MECR Mutations Cause Childhood-Onset Dystonia and Optic Atrophy, a Mitochondrial Fatty Acid Synthesis Disorder

Abstract

Mitochondrial fatty acid synthesis (mtFAS) is an evolutionarily conserved pathway essential for the function of the respiratory chain and several mitochondrial enzyme complexes. We report here a unique neurometabolic human disorder caused by defective mtFAS. Seven individuals from five unrelated families presented with childhood-onset dystonia, optic atrophy, and basal ganglia signal abnormalities on MRI. All affected individuals were found to harbor recessive mutations in MECR encoding the mitochondrial trans-2-enoyl-coenzyme A-reductase involved in human mtFAS. All six mutations are extremely rare in the general population, segregate with the disease in the families, and are predicted to be deleterious. The nonsense c.855T>G (p.Tyr285^∗), c.247_250del (p.Asn83Hisfs^∗4), and splice site c.830+2_830+3insT mutations lead to C-terminal truncation variants of MECR. The missense c.695G>A (p.Gly232Glu), c.854A>G (p.Tyr285Cys), and c.772C>T (p.Arg258Trp) mutations involve conserved amino acid residues, are located within the cofactor binding domain, and are predicted by structural analysis to have a destabilizing effect. Yeast modeling and complementation studies validated the pathogenicity of the MECR mutations. Fibroblast cell lines from affected individuals displayed reduced levels of both MECR and lipoylated proteins as well as defective respiration. These results suggest that mutations in MECR cause a distinct human disorder of the mtFAS pathway. The observation of decreased lipoylation raises the possibility of a potential therapeutic strategy.

Figure 1

Pedigrees of the Families Described in This Work For all tested individuals: Mut/mut refers to compound heterozygous affected individuals with the exception of subject II:1 from family D who is a homozygous affected individual and is specified with an asterisk (^∗); Mut/WT refers to heterozygous healthy carriers; and WT/WT refers to healthy non-carriers.

Figure 2

T2 Axial MRI Sections of Affected Individuals Are Suggestive of Basal Ganglia Necrosis (A) Hyper intense signal in the putamen and mild pallidal hypo intense signal in affected individual 1 (family A, II:1). (B) Hyper intense pallidal signal in affected individual 2 (family B, II:2). (C) Hyper intense signal in both putamen and caudate in affected individual 4 (family C, II:8). (D) Selective damage to posterior putamen with apparent preservation of the anterior portion in affected individual 5 (family D, II:1). (E) Hyper intense pallidal signal in affected individual 6 (family E, II:1). (F) Follow-up MRI 2 years later in the same affected individual demonstrates cystic left pallidal changes. (G) Hyper intense pallidal signal in affected individual 7 (family E, II:3). (H and I) A lactate peak (see arrows) is demonstrated in the spectroscopy of affected individuals 6 and 7 (family E, II:1 and II:3), respectively.

Figure 3

Structural Context of the Variants Detected in This Study (A) Sequence presentation that includes the domain organization, exon boundaries, secondary structures, and conservation data. In the secondary structure, track helices are indicated in dark color and strands in brighter color. The variants detected in this study are indicted on the coordinates ruler by the numbers 1–6, with the corresponding variant represented by each number given on the side. (B) Global view of the homodimer (PDB: 2vcy). The NADPH cofactor-binding site is shown in blue and the suspected fatty acid binding site is shown in dark green. The p.Gly232Glu, p.Tyr285Cys, and p.Arg258Trp missense mutations are shown in red. Positions 232 and 258 are close to the surface and although located in the cofactor binding domain, are not parts of the binding site itself while position 285 is part of the cofactor binding pocket. (C) Structure of the monomer colored by structural domains. The cofactor-binding domain is shown in pink, the catalytic domain in blue, and the transition peptide in green. (D) The region expected to be missing (black) or altered (gray) due to the p.Asn83Hisfs^∗4 frameshift deletion variant (if the transcript survives decay and is translated). (E) The region expected to be altered (gray) or missing (black) due to the c.830+2_830+3insT splicing variant assuming no alternative splicing donor site is utilized. (F) The region expected to be missing (black) due to p.Tyr285^∗ nonsense variant. (G) Detailed view of the region around residue 232. The side chain introduced by the p.Gly232Glu mutation creates severe steric clashing with residues 231 and 205 (shown in yellow), resulting in reduced stability and/or significant local conformation changes in this region. (H) Detailed view of the region around residue 285. The NADPH is shown in blue. Favorable interactions formed with Tyr285 are shown in green lines. Putative disulfide bond formed between the Cys285 mutant residue and Cys263 is shown in yellow line. (I) Detailed view of the region around residue 258. The p.Arg258Trp mutation abolishes several salt bridges and hydrogen bonds (shown in green lines) with side chains of residues Asp185 and Gln180. The tryptophan side chain (shown in semitransparent display) makes unfavorable interactions with polar groups in the area (yellow lines).

Figure 4

Modeling of the c.695G>A and c.855T>G Mutated MECR Alleles in the Respiratory-Deficient Yeast BJ1991etr1Δ mtFAS Defective Strain (A and B) Analysis of rescue of respiratory growth of the etr1Δ mutant by the MECR c.695G>A and c.855T>G allele variants, respectively. Yeast cells transformed with plasmids carrying the indicated constructs were grown on liquid media, normalized according to cell density, and serial diluted (1×, 1/10×, 1/100×, and 1/1,000×). For each mutation two independent clones transformed with mutation plasmids were tested. Equal volumes of cell suspensions were spotted on synthetic media containing only a non-fermentable carbon source (glycerol or lactate) or plates containing the fermentable carbon source glucose as growth control, and grown for several days. Growth on lactate or glycerol indicates respiratory competence. (C) Western blotting analyses of yeast extracts from BJ1991etr1Δ cells carrying YEp195-ETR1(yETR1), YEp352 (empty), YEp352-MTS-HsMECR (HsMECR), YEp352-MTS-HsMECR-c.695G>A (HsMECR-c.695G>A), or YEp352-MTS-HsMECR-c.885T>G (HsMECR-c.885T>G). Whole yeast cell extract was separated by SDS-page, transferred to a solid support, and probed for the relevant antigens. Antisera used for protein detection are indicated. Lat1: E2 subunit of yeast pyruvate dehydrogenase; Kgd2: E2 subunit of yeast α-ketoglutarate dehydrogenase. Actin: Loading control. An additional loading control (Ponceau S staining of the blotting membrane) can be found in the Supplemental Data.

Figure 5

Western Blotting Analysis for MECR and Lipoylated Protein Content of Cell Extracts from Several Affected Individuals’ Fibroblast Cell Lines (A) Western blotting of whole-cell extract from cell lines of affected individuals 1–3 (family A, II:1; family B, II:2; family C, II:2) demonstrates reduced lipoylation and decreased level of MECR compared to four control fibroblast cell lines. DLAT-LA: lipoyated E2 subunit of human pyruvate dehydrogenase. DLST-LA: lipoylated E2 subunit of human α-ketoglutarate dehydrogenase. β-Actin: loading control. (B) Western blotting of whole-cell extract from cell lines of affected individual 6 (family E, II:1) shows decreased level of MECR compared to control fibroblast cell lines. (C and D) Densitometric quantitation of the DLAT-LA (C) and DLST-LA (D) signals show significantly reduced lipoylation in affected individuals 1–3 fibroblasts compared with controls. For the DLAT/DLST control intensity bars, the average intensity of the luminescence signal of the four samples from healthy controls was calculated. The mean value was defined as 1.0 (100% intensity) and the error bars represent the standard error of the mean (SEM).

Figure 6

Respirometry Analysis of Affected Individuals’ Fibroblast Cell Lines Analysis was carried out measuring oxygen consumption using the Oroboros 2k respirometer; (A) ETS (electron transport system activity); (B) routine level respiration; (C) leak level respiration.