A Comprehensive Genomic Analysis Reveals the Genetic Landscape of Mitochondrial Respiratory Chain Complex Deficiencies

Abstract

Mitochondrial disorders have the highest incidence among congenital metabolic disorders characterized by biochemical respiratory chain complex deficiencies. It occurs at a rate of 1 in 5,000 births, and has phenotypic and genetic heterogeneity. Mutations in about 1,500 nuclear encoded mitochondrial proteins may cause mitochondrial dysfunction of energy production and mitochondrial disorders. More than 250 genes that cause mitochondrial disorders have been reported to date. However exact genetic diagnosis for patients still remained largely unknown. To reveal this heterogeneity, we performed comprehensive genomic analyses for 142 patients with childhood-onset mitochondrial respiratory chain complex deficiencies. The approach includes whole mtDNA and exome analyses using high-throughput sequencing, and chromosomal aberration analyses using high-density oligonucleotide arrays. We identified 37 novel mutations in known mitochondrial disease genes and 3 mitochondria-related genes (MRPS23, QRSL1, and PNPLA4) as novel causative genes. We also identified 2 genes known to cause monogenic diseases (MECP2 and TNNI3) and 3 chromosomal aberrations (6q24.3-q25.1, 17p12, and 22q11.21) as causes in this cohort. Our approaches enhance the ability to identify pathogenic gene mutations in patients with biochemically defined mitochondrial respiratory chain complex deficiencies in clinical settings. They also underscore clinical and genetic heterogeneity and will improve patient care of this complex disorder.

Fig 1. Schematic of comprehensive genomic analysis of 142 patients.

All 142 patients were subjected to mtDNA amplicon-based sequencing, WES, and high-density oligonucleotide array analysis in parallel. Variants were filtered on the basis of their rarity in public databases and population-matched datasets. For each analysis, candidate variants were prioritized on the basis of the type of variant. Candidate variants were validated by Sanger sequencing and tested for segregation within the family if DNA was available. mtDNA, mitochondrial DNA; pVUS, prioritized variant of unknown significance; CNV, copy number variation

Fig 2. Breakdown of 142 patients categorized by the type of mutations/variants in comprehensive genomic analysis.

Numerals in each colored box indicate the number of patients. Patients harboring multiple variants were assigned to each box on the basis of the highest priority variant in all cases.

Fig 3. Newly identified causative genes via whole-exome sequencing analysis.

(A) Family pedigrees of Pt276. (B and C) Complementation assay in Pt276 fibroblasts and normal control (fHDF). Mitochondrial fractions were isolated from fibroblasts with established stable expression of MRPS23 using a lentiviral expression system. Assembly levels and 12s rRNA stability were compared between control and MRPS23 expressing fibroblasts by BN-PAGE /Western blotting (B) and qRT-PCR (C). fHDF: normal fetal human dermal fibroblast, RFP: mito-TurboRFP-V5, MRPS23: MRPS23-V5. Significance was calculated in comparison with controls using Student's t-test (*; p < 0.01) (D and E) Family pedigrees of Pt250 (D) and Pt860 (E). (F) Time-course analysis of in vitro amidotransferase activity of wild-type hGatCAB and mutated hGatCAB (hGatA p.G133V and p.G117A). In vitro amidotransferase assay was performed following the protocol provided in Methods. [¹⁴C]-labeled Gln and Glu deacylated from aa-tRNAs were analyzed by TLC. Positions of Gln and Glu on TLC were confirmed by [¹⁴C]Gln and [¹⁴C]Glu. Results of time-dependent amidotransferase activity are presented graphically in the lower panel.

Fig 4. The effect of PNPLA4 variant on mitochondrial function.

(A and B) Family pedigrees of Pt712. A hemizygous variant c.298C>T (p.R100X) in PNPLA4 (NM_001172672) was found in Pt712. The healthy elder daughter and elder brother harbored wild-type PNPLA4. The c.298C>T variant was inherited from the mother by Pt712. (C) Immunofluorescence microscopy confirmed the colocalization of PNPLA4 (green) with MitoTracker Orange CMTMRos (red). Pt712 cells showed a decrease in the endogenous PNPLA4 protein level. The lentiviral-mediated exogenous expression of PNPLA4-V5 also confirmed mitochondrial localization of PNPLA4. Scale bar, 100 μm. (D and E) PNPLA4 mRNA and protein expression levels were determined by quantitative real-time PCR and SDS-PAGE/Western blotting. PNPLA4 mRNA (D) and protein (E) expression levels were apparently decreased in total cell lysates of Pt712 fibroblasts. β-actin was used as a loading control. Significance relative to controls was calculated using Student’s t-test (*; p < 0.01). (F and G) Complementation assay using Pt712 fibroblasts. (F) mito-TurboRFP-V5 and PNPLA-V5 proteins in the mitochondria of patient cells with stable expression of mito-TurboRFP-V5 or PNPLA4-V5 cDNA were detected by SDS-PAGE/Western blotting. HSP60 was used as a loading control. (G) BN-PAGE/ Western blotting under high (4.5 g/l) and low (1.0 g/l) glucose medium conditions (abbreviated to HG and LG). In low glucose medium conditions, BN-PAGE/ Western blotting analysis revealed complex IV deficiency in patient fibroblasts. Complementation with PNPLA4 restored the complex IV assembly level in patient fibroblasts. RFP: mito-TurboRFP-V5, PNPLA4: PNPLA4-V5.

Fig 5. Chromosomal microdeletions contribute to mitochondrial respiratory chain complex deficiencies.

(A) The 17p12 deletion was detected by high-density oligonucleotide arrays. This heterozygous deletion of 17p12 involved the last 2 exons of COX10. (B and C) Family pedigrees of Pt657 and Pt369. In Pt369, the 17p12 deletion was paternally inherited (C). Eu means uninformative DNA test. (D) Analysis of endogenous COX10 mRNA expression relative to that in normal cells (fHDF and NHDF; Normal neonatal human dermal fibroblast); Pt223 harbored compound heterozygous non-synonymous mutations in COX10. The COX10 expression in Pt369 and Pt657 was decreased by 50%. (E–G) Wild type COX10 cDNA rescue of complex IV assembly and activity in Pt657 fibroblasts. Mitochondria were isolated from control or Pt657 fibroblasts, and mitochondrial respiratory complex assembly was analyzed by BN-PAGE and Western blotting. Wild-type COX10-V5 cDNA expression rescued complex IV assembly in Pt657 fibroblasts (E). Mitochondrial TurboRFP-V5 (RFP) and COX10-V5 (COX10) were detected in control and Pt657 fibroblasts (F). Mitochondrial respiratory chain complex activities were measured twice. Compared with TurboRFP-V5 expressing Pt657 fibroblasts, expression of wild-type COX10-V5 in Pt657 fibroblasts resulted in a significant increase in complex IV activity (G).