Homozygous COQ7 mutation: a new cause of potentially treatable distal hereditary motor neuropathy

Abstract

Distal hereditary motor neuropathy represents a group of motor inherited neuropathies leading to distal weakness. We report a family of two brothers and a sister affected by distal hereditary motor neuropathy in whom a homozygous variant c.3G>T (p.1Met?) was identified in the COQ7 gene. This gene encodes a protein required for coenzyme Q10 biosynthesis, a component of the respiratory chain in mitochondria. Mutations of COQ7 were previously associated with severe multi-organ disorders characterized by early childhood onset and developmental delay. Using patient blood samples and fibroblasts derived from a skin biopsy, we investigated the pathogenicity of the variant of unknown significance c.3G>T (p.1Met?) in the COQ7 gene and the effect of coenzyme Q10 supplementation in vitro. We showed that this variation leads to a severe decrease in COQ7 protein levels in the patient's fibroblasts, resulting in a decrease in coenzyme Q10 production and in the accumulation of 6-demethoxycoenzyme Q10, the COQ7 substrate. Interestingly, such accumulation was also found in the patient's plasma. Normal coenzyme Q10 and 6-demethoxycoenzyme Q10 levels were restored in vitro by using the coenzyme Q10 precursor 2,4-dihydroxybenzoic acid, thus bypassing the COQ7 requirement. Coenzyme Q10 biosynthesis deficiency is known to impair the mitochondrial respiratory chain. Seahorse experiments showed that the patient's cells mainly rely on glycolysis to maintain sufficient ATP production. Consistently, the replacement of glucose by galactose in the culture medium of these cells reduced their proliferation rate. Interestingly, normal proliferation was restored by coenzyme Q10 supplementation of the culture medium, suggesting a therapeutic avenue for these patients. Altogether, we have identified the first example of recessive distal hereditary motor neuropathy caused by a homozygous variation in the COQ7 gene, which should thus be included in the gene panels used to diagnose peripheral inherited neuropathies. Furthermore, 6-demethoxycoenzyme Q10 accumulation in the blood can be used to confirm the pathogenic nature of the mutation. Finally, supplementation with coenzyme Q10 or derivatives should be considered to prevent the progression of COQ7-related peripheral inherited neuropathy in diagnosed patients.

Keywords: COQ7; Coenzyme Q10; distal hereditary motor neuropathy.

Figure 1

Presentation of the family and COQ7 protein. (A) Pedigree of the family. Squares are males and circles are females. Filled symbols represent affected subjects and empty symbols unaffected subjects (B) Amyotrophy of Patient II-2 legs. (C) Sanger sequencing confirmation of the genotypes. (D) Schematic representations of the different isoforms showing the three possible ATG start codons. The orange triangle shows the point mutation c.3G>T on isoform 1 (NM_016138.5), which is the main isoform expressed in the spinal cord. (E) Schematic representation of the three COQ7 isoforms aligned on the major isoform 1 (NP_057222.2). Isoform 1 is composed of 217 AA, including a 35-AA signal peptide which will be cleaved, and a 182-AA mature protein. Isoforms 2 and 3 are respectively composed of 179 and 203 AA. The variant from the patients is localized in the isoform 1 start codon (orange triangle), leading to the disruption of this codon. Other pathogenic mutations previously described are localized within the catalytic site (red triangle). Translation of the coding DNA sequence (CDS) is in yellow. The nuclear and mitochondrial targeting signal in the isoform 1 signal peptide is in orange. The cleavage site of the mitochondrial processing peptidase is shown by a dashed line. The diiron binding site of the catalytic domain is in grey. In-frame start codons are in blue. (F) COQ7 isoform alignment showing the variable AA in the N-terminal region.

Figure 2

COQ7 c.3G>T mutation reduces COQ7 protein expression in SH-EP cells. (A) Confocal images of SH-EP cells transfected with expression vectors for wild-type (WT) or mutant COQ7 fused to c-terminal FLAG-tag and counterstained for mitochondria in orange (MitoTracker), COQ7 in green (anti-FLAG), and nucleus (DAPI) in blue. Scale bar = 10 µm. (B) Western blot analysis of SH-EP transfected with COQ7 expression vectors. COQ7 was detected with anti-FLAG and anti-COQ7 antibodies. The mitochondrial protein TOM20 is detected with an anti-TOM20 antibody. Protein sample dilution ratio: 1, 1:2, and 1:4 for each condition. The two bands observed with the anti-COQ7 antibody and anti-Flag (Supplementary Fig. 3A and B) probably correspond to mature and immature isoform 1 with 36 additional AA before cleavage of the signal peptide. (C) Quantification of COQ7 protein by western blot (mean ± standard error of the mean). (D) Quantification of COQ7 mRNA in SH-EP cells transfected with expression plasmids for wild-type or mutant COQ7, using two primer pairs localized in exons 2–3 and exons 4–5 respectively. Values represent mean ± standard error of the mean.

Figure 3

COQ7 c.3G>T mutation decreases COQ7 protein level in the patient’s fibroblasts. (A) Quantification of COQ7 mRNA in the control (CTL) and patient’s fibroblasts using two primer pairs targeting exon 2–3 and exon 4–5. Values represent mean ± standard error of the mean. *P < 0.05; **P < 0.01; n = 3; one-way ANOVA followed by Bonferroni’s multiple comparison test. (B) Western blot analysis of control and patient’s fibroblasts using anti-COQ7 antibodies and α-tubulin gene to normalize. Protein loading 4×, 2×, and 1× for each condition. Stars show the non-specific histone band (Supplementary Fig. 3C and D). (C) Nuclear extract (NE), benzonase soluble (Chrom), and insoluble (Chrom & Mito Pellet) fractionation revealed by western blotting using anti-COQ7 antibodies to stain COQ7 protein, anti-TOM20 antibodies to stain mitochondria, and anti-H4 antibodies to stain H4 histones in the control and patient’s fibroblasts.

Figure 4

Metabolic changes in the patient’s plasma and fibroblasts. Representative HPLC-MS/MS chromatograms for the detection of CoQ10 and 6-DMQ (A) in fibroblasts, (B) in the plasma of healthy controls and Patient II-2, and (C) in the patient’s fibroblasts treated with 1 mM 2,4-dHB. The top chromatogram represents the detected signal for CoQ10 (indicated by asterisk) and the bottom chromatogram represents the detected signal for 6-DMQ (indicated by double asterisk). (D and E) Graphical representation of the rescue of CoQ10 and 6-DMQ levels by 1 mM 2,4-dHB in the patient’s fibroblasts (one-way ANOVA followed by Bonferroni tests ***P < 0.001; n = 3). (F and G) Graphical representation of ATP production by mitochondrial respiration (mitoATP) and glycolysis (glycoATP) measured in real-time using the Seahorse Bioscience Extracellular Flux Analyzer on control (#AB249 and #V972) or patient’s (#AF400) fibroblasts (one-way ANOVA followed by Bonferroni tests ***P < 0.001; n = 3). (H) Seahorse Mitostress analysis. OCR traces, expressed as pmol O₂/min/10³ cells of fibroblast cell lines from the patient and controls. The dashed lines indicate the time of addition of oligomycin, FCCP, and antimycin A/rotenone. The OCR profile is representative of three independent experiments. (I) Effect of 10 μM CoQ10 supplementation on OCR traces of fibroblast cell lines from the patient and control. The OCR profile is representative of three independent experiments.

Figure 5

The cell growth of the patient’s fibroblasts is lowered in galactose medium and is partially rescued by CoQ10 supplementation. (A) Fibroblast cultures from two controls and Patient II-2 after 4 days of cell growth, stained for nucleus (DAPI) in blue and actin (Phalloidin) in green. Scale bar = 300 μm. (B) Number of cells per field in the galactose medium after 4 days of growth, normalized to the number of cells in glucose medium, in the fibroblasts of the patient (#AF400) and controls (#AB249 and #V972) (Kruskal–Wallis test followed by Dunn’s multiple comparison test; ***P < 0.001; n = 3). (C) Number of cells per field in the optimal medium during 4 days of growth, normalized to the number of cells at Day 0 in the optimal medium, in the fibroblasts of the patient (#AF400) and controls (#AB249 and #V972). (D) Change in the number of cells per field in galactose medium over 4 days, normalized to the number of cells at Day 0 in glucose medium, in the fibroblasts of the patient (#AF400) and controls (#AB249 and #V972) (two-way ANOVA followed by Bonferroni tests **P < 0.01; n = 3). (E) Number of cells per field in glucose, galactose, and galactose with 10 μM CoQ10 medium after 3 days in culture, normalized to the number of cells at Day 0 in glucose medium, in the fibroblasts of the patient (#AF400) and controls (#V972) (two-way ANOVA followed by Bonferroni tests **P < 0.01; n = 3).