A biallelic variant in COX18 cause isolated Complex IV deficiency associated with neonatal encephalo-cardio-myopathy and axonal sensory neuropathy

Abstract

Pathogenic variants impacting upon assembly of mitochondrial respiratory chain Complex IV (Cytochrome c Oxidase or COX) predominantly result in early onset mitochondrial disorders often leading to CNS, skeletal and cardiac muscle manifestations. The aim of this study is to describe a molecular defect in the COX assembly factor gene COX18 as the likely cause of a neonatal form of mitochondrial encephalo-cardio-myopathy and axonal sensory neuropathy. The proband is a 19-months old female displaying hypertrophic cardiomyopathy at birth and myopathy with axonal sensory neuropathy and failure to thrive developing in the first months of life. Serum lactate was consistently increased. Whole exome sequencing allowed the prioritization of the unreported homozygous substitution NM_001297732.2:c.667 G > C p.(Asp223His) in COX18. Patient's muscle biopsy revealed severe and diffuse COX deficiency and striking mitochondrial abnormalities. Biochemical and enzymatic studies in patient's myoblasts and in HEK293 cells after COX18 silencing showed a severe impairment of both COX activity and assembly. The biochemical defect was partially rescued by delivery of wild-type COX18 cDNA into patient's myoblasts. Our study identifies a novel defect of COX assembly and expands the number of nuclear genes involved in a mitochondrial disorder due to isolated COX deficiency.

Fig. 1. Instrumental findings and sequence analysis in the Patient.

A Pedigree of the investigated family. Affected patients are indicated with black symbols. Arrow indicates the proband described in the report. The genotype for the COX18 variant c.667 G > C is indicated in the symbol of genotyped subjects (+/+ homozygous, +/– heterozygous carrier, –/– absence of the variant). B Sequence electropherograms showing the COX18 c.667 G > C variant in the patient and her unaffected parents. C Conservation of the human Asp223 residue across species and in the human OXA1L insertase protein. D Scheme of the COX18 gene and its encoded gene product displaying transmembrane (TM) domains. E Relative gene expression levels in human control tissues of COX18 compared to ECHS1 (encoding a mitochondrial matrix protein).

Fig. 2. Muscle histology and biochemical studies in myoblasts of the COX18-mutated patient.

A Modified Gomori Trichrome B Severe reduction of muscle fibers staining positive for cytochrome c oxidase (COX) is seen in the COX18 subject, whereas normal COX staining is shown in a healthy control (C) for comparison (Scale bar 25 µm). D–F Representative images of the alterations observed in mitochondria. Inner cristae were arranged to form concentric layers. (Asterisk in F indicates an osmiophilic inclusion. Scale bars D = 926 nm, E–F = 463 nm). G Spectrophotometric analysis of respiratory chain complexes activities normalized to citrate synthase levels in patient’s and control myoblasts. H Immunoblot analysis of myoblasts protein with antibodies directed against respiratory chain subunits. The mitochondrial porin (VDAC) and the cytosolic Actin proteins were used as a loading control. Densitometry (right) shows the reduction of steady state levels of COX subunits COX-II, COX-III and COX-IV in patient’s compared to controls’ myoblasts. I Blue-native polyacrylamide gel electrophoresis (BN-PAGE) analysis of myoblasts protein shows absence of fully assembled COX in the COX18 subject. Each of the five OXPHOS complexes (I–V) was visualised with a subunit-specific antibody that recognizes the native complex as follows: Complex I (NDUFA9), Complex II (SDHA), Complex III (UQCRC1), Complex IV (COX4), Complex V (ATP5A1). Densitometry after SDHA normalization (right) shows the prevalent reduction of fully assembled Complex IV. J In-gel activity assay showing the severe reduction of Complex IV activity in patient’s cells compared to controls.

Fig. 3. Biochemical effects of COX18 downregulation and overexpression.

A Quantitative gene expression levels of COX18 in HEK293 treated with scrambled or RNAi sequences directed against COX18. B Immunoblot analysis of selected respiratory chain subunits in COX18-silenced HEK293 cells, showing the reduction of COX subunits COX-II and COX-IV (densitometry showed on the right). C Activities of respiratory chain Complex II and IV in control or COX18-silenced HEK293 cells. D BN-PAGE analysis and (E) in-gel activity assay of respiratory chain complexes in control or COX18-silenced HEK293 cells, documenting a severe Complex IV reduction. F Immunoblot analysis showing the rescue of COX-II protein levels and Complex IV activity (G) in patient’s myoblasts after COX18 overexpression (visualized by fusion tag FLAG-DDK band, Patient^COX18wt) compared to mock-transfected patient’s myoblasts and control cells. H Profound decrease of COX histochemical reaction, visualized in patient’s myoblasts, compared to control cells, was partially rescued after COX18 overexpression (Patient^COX18wt). Scale bar 20 µm.