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. Author manuscript; available in PMC: 2022 Jan 1.
Published in final edited form as: Am J Med Genet A. 2021 Oct 8;188(1):357–363. doi: 10.1002/ajmg.a.62520

D-bifunctional protein deficiency caused by splicing variants in a neonate with severe peroxisomal dysfunction and persistent hypoglycemia

Kelly M Werner 1, Allison J Cox 2,3, Emily Qian 2, Preti Jain 2,4, Weizhen Ji 1, Irina Tikhonova 2, Christopher Castaldi 2, Kaya Bilguvar 2, James Knight 2, Sacha Ferdinandusse 5, Rima Fawaz 1, Yong-Hui Jiang 2, Patrick G Gallagher 1,2,6, Matthew Bizzarro 1, Jeffrey R Gruen 1,2, Allen Bale 2, Hui Zhang 2
PMCID: PMC8678290  NIHMSID: NIHMS1747879  PMID: 34623748

Abstract

D-bifunctional protein (DBP) deficiency is a rare, autosomal recessive peroxisomal enzyme deficiency resulting in a high burden of morbidity and early mortality. Patients with DBP deficiency resemble those with a severe Zellweger phenotype, with neonatal hypotonia, seizures, craniofacial dysmorphisms, psychomotor delay, deafness, blindness, and death typically within the first 2 years of life, although patients with residual enzyme function can survive longer. The clinical severity of the disease depends on the degree of enzyme deficiency. Loss-of-function variants typically result in no residual enzyme activity; however, splice variants may result in protein with residual function. We describe a full-term newborn presenting with hypotonia, seizures, and unexplained hypoglycemia, who was later found to have rickets at follow up. Rapid whole genome sequencing identified two HSD17B4 variants in trans; one likely pathogenic variant and one variant of uncertain significance (VUS) located in the polypyrimidine tract of intron 13. To determine the functional consequence of the VUS, we analyzed RNA from the patient’s father with RNA-seq which showed skipping of Exon 14, resulting in a frameshift mutation three amino acids from the new reading frame. This RNA-seq analysis was correlated with virtually absent enzyme activity, elevated very-long-chain fatty acids in fibroblasts, and a clinically severe phenotype. Both variants are reclassified as pathogenic. Due to the clinical spectrum of DBP deficiency, this provides important prognostic information, including early mortality. Furthermore, we add persistent hypoglycemia to the clinical spectrum of the disease, and advocate for the early management of fat-soluble vitamin deficiencies to reduce complications.

Keywords: D-bifunctional protein deficiency, peroxisomal biogenesis disorders, rapid whole genome sequencing, very-long-chain fatty acids, Zellweger spectrum disorders

1 |. INTRODUCTION

D-bifunctional protein (DBP) deficiency (MIM 261515) is a single-enzyme deficiency causing an inborn error of peroxisomal metabolism with a high burden of morbidity and early mortality caused by biallelic pathogenic variants in the HSD17B4 gene. The estimated prevalence of this disorder in the general population is 1 in 100,000 (Ferdinandusse, Denis, et al., 2006). Clinically, these patients resemble those diagnosed with a peroxisomal biogenesis disorders in the Zellweger spectrum (PBD-ZSD), which include Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease (Gould et al., 2001). Less deleterious variants in the same HSD17B4 gene can cause a milder disorder, Perrault syndrome. This sex-influenced disorder is characterized by sensorineural deafness and ovarian dysgenesis in females (Type I); when progressive neurologic degeneration is observed, it is considered Type II disease (Pierce et al., 2010).

Most patients with DBP deficiency present with neonatal hypotonia and seizures within the first month of life, and many have visual system failure including nystagmus, loss of hearing, external dysmorphia including enlarged fontanelle, delayed maturation of brain white matter, and hepatomegaly. Few patients survive beyond 2 years or achieve any cognitive or motor development (Ferdinandusse, Denis, et al., 2006).

DBP is involved in the breakdown of very-long-chain fatty acids (VLCFAs; ≥C22:0), α-methyl branched-chain fatty acids like pristanic acid, and the C27-bile acid intermediates di- and trihydroxycholestanoic acid (DHCA/THCA). After β-oxidation of DHCA and THCA in the peroxisome, the primary bile acids chenodeoxycholic acid and cholic acid (CA) are formed (Ferdinandusse et al., 2009). DBP is also involved in the synthesis of long-chain polyunsaturated fatty acids. DBP contains three domains: an enoyl-CoA hydratase domain, a 3-hydroxyacyl-CoA dehydrogenase domain, and a sterol-carrier domain. The two catalytic domains are used to characterize the nature of the defect in enzymatic function into three subtypes. In Type I, DBP activity is completely absent, representing a loss of activity of both the hydratase and dehydrogenase enzymatic domains. In Type II, only hydratase activity is deficient. In Type III, only the dehydrogenase activity is deficient. (Wanders et al., 2001). In Type IV, there are compound heterozygous mutations, one in each domain (McMillan et al., 2012). No clinical variances differentiate the subtypes, although patients with Type I disease typically have the shortest lifespan.

Diagnostic evaluation of peroxisomal biogenesis disorders and disorders of peroxisomal fatty acid oxidation begins with clinical observation of seizures and hypotonia in a neonate or elevated VLCFAs on newborn screening. The next step in evaluation requires a measurement of biochemical biomarkers in body fluids, often revealing elevated VLCFAa, pristanic acid, plasmalogen, and bile acid intermediates levels (Braverman et al., 2016). Enzyme testing in cultured skin fibroblasts obtained via biopsy can further characterize the defect and can help distinguish PBD-ZSD from single enzyme deficiencies such as DBP deficiency (Gloerich et al., 2003). Finally, a diagnosis of DBP deficiency can be established via detection of biallelic pathogenic variants through molecular analysis of the HSD17B4 gene on chromo-some 5.

A variety of known variants in the HSD17B4 gene have been documented to cause clinical disease consistent with DBP deficiency. Previous molecular analysis of complementary DNA (cDNA) of 110 patients with DBP deficiency identified 61 different variants. Notably, the variants which cause more detriment to the protein structure correlated with a more severe clinical presentation (Ferdinandusse, Ylianttila, et al., 2006). Our patient inherited one copy of a likely pathogenic variant from her mother and one copy of a variant of uncertain significance (VUS) from her father. Our report details further molecular and functional analyses in an attempt to correlate these two variants with her severe clinical presentation and early demise. In addition, our report details hypoglycemia as a novel presenting symptom which has not been previously described in the literature, as well as less described rickets associated with bile acid synthesis abnormality.

2 |. CLINICAL REPORT

A baby girl born at 40-week gestation was brought to the Neonatal Intensive Care Unit at 6 h of life for treatment of hypoglycemia despite twice administration of 40% glucose gel for glucose levels of 39 and 25 mg/dl, respectively. She was born to a nonconsanguineous African American couple, 35-year-old gravida 5, para 2, mother and father, via vaginal delivery without instrumentation. Maternal serologies and Group B streptococcus were negative. Cell-free fetal DNA sent prenatally due to advanced maternal age was negative. APGAR scores were 8 and 9 at 1 and 5 min, respectively. Birth weight was 2760 g (14th percentile), head circumference was 32.5 cm (12th percentile), and length was 52.5 cm (96.4th percentile). She was admitted on room air, having occasional episodes of oxygen desaturation, necessitating support with nasal cannula at 2 L/min of room air. The physical exam was significant for lethargy with minimal spontaneous activity, overriding coronal sutures, widely spaced sagittal suture, malalignment of posterior skull bones, severe hypotonia, and hyporeflexia. Laboratory results for complete blood count and basic metabolic panel were normal. On the second day of life, rhythmic lateral nystagmus and myoclonic jerks were noted. Electroencephalogram showed independent left and right frontal onset seizures correlating with contralateral arm and leg clonic movements. Brain magnetic resonance imaging showed hypoplastic corpus callosum and absent septum pellucidum. Blood gas analysis was normal. Lactate, pyruvate, and ammonia were normal, as was plasma amino acids. Urine organic acids showed increased excretion of 4-hydroxy-phenyllactic and 4-hydroxy-phenylpyruvic acids suggesting immature/impaired hepatocellular function. Abdominal ultrasound showed mild hepatosplenomegaly and no evidence of renal cysts.

The patient was fed via nasogastric tube due to poor suck and high risk of aspiration. Due to persistent hypoglycemia with glucose levels less than 60 mg/dl, she was transitioned to continuous feeds. Results of insulin, cortisol level, β-hydroxybutyrate, free fatty acids, C-peptide, and growth hormone levels from a critical sample taken at the time of hypoglycemia were unremarkable. Adrenocorticotropic hormone (ACTH) stimulation test produced an appropriate cortisol response.

Newborn screening showed elevated C24:0-LPC and C26:0-LPC, raising concern for a peroxisomal disorder. During the first 2 weeks of life, VLCFA studies revealed a markedly elevated C26:0 (6.62 μmol/L, ref. range: 0.17–0.73 μmol/L) and abnormal C26:0/C22:0 and C24:0/C22:0 ratios, showing a deficiency in the breakdown of VLCFAs. Pristanic acid and phytanic acid were normal. Total bile acids and CA were slightly elevated. Plasmalogen level was normal.

Rapid whole genome sequencing (WGS) of patient genomic DNA identified two variants in the HSD17B4 gene, c.1210-11C>G and c.350-2A>T (Table 1). Parental genetic testing verified that the two HSD17B4 variants were inherited in trans; the patient’s mother carried the likely pathogenic canonical splice variant, c.350-2A>T and her father carried the VUS, c.1210-11C>G.

TABLE 1.

HSD17B4 variants identified in the patient

Chr. Base pair [hg19] Reference allele Alternate allele Nucleotide changea Predicted protein changeb Initial ACMG classification Final ACMG classification MAF (gnomAD)
5 118,837,725 C G c.1210-11C>G Uncertain VUS Pathogenic 1.20E–05
5 118,813,110 A T c.350-2A>T Splice acceptor Likely pathogenic Pathogenic 0
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Abbreviations: Chr., chromosome; MAF, minor allele frequency.

a

Nucleotide change for transcript NM_000414.

b

Predicted change prior to phasing of variants or RNA-seq.

During hospitalization, multifocal seizures were difficult to control and unresponsive to levetiracetam, but eventually subsided with phenobarbital and clobazam. The patient had a percutaneous gastrostomy tube placed at 3 weeks of age. The patient was unable to tolerate bolus feedings due to persistent hypoglycemia so was maintained on continuous feeds. Due to hypotonia, the patient required continuous positive airway pressure which she weaned off postdischarge. She failed newborn hearing screening and acoustic brainstem response tests confirmed severe sensorineural hearing loss. She was referred for hearing aids. On outpatient follow-up at 4.5 months of age, she was able to move all four limbs against gravity and suck her thumb but could not roll or sit with support. Frontal bossing, hypoplastic midface, and tongue protrusion were observed.

The patient returned to the hospital at 6 months of age for respiratory distress and was found to have several rib fractures and a right humeral fracture. A skeletal survey fraying and cupping of the long bone metaphyses consistent with rickets and severe bone demineralization (Figure 1). Laboratory assessments showed hypocalcemia, appropriate rise in parathyroid hormone, elevated alkaline phosphatase, and very low 25-hydroxy vitamin D level (<4 ng/ml), consistent with vitamin D deficiency that resulted in metabolic bone disease. Daily intake was calculated to be adequate, suggesting malabsorption of fat-soluble vitamins. Total and fractionated bile acids were undetectable. She was supplemented with calcium and vitamin D. At 8 months of life, physical exam was significant for weight at the 11th percentile, dysmorphism, hypotonia, massive hepatomegaly, with soft liver edge felt 7 cm below the costal margin and no splenomegaly. Amino transaminase levels and gamma-glutamyl transferase levels were normal. Fat-soluble vitamin levels showed persistent severe vitamin D deficiency (4 ng/ml), severe vitamin E deficiency (undetectable at <1 mg/L; normal >9 mg/L), and a normal vitamin A level. She had coagulopathy with international normalized ratio of 1.98 that corrected with intramuscular vitamin K injection. Serum bile acids were not elevated as would be expected and urinary bile acids by Fab-mass spectrometry were qualitatively normal. CA of 15 mg/kg/day was started at 9 months of age. There was rapid improvement in weight gain with normalization of vitamin D 3 months later. Vitamin E level became high at 14.3 mg/L after 4 months of supplementation. Outpatient growth parameters closest to her final hospitalization included weight at the 44th percentile, length at the 40th percentile, and head circumference at the 5th–10th percentile.

FIGURE 1.

FIGURE 1

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X-ray of right leg. Long bone metaphyses are symmetrically frayed and cupped in the bilateral upper and lower extremities, most striking around the knees and ankles. The zone of provisional calcification is obliterated throughout the long bone metaphyses

She presented at 12 months of age with severe respiratory distress due to presumed viral infection (COVID-19 negative) requiring intubation for 17 days. She was extubated to high flow nasal canal but was reintubated 2 weeks later due to further respiratory decompensation and hypotonia. She was unable to be extubated. Ultimately, care was redirected and the patient died at 13.5 months of age.

3 |. METHODS

3.1 |. WGS, whole exome sequencing, and RNA-seq

Genomic DNA was isolated from blood using the Maxwell 16 Blood DNA Purification Kit (Promega), and RNA was isolated using the PAXgene Blood RNA Kit (Qiagen). WGS, whole exome sequencing (WES), and RNA-seq were performed at the Yale Center for Genome Analysis (West Haven, CT). Globin molecules were removed from RNA using the Globin-Zero Gold rRNA Removal Kit (Illumina). The cDNA library was prepared using random primers followed by second-strand synthesis with dUTP and digestion with UDG. Paired-end libraries for all samples were prepared using KAPA Hyper Kit (Roche Diagnostics). For WES, following amplification with IDT UDI primers, enrichment of the exome was performed by IDT xGen Human Exome V1 reagents. All libraries were sequenced using an Illumina NovaSeq-6000 with 101 bp (WES and RNA-seq) or 151 bp (WGS) paired-end reads. FASTQ files were aligned to the hg19/GRCh37 reference genome and variants were called, recalibrated, and filtered using GATK best practices (Van der Auwera et al., 2013). Variants were annotated with allele frequency, variant effects, OMIM and ClinVar database information, and numerous in silico attributes using ANNOVAR (Wang et al., 2010), and alignments were visualized using Integrative Genomics Viewer (Thorvaldsdóttir et al., 2013). Variants identified in the proband were filtered using a minor allele frequency cut-off of 2.5% and including only variants within or near exons.

3.2 |. Fibroblast enzymatic and functional studies and immunofluorescence microscopy analysis

A skin biopsy was performed to perform enzymatic and functional studies to characterize the defect. Cultured skin fibroblasts were sent to Amsterdam University Medical Center for subsequent testing (Amsterdam, the Netherlands). Both the enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities of DBP were measured in the cultured skin fibroblasts. In addition, VLCFA metabolism was studied with a D3-C22 loading test, VLCFA and C26-LPC measurement. Morphology of peroxisomes was studied by immunofluorescence microscopy analysis using antibodies against catalase, a peroxisomal matrix protein (Ferdinandusse et al., 2016).

4 |. RESULTS

Rapid WGS identified two variants in trans in HSD17B4; c.350-2A>T, which was maternally inherited and c.1210-11C>G which was paternally inherited. Using ACMG/AMP classification guidelines (Richards et al., 2015), c.350-2A>T was classified as likely pathogenic and c.1210-11C>G was classified as a VUS.

To determine the functional consequence of the c.1210-11C>G variant classified as being of uncertain significance, whole blood RNA from the patient’s father carrying the c.1210-11C>G was analyzed by RNA-seq. Results indicated that the dosage of exon 14 was reduced by half and exon-skipping was observed in many reads (Figure 2). Exon 14 is essential for all isoforms of the gene, and the exon skipping is predicted to cause a frameshift resulting in premature termination of three amino acids downstream (p.Val404Glufs*3). The effect on splicing was therefore validated for the HSD17B4 c.1210-11C>G variant.

FIGURE 2.

FIGURE 2

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Integrated Genomics Viewer (IGV) image of RNA-seq alignments from the patient’s father and whole genome sequence (WGS) from the patient. (a) Coverage of alignments for exons 13 and 14 of HSD17B4. The number of reads for exon 14 (26 reads) is half that of exon 13 (52 reads). The total coverage for exon 15 is 92 reads (not shown). (b) Exon junctions track show that for some reads, exon 14 is spliced in the transcript, and for others (red arrow), exon 14 is skipped. (c) Alignments of RNA-seq reads from the father. (d) Alignments of WGS data from the patient. The red arrow points to the c.1210-11C>G variant. (e) Exon boundaries for various HSD17B4 transcripts, in the canonical transcript NM_000414, they are exons 13 and 14

Results from patient fibroblast enzyme studies showed markedly reduced enoyl-CoA hydratase activity (6 pmol/[min.mg protein], ref. range 115–600) and absent 3-hydroxyacyl-CoA dehydrogenase activity of DBP (<5 pmol/[min.mg protein], ref. range: 25–300). VLCFA profile was clearly abnormal with increased C26:0 level (1.3 μmol/g protein, ref. range 0.16–0.41) and increased C26:0/C22:0 ratio, and increased C26-LPC level (48 pmol/mg protein, ref. range 2–14). The D3-C22 loading test was abnormal showing abnormal VLCFA metabolism with deficient peroxisomal β-oxidation and increased chain elongation of D3-C22. Catalase immunofluorescence microscopy analysis revealed that peroxisomes were both reduced in number and enlarged in size.

These functional studies showing a deficiency of DBP and resulting in abnormal VLCFA metabolism allow the reclassification of both HSD17B4 variants identified in the patient as pathogenic.

5 |. DISCUSSION

The patient described here exhibited a severe phenotype at birth with seizures, hypotonia, and persistent hypoglycemia as well as abnormal newborn screening results which prompted rapid molecular diagnostic assessment. The identified VUS, c.1210-11C>G, was further characterized with RNA-seq analysis. Primarily, we provide supportive evidence in reclassifying this VUS as pathogenic. Secondarily, the novel clinical finding of persistent hypoglycemia remains an important element of the spectrum of disease not previously described in the literature. Finally, the finding of metabolic bone disease from vitamin D deficiency highlights the importance of early screening, supplementation of fat-soluble vitamins, and supplementation of bile acids to improve quality of life and weight gain.

Rapid WGS identified two variants in HSD17B4. One variant, c.350-2A>T, impacted the canonical splice acceptor of exon 7 of the transcript NM_000414 and was classified as likely pathogenic based on its predicted loss-of-function impact and its absence from control populations (gnomAD) (Karczewski et al., 2020; Richards et al., 2015). The second variant impacted a nucleotide 11 base pairs upstream of exon 14 (c.1210-11C>G) and was reported as a VUS. While not at a canonical splice site, the variant was predicted to alter splicing by dbscSNV (Jian et al., 2014) as this mutation disrupts the polypyrimidine tract (Anna & Monika, 2018). At the time of testing, the c.1210-11C>G variant had been reported twice in ClinVar (ID:559071), once as a VUS and once as likely benign. This variant had been described only once in the literature, inherited in trans with a truncating mutation (Meng et al., 2017); this patient was confirmed to have died at 26 months of age (personal communication). In coincidence with our clinical reclassification of c.1210-11C>G as pathogenic, there is now a third entry in ClinVar (VarID:559071, accession#:SCV001424305.1) with a likely pathogenic classification, although supportive evidence is not provided.

RNA-seq analysis confirmed that the C>G base substitution upstream of exon 14 resulted in exon-skipping. We corroborated this analysis of RNA with the fibroblast enzyme studies, which showed that both the hydratase and dehydrogenase activities of DBP were deficient. Based on this RNA and enzymatic data, our patient carries compound heterozygous pathogenetic variants in the HSD17B4 gene, confirming the diagnosis of DBP deficiency. Biochemical studies showed significantly elevated C26:0 level in both fibroblasts and plasma. In the largest case series of 110 patients with DBP deficiency, no patients with plasma C26:0 levels greater than 5 μmol/L survived greater than 30 months and all patients with Type I disease died within the first 14 months of life (Ferdinandusse, Denis, et al., 2006). More recently, cases surviving beyond infancy have been reported, where one patient with Type III DBP deficiency survived (Chapel-Crespo et al., 2020).

We add the novel clinical finding of early and persistent hypoglycemia to the phenotypic spectrum of DBP deficiency. In neonates, hypoglycemia in the first day of life is most commonly caused by maternal hyperinsulinism or fetal growth restriction, both typically resolving in hours to days. Hypoglycemia that persists after the first few days of life is much less common, especially in full-term infants. Causes of persistent hypoglycemia include endocrine disorders, namely neonatal hyperinsulinism, hypopituitarism, isolated growth hormone deficiency, and adrenal disorders (Yadav, 2016). These are diagnosed by measuring serum hormone levels and were normal in our patient. Inborn errors of metabolism encompass rare causes of hypoglycemia. Peroxisomal disorders and DBP deficiency do not fall into this category of inborn errors of metabolism which cause hypoglycemia. Genes associated with neonatal hypoglycemia including KCNJ11, GCK, HADH, INSR, GLUD1, and SLC16A1 were normal on WGS analysis.

Adrenal insufficiency is another rare cause of neonatal hypoglycemia and occurs in conjunction with hyponatremia. Adrenal function impairment is known to be present in a significant portion of patients with DBP deficiency. The adrenal pathology of patients with ZSDs appears to be caused by VLCFA accumulation, which are thought to be toxic to adrenal cortical cells (Gould et al., 2001). However, adrenal cortical atrophy slowly develops over time, often only observed at autopsy. An ACTH stimulation test in our patient was normal in the neonatal period and again normal at 11 months of age. We suspect that our patient had some component of adrenocortical impairment that was not detected by the ACTH stimulation test. There are no prior reports of hypoglycemia associated with the HSD17B4 gene abnormality. However, due to this risk, we recommend considering stress-dose steroids for these patients in the setting of acute illness.

Our patient developed rickets from vitamin D deficiency. While many case reports detail the neurologic sequelae of DBP deficiency, few descriptions of malabsorption of fat-soluble vitamins are reported, although bile acid synthesis defects are recognized as a complication of DBP deficiency (Wanders et al., 2001). In the case series by Ferdinandusse, Denis, et al. (2006), they report scoliosis without mentioned cause in four of five patients with prolonged survival, but do not mention osteopenia in the neonatal period. In addition, there is no mention of hematologic abnormalities from vitamin K deficiency. However, there is a report of a patient with DBP deficiency surviving until at least 6 years of age who required growing rods for metabolic bone disease and was noted to have “soft bone like cardboard” (Buckwalter & Weinstein, 2014). In patients with ZSD, osteopenia can be noted in childhood. Rush et al. (2016) described 13 patients with ZSD who had low bone mineral density but no rickets or vitamin D deficiency; they hypothesized that VLCA and/or phytanic acid contribute to inhibition of osteogenesis or deficient plasmalogens impair endochondral bone ossification. Hypoprothrombinemia responsive to vitamin K was noted in 18 of 19 patients in an early study (Heymans, 1984). In ZSD patients, it is recommended to screen for deficiencies of fat-soluble vitamins A, D, E, and K (Klouwer et al., 2018). Just as in ZSD patients, patients with DBP deficiency can have defects in bile acid synthesis, as DBP plays a role in the peroxisomal β-oxidation of bile acid intermediates DHCA and THCA (Ferdinandusse et al., 2009). We believe it is important to note that bile acid synthesis defects can result in further reduced quality of life over time, particularly as medical care improves and these patients may be surviving beyond the initially quoted life expectancy of 2 years. Due to the effect on bile acid synthesis, we recommend screening for vitamin D deficiency and empiric supplementation of fat-soluble vitamins and bile acids to improve quality of life and prevent severe complications.

6 |. CONCLUSION

In this patient, VUS results on rapid WGS prompted parental RNA-seq analysis, which suggested the VUS to be pathogenic. Clinical findings, biochemical studies, WGS, and RNA-seq analysis, corroborated with fibroblast enzyme studies, explain the patient’s severe disease course. While there is no curative treatment for this condition at this time, we believe that rapid molecular diagnostics such as those utilized for this patient are necessary to clarify VUS with potential splice affects, and may be useful particularly for conditions with available treatment in the neonatal period. We add persistent hypoglycemia to the phenotypic spectrum of DBP deficiency and describe vitamin D-deficient rickets in this patient.

ACKNOWLEDGMENTS

WGS, WES, and RNA-seq were performed at the Yale Center for Genome Analysis. Testing of very-long-chain fatty acids, urine organic acids, and plasma amino acids were performed at ARUP Laboratories, Salt Lake City, Utah. Fibroblast studies were performed at Amsterdam University Medical Center, the Netherlands. The authors thank the patient’s family for their contributions.

Footnotes

CONFLICT OF INTEREST

The authors have no conflicts to declare.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

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