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. 2014 Jul 2;312(1):68-77.
doi: 10.1001/jama.2014.7184.

Use of whole-exome sequencing to determine the genetic basis of multiple mitochondrial respiratory chain complex deficiencies

Robert W Taylor  1 , Angela Pyle  2 , Helen Griffin  2 , Emma L Blakely  1 , Jennifer Duff  2 , Langping He  1 , Tania Smertenko  2 , Charlotte L Alston  1 , Vivienne C Neeve  2 , Andrew Best  2 , John W Yarham  1 , Janbernd Kirschner  3 , Ulrike Schara  4 , Beril Talim  5 , Haluk Topaloglu  5 , Ivo Baric  6 , Elke Holinski-Feder  7 , Angela Abicht  7 , Birgit Czermin  7 , Stephanie Kleinle  7 , Andrew A M Morris  8 , Grace Vassallo  9 , Grainne S Gorman  1 , Venkateswaran Ramesh  10 , Douglass M Turnbull  1 , Mauro Santibanez-Koref  2 , Robert McFarland  11 , Rita Horvath  2 , Patrick F Chinnery  2
Affiliations

Use of whole-exome sequencing to determine the genetic basis of multiple mitochondrial respiratory chain complex deficiencies

Robert W Taylor et al. JAMA. .

Abstract

Importance: Mitochondrial disorders have emerged as a common cause of inherited disease, but their diagnosis remains challenging. Multiple respiratory chain complex defects are particularly difficult to diagnose at the molecular level because of the massive number of nuclear genes potentially involved in intramitochondrial protein synthesis, with many not yet linked to human disease.

Objective: To determine the molecular basis of multiple respiratory chain complex deficiencies.

Design, setting, and participants: We studied 53 patients referred to 2 national centers in the United Kingdom and Germany between 2005 and 2012. All had biochemical evidence of multiple respiratory chain complex defects but no primary pathogenic mitochondrial DNA mutation. Whole-exome sequencing was performed using 62-Mb exome enrichment, followed by variant prioritization using bioinformatic prediction tools, variant validation by Sanger sequencing, and segregation of the variant with the disease phenotype in the family.

Results: Presumptive causal variants were identified in 28 patients (53%; 95% CI, 39%-67%) and possible causal variants were identified in 4 (8%; 95% CI, 2%-18%). Together these accounted for 32 patients (60% 95% CI, 46%-74%) and involved 18 different genes. These included recurrent mutations in RMND1, AARS2, and MTO1, each on a haplotype background consistent with a shared founder allele, and potential novel mutations in 4 possible mitochondrial disease genes (VARS2, GARS, FLAD1, and PTCD1). Distinguishing clinical features included deafness and renal involvement associated with RMND1 and cardiomyopathy with AARS2 and MTO1. However, atypical clinical features were present in some patients, including normal liver function and Leigh syndrome (subacute necrotizing encephalomyelopathy) seen in association with TRMU mutations and no cardiomyopathy with founder SCO2 mutations. It was not possible to confidently identify the underlying genetic basis in 21 patients (40%; 95% CI, 26%-54%).

Conclusions and relevance: Exome sequencing enhances the ability to identify potential nuclear gene mutations in patients with biochemically defined defects affecting multiple mitochondrial respiratory chain complexes. Additional study is required in independent patient populations to determine the utility of this approach in comparison with traditional diagnostic methods.

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Conflict of interest statement

Conflicts of Interest disclosures

The authors report no conflicts of interest

Figures

Figure 1
Figure 1. Molecular haplotypes flanking RMND1, AARS2 and MTO1 in the patients studied here.
The grey highlighted box in (A), (B) and (C) shows haplotype blocks generated from the selected markers using 62 in-house control exomes and the patients found to harbor mutations in RMND1, AARS2 and MTO1 in this study. Population frequencies are shown next to each haplotype and lines show the most common crossings from one block to the next. The thicker lines show more common crossings than thinner lines with multilocus D’ below, which is a measure of the linkage disequilibrium between two blocks. The closer the value is to zero, the greater the amount of historical recombination between the two blocks. The large blue boxes group together the single nucleotide variants (SNV), which make up the shared RMND1, AARS2 and MTO1 haplotypes. The red and orange boxes show the alleles at each SNV that make up the shared haplotype. (A) Molecular haplotypes flanking RMND1 in P1, P2, P3, P4 & P5 (blue box). The red boxes show the alleles at each SNV that make up the shared haplotype. The light grey solid boxes represent the two different haplotypes shared between P1/P2, and P4/P5, which are situated 3’ to the RMND1 gene. The dark grey boxes represent the haplotype shared by P3, P4 and P5. Regions of extended homozygosity surrounding the RMND1 gene are represented by green lines. The RMND1 mutation, c.1349G>C (p.*450Serext*32) (between Haploview markers 335 and 338, shown by an asterisks *), violates Hardy-Weinberg equilibrium because there are no heterozygotes and so is not included by Haploview in the blocks. The position of the RMND1 gene is highlighted by a solid orange box in chromosome 6. (B) Molecular haplotype flanking the AARS2 in P7, P8, P8, P10 and P11 (0.3Mb, blue box) including the c.1774C>T (p.Arg592Trp) mutation. A haplotype spanning exons 10-22 is shown by orange boxes. The red boxes represent the further six haplotype blocks which appear to be shared between p.Arg592Trp AARS2 mutation carriers, however, for the carriers of the discrete heterozygous AARS2 mutations (P7, P8 and P10), it was not possible to resolve the phase of these blocks. Regions of extended homozygosity surrounding the AARS2 gene are represented by green lines in patients P9 and P11. The dashed boxes represent the alternative haplotype blocks represent the alternative haplotype blocks in the patients where the mutation was heterozygous (P7, P8 and P10). The AARS2 gene is represented in chromosome 6 by a solid purple box. (C) Molecular haplotype flanking the MTO1 gene in 2 of the 4 affected individuals (P13 and P14, blue box) showing the shared haplotype defining a founder allele. Regions of extended homozygosity surrounding the MTO1 gene are represented by green lines. The homozygous MTO1 mutation, c.1232C>T:p.Thr411Ile is located between Haploview markers 382 and 392 and is shown by a purple asterisks (*). The MTO1 gene is represented in the chromosome by a solid purple box.
Figure 2
Figure 2. Intra-mitochondrial protein synthesis. Genes present within the cell nucleus encode proteins critical for intra-mitochondrial protein translation.
These are synthesised in the cytoplasm (upper half), and transported through the double mitochondrial membrane into the mitochondrial matrix. Here they regulate (from left to right) mtDNA maintenance; mitochondrial tRNA synthases or tRNA modifications; mitochondrial ribosomal protein components or ribosomal assembly; the initiation, elongation or termination of protein translation; or the protein subunits themselves and their assembly factors. Genes known to cause defects of intra-mitochondrial protein translation are shown in capitals and italics. Genes in red are the novel predicted mitochondrial disease genes described here. mtDNA = mitochondrial DNA, mt = mitochondrial, tRNA = transfer RNA.
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