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. Author manuscript; available in PMC: 2026 Sep 2.
Published in final edited form as: Rare. 2026 Jul 21;5:100129. doi: 10.1016/j.rare.2026.100129

Unusual Variants in NDUFAF6-Associated Mitochondrial Disease

Yuka Aoyama 1,*, Julia Grafstein 1,*, Parker Houston 1, Marisa W Friederich 2,3, Roxanne A Van Hove 2, Cole R Michel 4, Richard Reisdorph 4, Johan Van Hove 2,3, William Gahl 1,5, David R Adams 1,5, Lynne Wolfe 1, Ellen F Macnamara 1
PMCID: PMC13533396  NIHMSID: NIHMS2199116  PMID: 42683144

Abstract

A 6-year-old female with global developmental delay, chronic kidney disease (stage III), and renal tubular dysfunction was evaluated in the National Institutes of Health Undiagnosed Diseases Program. Although exome sequencing did not yield a diagnosis, family genome sequencing revealed biallelic variants in NDUFAF6, i.e., a paternally inherited intronic variant (NM_152416.3:c.298-768T>C) and a maternally inherited 1.6 kb deletion (NC_000008.11:g.95044573_95046180del, spanning exon 5). NDUFAF6 plays an important role in mitochondrial complex I assembly by regulating ND1 biogenesis and facilitating the incorporation of NDUFS8. Variants in NDUFAF6 are associated with two OMIM disorders i.e., Fanconi renotubular syndrome 5 (OMIM #618913) and Mitochondrial complex I deficiency, nuclear type 17 (OMIM #618239). The associated phenotypes include proximal tubule dysfunction and degeneration of the central nervous system. The intronic single nucleotide variant in this case (sometimes referred to as the Acadian variant) has been reported to cause aberrant splicing. This case highlights the need to consider comprehensive sequencing methods, such as genome sequencing, to identify atypical variants in planning a comprehensive diagnostic strategy.

Keywords: Mitochondrial disease, complex 1 deficiency, NDUFAF6

1. Introduction

NADH:ubiquinone oxidoreductase (complex I), the first enzyme complex in the mitochondrial respiratory chain, plays a crucial role in ATP production via oxidative phosphorylation. Complex I (C1) maintains the electrochemical gradient by transferring protons across the inner mitochondrial membrane while transferring electrons from reduced NADH to coenzyme Q10. C1 electron leaks are a major source of reactive oxygen species [1]. Alterations to C1 and ATP synthesis can significantly impact health; affected individuals typically develop symptoms during the first year of life [1]. C1 deficiency is clinically and genetically heterogeneous but defects in C1 are the most common respiratory chain disorders, accounting for up to 30% of mitochondrial diseases presenting in childhood [2].

C8orf38, now known as NDUFAF6, is widely recognized to play a role in C1 functionality [2–20]. The first evidence of the disease was derived from homozygosity mapping of variants in two siblings with both Leigh syndrome, a progressive neurodegenerative disease, and complex I deficiency [3]. RNA interference studies found that a knockdown of 80% in NDUFAF6 mRNA expression led to an 80% reduction of C1 activity [3]. Later studies showed that NDUFAF6 functions in the synthesis and stabilization of the C1 subunit, ND1, and the incorporation of another subunit, NDUFS8 [2].

Recently, researchers discovered an intronic variant within NDUFAF6 among Acadian patients [21]. This variant affects splicing and synthesis of NDUFAF6 isoforms and manifests with proximal tubule dysfunction but without neurological symptoms, expanding the phenotype associated with NDUFAF6.

We utilized genome sequencing to diagnose NDUFAF6-related mitochondrial disease caused by compound heterozygous variants in an individual with both global developmental delay and chronic kidney disease (stage III). The patient’s atypical variants, an intronic variant and a novel 1.6kb deletion spanning exon 5, were missed by exome sequencing, emphasizing the importance of genome sequencing in identifying potential variants in mitochondrial disease. This case demonstrates the phenotypic spectrum of NDUFAF6 disease and complex I deficiency, as the patient presented with both neurological and renal involvement.

2. Materials and methods

2.1. Whole Genome Sequencing

Clinical genome sequencing was performed by Baylor Genetics on the proband and both parents. The library was prepared according to the KAPA Hyper Prep Kit protocol and was subjected to sequence analysis on the Illumina NovaSeq 6000 platform. Variant calling was performed on the BAM file using the Illumina Dragen haplotype-based variant calling system while copy number analysis uses Illumina Dragen genome wide depth-based CNV caller. Structural variant calling used the Illumina Manta Structural Variant Caller.

Prior exome testing included 96% coverage of NDUFAF6, and exonic deletions were called using an “in-house algorithm” that compares read depth of the proband with mean read-depth and read-depth distribution.

2.2. Mitochondrial functional assays

Respiratory chain enzyme activities of complexes I, II, III, II-III combined, IV and citrate synthase were assayed in skin fibroblasts on post-600 x g homogenates using spectrophotometric assays at 30° C on a Cary 300 spectrophotometer as described in [22]. Solubilized mitochondrial membranes isolated by differential centrifugation from the homogenate of skin fibroblasts were separated by nondenaturing blue native gel electrophoresis, and the activity identified by colorimetric in-gel activity staining [22]. Complex I assembly was analyzed using a nondenaturing gel of the mitochondrial inner membrane fraction and the complex I intermediates visualized after western blotting and visualization using an antibody against NDUFS2 (Abcam ab110249, at 1:2000 dilution) [23]. Unlabeled whole-cell proteomic analysis was performed in fibroblasts from the patient in triplicate and five controls [24]. Proteins were solubilized with sodium dodecyl sulfate, loaded on an S-trap column™ (ProtiFi, Newport, NY); after reduction the disulfide bonds were stabilized using chloroacetamide, and proteins digested overnight with trypsin. After washing, the peptides were eluted and analyzed by reverse phase liquid chromatography and label-free data-independent acquisition on an OrbiTrap Eclipse Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA) using conditions as previously described [24]. Raw data files were processed on Spectronaut® 19.9 software (Biognosys AG, Schlieren, Swtizerland) [25], excluding single hit proteins, and identified on the UniProt human database (canonical peptides + isoforms, reviewed, 42,474 entries) for label-free quantification. The total number of proteins was reviewed for QC, and MS2 quantity of proteins were then analyzed on Perseus software v.2.0.11 (Max Planck Institute for Biochemistry) [26]. Log2-transformed label-free quantification intensities were grouped in patient in triplicate and 5 in-assay controls, two-sample t-tests run between groups, and Volcano plots were generated using the built-in scatter plot function with p-value set to 0.05 equivalent (−log10=1.301). Log2 transformed data was generated in Perseus using MitoCarta3.0 [27] + entries and normalization via subtract row cluster means prior to a two-sample t-test using p-value = 0.05 for truncation. The relative complex abundance (RCA) was plotted for each complex of the respiratory chain, the small and the large subunit of the mitoribosome and the pyruvate dehydrogenase complex in R (version 4.3.0) and RStudio (version 2023.03.1+446) using a custom build program [18].

3. Case Report

3.1. Clinical Report

The female proband was born at 35 weeks and 5 days via cesarean delivery for oligohydramnios. She weighed 2100 g, below the first centile, and had Apgar scores of 8 & 9. Before delivery, the mother underwent treatment with Lovenox for deep vein thrombosis and was on bed rest for two months.

The proband was admitted to the neonatal intensive care unit (NICU) for 29 days because of respiratory distress and received continuous positive air pressure. Poor weight gain prompted increased calorie feeds. During the NICU stay, the peak total bilirubin was 12.7 mg/dL (<10 mg/dl), and she was treated with phototherapy.

At 3.5 months, the patient refused feedings and exhibited frequent emesis. She was diagnosed with failure to thrive and gastroesophageal reflux. At 19 months, a gastrostomy tube was placed; vomiting frequency decreased and feeding improved.

At 14 months, plasma tyrosine and methionine elevations suggested liver disease. A liver biopsy showed mild microvesicular and macrovesicular steatosis, focally increased hepatocellular cytoplasmic eosinophilia, scattered acidophilic bodies, and mild sinusoidal fibrosis. Urine amino acid analysis showed generalized aminoaciduria, and urine organic acid analysis indicated persistent lactic aciduria and ketonuria. Two weeks later, the growth differentiation factor 15 (GDF15) level was 2727 pg/mL (normal, <750), considered related to her renal and hepatic condition. At 22 months, chronic kidney disease (stage III) was diagnosed, with a glomerular filtration rate of less than 30 mL/min/1.73m2.

Plagiocephaly was diagnosed at 4 months of age. At 13 months, dysmorphic features included relative macrocephaly, a large anterior fontanelle, deep-set eyes, down-slanting palpebral fissures, low set, protruding ears, mild-moderate retrognathia, and a tented upper lip. Brain MRI at 14 months showed mild and generalized ventriculomegaly; other brain structures appeared normal.

Developmental delay was evident by 3 months of age; occupational and physical therapy resulted in notable improvements in gross motor development. The patient walked at 18.5 months but exhibited fine motor and speech delays during her NIH evaluation.

At 2 years and 2 months, the proband was enrolled in the NIH Undiagnosed Diseases Program through clinical protocol 15-HG-0130, “Clinical and Genetic Evaluation of Individuals with Undiagnosed Disorders Through the Undiagnosed Diseases Network” (ClinicalTrials.gov Identifier: NCT02450851). During this evaluation, her GDF15 level was 4658 pg/mL (normal, ≤750), plasma lactate was 4.2 mmol/L (normal, 0-2.2 mmol/L), and plasma pyruvate was 0.23 mmol/L (normal 0.08-0.16 mmol/L).

3.2. Genetic Testing

Prior genetic testing, including a CFTR gene variant panel, H19 and UPD7 panel testing for Russel-Silver syndrome, chromosomal microarray analysis, and mitochondrial genome sequencing, was negative. Clinical exome sequencing identified two variants of unknown significance in SLX4, associated with Fanconi Anemia. These variants, NM_032444.2:c.1192C>T(p.Arg398Trp) and c.4057C>A(p.His1353Asn), were considered nondiagnostic since the patient’s phenotype did not match.

Clinical genome sequencing of the proband and unaffected parents by the NIH UDP identified compound heterozygous variants in NDUFAF6. The paternally inherited NM_152416.3:c.298-768T>C (chr8:95034686T>C) is an intronic splice variant previously reported as disease-causing in several patients [21]. The maternally inherited NC_000008.11:g.95044573_95046180del causes a 1.6 kb in-frame deletion in exon 5 not previously reported but considered likely pathogenic.

3.3. Functional Testing

Fibroblast studies showed a clear decrease in C1 activity on respiratory chain enzyme analysis (Table 1). Blue native PAGE with in-gel activity staining also showed an isolated substantial decrease in the activity of complex I but did not show lower molecular weight bands in complex V as would be expected for a combined deficiency (Fig 1). Tracing the assembly of C1 showed a strong decrease in the holocomplex at 1 MDa, without noticeably abnormal assembly intermediates (Fig 2). These changes constitute a major diagnostic criterion for isolated complex I deficiency.

Table 1.

Mitochondrial respiratory chain enzyme activities in skin fibroblasts of Patient 1.

Activity (controls) Z-score Ratio /CS Z-score Ratio / complex II Z-score
Complex I 4.9 (49.3-131.1) −9.5 9 (145-396) −10.7 15 (237-754) −8.9
Complex II 323.1 (130.9-364.4) 1.1 600 (297-863) 0.2 NA NA
Complex III 13.5 (8-29.2) −0.1 25 (19-65) −0.4 42 (36-114) −0.5
Complex II-III 137.4 (61.8-158.8) 0.9 255 (131-376) 0.4 425 (263-1100) −0.2
Complex IV 8.2 (2.2-7.1) 1.7 15 (6-23) 1.5 25 (12-35) 1.5
Citrate synthase 538.8 (253.5-554.1) 1.1 NA NA NA NA
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Legend: Mitochondrial respiratory chain enzyme activities from the patient’s primary skin fibroblasts. The activities of complexes I, II, combined II-III, and citrate synthase (CS) are expressed as nmol.min−1.protein−1. The activities of complexes III and IV are shown as first order rate constants expressed as nmol.min−1. Activities are also shown as ratios to the activity of citrate synthase (CS) and complex III. The values are also expressed as the standard deviations from the mean (Z-score) of the log transformed values of controls, which are normally distributed. The results show an isolated deficiency of complex I.

Figure 1.

Figure 1.

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Blue native gel electrophoresis with in-gel activity staining of mitochondrial complexes. Legend: The activity of mitochondrial complexes I, II, IV, V is visualized after separation by electrophoresis on a blue native gel, and visualization of the activities using in-gel activity staining. There is a large decrease in patient’s complex I activity, with normal activity in complexes II-V and normal assembly of complex V.

Figure 2.

Figure 2.

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Complex I assembly assay. Legend: After electrophoresis on a native gel and immunoblotting, complex I intermediates are visualized. There is a strong decrease in in the holocomplex I band and absence of intermediate forms. A normal control is shown and as positive control an immortalized hepatic cell line HepG2 treated with the mitochondrial translation inhibitor chloramphenicol (CAM) is shown (denoted HepG2/CAM) for use as a positive control for Complex 1 assembly intermediates.

Label-free proteomics analysis identified 7,530±50 proteins in each run. The NDUFAF6 protein was consistently identified by 2 or 3 peptides in all 20 samples in this experiment, but in each of the patient’s triplicate runs, no peptide was identified, indicating that this protein was decreased to below the detectable limit. On the volcano plot without or with correction for abundance of mitochondrial proteins, the subunits of complex I were all decreased (Fig 3). The relative complex abundance (RCA) of complex I was decreased to 35% of in-assay controls, well below the range observed in 21 control fibroblasts (87-114%) (Fig 4). In contrast, the RCA of all other complexes remained within the control range. This confirms isolated complex I deficiency due to loss of the stability of complex I.

Figure 3.

Figure 3.

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Abundance of mitochondrial proteins in the fibroblast of the patient. Legend: A Volcano plot of the abundance of the mitochondrial proteins in fibroblasts of patient is shown. On proteomics analysis after correction of mitochondrial abundance, the volcano plot shows the differential abundance results. The x-axis represents the difference between the patient and control mean values, and the horizontal line shows a cutoff p-value <0.05. This shows a decrease in the abundance of the subunits of complex I (red) whereas subunits of complex IV have normal abundance (blue). Subunits with the most decreased abundance were NDUFS8, NDUFB8, NDUFA13 and NDUFB7.

Figure 4.

Figure 4.

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Relative complex abundances on proteomics analysis in fibroblasts of patient. Legend: Quantification of mitochondrial complex abundance derived from whole cell label-free proteomics shows a decrease in complex I without changes outside the normal range for the other respiratory chain complexes, mitoribosomal subunits or the pyruvate dehydrogenase complex. These data represent an aggregation of the data presented in Fig 3. The p-value and the relative % of the complex in the proband compared to the controls, and the normal range derived from 21 normal control fibroblasts are provided in sequential rows above the figure. Abbreviations: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, ns = non-significant. CI = complex I, CII = complex II, CIII = complex III, CIV = complex, IV, CV = complex V, LSU = large subunit of the mitoribosome, SSU = small subunit of the mitoribosome, PDH = pyruvate dehydrogenase complex.

4. Discussion

We describe a patient with compound heterozygous loss of function variants in NDUFAF6 and mitochondrial disease. She presented with global developmental delay, stage III kidney disease, liver biopsy findings, elevated GDF15 levels, and lab results consistent with a metabolic disease. Exome sequencing failed to identify the disease-causing variants so functional mitochondrial testing was not initially performed.

Genome sequencing confirmed pathogenic compound heterozygous variants in NDUFAF6, emphasizing the value of investigating non-coding regions, including introns, untranslated regions, and intergenic regions in testing for mitochondrial diseases.

The maternally inherited, 1.6kB deletion NC_000008.11:g.95044573_95046180del was not previously reported, nor were other large deletions previously reported in this gene (Table 2). The deletion encompasses exon 5 and was considered likely pathogenic. NDUFAF6 has multiple isoforms, 13 of which have a deleted exon 5. However, the mitochondrial isoform, associated to the inner mitochondrial matrix and demonstrated to restore C1 activity in patient fibroblasts, includes exon 5 [2], suggesting its importance in mitochondrial function.

Table 2:

Summary of all currently published papers that describe variants in NDUFAF6 in affected individuals.

Authors PMID Numbe r of Affecte d Individ uals Variants in NDUFAF6 Inheritance Diagnosis
Pagliarin i et al.(2008) 18614 015 2 c.296A>G (p.Gln99Arg)*c
.296A>G (p.Gln99ArgfsX3)		 Homozygo us Leigh Syndrome
Kohda et al.(2016) 26741 492 4 1)c.226T>C (p.Ser76Phe), c.805C>G (p.His269Asp)
2) c.206A>T (p.Asp69Val), c.371T>C (p.Ile124Thr)
3) c.371T>C (p.Ile124Thr), c.805C>G (p.His269Asp)
4) c.820A>G (p.Arg274Gly)		 1)Comp. Het.
2) Comp.Het.
3) Comp. Het.
4) Homozygou s** 		1)Leigh Syndrome
2) Leigh Syndrome
3) Leigh Syndrome
4)Mitochondrial Cytopathy
Hartman nová et al.(2016) 27466 185 9 c.298-768T>C Homozygo us Renal Fanconi Syndrome
Bianciar di et al.(2016) 27623 250 1 c.532G>C (p.Ala178Pro) Heterozygo us*** Leigh Syndrome
Ogawa et al. (2017) 28429 146 5 1)c.371T>C (p.Ile124Thr), c.805C>G (p.His269Asp)
2)c.820A>G (p.Arg274Gly)
3)c.226T>C (p.Ser76Pro), c.805C>G (p.His269Asp)
4)c.206A>T (p.Asp69Val), c.371T>C (p.Ile124Thr)
5) c.371T>C (p.Ile124Thr), c.805C>G (p.His269Asp)		 1) Comp Het.
2) Homozygous
3) Comp Het.
4)Comp Het.
5)Comp Het.** 		1) Leigh Syndrome
2) Leigh Syndrome
3) Leigh Syndrome
4)Leigh-like Syndrome
5) Leigh Syndrome
Fang et al.(2017) 28639 102 1 c.337C>T (p.Arg113X), c.265G>A (p.Ala89Thr) Comp. Het. C1 Deficiency
Catania et al.(2018) 29531 337 4 c.532G>C (p.Ala178Pro) and c.420+748C>T Comp. Het. Leigh Syndrome
Baide-Mairena et al.(2019) 30642 748 3 c.554_558delTTCTT and c.371T>C (p.Ile124Thr) Comp. Het. Leigh Syndrome
Ogawa et al. (2020) 31967 322 8 1)c.206A>T (p.Asp69Val), c.371T>C (p.Ile124Thr)
2)c.226T>C (p.Ser76Pro), c.805C>G (p.His269Asp)
3)c.226T>C (p.Ser76Pro), c.337C>T (p.Arg113fsX4)
4)c.337C>T (p.Arg113X), c.371T>C (p.Ile124Thr)
5) c.371T>C (p.Ile124Thr), c.805C>G (p.His269Asp)
6)c.371T>C (p.Ile124Thr), c.578T>A (p.Leu193X)
7)c.371T>C (p.Ile124Thr), c.805C>G (p.His269Asp)
8) c.820A>G (p.Arg274Gly)		 1)Comp. Het.
2) Comp. Het.
3) Comp. Het.
4)Comp. Het.
5) Comp. Het.
6) Comp. Het.
7)Comp. Het.
8) Homozygou s		 1) Leigh Syndrome
2) Leigh Syndrome
3) Leigh Syndrome
4) Leigh Syndrome
5) Leigh Syndrome
6) Leigh Syndrome
7) Leigh Syndrome
8) Leigh Syndrome
Johnston e et al.(2020) 33097 395 1 c.371T>C (p.Ile124Thr) and c.420 + 2_420 + 3insTA Comp. Het. Leigh Syndrome
Hu et al. (2020 ) 32348 839 1 c.337C>T (p.Arg113X) and c.371T>C (p.Ile124Thr) Comp. Het. Leigh Syndrome
Kim, Lee, Jang (2022) 35664 867 1 c.371T>C (p.Ile124Thr) and c.233_242del (p.Leu78GInfsX10) Comp. Het. Leigh-like Syndrome
Gedikba si et al.(2023) 37377 599 1 c.479delA (Asn162IlefsX27) and 420+784C>T Comp. Het. Leigh-like Syndrome
Sung et al.(2024) 38720 117 18 1)c.805C>T (p.His269Tyr)
2) c.805C>T (p.His269Tyr), c.581-7A>G
3) c.659C>A (p.Thr220Lys)
4) c.659C>A (p.Thr220Lys)
5)c.226T>C (p.Ser76Pro)
6)c.226T>C (p.Ser76Pro)
7)c.226T>C (p.Ser76Pro), c.337C>T (p.Arg113X)
8)c.928C>T (p.His310Tyr), c.907C>T (p.Arg303X)
9)c.371T>C (p.Ile124Thr), c.337C>T (p.Arg113X)
10)c.371T>C (p.Ile124Thr), c.578T>A (p.Leu193X)
11)c.371T>C (p.Ile124Thr), c.873+5G>A
12) c.820A>G (p.Arg274Gly)
13)c.820A>G (p.Arg274Gly)
14) c.371T>C (p.Ile124Thr), c.337C>T (p.Arg113X)
15) c.236T>C (p.Leu79Pro), c.532G>C (p.Ala178Pro)
16)c.536A>G (p.Glu179Gly), c.532G>C (p.Ala178Pro)
17) c.920A>G (p.Asp307Gly), c.920A>G (p.Asp307Gly)
18)c.266C>T (p.Ala89Val), c.266C>T (p.Ala89Val)		 1) Homozygous
2) Comp. Het.
3)Homozygous
4)Homozygous
5) Homozygous
6)Homozygous
7)Comp. Het.
8)Comp. Het.
9)Comp. Het.
9)Comp. Het.
10)Comp. Het.
11)Comp. Het.
12) Homozygous
13) Homozygous
14)Comp. Het.
15)Comp. Het.
16)Comp. Het.
17)Comp. Het.
18)Comp. Het.		 1) Leigh Syndrome
2) Leigh Syndrome
3) C1 Deficiency
4)C1 Deficiency
5) Leigh Syndrome
6)Leigh Syndrome
7)C1 Deficiency
8) C1 Deficiency
9) Leigh Syndrome
10) Leigh Syndrome
11) Leigh Syndrome
12) C1 Deficiency
13) Leigh Syndrome
14) Leigh Syndrome
15) C1 Deficiency
16) Leigh Syndrome
17) Leigh Syndrome
18) Leigh Syndrome
Rouzier et al.(2024) 38703 036 2 1)c.532G>C (p.Ala178Pro), c.536A>G (p.Glu179Gly)
2) c.275C>T (p.Ala92Val), c.694C>A (p.Pro232Thr)		 1)Comp. Het.
2) Comp. Het		 1)C1 Deficiency
2) C1 Deficiency
Zhou et al.(2024) 39720 739 2 1)c.371T>C (p.Ile124Thr), c.923T>C (p.Ile308Thr)
2) c.371T>C (p.Ile124Thr), c.920A>T (p.Asp307Val)		 1)Comp. Het.
2) Comp. Het.		 1) Leigh Syndrome
2) Leigh Syndrome
Hock et al.(2025) 40400 026 1 c.967delT (p.Tyr323IlefsX18), c.298-768T>C Comp. Het Leigh Syndrome and proximal renal tubular dysfunction
Torraco et al. (2026) 41924 699 27 1)c.532G>C (p.Ala178Pro), c.298-768T>C
2) c.532G>C (p.Ala178Pro), c.298-768T>C
3) c.485del (p.Asn162IlefsX27), c.420+784C>T
4)c.485del (p.Asn162IlefsX27), c.420+784C>T
5)c.967del (p.Tyr323IlefsX18), c.420+784C>T
6)c.2T>C (p.Met1Thr), c.532G>C (p.Ala178Pro)
7) c.337C>T (p.Arg113X), c.420+784C>T
8) c.337C>T (p.Arg113X), c.420+784C>T
9) c.337C>T (p.Arg113X), c.420+784C>T
10)c.532G>C (p.Ala178Pro), c.536A>G (p.Glu179Gly)
11) c.237del (p.Pro80LeufsX11), c.920A>G (p.Asp307Gly)
12) c.266C>T (p.Ala89Val)
13) c.420+784C>T, c.532G>C (p.Ala178Pro)
14) c.226T>C (p.Ser76Pro)
15) c.226T>C (p.Ser76Pro)
16) c.581-7A>G (p.Leu193_Gly194insValIle), c.805C>T (p.His269Tyr)
17) c.659C>A (p.Thr220Lys)
18) c.659C>A (p.Thr220Lys)
19) c.659C>A (p.Thr220Lys)
20) c.659C>A (p.Thr220Lys)
21) c.659C>A (p.Thr220Lys)
22) c.805C>T (p.His269Tyr)
23) c.532G>C (p.Ala178Pro)
24) c.206A>T (p.Asp69Val)
25) c.206A>T (p.Asp69Val)
26) c.820A>G (p.Arg274Gly)
27) c.230T>C (p.Leu77Pro), c.371T>C (p.Ile124Thr)		 1)Comp. Het.
2) Comp. Het.
3) Comp. Het.
4) Comp. Het.
5) Comp. Het.
6) Comp. Het.
7) Comp. Het.
8) Comp. Het.
9)Comp. Het.
10) Comp. Het.
11) Comp. Het.
12) Homozygou s
Comp. Het.
14) Homozygou s
15) Homozygou s
16) Comp. Het.17)
Homozygou s
18) Homozygou s
19) Homozygou s
20) Homozygou s
21) Homozygou s
22)Homozygou s
23) Homozygou s
24) Homozygou s
25) Homozygou s
26)Homozygou s
27) Comp. Het.		 1) Renal Fanconi Syndrome
2) C1 Deficiency
3) Leigh Syndrome
4) Leigh Syndrome
5) Leigh Syndrome
6) Leigh Syndrome
7) Leigh Syndrome
8) Leigh Syndrome
9) Leigh Syndrome
10) Leigh Syndrome
11) Leigh Syndrome
12) Leigh Syndrome
13) Leigh Syndrome
14) Leigh Syndrome
15) Leigh Syndrome
16) Leigh Syndrome
17) C1 Deficiency
18) C1 Deficiency
19) C1 Deficiency
20) C1 Deficiency
21) C1 Deficiency
22) Leigh Syndrome
23) Leigh Syndrome
24) C1 Deficiency
25) C1 Deficiency
26) C1 Deficiency
27) Leigh Syndrome
Yang et al.(2026) 41982 415 2 1)c.362_364dupTGG (p.Val121dup) c.362_364dup
2)TGG (p.Val121dup), c.169_190dup (p. Leu64fsX2)		 1) Homozygou s
2) Comp. Het.		 1)Leigh Syndrome
2) Leigh Syndrome
Present Case - 1 g.95044573_95046180del, c.298-768T>C Comp. Het. C1 deficiency and proximal renal tubular dysfunction
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Legend: This table includes the authors of the published paper, the Pubmed ID (PMID), how many affected individuals are described in the paper, the variants described (as well as variant allele pattern), as well as the patients’ diagnoses. Note compound heterozygous variants are shorted to “Comp Het.”

*

This secondary effect, a splicing defect, caused by the same variant was identified by McKenzie et al. (2011) [2]

**

In these papers each individual was identified with different variants and in some instances different diseases. Each patient is represented from a-e along with their associated diagnosis following the same format.

***

In this paper the patient was heterozygous for this variant, but the researchers believe that there is a second variant affecting the maternally inherited copy of NDUFAF6 that they could not discover.

Functional studies identified an isolated decrease in C1 activity. The C1 assembly assay revealed lack of the holocomplex without identifiable intermediates, compatible with a very early assembly defect at the level of the Q or the ND1 module [15, 28], where NDUFAF6 functions [18]. Proteomics data showed the lowest C1 complex abundance ever found in our laboratory. Hock et al. [18] reported a similarly low abundance for cases of NDUFAF6 and NDUFS8. NDUFAF6 interacts with NDUFS8 [15], and this was also the lowest quantifiable protein in this case. Despite this low C1 abundance (33%) and low enzyme activity (−10 SD), this patient did not have overwhelming neonatal mitochondrial disease, reflecting the complex relation between biochemical defect and clinical symptomatology and illustrating the utility of functional studies. The novel methodology applying proteomics to diagnose mitochondrial disorders offers rapid turnaround and the ability to detect specific changes [18].

This case expands our understanding of NDUFAF6 and its associated phenotypes and suggests that specific variants may result in more complex, or blended, phenotypes. Most cases have Leigh syndrome, an early onset progressive neurodegenerative disorder (Table 2) [2–20]. A homozygous non-coding variant in NDUFAF6 was found in patients of Acadian origin presenting with Fanconi syndrome, chronic kidney disease, and pulmonary fibrosis [21]. Our patient, whose NDUFAF6 variants have not been reported in combination before, presented with global developmental delay and chronic kidney disease, and liver disease. A previously described patient with one Acadian variant (NM_152416.:c.298-768T>C) had a similarly blended phenotype with Leigh syndrome phenotype and proximal renal dysfunction, suggesting that the combination of variants determines the phenotype [18, 19, 21]. This case demonstrates the need to consider atypical variants in mitochondrial disease, illustrates the limitations of exome sequencing in diagnosing diseases caused by complex variants, and highlights the benefit of genome sequencing in the diagnostic testing of these patients.

Acknowledgments

We thank our patient and their family for their participation in this study. This work was supported by the National Human Genome Research Institute, National Institutes of Health [HG000215-20]. The contributions of the NIH authors are considered Works of the United States Government. The findings and conclusions presented in this paper are those of the authors and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services. In Colorado, the study was supported by philanthropic support from the Children’s Hospital Colorado Summits for Samantha, and the University of Colorado Foundation, and by a grant from the National Institutes of Health, NIH U54NS078059 for the North American Mitochondrial Disease Consortium (NAMDC) to J.L.K.V.H. NAMDC is part of Rare Diseases Clinical Research Network (RDCRN), an initiative of the Office of Rare Diseases Research (ORDR), NCATS. This consortium is funded through collaboration with NCATS. The work performed in the lab of R.R. is supported by NIH/NCCR 1 S10 OD028538-01A1 to Nichole Reisdorph.

Footnotes

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Conflict of Interest

The authors declare no conflict of interest

Ethics Statement

This study was conducted as part of a National Human Genome Research Institute, Institutional Review Board-approved protocol [15-HG-0130].

There are no conflicts of interest to disclose.

CRediT Author Statement

Yuka Aoyama: Writing - Original Draft; Julia Grafstein: Writing - Original Draft; Parker Houston: Writing - Original Draft; Marisa W. Friederich: Investigation, Writing- Review and Editing; Johan Van Hove: Investigation, Writing- Review and Editing; Roxanne A. Van Hove: Investigation; Cole Michel: Investigation; Richard Reisdorph: Investigation; William Gahl: Writing- Review and Editing; David R. Adams Investigation, Writing- Review and Editing; Lynne Wolfe: Investigation, Writing- Review and Editing; Ellen F. Macnamara: Supervision, Investigation, Writing- Review and Editing.

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