Typically, patients with LHON and adOA have no relevant health problems apart from optic atrophy. However, the disease may be associated with a wide range of extraocular features of mitochondrial dysfunction, including brain, cardiac, muscle, or auditive signs. The clinical expression of extraocular manifestations varies from absence of expression to severe dysfunction, which defines the “plus” forms of LHON and OPA1.1–4

In contrast with LHON and adOA, autosomal-recessive optic atrophies (arOAs) usually involve the central nervous system and other organs. Nonsyndromic arOA are less common. Hitherto, one locus has been mapped but no disease gene has been identified (OPA6, ROA1 [MIM 258500]).5

Using a combination of Affymetrix GeneChip Human Mapping 10K 2.0 Arrays and microsatellite markers, we performed homozygosity mapping in a multiplex inbred nonsyndromic arOA family of Algerian ancestry. This approach identified a unique region of homozygosity on chromosome 11q14.1-q21 (ROA2, family 1, Figures S1 and S2 available online). The critical interval spanned 14.4 Mb and contained 40 known genes (Figure 1; maximum LOD score Zmax = 3.73 with no recombination event at the D11S4187 locus).

Considering that all known optic atrophy genes encode mitochondrial proteins,6 we selected in the interval three genes predicted to encode mitochondrial products7 (Figure 1). Systematic sequence analysis identified a homozygous nonsense mutation in the gene encoding TMEM126A, a transmembrane protein (c.163C→T; p.Arg55X; GenBank accession number NM_032273; Figure 1 and Figure S2). The mutation occurred in a CpG doublet, segregated with the disease in the family (Figure S2) and was not found in 700 control chromosomes (European, n = 600; Algerian, n = 100).

We screened 48 additional unrelated patients affected with nonsyndromic OA (arOA n = 14; adOA n = 10; sporadic n = 24) for TMEM126A mutations. All affected patients and their relatives, including all members of family 1, were recruited with their informed and written consent, as prescribed by the law on bioethics of the European Community and after approval by the local ethics committee (DC-2008-512, Paris-Necker). In all cases, OPA1 mutations and the most frequent LHON mutations (mtDNA G11778A, G3460A, T14484C, and G15257) were previously excluded by direct sequencing. None of the sporadic and adOA cases carried TMEM126A mutations. Conversely, the p.Arg55X mutation was identified in three additional arOA families of Maghrebian origin (Tunisa, family 2; Morocco, Families 3 and 4; Figure S2).

Segregation analysis of microsatellite markers flanking the mutation supported the hypothesis of a founder effect by showing the transmission of a small common haplotype with the disease in the four families (Figure S2). Haplotype studies as well as Bayesian calculations8 suggested that the TMEM126A c.163C→T mutation occurred 80 generations ago, i.e., about 2400 years ago (95% credible interval 35–150 generations).

The p.Arg55X mutation caused an early-onset severe bilateral deficiency in visual acuity (VA), optic disc pallor, and central scotoma (onset between 4 and 6 years; VA = counting fingers to 20/200; Figure S3). The peripheral visual field was strictly normal in all but the oldest patient, who lost it between the age of 30 and 37 (III6, family 1; Figure S3).

The phenotype differs from that of ROA1 patients (OPA6 [MIM 258500]) presenting with an early-onset but slowly progressive OA affecting the macular beam of optic nerve with preservation of the peripheral bundles in advanced stage.5

Polarographic tests and spectrophotometric assays on cultured skin fibroblasts9 showed normal respiratory chain function in patient III6 - family 1 but partial deficiency of Complex I in patient V1 - family 2 (17 nmol/min/mg of protein, mean = 37 ± mean standard deviation 5 nmol/min/mg of protein). Patient V1 - family 2 presented with normal brain MRI but moderate hypertrophic cardiomyopathy. These features, along with the minor brain MRI alterations (Figure S4) and mild hearing loss in patient III6 - family 1, are suggestive of a mitochondrial dysfunction as previously noted in patients with LHON or OPA1 mutations.1–4,10

The TMEM126A gene spans 8.5 kb on chromosome 11 and encodes a single ubiquitous transcript (770 bp) made of one noncoding and four coding exons (Figure 1).

The TMEM126 protein includes the Pfam domain of unknown function defined by the homology between the TMEM126A p.Lys5-Gly191 and TMEM126B p.Asn45-229Glu amino acid sequences, respectively. Four transmembrane domains with the N-terminal and C-terminal sequences on the outside of the membrane were predicted in the TMEM126A DUF1370 domain (PredictProtein, Figure 1). Domain of unknown function (DUF) proteins are hypothetical eukaryotic proteins of around 200 residues in length. Members of this family are regarded as specific to mammals, but phylogenetic analyses contradict this notion. Indeed, orthologs of several DUF proteins, including TMEM126A, have been found in many vertebrate and invertebrate species as well as in plant organisms.11,12 No ortholog could be identified in yeast and fungi.

To determine its subcellular localization, we overexpressed a TMEM126A-myc fusion protein into COS-7 cells. Epitope-tagged wild-type TMEM126A colocalized with mitochondrial Complex II subunit 70 kDa Fp (SDHA [MIM 600857]), Complex IV subunit 1 (MTCO1 [MIM 516030]), ATP synthase subunit beta (ATP5B [MIM 102910]), and ATP synthase subunit alpha (ATP5A [MIM 164360]), supporting the mitochondrial localization of the protein (Figure 2). The fusion protein did not significantly colocalize with the markers of the Golgi apparatus, lysosomes, early endosomes, cytoskeleton, microtubule, and centrosome (Figure S5).

RT-PCR on total RNA from various adult and fetal human tissues showed that TMEM126A is strongly expressed in the brain (whole), cerebellum, fetal brain, skeletal muscle, testis, fetal retinal pigmentary epithelium (RPE), and fetal retina (Figure S6). In situ hybridization on adult mouse retina (8 months) detected significant levels of specific mRNA in the ganglion cell layer (GCL), the optic nerve head (ON), the outer plexiform layer (OPL), and in the outer ellipsoide (oe) length of photoreceptor inner segments (IS). Faint to no labeling was noted in the outer nuclear layer (ONL) and photoreceptor outer segments. Immunolocalization of the mitochondria-specific alpha subunit of the ATP synthase (ATP5A) on retinal sections of the same animal showed the same pattern of expression, supporting the view that TMEM126A transcripts colocalized with mitochondria (Figure 3). This result suggests that similarly to the dynamin-related GTPase protein Mgm1, of which OPA1 is the human ortholog, TMEM126A is a mitochondria-localized mRNA (MLR) proteins and may be essential in the early nucleation process of large mitochondrial complexes.13–16

In contrast to OPA1 mutations, we found no fragmentation of the mitochondrial network and/or in depletions of the mitochondrial DNA (mtDNA)17,18 in the fibroblasts of patients III6 - family 1 and V1 - family 2 (data not shown), suggesting that TMEM126A and OPA1 may not be functionally related.

Recently, mutations in another TMEM protein have been reported to cause neonatal mitochondrial encephalocardiomyopathy.12 Interestingly, the absence of TMEM70 in patients resulted in the loss of ATP synthase,12 whose subunits have also been shown to be MLR proteins.16

As a probable MLR protein, TMEM126A may be important to the assembly of a protein complex required for the function of retinal ganglion cells in higher eukaryotes. In other tissues, it is possible that another protein of the TMEM family could adequately substitute for the defective function of TMEM126A. It is also possible that TMEM126A may be required to accelerate the assembly of a protein complex. In the absence of TMEM126A, the complex would constitute slowly, and only high-energy-demanding retinal ganglion cells would be affected.