Recessive MECR pathogenic variants cause an LHON-like optic neuropathy

Abstract

Background: Leber's hereditary optic neuropathy (LHON) is a mitochondrial disorder characterised by complex I defect leading to sudden degeneration of retinal ganglion cells. Although typically associated with pathogenic variants in mitochondrial DNA, LHON was recently described in patients carrying biallelic variants in nuclear genes DNAJC30, NDUFS2 and MCAT. MCAT is part of mitochondrial fatty acid synthesis (mtFAS), as also MECR, the mitochondrial trans-2-enoyl-CoA reductase. MECR mutations lead to a recessive childhood-onset syndromic disorder with dystonia, optic atrophy and basal ganglia abnormalities.

Methods: We studied through whole exome sequencing two sisters affected by sudden and painless visual loss at young age, with partial recovery and persistent central scotoma. We modelled the candidate variant in yeast and studied mitochondrial dysfunction in yeast and fibroblasts. We tested protein lipoylation and cell response to oxidative stress in yeast.

Results: Both sisters carried a homozygous pathogenic variant in MECR (p.Arg258Trp). In yeast, the MECR-R258W mutant showed an impaired oxidative growth, 30% reduction in oxygen consumption rate and 80% decrease in protein levels, pointing to structure destabilisation. Fibroblasts confirmed the reduced amount of MECR protein, but failed to reproduce the OXPHOS defect. Respiratory complexes assembly was normal. Finally, the yeast mutant lacked lipoylation of key metabolic enzymes and was more sensitive to H₂O₂ treatment. Lipoic Acid supplementation partially rescued the growth defect.

Conclusion: We report the first family with homozygous MECR variant causing an LHON-like optic neuropathy, which pairs the recent MCAT findings, reinforcing the impairment of mtFAS as novel pathogenic mechanism in LHON.

Keywords: Genetics, Medical; Neuromuscular Diseases; Ophthalmology.

Figure 1

Clinical and pedigree data. Fundus pictures showing temporal optic disc pallor, visual field showing central scotoma, and optical coherence tomography revealing temporal retinal nerve fibre layer thinning and diffuse macular ganglion cell layerthinning for (A) patient II-1 and (B) patient II-2. (C) Family pedigree.

Figure 2

Yeast modelling of MECR^R258W variant. (A) Alignment of MECR proteins from different organisms, including Mus musculus (mammals), Gallus gallus (birds), Chelonia mydas (reptiles), Xenopus laevis (amphibians), Danio rerio (bony fish), Scyliorhinus canicula (cartilaginous fish), Asterias rubens (echinoderms), Drosophila melanogaster (arthropods), Ophiocordyceps sinensis (mollusks), Caenorhabditis elegans (roundworms), Schizosaccharomyces pombe and Saccaromyces cerevisiae (fungi). (B) Spot assay of W303-1B etr1Δ strain transformed with pFL38ETR1, empty YEplac112TEToff and YEPlac112TEToffMECR, either wild-type or harbouring R258W mutation, on YP medium supplemented with either 2% glucose or 2% ethanol. Pictures were taken after 3 days. (C) Oxygen consumption rate (OCR) on strains transformed with MECR ^wt or MECR^R258W . OCR was measured on four independent clones for each strain and normalised to the OCR of the strain transformed with MECR^wt and reported as mean±SD. Statistical analysis was performed by one-way analysis of variance followed by a post hoc Bonferroni test. ***p<0.001. (D) Representative image of a Western blot on the same strains reported in panel C for MECR and Por1 as loading control, with MECR/Por1 levels normalised to the strain harbouring MECR^wt .

Figure 3

Characterisation of patient-derived fibroblasts carrying MECR^R258W variant. (A) Western blot of MECR; GAPDH was used as loading control. A representative blot of three independent experiments (biological replicates) is shown. (B) Densitometry of MECR content. All data are means and SD Statistical analysis was performed by t-test. **: p<0.01. (C) OCR expressed as picomoles O2/min, normalised for protein content, under basal conditions and after injection of oligomycin (O), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; F), rotenone (R) and antimycin A (A). Data are expressed as means ± SD of three independent experiments (biological replicates). (D) Basal, ATP-linked, maximal and CI respiration. All values are means and SD of three independent experiments (biological replicates). (E) Western blot of OXPHOS subunits; GAPDH was used as loading control. A representative blot of three independent experiments is shown for each protein. (F) Densitometry of OXPHOS subunits of three independent experiments (biological replicates). All data are means and SD. (G) CI-IGA of SCs from mitoplasts obtained from control and mutant cells. One representative experiment of three is shown. IGA, in gel activity.

Figure 4

Oxidative stress and LA supplementation on yeast model. (A) Representative image of a Western blot of lipoylated Lat1, Kgd2 and Por1 as loading control on the same proteins extract as in figure 2, panel D. (B) Growth phenotype of the same strains as in panel A on YPD medium without or with 2 mM H₂O₂. Pictures were taken after 3 days. (C) Viability test assay after treatment with different concentrations of H ₂ O ₂ for different times on MECR^wt and MECR^R258W strains. Viability was measured on three independent clones for each strain, at least 5000 cells plated for each clone, and reported as mean±SD. **p<0.01; ***p<0.001. (D) Cell concentration of the same strains as in panel C, after 48-hour, 72-hour and 96-hour growth in liquid YP medium supplemented with 2% ethanol and, for the MECR^R258W strain, without or with 40 µg/mL LA. Cell concentration was measured on three independent replicates for each strain/condition and reported as mean±SD. *p<0.05; ***p<0.001. (E) Representative image of a Western blot of lipoylated Lat1 and Kgd2 proteins extracted from untreated MECR^wt strain and untreated or LA-supplemented MECR^R258W strain. (F) Viability test assay after treatment with 1 mM H₂O₂ for 2 hours and rescue with different concentrations of LA on MECR^R258W strain. Viability was measured on three independent replicates for each condition, at least 5000 cells plated for each replicate, and reported as mean±SD. ***p<0.001. LA, lipoic acid.