Biallelic Mutations in TMEM126B Cause Severe Complex I Deficiency with a Variable Clinical Phenotype

Abstract

Complex I deficiency is the most common biochemical phenotype observed in individuals with mitochondrial disease. With 44 structural subunits and over 10 assembly factors, it is unsurprising that complex I deficiency is associated with clinical and genetic heterogeneity. Massively parallel sequencing (MPS) technologies including custom, targeted gene panels or unbiased whole-exome sequencing (WES) are hugely powerful in identifying the underlying genetic defect in a clinical diagnostic setting, yet many individuals remain without a genetic diagnosis. These individuals might harbor mutations in poorly understood or uncharacterized genes, and their diagnosis relies upon characterization of these orphan genes. Complexome profiling recently identified TMEM126B as a component of the mitochondrial complex I assembly complex alongside proteins ACAD9, ECSIT, NDUFAF1, and TIMMDC1. Here, we describe the clinical, biochemical, and molecular findings in six cases of mitochondrial disease from four unrelated families affected by biallelic (c.635G>T [p.Gly212Val] and/or c.401delA [p.Asn134Ilefs(∗)2]) TMEM126B variants. We provide functional evidence to support the pathogenicity of these TMEM126B variants, including evidence of founder effects for both variants, and establish defects within this gene as a cause of complex I deficiency in association with either pure myopathy in adulthood or, in one individual, a severe multisystem presentation (chronic renal failure and cardiomyopathy) in infancy. Functional experimentation including viral rescue and complexome profiling of subject cell lines has confirmed TMEM126B as the tenth complex I assembly factor associated with human disease and validates the importance of both genome-wide sequencing and proteomic approaches in characterizing disease-associated genes whose physiological roles have been previously undetermined.

Figure 1

Autosomal-Recessive TMEM126B Variants Are Identified in Six Unrelated Subjects from Four Families Affected by an Isolated Complex I Deficiency (A) Pedigrees and genotype of affected individuals harboring TMEM126B variants. Subject 1 harbors a homozygous c.635G>T (p.Gly212Val) TMEM126B variant; his parents and unaffected sister are heterozygous carriers of this variant. Subjects 2 and 3 harbor compound-heterozygous TMEM126B variants—a paternal c.401delA (p.Asn134Ilefs^∗2) variant and a maternal c.635G>T (p.Gly212Val) variant. Subjects 4 and 5 also harbor compound-heterozygous c.401delA (p.Asn134Ilefs^∗2) and c.635G>T (p.Gly212Val) TMEM126B variants; carrier testing confirmed that the subjects’ mother harbors a heterozygous c.401delA (p.Asn134Ilefs^∗2) variant, but paternal DNA was unavailable for confirmatory testing. Subject 6 harbors a homozygous c.635G>T (p.Gly212Val) TMEM126B variant; both her parents are carriers, and her unaffected brother does not harbor the mutation. (B) Sequencing chromatograms depict the recurrent c.635G>T (p.Gly212Val) and c.401delA (p.Asn134Ilefs^∗2) TMEM126B variants, which represent the disease alleles identified in our cohort of six affected subjects. (C) Clustal Omega sequence alignment shows the evolutionary conservation of the p.Gly212 residue (marked with an asterisk). (D) Shared maternal and paternal haplotypes in the region of interest for subjects 1–6, as inferred by SHAPEIT2. Subject 1 has a ∼0.5 Mb homozygous region from 91.67 to 91.74 cM, whereas subject 6 has a ∼2 Mb homozygous region from 91.51 to 92.46 cM (blue boxes). The two Belgian sibling pairs (subjects 2 and 3 and subjects 4 and 5) share the p.Gly212Val haplotype over a ∼1.75 Mb region (91.31–92.12 cM: blue diagonal shade) and the p.Asn134Ilefs^∗2 haplotype over a ∼4.6 Mb region (89.35–92.2 cM: red diagonal shade). Both haplotypes are shared over the 91.31–92.12 cM region. Boxed white areas represent regions shared with at least one other allele from a different family. The Polish subject 6 shares a ∼2.2 Mb (91.31–92.46 cM) p.Gly212Val haplotype region with siblings 2 and 3 and a ∼2.5 Mb (90.75–92.12 cM) region with siblings 4 and 5.

Figure 2

Biochemical Analysis of Subject Samples Demonstrates Tissue-Specific Complex I Deficiency (A and B) Mitochondria isolated from cultured skin fibroblasts of control subjects and subjects 1–3 were analyzed by BN-PAGE after solubilization in (A) digitonin for maintaining supercomplex interactions or (B) Triton X-100 (TX100) for isolating holo-complexes according to published methodologies. Immunoblotting was performed with antibodies as indicated. The blot probed with an antibody raised against NDUFS3 revealed the presence of additional, partially assembled complex I intermediates in the samples from subjects 2 and 3 (A, indicated by an asterisk). (C) Mitochondria were isolated as described in (A) and (B) and analyzed by SDS-PAGE. Immunoblotting was performed with antibodies against complex I subunits or control proteins as indicated. (D) Muscle samples derived from subject 1 and two control subjects were solubilized in n-dodecyl β-D-maltoside (DDM) and subjected to BN-PAGE and immunoblot analysis using antibodies directed to various OXPHOS complexes as indicated. (E) Respiratory-chain enzyme activities in fibroblast mitochondria were assayed spectrophotometrically as described and expressed as percentages of residual activity in relation to citrate synthase for subject 1 (white bars), subject 2 (light-gray bars), and subject 3 (dark-gray bars). Vertical lines represent the observed normal ranges for either 8 (subject 1) or 36 (subjects 2 and 3) normal control cell lines determined in Newcastle or Melbourne, respectively. The following abbreviation is used: ND, not detected.

Figure 3

Re-expression of Wild-Type TMEM126B Can Lead to Increased Complex I Assembly and Activity in Subject Cells (A) Wild-type TMEM126B mRNA was generated by retroviral expression in control and subject 2 fibroblasts as described previously. After transduction and puromycin selection of cells, whole-cell lysates were solubilized in 1% Triton X-100 and analyzed by BN-PAGE and immunoblotting using antibodies against NDUFA9 (complex I) and SDHA (complex II) as a loading control. (B) Wild-type TMEM126B mRNA was generated by lentiviral expression, and activities of complexes I and IV were assessed by enzyme dipstick analyses as described previously. Barplots show complex I (CI) activity, normalized by complex IV (CIV) activity in control and subject fibroblasts, after transduction with (gray bars) and without (white bars) wild-type TMEM126B mRNA. Data shown are means of three independent transfections ± SEM. ^∗p < 0.05, ^∗∗p < 0.01.

Figure 4

Complexome Profiling of Fibroblasts from Subjects 2 and 3 Identifies Stalled Complex I Assembly Intermediates Prior to mitochondrial isolation, skin fibroblasts were cultured for 48 hr in medium supplemented with galactose as a carbon source. Mitochondrial protein complexes were solubilized with digitonin and separated by BN-PAGE. Native gels were fixed and stained with Coomassie and cut into 60 equal fractions; proteins were digested with trypsin and analyzed by quantitative mass spectrometry. For direct comparison of protein-abundance profiles in control and affected subjects, intensity-based absolute quantification values calculated by MaxQuant proteomics software were normalized to the maximum over datasets (left part of each sample). Less abundant complex I assembly intermediates were normalized to the maximum within the mass region below 1,200 kDa (right part of each sample) for enabling better visualization within a heatmap. The native masses of gel slices were calibrated by exponential regression using positions of the human OXPHOS complex in the gel. The left lane indicates assembly factors (orange), MCIA components (blue), and structural subunits of complex I (yellow), complex III (red), and complex IV (green). Abbreviations are as follows: MCIA, mitochondrial complex I assembly complex; CIII₂, complex III dimer; CIV, complex IV; and S, supercomplex containing complex I, a dimer of complex III, and one to four copies of complex IV.