COQ4 mutations cause a broad spectrum of mitochondrial disorders associated with CoQ10 deficiency

Abstract

Primary coenzyme Q10 (CoQ10) deficiencies are rare, clinically heterogeneous disorders caused by mutations in several genes encoding proteins involved in CoQ10 biosynthesis. CoQ10 is an essential component of the electron transport chain (ETC), where it shuttles electrons from complex I or II to complex III. By whole-exome sequencing, we identified five individuals carrying biallelic mutations in COQ4. The precise function of human COQ4 is not known, but it seems to play a structural role in stabilizing a multiheteromeric complex that contains most of the CoQ10 biosynthetic enzymes. The clinical phenotypes of the five subjects varied widely, but four had a prenatal or perinatal onset with early fatal outcome. Two unrelated individuals presented with severe hypotonia, bradycardia, respiratory insufficiency, and heart failure; two sisters showed antenatal cerebellar hypoplasia, neonatal respiratory-distress syndrome, and epileptic encephalopathy. The fifth subject had an early-onset but slowly progressive clinical course dominated by neurological deterioration with hardly any involvement of other organs. All available specimens from affected subjects showed reduced amounts of CoQ10 and often displayed a decrease in CoQ10-dependent ETC complex activities. The pathogenic role of all identified mutations was experimentally validated in a recombinant yeast model; oxidative growth, strongly impaired in strains lacking COQ4, was corrected by expression of human wild-type COQ4 cDNA but failed to be corrected by expression of COQ4 cDNAs with any of the mutations identified in affected subjects. COQ4 mutations are responsible for early-onset mitochondrial diseases with heterogeneous clinical presentations and associated with CoQ10 deficiency.

Figure 1

Pedigrees of Investigated Families and COQ4 Structure and Conservation of Identified Mutations (A) Pedigrees of four families affected by mutations in COQ4. The mutation status of affected and unaffected family members is indicated by closed and open symbols, respectively. (B) COQ4 structure showing the identified mutations. The structure of the gene product, COQ4, is also shown with known domains and localization and conservation of amino acid residues affected by the mutations. Intronic regions are not drawn to scale.

Figure 2

Biochemical Studies in COQ4 Mutant Muscle and Fibroblasts (A) CoQ₁₀ in muscle from affected subjects S1 and S3–S5 is reported as a percentage of the mean of control values (the analyses were performed in different laboratories, and the reference values are diverse; see text). Data are reported after normalization to protein content or CS activity. (B) Maximal respiration rate (MRR) measured in fibroblasts from subjects S1, S4, and S5; MRR values are expressed as percentages of MRR values obtained in control fibroblasts. The graphs represent the mean values from two independent experiments, each with six to eight replicates. Error bars represent the SD. (C) Immunoblot analysis of COQ4 in fibroblasts from subjects S1, S4, and S5 and control individuals (Ct). Arrowheads indicate the band corresponding to COQ4. An antibody against tubulin was used as a loading control.

Figure 3

Yeast Studies (A) Glycerol (YPG) growth of transformed ΔCOQ4 yeast with the different mutated versions of human COQ4 (pYES2.1, empty vector; hCOQ4, pYES:hCOQ4^WT; yCOQ4, pYES:yCOQ4^WT; c.433C>G, pYES:hcoq4^p.Arg145Gly; c.421C>T, pYES:hcoq4^p.Arg141∗; c.718C>T, pYES:hcoq4^p.Arg240Cys; c.155T>C, pYES:hcoq4^p.Leu52Ser; c.521_523delCCA, pYES:hcoq4^p.Thr174del; c.190C>T, pYES:hcoq4^p.Pro64Ser; c.155T>C and c.521_523delCCA, pYES:hcoq4^{p.Leu52Ser/p.Thr174del}; and c.421C>T and c.718C>T, pYES:hcoq4^{p.Arg141∗/p.Arg240Cys}). WT indicates the wild-type yeast transformed with the YES2.1 empty vector. Cells were grown in selective medium for 16 hr, induced in galactose for 4 hr, and inoculated in YPG at 0.1 U of optical density (OD) at 600 nm. Growth at 30°C was monitored over 5 days by measurement of OD cultures at 600 nm. (B) Yeast mitochondrial CoQ₆ levels. Purified mitochondria lipid extraction and high-performance-liquid-chromatography quantification of CoQ₆ was performed in the ΔCOQ4 strain transformed with the empty vector (pYES2.1), WT yeast (yCOQ4), or human (hCOQ4) or hcoq4^p.Arg145Gly (c.433C>G) COQ4 genes. A WT strain transformed with the empty vector was included as a positive control. Error bars represent the SD.