTEMPO, will improve mitochondrial function in the colons of HAF rats. At 4 weeks of age, female rats received dihydrotestosterone (DHT, 7.5mg/90 days) pellets. MitoTMEPO (1mg/kg i.p daily) is administered 10 days before tissue collection. At 15 weeks of age, colon tissues were collected for mitochondria isolation. Intact mitochondrial respiration was measured simultaneously with reactive oxygen species (mtROS) using the Oroboros O2k-FluoRespirometer. Data was normalized to mitochondrial content using citrate synthase (CS) activity. HAF rats had a 52% increase in body weight compared to controls. MitoTEMPO treatment decreased body weight by 16% compared to HAF rats. Mitochondria from the colon of HAF rats showed a 25% decrease in complex I-driven respiration while mitoTEMPO improves respiration to the same as control. Complex II-driven respiration showed a 34% decrease that is recovered by mitoTEMPO to the same rate as control. Fatty acid oxidation using long-chain fatty acid (LCFA) or medium-chain fatty acid (MCFA) both show a 24% decrease. However, mitoTEMPO only improves MCFA oxidation to 10% of control respiraiton. The colons of HAF rats also showed a 170% increase in mtROS that is decreased by 30% with mitoTEMPO. The observed colonic mitochondrial dysfunction in the HAF model of PCOS suggests that mitochondrial dysfunction is involved in the development of IBS symptoms in PCOS. The improvement in body weight and colonic mitochondrial function in the HAF rat model of PCOS suggests that targeting mitochondria may provide a new avenue for treatment options for PCOS suffering from obsesity and IBS. Further studies are ongoing to determine in colon inflammation is decreased in mitoTEMPO treated HAF rats.

Supported by NIH grants: P20PGM121334

Abstract #: 2024PA-0000000126

Presenter: Eiko Nakamaru-Ogiso, PhD

mtDNA mutation heteroplasmic zebrafish models created by mitochondrial base editor tools

Jun Morisue¹, Ankit Sabharwal², Marni J. Falk^1,3, Stephen C. Ekker², Eiko Nakamaru-Ogiso^1,3*

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA, ²Department of Pediatrics, Dell Medical School, University of Texas at Austin, TX, ³Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA

*ogisoe@chop.edu

Abstract: Mitochondrial DNA (mtDNA) pathogenic variants are a major cause of primary mitochondrial disease (PMD) across all ages. A major challenge limiting both the study and the development of effective therapeutic strategies for mtDNA diseases has been the lack of representative animal models. We have recently established novel transgenic zebrafish models harboring pathogenic mtDNA mutations at 50-70% heteroplasmy levels, created using mitochondria-targeted base editing strategies with a FusX TALE Base Editor (FusXTBE) to enable precise manipulation of mtDNA with high editing efficiency¹. Here, we report the creation of a novel MT-ND5 m.13311C>T heteroplasmic zebrafish model, including its phenotypic characterization at the level of behavior and biochemistry in 7 days post fertilization (dpf) larvae. In addition, we developed a novel approach to profile mitochondrial energy metabolic substrate utilization in zebrafish.

A mitoFusXTBE targeting MT-ND5 (mtND5BE) predicted by TALE Writer was generated. 40 pg mRNA of mtND5BE was injected into single-cell zebrafish embryos, which were then raised at 29°C. Swimming activity was measured at 7 dpf (Zebrabox, ViewPoint). For biochemical assays, 20 embryos at 7 dpf were collected per tube, flash-frozen, and stored at −80°C until use. For respiratory chain complex and citrate synthase enzyme activity assays, crude mitochondrial-enriched fractions were isolated by differential centrifugation. For ATP and lactate/pyruvate assays, neutralized supernatant from perchloric acid extraction was used, while NADH was extracted with alkaline-acetonitrile solution. For GSH and GSSG analyses, fish homogenates in water were used and GSSG was isolated after N-ethylmaleimide treatment. ATP, NAD⁺, NADH, GSH, and GSSG were separated by HPLC-PDA or HPLC-ECD systems. Spectrophotometric assays were performed using a Tecan Infinite 200 PRO plate reader. Mitochondrial energy substrate metabolic flux profiling was carried out using Biolog MitoPlate S-1 with modifications. High-resolution respirometry was performed by Oxygraph-2K (Oroboros) and Blue-Native-PAGE to verify the function and integrity of each ETC protein.

mtND5BE injected animals (MT-ND5) consistently showed ~50% m.13311C>T heteroplasmy levels and reduced swimming activity by 24% relative to wild-type control larvae at 7 dpf. Complex I enzyme activity was significantly reduced by 25%, with 32% increased complex II enzyme activity in MT-ND5 animals. Whole fish lactate levels and lactate/pyruvate ratio were significantly increased in the MT-ND5 mutant larvae, successfully recapitulating PMD phenotypes. GSSG levels were increased by 185%, resulting in increased GSSG/GSH, in MT-ND5 mutant larvae. No difference was seen in NAD⁺ and NADH levels, nor in the NADH/NAD⁺ ratio, relative to uninjected wild-type control larvae. High-throughput mitochondrial energy metabolic flux profiling by MitoPlate S1 analysis demonstrated increased succinate (CII substrate) flux with decreased malate or glutamate/glutamine (CI substrate) flux, reflecting the altered energy metabolism of MT-ND5 mutant zebrafish.

Overall, our translational zebrafish model of a heteroplasmic mtDNA mutation in MT-ND5 successfully recapitulated the expected behavioral and biochemical disease phenotypes of complex I-based PMD. Further, we demonstrate the utility of a novel mitochondrial energy substrate metabolic flux profiling method. This vertebrate animal modeling and characterization approach will readily facilitate future development and validation of effective therapeutic strategies for mtDNA diseases.

Reference

 1. Sabharwal A, et. al. CRISPR J. 2021:799-821

Abstract #: 2024PA-0000000129

Presenter: Suraiya Haroon

High-throughput drug screening in a humanized C. elegans model identified potential therapeutic compounds for OPA1 disease

Mendel R¹, O’Hara T¹, Tara Z¹, Lu A¹, Wei S¹, Mathew N¹, Keith K², Seiler C³, Chen S⁴, Ogiso E^1,5, Falk MJ^1.5, and Haroon S^1,5

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA, ²Department of Biomedical & Health Informatics (DBHi), Children’s Hospital of Philadelphia, Philadelphia, PA, USA, ³Aquatics Core Facility, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA, ⁴Medical University of South Carolina, Charleston, SC, USA, ⁵Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA

*HAROONS@chop.edu

Abstract: Background: Pathogenic OPA1 variants primarily lead to progressive vision loss and 20% of patients exhibit addition neuronal and muscular deficiencies. At the molecular level, these variants induce fragmented mitochondria, mitochondrial dysfunction, and mtDNA depletion that lead to bioenergetic dysfunction and subsequent mitochondrial degradation via mitophagy. One approach to prevent the continual cycling of mitochondrial degradation and biogenesis is to modulate mitophagy, which we postulate may reduce ATP consumption, preserve mitochondrial content, and stabilize mtDNA content. Using this molecular understanding of the disease, our aim is to develop therapies for OPA1 disease.

Results: We generated a worm strain carrying a R289Q missense mutation, orthologous to the known OPA1 pathogenic variant R290Q, and the other strain carries a V328I missense mutation. Both strains showed defects in mtDNA content, respiration, fecundity, larval development, and neuromuscular function. Both strains exhibit an increased mitochondrial unfolded protein stress response (mtUPR) and this phenotype in the humanized R290Q strains was used to screen (i) potential therapies currently in empiric use for complex I disease worm model, (ii) 62 mitophagy modulating drugs, and (iii) 2,560 FDA-approved compounds. The first two screens have led to the identification of Thiamine and Celastrol that reproducibly reduced mitochondrial stress in the worms and show promise in rescuing movement in both worm models of OPA1 disease. The FDA-approved drug library screen has identified 16 compounds that require further validation to assay reproducibility and efficacy in improving mitochondrial and organismal health. Preliminary studies with Thiamine, Tripterin and Hemin treatment in the OPA1(R290Q/+) and OPA1(I403T) patient derived cell lines with defects in mtDNA content (qPCR) and mitochondrial respiration (Seahorse) show improvement in ATP production (CellGlo).

Conclusions: Overall, we describe a novel worm mutant strain (R289Q), a known worm mutant strain (V328I) and two patient fibroblast cell (R289^+/- and I403T^+/-) models of OPA1 disease. The zebrafish OPA1^-/- model exhibits impaired vision, a hallmark of OPA1 disease. Compound screens using the R290Q worm model have identified 19 different therapeutic candidates. Future validations will include testing the efficacy of the candidate compounds on multiple fitness outcomes in both C. elegans models, a zebrafish knockout model that exhibits optokinetic response defects, and both patient derived cell lines.

Abstract #: 2024PA-0000000130

Presenter: Camille Brown¹

Burden, coping, and resilience in caregivers of individuals with primary mitochondrial disease: Exploration, assessment, and implications for an intervention trial

Brown, C¹*, McCormick, EM², Valverde, KD^1,3, Falk, MJ^2,3

¹Master of Science in Genetic Counseling Program, University of Pennsylvania, Philadelphia, PA, ²Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA, ³Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA

*Camille.brown1@pennmedicine.upenn.edu

Abstract: Primary mitochondrial dhasse (PMD) encompasses a diverse group of inherited disorders impairing energy production that have limited treatment options. PMD patients typically endure an average of 16 unique clinical symptoms across multiple organ systems, significantly impacting daily life and increasing caregiver responsibilities. Caregivers of PMD patients commonly experience guilt, worry, sorrow, anger, and uncertainty regarding their child’s condition. Parental distress can impact child health outcomes if it interferes with their ability to successfully manage their child’s chronic condition. Therefore, gaining improved understanding of the psychosocial burden, existing resilience strategies, and coping skills of parents of indivduals with PMD is a critical first step in exploring ways to improve their coping, resilience, overall well-being, and potentially their children’s health outcomes.

Unaffected caregivers of pediatric and adult individuals with PMD having a confirmed genetic etiology recruited from Children’s Hospital of Philadelphia (CHOP) Mitochondrial Medicine Frontier Program (MMFP) Institutional Review Board (IRB) #08-6177 were invited to participate in discussion groups exploring daily living and existing coping strategies, and complete validated surveys (Zarit Caregiver Burden inventory, ZBI; Family Crisis Oriented Personal Scales, F-COPES; and Connor-Davidson Resilience Scale, CD-RISC). All discussion groups were Web-based, recorded, transcribed, and facilitated by a semi-structured interview guide. Transcripts were analyzed for common themes and topics via a grounded theory approach. The validated surveys were sent to all eligible caregivers regardless of discussion group participation.

One hundred ninety-two caregivers were invited to participate in the discussion groups and complete the surveys. Three discussion groups were held between December 2020 and February 2021 and an additional fourteen groups were held between June and October 2023. Forty individuals took part (10 men, 30 women), including three that had two affected children. Common themes highlighted by focus group participants included lifestyle adjustments post-diagnosis, diagnostic uncertainty, and finding positive aspects. Caregivers of individuals with PMD also discussed unique challenges such as adapting daily routines to their needs and coping with an uncertain prognosis, while finding silver linings in their situations. Forty-six caregivers completed all three scales, one caregiver completed only the CD-RISC and F-COPES, and eight caregivers completed only the CD-RISC (as surveys were administered sequentially, some caregivers completed only the first (CD-RISC), or first and second (CD-RISC and F-COPES) scales. CD-RISC average score was 73.4 (range: 0-100, higher scores indicate greater resilience). F-COPES average score was 100.5 (range: 29-145, higher scores signify better coping). ZBI average score was 31.5 (range: 0-88, higher scores indicate increased burden).

Overall, exploratory discussion groups and validated assessments in caregivers of individuals with PMD demonstrated they experience both acute and daily challenges and uncertainty and have reduced coping and resilience strategies with increased caregiver burden. As resilience can be taught and harnessed, outcomes of these discussion groups and measures are critical to establish baseline metrics in this population by which to assess the impact of techniques to improve the coping, resilience, and overall well-being of caregivers of individuals living with PMD.

Abstract #: 2024PA-0000000131

Presenter: Ibrahim Elsharkawi, MD

Use of Dichloroacetate as novel therapy in ECHS1 deficiency: A comparison of two cases

Jacob N¹, Ganesh J¹, Lopiccolo MK¹, Breilyn M¹, Morava E¹, Ganetzky R², Ginevic I¹, Waite J, Elsharkawi I¹*

¹Division of Medical Genetics, Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai. New York, New York, USA, ²Division of Human Genetics, Department of Pediatrics, Children’s Hospital of Philadelphia, University of Pennsylvania. Philadelphia, PA, USA

*Corresponding author: Ibrahim Elsharkawi, Ibrahim.Elsharkawi@mssm.edu

Abstract: ECHS1 (Mitochondrial Short Chain Enoyl-CoA hydratase) deficiency is a genetic defect in valine and fatty acid oxidation that led to disruption of oxidative phosphorylation and signs and symptoms of primary mitochondrial disease, often presenting with multi-systemic disease across a phenotypic spectrum. In neonates it may present with overwhelming primary lactic acidosis leading to loss of cardiovascular tone, with a reported mortality approaching 100% in the immediate neonatal period. Dichloroacetate (DCA) is a hitherto investigational agent which acts upon pyruvate dehydrogenase, maximizing its activation by inhibition of its inhibitor. Given evidence of secondary pyruvate inhibition in patients with neonatal ECHS1, and clinical similarities between ECHS1 and pyruvate dehydrogenase deficiency, we hypothesized utilizing DCA in ECHS1 may improve clinical outcomes.

Here, we report two sibling cases of ECHS1 with primary lactic acidosis phenotype. The first patient suffered progressive metabolic lactic acidosis refractory to standard medical therapies, and despite extra corporeal membrane life support, succumbed to cardiovascular collapse due to overwhelming metabolic acidosis and died in the newborn period. A molecular diagnosis of ECHS1 was confirmed prior to death. The second case, our proband, was the second child born to the same parents, presented a few hours after birth with respiratory distress with worsening lactic acidosis (peak lactate of 30.45 nmol/L) and developing cardiac dysfunction and cardiac arrest, refractory to aggressive sodium bicarbonate therapy. On DOL-3 the patient was started on DCA via emergent IND, with subsequent improvement and normalization of lactate levels. A valine restricted diet was also instituted. He exhibited prolonged stability, some developmental progress, and was discharged home.

At the time of writing, the patient is 7 months of age, with overall stable acid-base status on DCA and supplemental sodium bicarbonate, a valine restricted diet, N-acetylcysteine, and thiamine. He is largely PO fed and has made some developmental milestones despite significant global developmental delay. To our knowledge, this is the first case of ECHS1 managed with dichloroacetate. We posit that dichloroacetate may be of particular benefit in ECHS1 as well as primary PDH deficiency and may be of utility in other molecular etiologies of primary lactic acidosis.

Abstract #: 2024PA-0000000132

Presenter: Kristin Edwards

Colonic mitochondrial dysfunction and sub-acute gastrointestinal inflammation in the hyperandrogenemic rat model of PCOS is exacerbated by metformin

Edwards*, K., Hoang, N.H., Rezq, S., Shawky, N.M., Brooks, K., Quin, R.M., Yanes Cardozo, L.L., and Romero, D.G

Department of Pharmacology and Toxicology, The Women’s Health Research Center, Mississippi Center of Excellence in Perinatal Research, University of Mississippi Medical Center Jackson, MS, USA

*Corresponding author’s email: kedwards1@umc.edu

Abstract: Polycystic ovarian syndrome (PCOS) is the most common endocrine disorder in women during their reproductive years. Approximately 80% of PCOS women suffer from hyperandrogenemia. PCOS women often exhibited symptoms of irritable bowel syndrome (IBS) that is associated sub-acute gastrointestinal (GI) inflammation. However, the underlying pathophysiology for the development of IBS is unknown. Mitochondrial dysfunction is associated with the development of inflammation and hyperandrogenemia can alter mitochondrial function. The common treatment for PCOS, metformin, has been shown to cause GI side effects. This study aims to investigate the mechanisms linking PCOS and mitochondrial dysfunction to the development of IBS symptoms that are exacerbated by metformin. The hyperandrogenemic female (HAF) rodent models exhibit characteristics similar to PCOS women such as increased body weight, fat mass, and food intake. At 4 weeks of age, female rats received dihydrotestosterone (DHT, 7.5mg/90 days) pellets. Rats received metformin (300 mg/kg/day in food) starting at 12 weeks of age. At 15 weeks of age, colon tissues were collected for histology (H&E staining) and mitochondria isolation. Intact mitochondrial respiration was measured simultaneously with reactive oxygen species (mtROS) using the Oroboros O2k-FluoRespirometer. Complex IV activity, a marker for oxidative phosphorylation capacity, was also measured using the Oroboros O2k-FluoRespirometer. Complex I, II, III, citrate synthase, and aconitase were measured spectrophotometrically. Data was normalized to mitochondrial content using citrate synthase (CS) activity. In the colons from HAF rats, histology showed an increase in immune cell infiltration and structural derangement that was exacerbated by metformin treatment. These colons also showed a decrease in mitochondrial complex I-driven respiration (25% decrease), complex II-driven respiration (34% decrease), LCFA oxidation (26% decrease), and MCFA oxidation (22% decrease) compared to controls. Metformin treatment further decreased complex I-driven respiration (40% further decrease) and MCFA oxidation (23% further decrease) compared to HAF without metformin. The colons of HAF rats also showed an increase in mtROS (170% increase) that was further increased by metformin treatment (22% further increase). Colon mitochondria from the HAF rats showed a decrease in all of the mitochondrial electron tranport complexes activities (40-50% decrease) but metformin only further decreased complex I activity (30% further decrease). Aconitase activity was decreased (68%) in the colons from the HAF rats indicating potential oxidative damage. However, metformin treatment showed no further change. The observed colonic inflammation and associated mitochondrial dysfunction in HAF model of PCOS suggests that mitochondrial dysfunction is involved in the development of IBS symptoms in PCOS. The metformin-mediated mitochondrial dysfunction suggests that mitochondria play a role in the GI side effects with this medication. This study provides a better understanding of the role of mitochondria in the development of IBS with PCOS and metformin treatment while providing an avenue for the development of strategies to re-establish normal mitochondrial function. This could provide options for preventive and therapeutic interventions for IBS where there are limited treatment options in PCOS women.

Supported by NIH grants: P20PGM121334

Abstract #: 2024PA-0000000135

Presenter: Schlein ML^★

Antibody response to the pneumococcal vaccine in children with mitochondrial disease

Schlein ML¹*, Gordon-Lipkin EM¹, Kruk S¹, McGuire PJ¹

¹Metabolic Medicine Branch, NHGRI, NIH, United States

*melissa.schlein@nih.gov

Abstract: Mitochondrial diseases (MtD) are the most common inborn errors of metabolism. Mutations in mtDNA or nDNA result in disorders of oxidative phosphorylation and mitochondrial maintenance. Mitochondria have been shown to play a role in immunity, and a weakened immune phenotype has been observed in patients with MtD. Infection is a major cause of morbidity in patients with MtD, and the two most common causes of death for these patients are sepsis and pneumonia. Previous work of the group has found that children with MtD frequently fail to produce antibodies against common childhood vaccinations (MMR, varicella). With this in mind, we analyzed pneumococcal titers collected from our patients to determine if MtD patients struggle to mount an immune response to the pneumococcal vaccine. Children with MtD participated in a natural history study. Eligibility criteria included “probable” or “definite” MtD by revised Walker criteria. Patients were seen at the NIH Clinical Center in Bethesda, Maryland, where serum was evaluated for presence of the 23 serotypes included in the pneumococcal polysaccharide vaccine (PPSV23) as well as levels of immunoglobulins IgG, IgA, and IgM. This data was extracted from clinical patient records. A sufficient immune response against the pneumococcal vaccine was defined as having positive titers for greater than 50% of the serotypes included in the vaccine. We also used data from a previous analysis of bacterial epitopes via PhIPSeq analysis to measure environmental exposure to pneumococcus bacteria in 2020 and 2021. A subset of our cohort (n = 17) had this data available. Cohabiting family members were used as healthy controls. 43 patients were included, 26 males and 16 females with an average age of 9.3 years old. The mitochondrial phenotypes were identified as Kearns Sayre Syndrome (N = 3), Leigh Syndrome (N = 14), Leigh-like mitochondrial disease (N = 6), and MtD, not otherwise specified (N = 20). Out of all patients, 58.1% (N = 25) had a sufficient response to the 23-valent vaccine, out of all patients, 58.1% (N = 25) had a sufficient response to the 23-valent vaccine, and 41.9% (N = 18) did not. We did not identify a correlation between percentage of positive titers and age, sex, disease subtype, or IgG levels. The antibody binding of all measured pneumococcus proteins decreased on average by 61.7% in MtD patients and by 56.7% in their cohabiting family members between 2020 and 2021. While most patients produced titers in response to the pneumococcal vaccine, up to one-third of them were lacking an appropriate antibody response. These patients may be vulnerable to pneumococcal disease and require revaccination with the PPSV23 vaccine. Longitudinal and revaccination studies will determine whether they fail to produce versus fail to maintain antibodies. Children with MtD may become more severely ill from pneumococcal infection, making vigilance to vaccine response important. Additionally, compared to their cohabiting family members, the rate of loss of antibodies to pneumococcal protein antigens was greater in children with MtD. Providers who care for these patients should consider including pneumococcal titers in their clinical evaluation.

Abstract #: 2024PA-0000000138

Presenter: Yue Wang

Low- Level Large Deletions in Mitochondria Genomes: A Potential Diagnosis of Mitochondrial Diseases

Jun Yang¹, Tiansheng Chen¹, Chung-Yang Kao¹, Jie Dong¹, John Lattier¹, Hongzheng Dai^1,2, Linyan Meng^1,2, Fan Xia^1,2, Eric S. Schmitt¹, Sandra Peacock¹, William J Craigen^1,2, Robert Rigobello¹, Lee-Jun C Wong^1,2†, Christine M. Eng^1,2, Yue Wang^1,2

¹Baylor Genetics, 2450 Holcombe Blvd, Houston, TX 77021, ²Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030

*ywang@baylorgenetics.com

Abstract: Background: Large deletions in the mitochondrial genome are a significant contributor to mitochondrial diseases. The proportion of mitochondrial DNA (mtDNA) carrying these large deletions is a critical factor in determining phenotype and clinical outcomes. In this retrospective study, we aim to explore age-related large deletions that may contribute to mitochondrial diseases.

Methods: A long-term study of mitochondrial genome wide variants was performed using a Next Generation Sequencing (NGS) based platform. Long-range PCR is used to enrich and amplify mitochondrial DNA followed by NGS. NGS data are processed using bioinformatic pipelines to call single nucleotide variants (SNV) and large deletions throughout the mitochondrial genome. SNVs and small indel variants can be confidently called at heteroplasmic levels as low as 1.5%. MLPA was used to confirm the large deletion and estimate the fraction of deficient mtDNA. We retrospectively analyzed the findings of large deletions from cases.

Results: Among tested samples, 6.5% cases exhibited large deletions in mitochondrial genomic regions, with 4.6% of these cases also tested positive for pathogenic or likely pathogenic single nucleotide variants (SNVs). Out of the cases solely positive for large deletions, 82% displayed phenotypes at least partially consistent with mitochondrial diseases. Among these, approximately 50% samples had low-level deletions detected by NGS sequencing and confirmed by MLPA.

While low-level deletions of muscle mtDNA occur with age in healthy individuals and can potentially be disease-causing in young patients, our study identified ~39% patient cases with low-level deletions who were younger than 30 years old. Some of them exhibited symptoms suggesting mitochondrial disorders. The cutoff for low-level deletions was approximately 10% or less. This subset included approximately 43% blood samples or DNA samples from blood, 51% muscle samples, and 6% samples from saliva, liver, or other tissue types.

Conclusion: It has been reported that a biochemical deficiency may not be observed unless the large deletion fraction exceeds 60%, and low-level deletions of muscle mtDNA are known to occur with age in healthy individuals. However, within our study cohort, a significant proportion of young patients exhibit low-level heteroplasmic large deletions. This phenomenon could result from the clonal effect causing biological impact being significantly diluted in the tested sample or may be attributed to tissue specificity. To ensure accurate diagnosis, affected tissues such as muscle instead of blood are recommended.

Considering that mitochondrial disorders may be caused by molecular defects in nuclear genes, sequence analysis of specific nuclear genes may also be indicated. Employing a combination test involving mitochondria-related nuclear gene testing along with mitochondrial genome-wide testing or utilizing whole-genome sequencing (WGS) that sequences the entire human nuclear genome and mitochondrial genomes simultaneously could provide opportunities to reveal the etiologies of dual genomes in the patients.

Abstract #: 2024PA-0000000140

Presenter: Margaret Moore, MS

Patient Perspectives on Myopathy Associated With the m.3243A>G Mitochondrial Disorders Variant: A Qualitative Research Study

Wilson N¹, Moore MA¹, Yeske P¹, Arora T², Bayley J², Mehta A², Leavitt E²*, Karaa A³

¹The United Mitochondrial Disease Foundation, USA; ²Trinity Life Sciences, USA; ³Division of Genetics, Massachusetts General Hospital, Harvard Medical School, USA

*Corresponding author eleavitt@trinitylifesciences.com

Abstract: The mitochondrial DNA (mtDNA) m.3243A>G mutation is a common and complex pathogenic mtDNA variant associated with heterogeneous diseases including mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), maternally inherited diabetes and deafness (MIDD), and primary mitochondrial myopathy (PMM). There is little information available about the impact of m.3243A>G-associated myopathy on quality of life. To better understand the patient’s perspective on the diagnostic journey and experience with disease management, we performed qualitative interviews of patients with the m.3243A>G mutation experiencing symptoms of myopathy. Eligible patients were aged >18 years, diagnosed with a primary mitochondrial disorder, positive for the m.3243A>G mutation, and experienced muscle weakness, exercise intolerance, and/or muscle fatigue. The survey consisted of a 1-hour conversation discussing patient background, the impact of disease on daily life, current symptom management and unmet needs, and openness to new therapies. Nine patients met the criteria for inclusion in the study: 6 self-reported having MELAS, and 1 each MIDD, PMM, or MIDD and PMM. The mean age was 53 years and mean time since diagnosis was 5 years. In addition to myopathy, 7 of 9 (78%) patients had hearing loss. Patients reported that their symptoms appeared in young adulthood and progressively worsened; however, the majority did not present for diagnosis until symptoms interfered with their personal responsibilities or relationships. Given the variety of symptoms such as exercise intolerance, hearing loss, droopy eyelids, gastrointestinal issues, and brain fog, patients are often believed to have different independent conditions and are referred to various specialists. Misdiagnosis and delay in diagnosis are common (average 4-5 years before referral to genetic specialist); patients with a family history of similar conditions may be diagnosed more quickly, whereas those with limited access to specialists could experience longer delay. Although patients report experiencing psychological relief upon receiving a diagnosis, feelings are complicated by learning they have a progressive, uncurable disease that could be passed on to their children (“I know what the future holds so it’s hard to go on like this”). As a result of the exploratory and often prolonged diagnosis process, patients report reduced trust in the healthcare system, furthered by a lack of disease-modifying treatments. All patients cited routine use of 4-10 supplements that do not result in symptomatic relief (satisfaction levels of 1-2); however, they fear stopping could have negative consequences. Five patients reported going to a physical therapist; 2 experienced relief from muscle spasms, whereas 3 did not experience benefit. Patients often resort to self-treatment with monitoring devices and home remedies. Regarding potential new therapies, the ideal scenario would be curative treatment; otherwise, treatment that stopped or slowed disease progression and reduced daily pill burden was viewed positively. This qualitative study of 9 patients with m.3243A>G provides insight into the diagnostic and treatment journey of those experiencing m.3243A>G-related myopathy. Delay in diagnosis results in delayed symptom management and increased burden on the patient. Current management options are ineffective at controlling symptoms. There is an unmet need for a therapy that is curative or delays/halts progressive symptoms of myopathy.

Abstract #: 2024PA-0000000142

Presenter: Sohum Purao, B.S.

Heterozygosity for HOGA1 variants is associated with an increased risk for kidney stones

Purao S^1,2*, Chang A³, Strande NT⁴ and Elsea SH^2,5,6

¹Texas A&M School of Medicine, Bryan, TX 77807 USA ²Dept. of Molecular & Human Genetics, Baylor College of Medicine, Houston, TX 77030 USA, ³Dept. of Nephrology, Geisinger Medical Center, Danville, PA 17822 USA, ⁴Autism and Developmental Medicine, Geisinger Medical Center, Danville, PA17822 USA, ⁵Baylor Genetics, Houston, TX 77030 USA, and ⁶BCM-Human Genome Sequencing Center, Houston, TX 77030 USA

*Corresponding author: sohumpurao@tamu.edu

Abstract: Primary hyperoxaluria (PH) is a rare autosomal recessive hereditary disorder of glyoxylate metabolism that is characterized by excessive endogenous oxalate production due to impaired oxalate catabolism. Excess oxalate is excreted through the kidneys and over time, typically results in nephrolithiasis, including calcium oxalate renal stone formation. Insufficient activity of any one of three primary enzymes involved in glyoxylate metabolism (AGT [PH1], GRHPR [PH2], or HOGA [PH3]) can result in similar clinical outcomes. PH3 is associated with bi-allelic pathogenic variants in HOGA1, coding for 4-hydroxy-2-oxoglutarate aldolase, a mitochondrial enzyme critical for managing oxalate levels. When deficient, elevated oxalate leads to nephrolithiasis and for some patients, end-stage renal disease. Both genetic and clinical heterogeneity of PH3 greatly contribute to the difficulties in diagnosis and further understanding this disease. Although the recessive form has been well-documented to lead to severe renal complications, risk for kidney stones and kidney disease in individuals heterozygous for HOGA1 pathogenic variants has not been described. In this study, HOGA1 variants clinically reported in ClinVar (www.ncbi.nlm.nih.gov/clinvar/) and pathogenic (P) and likely pathogenic (LP) variants reported in the literature were curated. Variant prevalence was assessed using the Genome Aggregation Database (gnomAD v4.0.0) to determine allelic frequencies across ethnic populations and to estimate disease prevalence. Stringent ACMG/AMP pathogenicity guidelines were followed to curate 130 reported disease associated HOGA1 variants. The pan-ethnic disease prevalence was estimated at ~1/70,000, with the carrier frequency estimated at ~1/130 individuals, also indicating higher carrier rates in East Asian and Ashkenazi Jewish populations. Furthermore, while previous studies have shown intermittent levels of urine oxalate in some heterozygous individuals, carriers have not been described with phenotypic consequences, and few studies have broadly assessed the impact of carrier status in healthcare populations. Using exome sequencing and electronic health record data from the Geisinger MyCode DiscovEHR study, we investigated prevalence and phenotypic spectrum of kidney disease in individuals who were carriers of known P/LP HOGA1 variants. Analysis revealed a higher risk for nephrolithiasis in patients heterozygous for a P/LP HOGA1 variant when compared to controls without variants in PH-associated genes (7.9% vs. 5.8%; p=0.002). The P/LP variants most commonly observed in association with nephrolithiasis included: c.700+5C>T (70/844; 8.3%; p=0.002), p.Glu315del (5/41; 11.0%;p=0.002), p.Ala36Val (6/45; 13.3%; p=0.03), and p.Pro190Leu (7/41; 17.1%; p=0.002). While no associations with glomerular filtration rate or end-stage renal disease were identified in HOGA1 carriers in this population, the increased risk for kidney stones poses a significant burden on quality of life. This preliminary analysis showed association between three heterozygous HOGA1 variants and a common characteristic of PH3; however, variants not present in this population may pose risks for other groups. Adult-onset of nephrolithiasis in heterozygotes also supports the need for additional studies in healthcare populations to determine other associated factors leading to clinical presentation in carriers. Our analyses show that while the recessive form of PH3 is rare, it is likely underdiagnosed, and individuals heterozygous for pathogenic variants in HOGA1 have an increased risk to develop kidney stones in adulthood, which may be preventable through early identification and intervention.

Abstract #: 2024PA-0000000143

Presenter: Sarah H Elsea

Succinic semialdehyde dehydrogenase deficiency: Disease prevalence, plasma biomarkers, and a proposed integrated metabolomic and genomic approach to rapid diagnosis

Gijavanekar C.^1,2, Glinton K.E.^1,3, Rajagopal A.¹, Mackay L.P.⁴, Martin K.A⁵, Pearl P.L.⁶, Gibson K.M.⁷, Wilson T.A.¹, Sutton V.R.^1,3,8, and Elsea S.H.^1,2,8*

¹Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA, ²Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX, USA, ³Texas Children’s Hospital, Houston, TX, USA, ⁴Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA, ⁵NeoGenomics Laboratories, Aliso Viejo, CA, USA, ⁶Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA, ⁷Department of Pharmacotherapy, College of Pharmacy and Pharmaceutical Sciences, Washington State University, Spokane, Washington, USA, ⁸Baylor Genetics Laboratories, Houston, TX, USA

*Corresponding author: elsea@bcm.edu

Abstract: Genomic sequencing offers an untargeted, data-driven approach to genetic diagnosis; however, variants of uncertain significance often hinder the diagnostic process. The discovery of rare genomic variants without previously known functional evidence of pathogenicity often results in variants being overlooked as potentially causative, particularly in individuals with undifferentiated phenotypes. Consequently, many neurometabolic conditions, including those in the GABA (gamma-aminobutyric acid) catabolism pathway, are underdiagnosed. Succinic semialdehyde dehydrogenase deficiency (SSADHD, OMIM #271980) is a neurometabolic disorder in the GABA catabolism pathway. The disorder is due to bi-allelic pathogenic variants in ALDH5A1 and is usually characterized by moderate-to-severe developmental delays, hypotonia, intellectual disability, ataxia, seizures, hyperkinetic behavior, aggression, psychiatric disorders, and sleep disturbances. GABA is catabolized by a 2-step enzymatic pathway involving GABA-transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH) in the astrocyte mitochondria. Deficiency of either enzyme leads to accumulation of GABA. Accurate genetic diagnosis requires comprehensive knowledge of disease - including pathogenic genetic variants and associated biomarkers. Both metabolite and genomic testing may be required for accurate diagnosis but are currently clinically underutilized due to invasiveness of specialty procedure of cerebrospinal fluid neurotransmitter analysis and poor characterization of genomic variants. In this study, we utilized an integrated approach to diagnosis of SSADHD by examining molecular, clinical, and metabolomic data from a single large commercial laboratory. Our analysis led to the identification of 16 patients with SSADHD, along with three novel variants. We performed plasma untargeted metabolomic analysis and genome sequencing to identify key biomarkers of altered GABA catabolism and associated genomic variants. We identified a clear metabolomic signature of plasma biomarkers for GABA-TD (2-pyrrolidinone, succinamic acid, 4-guanidinobutanoate) and SSADHD (2-pyrrolidinone, 4-guanidinobutanoate, argininate) that also distinguish individuals treated with vigabatrin, an anti-seizure medication that blocks GABA-T. We further surveyed all available pathogenic/likely pathogenic variants and used this information to estimate the global prevalence of this disease to be between 1/223,000 to 1/564,000. We also assessed the clinically referred population in our laboratory from 2014-2022 and identified 16 individuals with SSADHD and 6 individuals with GABA-TD, suggesting that the disease frequency of SSADHD in this population is ~1 in 1,375 and that of GABA-TD is ~1 in 3,500. The cumulative disease frequency of these GABA metabolism disorders in our laboratory during this period is estimated to be ~1/1,100. Genomic sequencing is now considered part of the first-line approach for evaluation of a child with developmental delays. Metabolomic profiling demonstrated that blood-based biomarkers effectively interrogate the GABA pathway, and CSF is not required for accurate diagnosis. Taken together, our comprehensive analysis allows for a global approach to the diagnosis of SSADHD and provides a pathway to improved diagnosis and potential incorporation into newborn screening programs. Furthermore, early diagnosis facilitates referral to genetic counseling, family support, and access to targeted treatments - taken together, these provide the best outcomes for individuals living with either GABA-TD or SSADHD, as well as other rare conditions.

Abstract #: 2024PA-0000000145

Presenter: Bryn D. Webb

MARS2 Deficiency: Where are the missing patients?

Webb BD^1*, Wheeler PG², and Houten SM³

¹Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA, ²Division of Genetics, Arnold Palmer Hospital, Orlando, FL, USA, ³Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA

*bdwebb@wisc.edu

Abstract: Previously we identified by whole exome sequencing that recessive single nucleotide variants (SNV) in mitochondrial methionyl amino-acyl tRNA synthetase (MARS2) cause a mitochondrial disease characterized by developmental delay, poor growth, and sensorineural hearing loss (MIM #616430; Combined oxidative phosphorylation deficiency 25). The compound heterozygous pathogenic variants c.550C>T;p.Gln184^★ and c.424C>T;p.Arg142Trp [NM_138395.3] were identified in two siblings and led to: decreased MARS2 protein levels in patient lymphoblasts; decreased Complex I and IV enzyme activities in patient fibroblasts; and reduced protein levels of NDUFB8 and COXII, representing Complex I and IV respectively, in patient fibroblasts and lymphoblasts. Overexpression of wild-type MARS2 in patient fibroblasts increased NDUFB8 and COXII protein levels¹. We have now established a Mars2 mouse model of disease and have identified differentially expressed gene signatures for homozygous mutant mice in liver, cortex, gastrocnemius, heart, and kidney. We also present the clinical follow-up of the original two affected siblings, the only family reported to date, and query the conference attendees whether others have identified patients with MARS2 SNV mutations.

References

 1. Webb BD, Wheeler PG, Hagen JJ, Cohen N, Linderman MD, Diaz GA, Naidich TP, Rodenburg RJ, Houten SM, Schadt EE. Novel, compound heterozygous, single-nucleotide variants in MARS2 associated with developmental delay, poor growth, and sensorineural hearing loss. Hum Mutat. 2015 Jun;36(6):587-92.

Abstract #: 2024PA-0000000146

Presenter: Graeme Preston

Induced Pluripotent Stem Cell Based Cardiac Model of MELAS Disease

Graeme P^1,2*, Tim E², Eva M^1,2, Tamas K^1,2

¹Ichan School of Medicine at Mount Sinai, Department of Genetics & Genomic Sciences, New York NY, USA, ²Mayo Clinic, Department of Clinical Genomics, Rochester MN, USA

*graeme.preston@mssm.edu

Abstract: Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke Like Episodes (MELAS) Syndrome is the most common primary mitochondrial disease (PMD), affecting ~1:20,000 The disease is caused by mutations in the mitochondrial leucyl-tRNA gene, most frequently by the single base pair variant m.3243A>G in the MT-TL1 gene. Mitochondria possessing the m.3243A>G variant experience a significant reduction in the activity of mitochondrial electron transport chain complex I (CI), resulting in profound energetic and metabolic dysfunction. One notable metabolic consequence of the reduced flux through the NADH dehydrogenase CI is the accumulation of NADH and the concomitant depletion in its reduced equivalent NAD+. This increased ratio of NADH:NAD+, termed “reductive stress”, has far reaching implications on the total oxidative/reductive metabolism of the cell, and can act as a functional readout for the efficacy of putative pharmacologic interventions to ameliorate MELAS symptomatology. We have previously utilized the live fluorescent probe of NADH:NAD+ “SoNar” to perform a high throughput screen of FDA-approved to identify putative compounds for the amelioration of MELAS symptomatology. Here we validate compounds identified in the initial screen, and performed subsequent oximetry analysis to further elucidate the effects of these compounds on iCM mitochondrial energy metabolism.

We reprogrammed induced pluripotent stem cells (iPSCs) from skin fibroblasts isolated from 3 individuals with MELAS syndrome precipitating from the m.3243A>G variant. This reprogramming results in multiple clones with varying levels of m.3243A>G heteroplasmy, ranging from 0% to over 90%. We differentiated “high” heteroplasmy (~65%) and “low” heteroplasmy (0%) isogenic controls to induced cardiomyocytes (iCMs) through treatment with the Wnt activator ChiR, followed by the Wnt inhibitor IWP2. We subsequently transduced these cells with the SoNar probe to validate the efficacy of our initial screen hits in ameliorating reductive stress. Finally, we performed Seahorse oximetry to further elucidate the effects on these compounds on the mitochondrial energy metabolism of MELAS iCMs.

We observed significant amelioration of reductive stress in MELAS m.3243A>G iCMs following incubation with the topoisomerase 2 (TOP2) inhibitors idarubicin and amrubicin, the angiotensin converting enzyme 2 (ACE2) inhibitor captopril, and the sympathomimetic clenbuterol. Subsequently Seahorse oximetry demonstrated increased oxygen consumption as well as glycolytic flux in iCMs following incubation clenbuterol and captopril, as well as another popular ACE2 inhibitor lisinopril. Conversely, we observed reduced oxygen consumption and glycolytic flux in iCMs incubated with the TOP2 inhibitors amrubicin and idarubicin, as well as the TOP1 inhibitor topetecan. Most notably, while high heteroplasmy MELAS iCMs predictably displayed a reduced oxygen consumption and glycolytic flux relative to low heterplasmy isogenic controls when oxygen consumption and glycolytic flux were normalized to mitochondrial mass (citrate synthase activity), these same high heteroplasmy iCMs displayed increased oxygen consumption and glycolytic flux relative to controls when normalized to cell number, due to an apparent compensatory increase in mitochondrial mass. This finding comports with the so-called “hypermetabolic” phenotype increasing recognized in disease-relevant PMD cell types, and fundamentally shifts the metric by which the efficacy of putative pharmacologic interventions for ameliorating the symptoms of MELAS syndrome, in particular MELAS cardiomyopathy.

Abstract #: 2024PA-0000000147

Presenter: Quinn Peterson

Modeling Endocrine Dysfunction in Mitochondrial Disease: The Promise of Stem Cell-Derived Beta Cells

Khashim Z¹, Schornack AR¹ and Peterson, QP^1,2*

¹Mayo Clinic, Department of Physiology and Biomedical Engineering, Rochester, MN, USA, ²Mayo Clinic, Center for Regenerative Biotherapeutics, Rochester, MN, USA

*Peterson.Quinn@mayo.edu

Abstract: One of the earliest clinical manifestations of MELAS syndrome are defects in endocrine metabolism. Although variable in clinical presentation, estimates suggest that as many as 80% of MELAS patients are diagnosed with diabetes mellitus often before diagnosis of MELAS syndrome. Despite this high degree of prevalence, little is known about the mechanism of pancreatic defect resulting from MELAS mutations in large part to the absence of suitable model systems. Toward this end, we sought to develop a cellular model system for the most common variant associated with MELAS, m.3243A>G. This variant occurs in over 80% of MELAS cases and affects the mitochondrial MT-TL1 gene. Our laboratory specializes in the development of pancreatic cell types from pluripotent stem cells. We recently reported protocols for the generation of stem cell-derived beta (SC-beta) and stem cell-derived alpha (SC-alpha) cells. Additionally, we have generated patient specific iPSC lines with varying levels of mitochondrial heteroplasmy. Here we report preliminary finding on the functional and differentiation implications of varying levels of heteroplasmy on pancreatic islet cell function. We demonstrate the ability to generate m.3243A>G specific SC-beta cells with heteroplasmy levels of 0%, 60% and 80% respectively. Increasing heteroplasmy level does not have a significant impact on the developmental process leading from pluripotent cells to differentiated beta cells, however, heteroplasmy level impacts the insulin secretion function of these cells. These results demonstrate the potential for this model system to elucidate early endocrine defects that proceed MELAS diagnosis and may identify early intervention points in evaluating and treating this disease.

Abstract #: 2024PA-0000000152

Presenter: Keri-Lyn Kozul

FBXL4-associated mtDNA Depletion Syndrome is characterized by elevated BNIP3/NIX-mediated mitophagy

Kozul K^1,2*, Nguyen-Dien GT^2,3, Taylor R^4,5, Pagan JK^2,6,7, Niemi NM¹

¹Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, USA, ²School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, Australia, ³Department of Biotechnology, Viet Nam National University-International University, Vietnam, ⁴Wellcome Centre for Mitochondrial Research, Newcastle University, UK, ⁵NHS Highly Specialised Service for Rare Mitochondrial Disorders, UK, ⁶Institute for Molecular Bioscience, The University of Queensland, Australia, ⁷Frazer Institute, The University of Queensland, Australia.

*kozul@wustl.edu (*Corresponding author’s email)

Abstract: Quality control mechanisms are the hallmark of mitochondrial homeostasis, ensuring stringent regulation of mitochondrial processes by balancing the biogenesis and turnover of mitochondrial proteins. The Ubiquitin Proteasome System (UPS) is canonically responsible for cellular protein turnover. SKP1-Cullin 1-F-box protein (SCF) complexes are multi-subunit E3 ubiquitin ligases that function within the UPS to control targeted protein degradation. SKP1 acts as an adaptor, CUL1 serves as the scaffolding backbone, and F-box proteins are the interchangeable, substrate-targeting components of the SCF complexes. However, although there are 69 known mammalian F-box proteins, most have not been well characterized and little is known regarding the role of the SCF ubiquitin ligases in regulating mitochondrial proteins. F-box proteins often recognize short linear motifs on their protein substrates, which are commonly phosphorylated residues known as degrons. Hence, kinases and phosphatases play integral roles in the fine-tuning of how SCF ubiquitin ligases recognize their substrates. One mitochondrially localized SCF ubiquitin ligase is SCF-FBXL4. Early studies show that mutated FBXL4 results in severe encephalopathic mitochondrial DNA Depletion Syndrome 13 (MTDPS13)^1,2. Along with encephalopathy, MTDPS13 patients present with symptoms ranging from metabolic dysfunction to developmental defects. Fbxl4 knockout (KO) mouse models exhibit severe MTPDS13-associated dysfunction, including reduced mtDNA levels and elevated levels of mitophagy³; a key mitochondrial quality control mechanism where selected mitochondria are removed by autophagy. Interestingly, knockout of the mitochondrial phosphatase Pptc7 in mice and cell models metabolically resembles the defects of the Fbxl4 KO model^4,5,6,7,8, suggesting the potential of PPTC7 fine-tuning FBXL4 function. However, prior to this investigation, specific mitophagy mechanisms remained poorly understood. Hence, by using CRISPR-Cas9 knockout and MTDPS13 patient-derived fibroblasts, we performed rescue experiments, immunoprecipitation assays and mitophagy assays to characterize the molecular phenotype of FBXL4-associated MTDPS13. First, we uncovered that FBXL4 is a key regulator of BNIP3 and NIX turnover^5,6,7. We then investigated the function of FBXL4 in mitophagy suppression and characterized how pathogenic FBXL4 variants, derived from MTDPS13 patients, affect the ability of FBXL4 to regulate the turnover of BNIP3 and NIX^5,6,7. Loss of FBXL4 function results in decreased ubiquitination, an increased accumulation of BNIP3 and NIX, and subsequently elevated mitophagy^5,6,7. We have found that MTDPS13-associated pathogenic variants of FBXL4 are unable to maintain basal mitophagy due to an inability to interact with core SCF ubiquitin ligase machinery and subsequently degrade BNIP3 and NIX^5,6,7. Thus, by studying the FBXL4-BNIP3/NIX axis in mitophagy, we reveal that FBXL4 mutations contribute to MTDPS13 via excessive BNIP3/NIX-mediated basal mitophagy. Lastly, we uncover that the mitochondrial phosphatase PPTC7 regulates the FBXL4-mediated degradation of BNIP3 and NIX^8,9,10,11. Overall, these findings describe the role of FBXL4, together with PPTC7, in the turnover of BNIP3 and NIX to suppress basal mitophagy. FBXL4 and PPTC7 may be used therapeutically in the treatment of MTDPS13 given their roles as critical regulators of basal BNIP3/NIX-mediated mitophagy.

References

 1. Gai, X., D. Ghezzi, M.A. Johnson, C.A. Biagosch, H.E. Shamseldin, T.B. Haack, A. Reyes, M. Tsukikawa, C.A. Sheldon, and S.J.T.A.J.o.H.G. Srinivasan. 2013. Mutations in FBXL4, encoding a mitochondrial protein, cause early-onset mitochondrial encephalomyopathy. 93:482-495.

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