Journal: Frontiers in Neurology

Article Title: Compound Heterozygous COX20 Variants Impair the Function of Mitochondrial Complex IV to Cause a Syndrome Involving Ophthalmoplegia and Visual Failure

doi: 10.3389/fneur.2022.873943

Figure Lengend Snippet: COX20 mRNA and protein expression. (A) mRNA expression of COX20 in the muscle tissues and fibroblasts of the proband and control. GAPDH was used as the loading control. Significant differences in the mRNA levels exist between the groups. (B) Gene and cDNA schematic. c.41A > G and c.222G > T mutations were identified in the affected siblings. According to an NCBI genome analysis, the full length cDNA corresponds to transcript variant 1 (NM_198076.6), and the coding sequence (CDS) is 357 bp in length. Transcript variant 4 (NM_001312873.1) lacks exon 2 and a part (20 bp) of exon 1 of NM_198076.6. The coding region is 222-bp long and shorter than the transcript variant 1 by 135 bp. (C) Sanger sequencing reveals errors in mRNA splicing in fibroblasts of the patient. The results of sequences ①/②/③ were aligned with the reference sequence NM_198076.6. Blanks in the sequence alignment depict deletion or mismatch of nucleotides. Sequences ① were obtained from fibroblasts of control. Alignment of sequence ② shows that the variant c.41A > G led to a 20-bp deletion in exon 1. Alignment of sequence ③ shows that the variant c.222G > T did not affect mRNA splicing. (D–F) Western blot analysis of COX4, COX20, and OXPHOS in muscle tissues and fibroblasts. GAPDH was used as the internal control for muscles, and β-actin for cells. Significant differences are observed in the protein expression of COX20 and CIV but not in the protein expression of CI, CII, CIII, and CV. (G) Blue native-PAGE analysis of mitochondria isolated from fibroblasts reveals lower COX2, COX4, and CIV levels; ** p < 0.01. All data are presented as mean ± SEM and p -values were calculated by the Mann–Whitney U -test.

Article Snippet: Human skin fibroblasts were obtained from iCell (Shanghai, China, h075).

Techniques: Expressing, Control, Variant Assay, Sequencing, Western Blot, Muscles, Blue Native PAGE, Isolation, MANN-WHITNEY

Journal: Frontiers in Neurology

Article Title: Compound Heterozygous COX20 Variants Impair the Function of Mitochondrial Complex IV to Cause a Syndrome Involving Ophthalmoplegia and Visual Failure

doi: 10.3389/fneur.2022.873943

Figure Lengend Snippet: (A,B) OCR of patient (II-1) fibroblasts was measured using a Seahorse XF24 Extracellular Flux Analyzer. Basal respiration, maximal respiration, ATP production, and spare respiratory capacity of fibroblasts from the patient are significantly lower than those of control. (C) To detect the enzymatic activities of complexes, mitochondria were isolated from fibroblasts, and citrate synthase was used as the internal control. Complex IV enzyme activities of the patient fibroblasts were lower than control. (D,E) Functional complementation assays demonstrate that COX20 variants are responsible for the deficiency of complex IV and mitochondria in patient fibroblasts. Fibroblasts transduced with adenovirus vector (ADM-FH) were used as a control group. Western blot analysis shows that compared with fibroblasts transduced with ADM-FH, the expression level of COX20 and CIV in fibroblasts from patients transduced with COX20 increased significantly. (F,G) Seahorse analysis shows that COX20 overexpression promoted maximal respiration and increased mitochondrial spare respiratory capacity. (H) Enzyme activity measurements show that COX20 overexpression can help mitochondria restore the enzymatic activity of complex IV; * p < 0.05, ** p < 0.01 vs. ADM-FH, # p < 0.05 vs. II-1 + ADM-FH and II-1 + AD-COX20. All data are presented as mean ± SEM and p -values were calculated by Kruskal–Wallis tests. ## p < 0.01 versus II-1+ADM-FH and II-1+AD-COX20.

Article Snippet: Human skin fibroblasts were obtained from iCell (Shanghai, China, h075).

Techniques: Control, Isolation, Functional Assay, Transduction, Plasmid Preparation, Western Blot, Expressing, Over Expression, Activity Assay