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Original Investigation |

Use of Whole-Exome Sequencing to Determine the Genetic Basis of Multiple Mitochondrial Respiratory Chain Complex Deficiencies

Robert W. Taylor, PhD, FRCPath1; Angela Pyle, PhD2; Helen Griffin, PhD2; Emma L. Blakely, PhD1; Jennifer Duff, PhD2; Langping He, PhD1; Tania Smertenko, BSc2; Charlotte L. Alston, BSc1; Vivienne C. Neeve, PhD2; Andrew Best, PhD2; John W. Yarham, PhD1; Janbernd Kirschner, MD3; Ulrike Schara, MD4; Beril Talim, MD5; Haluk Topaloglu, MD5; Ivo Baric, MD6; Elke Holinski-Feder, MD7; Angela Abicht, MD7; Birgit Czermin, MD7; Stephanie Kleinle, MD7; Andrew A. M. Morris, PhD, FRCPCH8; Grace Vassallo, FRCPCH9; Grainne S. Gorman, MD, FRCPI1; Venkateswaran Ramesh, MD, FRCPCH10; Douglass M. Turnbull, PhD, FRCP, FMedSci1; Mauro Santibanez-Koref, PhD2; Robert McFarland, PhD, FRCPCH1,10; Rita Horvath, MD, PhD2; Patrick F. Chinnery, PhD, FRCP, FMedSci2
[+] Author Affiliations
1Wellcome Trust Centre for Mitochondrial Research, Institute for Ageing and Health, The Medical School, Newcastle University, Newcastle upon Tyne, England
2Wellcome Trust Centre for Mitochondrial Research, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, England
3Division of Neuropediatrics and Muscle Disorders, University Medical Center Freiburg, Freiburg, Germany
4Department of Neuropediatrics, University of Essen, Essen, Germany
5Department of Pediatrics, Hacettepe University, Ankara, Turkey
6Department of Paediatrics, University Hospital Center Zagreb, School of Medicine, University of Zagreb, Zagreb, Croatia
7Medical Genetics Center, Munich, Germany
8Willink Biochemical Genetics Unit, Manchester Centre for Genomic Medicine, Central Manchester University Hospitals NHS Foundation Trust, Manchester, England
9Department of Paediatric Neurology, Central Manchester University Hospitals NHS Foundation Trust, Manchester, England
10Department of Paediatric Neurology, Newcastle upon Tyne Hospitals NHS Trust, Newcastle upon Tyne, England
JAMA. 2014;312(1):68-77. doi:10.1001/jama.2014.7184.
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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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Figure 1.
Molecular Haplotypes Flanking RMND1, AARS2, and MTO1 in Selected Study Patients

Haplotype blocks were generated from selected markers using exomes from 62 in-house controls and from the patients found to harbor mutations in RMND1, AARS2, and MTO1. Population frequencies are shown next to each haplotype; thicker connecting lines show more common crossings than thinner lines. Multilocus D′, a measure of the linkage disequilibrium between 2 blocks, is shown. The closer the value is to 0, the greater the amount of historical recombination. SNV indicates single nucleotide variant. A, Molecular haplotypes flanking RMND1 in patients 1-5. In addition to the main haplotype that includes the RMND1 mutation, patients 1 and 2 and patients 4 and 5 shared one of 2 different haplotypes situated 3′ to the RMND1 gene. Patients 3, 4, and 5 also shared a haplotype. The RMND1 mutation, c.1349G>C (p.*450Serext*32), is indicated between Haploview markers 335 and 338. The mutation is not included in the Haploview analysis because all patients were homozygous for the mutation. B, Molecular haplotype flanking AARS2 in patients 7-11. A shared haplotype spanning exons 10-22, including the c.1774C>T (p.Arg592Trp) mutation at Haploview marker 566, was identified. Six additional haplotype blocks appeared to be shared between p.Arg592Trp AARS2 mutation carriers; however, for the carriers of the discrete heterozygous AARS2 mutations (patients 7, 8, and 10), it was not possible to resolve the phase of these blocks. Alternative haplotype blocks in which the mutation was heterozygous were identified in patients 7, 8, and 10. C, Molecular haplotype flanking MTO1 in patients 13 and 14 show the shared haplotype defining a founder allele. The homozygous MTO1 mutation, c.1232C>T (p.Thr411Ile), is located between Haploview markers 382 and 392.

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Figure 2.
Nuclear Genes Associated With Multiple Mitochondrial Respiratory Chain Complex Defects

Genes present within the cell nucleus encode proteins critical for intramitochondrial protein synthesis. These proteins are transported through the double mitochondrial membrane into the mitochondrial matrix. Nuclear genes associated with multiple mitochondrial respiratory chain complex defects are shown at the top. nDNA indicates nuclear DNA; mtDNA, mitochondrial DNA; mt-tRNA, mitochondrial transfer RNA; mt-mRNA, mitochondrial messenger RNA; mt, mitochondrial.aNewly identified nuclear gene with possible pathogenic variants.

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