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

Abstract

Importance

Mitochondrial disorders have emerged as a common cause of inherited disease, but their diagnosis remains challenging. Patients with 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 intra-mitochondrial protein synthesis, with many not yet linked to human disease.

Objective

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

Design

We studied 53 patients referred to 2 national centers in the United Kingdom and Germany between 2005-2012. All had biochemical evidence of multiple respiratory chain complex defects, and no primary pathogenic mitochondrial DNA mutation. Whole exome sequencing was performed using 62Mb 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.

Participants

53 patients with biochemical evidence of multiple respiratory chain complex defects but no primary pathogenic mitochondrial DNA mutation.

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 account 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, “classical” features were absent 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 will be required in independent patient populations to determine the utility of this approach in comparison to traditional diagnostic methods.

Introduction

Defects of the mitochondrial respiratory chain have emerged as the most common cause of childhood and adult neurometabolic disease, with an estimated prevalence of 1 in 5000 live births.¹ Clinically they can present at any time of life, are often seen in association with neurological impairment, and cause chronic disability and premature death.² Major advances in understanding the molecular basis of mitochondrial disease have been mirrored by a complex, expanding phenotypic spectrum. Although some genetic defects appear to be seen in association with specific clinical features, this is not usually the case, and a systematic multidisciplinary approach is required to make a diagnosis.³ Biochemical and molecular genetic investigations are time consuming, expensive, and highly specialized, often involving a biopsy of an affected tissue or organ. With a growing list of mitochondrial diseases caused by different nuclear gene defects,⁴ achieving a comprehensive molecular diagnosis is now more labor intensive than ever. This can compromise clinical management through protracted and often repeated investigations, impeding reliable genetic counseling and prenatal diagnosis.

Approximately one third of patients with mitochondrial disease have a biochemical defect involving multiple respiratory chain complexes suggesting a defect of intra-mitochondrial protein synthesis⁵. With only a minority having a primary defect involving mitochondrial DNA (mtDNA), the remainder presents a particular challenge. The molecular mechanism potentially involves many different gene products affecting mtDNA replication and expression, including ribosomal structural and assembly proteins, aminoacyl-tRNA synthetases, tRNA modifying and methylating enzymes, and several initiation, elongation and termination factors of mitochondrial translation.⁶ Recent studies have shown that apparently unique genetic defects are common in this group, often involving proteins not previously thought to influence mitochondrial function, nor with clear mitochondrial localization. The objective of this study was to determine whether whole exome sequencing approach could be used to define the molecular basis in these patients.

Methods

Patients

Patients suspected of having mitochondrial disease referred to two nationally-accredited diagnostic laboratories (Newcastle upon Tyne Highly Specialised Service Mitochondrial Diagnostic Laboratory, UK and the Medical Genetics Centre, Munich, Germany) between 2005 and 2012 and meeting the inclusion criteria were included in this study. The inclusion criteria were: (1) histochemical and/or biochemical diagnosis of mitochondrial disease in a clinically-affected tissue (skeletal muscle, liver or heart), confirming decreased activities of multiple respiratory chain complexes based on published criteria (Table 1);⁷ (2) The absence of large-scale mtDNA rearrangements, mtDNA depletion and mtDNA point mutations,⁸ with the exception of patients P20, P21, P25, P43 and P45, where decreased levels of mtDNA were confirmed in muscle (mtDNA depletion); (3) The exclusion of major nuclear gene rearrangements by comparative genomic hybridisation arrays in patients with congenital structural abnormalities. Standardized clinical assessments were performed by the study authors. Clinical phenotypes were defined using local reference ranges for cardiomyopathy on echocardiography, abnormal renal and liver function tests, severe lactic acidosis as a blood level >5mM/L, and clinical neurophysiology for peripheral neuropathy. Informed consent was obtained from all participants in accordance with protocols approved by local institutions and research ethics committees.

Table 1. Clinical and molecular genetic characteristics of 53 patients with multiple respiratory chain complex defects.

Patients were categorized into four groups based on the molecular genetic results defined a priori (see Methods). The complete data set is show in eTable 1, including the results of biochemical analyses. Abbreviations: M: male, F: female; *: included in reference,⁵ C: consanguinity, N: no family history, 1S or 2S: number of other affected sibs, Clinical Presentation, Mu: muscle, C: central nervous system, H: heart, L: liver, Hom: homozygous. + = present. - = absent. Current age = if alive, age at last follow up.

A) Presumptive
Patient Information				Clinical Presentation				Genetic Analysis
ID/Sex	Country of Origin	Family History	Onset/Death(†) or current age	Mu	C	H	L	Gene	Variant
1/M	British Pakistani	C	6m/4y	+	-	-	-	RMND1	Hom. c.1349G>C: p.*450Serext*32
2/M*	British Pakistani	C	3m/1y†	+	+	-	-	RMND1	Hom. c.1349G>C: p.*450Serext*32
3/F	British Pakistani	C	18m/5y	+	+	+	-	RMND1	Hom. c.1349G>C: p.*450Serext*32
4/F*	Turkish	C	6m/10y†	+	-	+	-	RMND1	Hom. c.1349G>C: p.*450Serext*32
5/F	British Pakistani	C	<1m/18m	+	-	-	-	RMND1	Hom. c.1349G>C: p.*450Serext*32
6/M	British	N	18m/5y†	+	-	-	-	RMND1	c.713A>G:p.Asn238Ser c.829_830+2delGAGT:p.Glu277Glyfs*2
7/M*	British	N	birth/6w†	+	+	+	-	AARS2	c.1774C>T: p.Arg592Trp c.2882C>T: p.Ala961Val
8/M	German	N	birth/1m†	+	+	+	-	AARS2	c.1616A>G: p.Tyr539Cys c.1774C>T: p.Arg592Trp
9/F*	German	C	3w/2m†	+	-	+	-	AARS2	Hom. c.1774C>T: p.Arg592Trp
10/F	British	N	birth/3m†	-	-	+	-	AARS2	c.647_648insG: p.Cys218Leufs*6 c.1774C>T: p.Arg592Trp
11/F	British	N	6m/11m†	-	-	+	-	AARS2	Hom. c.1774C>T: p.Arg592Trp
12/F*	Croatian	1S	birth/1m	+	+	+	-	MTO1	c.631_631delG: p.Gly211Aspfs*3 c.1282G>A: p.Ala428Thr
13/M*	British Pakistani	C	birth/1y†	+	-	+	-	MTO1	Hom. c.1232C>T: p.Thr411Ile
14/M*	British Pakistani	C	1y/3y†	+	+	+	+	MTO1	Hom. c.1232C>T: p.Thr411Ile
15/M	British	N	<1y/2y	+	-	-	-	MTO1	c.122T>G: p.Val41Gly c.767A>G: p.His256Arg
16/M*	Turkish	N	birth/3m†	+	-	-	+	EARS2	Hom. c.193A>G: p.Lys65Glu
17/M*	British	N	2m/6m†	+	+	-	+	EARS2	c.322C>T: p.Arg108Trp c.814G>A: p.Ala272Thr
18/F*	German	1S	3y/16y	+	-	-	-	MTFMT	c.452C>T: p.Pro151Leu c.994C>T: p.Arg332*
19/F	British	N	birth/20y	+	+	+	-	MTFMT	c.626C>T: p.Ser209Leu c.1100_1101delTT: p.Phe367Serfs*22
20/M	British	N	2y/2.5y†	-	-	+	-	MGME1	c.532C>T: p.Arg178Trp c.794C>T: p.Thr265Ile
21/F	Irish	1S	2.5y/13	+	+	-	-	C12orf65	Hom. c.96_99dupATCC: p.Pro34Ilefs*25
22/M*	Lebanese	C	14y/37y†	+	-	-	-	YARS2	Hom. c.137G>A: p.Gly46Asp
23/F	Turkish	C	4y/17y	+	+	-	-	PUS1	Hom. c. 426C>A: p.Cys142*
24/M	British Pakistani	C	birth/1m†	+	+	+	-	TRMU	Hom. c.287A>G: p.Asn96Ser
25/F	British	C	birth/<1m†	+	+	-	-	TK2	Hom. c.1A>G: p.Met1Val
26/F	Polish	N	7m/18m†	+	+	-	-	SCO2	Hom. c.418G>A: p.Glu140Lys
27/F*	German	N	birth/3w†	+	-	+	-	ELAC2	c.1478C>T: p.Pro493Leu c.1621G>A: p.Ala541Thr
28/M*	Turkish	1S	4y/7y	+	+	-	-	ETHE1	Hom. c.3G>T: p.Met1Ile

B) Possible
Patient Information				Clinical Presentation				Genetic Analysis
ID/Sex	Country of Origin	Family History	Onset/Death(†) or current age	Mu	C	H	L	Gene	Variant
29/M	British	N	<1/10y	+	+	-	-	VARS2	c.1135G>A: p.Ala379Thr c.1877C>A: p.Ala626Asp
30/M*	Turkish	C	4m/8m†	+	-	-	-	FLAD1	Hom. c.397_400 delTTCT: p.Phe134Cysfs*8
31/F*	Turkish	C 2S	birth/1m†	+	-	+	-	GARS	Hom. c.2065C>T: p.Arg689Cys
32/F*	British	N	4m/8m†	-	-	+	-	PTCD1	c.337C>T: p.Arg113Trp c.388C>T: p.Arg130* c.550G>A: p.Gly184Arg

C) Variants of unknown significance
Patient Information				Clinical Presentation				Genetic Analysis
ID/Sex	Country of Origin	Family History	Onset/Death(†)or Current age	Mu	C	H	L	Gene	Variant
33/F*	Turkish	C	2y/6y	+	+	-	-	SLC25A12 METAP1D	Hom. c.1333G>A: p.Ala445Thr Hom. c.497+2T>A
34/M	British	N	17y/20y	-	+	-	-	ACSM5	c.1157A>C: p.Lys386Thr c.1273C>A: p.Pro425Thr
35/F	Georgian	C	7y/10y	+	+	-	-	PERP MEF2A ACSM5	Hom. c.206T>C: p.Met69Thr c.1262A>C: p.Gln421Pro c.1265A>C: p.Gln422Pro c.68A>G: p.His23Arg c.73A>C:p.Lys25Gln
36/M	German	C	10y/14y	+	-	-	-	HKDC1 ETFA IREB2 SMCR7	Hom. c.1276C>T: p.Arg426Cys Hom. c.20C>T: p.Pro7Leu Hom. c.2393C>T: p.Thr798Ile Hom. c.241C>T: p.Gln81*
37/F	British	N	<1m/5m	+	+	-	-	PC	c.1876C>T: p.Arg626Trp c.1892G>A: p.Arg631Gln
38/M	Turkish	C	4y/6y	+	+	-	+	TPO	Hom. c.443C>T: p.Ala148Val
39/M	British	N	<1y/5y	+	+	-	-	HERC2	c.6448C>G: p.Leu2150Val c.9979G>A: p.Val3327Met
40/M*	Turkish	C	2w/3w†	+	-	-	-	MAGI1 NDRG3 TPX2 TAF9	Hom. c.2290A>C: p.Thr764Pro Hom. c.469G>A: p.Gly157Ser Hom. c.505C>T: p.Pro169Ser Hom. c.406G>C: p.Glu136Gln
41/M*	Croatian	N	birth/9m†	+	+	+	-	SLC25A43 FAAH2	c.493C>T: p.Arg165* (X-linked) c.368T>C: p.Phe123Ser (X-linked)
42/M	Hungarian	N	6y/14y	+	-	-	-	DLAT SDHD POLRMT ARHGEF5	c.55G>C: p.Glu19Gln c.626A>G: p.Gln209Arg c.34G>A: p.Gly12Ser c.386T>C: p.Leu129Ser c.112C>T:p.Pro38Ser c.232G>A:p.Val78Met c.1738G>T:p.Gly580Cys c.4066A>G:p.Asn1356Asp
43/F	British	N	birth/<1m†	+	-	-	-	TYMP ACSM2A LRPPRC HTRA2 ALDH1L1 BCKDHB SLC25A4	c.242G>A: p.Arg81Gln c.1003G>A: p.Val335Ile c.4132A>G: p.Ser1378Gly c.1210C>T: p.Arg404Trp c.2143G>C:p.Glu715Gln c.23C>T:p.Ala8Val c.239G>A:p.Arg80His
44/M*	Turkish	1S	18m/2y	+	-	-	+	PPL SLC5A10	c.263A>G: p.Asp88Gly c.1003C>A: p.Leu335Met c.674A>G: p.Glu225Gly c.1799T>C: p.Leu600Pro
45/F	German	Healthy Dizygotic twin	1m/6m†	+	+	-	+	FASN FNDC1	c.1850C>T: p.Pro617Leu c.2657T>C: p.Phe886Ser c.4429A>G: p.Thr1477Ala c.4547C>A: p.Thr1516Asn
46/M	German	N	6y/21y	+	-	-	-	PDPR LONP1	c.616A>G: p.Ile206Val c.1774A>G: p.Thr592Ala c.79G>C: p.Ala27Pro c.2485G>A: p.Ala829Thr
47/M	Egyptian	C	2m/2y†	+	+	-	+	MIPEP STARD13	Hom. c.671A>G: p.Asn224Ser Hom. c.1186C>T: p.His396Tyr

D) Unresolved
Patient Information				Clinical Presentation				Genetic Analysis
ID/Sex	Country of Origin	Family History	Onset/Death(†) or Current age	Mu	C	H	L
48/M	Turkish	C	4m/1y	+	+	+	+	No candidate variants detected
49/F*	German	N	1y/3y	+	+	-	-
50/F*	British Pakistani	C	birth/4d†	-	-	+	-
51/F	British Pakistani	C	adult-onset	+	-	-	-
52/M	German	N	2y/5y†	+	+	-	+
53/F	German	N	birth/4y	+	+	-	-

Molecular genetics and bioinformatics

Exome sequencing, bioinformatic analysis, variant confirmation, and segregation analysis were performed in Newcastle upon Tyne. Genomic DNA was isolated from primary cell lines, muscle or circulating lymphocytes (DNeasy^®, Qiagen, Valencia, CA), fragmented and enriched by Illumina TruSeq™ 62 Mb exome capture, and sequenced (Illumina HiSeq 2000, 100 bp paired-end reads). The in-house bioinformatics pipeline involved the following steps: alignment to the human reference genome (UCSC hg19),⁹ removal of duplicate sequence reads (Picard v1.85 http://picard.sourceforge.net) and variant detection (Varscan v2.2¹⁰, Dindel v1.01¹¹). On-target variant filtering excluded those with minor allele frequency greater >0.01 in several databases: dbSNP135, 1000 genomes (February 2012 data release), the National Heart, Lung and Blood Institute (NHLBI, NIH, Bethesda, MD) Exome Sequencing Project (ESP) 6500 exomes, and 238 unrelated in-house controls. We used published and experimentally-validated bioinformatic tools to predict mitochondrial localization and probably impact on mitochondrial function.^12,13 Rare homozygous and compound heterozygous variants were defined, and protein altering and/or putative ‘disease causing’ mutations, along with their functional annotation, were identified using ANNOVAR.¹⁴ Candidate genes were filtered against a list of bioinformatically predicted mitochondrial proteins,^12,13 as well as genes which matched a Gene-Ontology (GO)-term of ‘mitoch’ and prioritized if previously seen in association with a disease phenotype (see eTable 3 for detailed methods). Putative pathogenic variants were confirmed by Sanger sequencing using custom-designed primers (http://frodo.wi.mit.edu) on an ABI 3130XL (BigDye, Applied Biosystems, Carlsbad, CA) and compared to transcripts available at the Nucleotide database at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/nuccore) allowing segregation analyses where possible.

Variants were classified into four groups, defined a priori: (1) Presumptive pathogenic - homozygous or compound heterozygous mutations in genes previously shown to cause multiple respiratory chain complex deficiencies; (2) Possible pathogenic - homozygous or compound heterozygous mutations in novel genes predicted to cause a mitochondrial translation defect based on their proposed function and similarity to known disease genes; (3) Variants of unknown significance - homozygous or compound heterozygous mutations in novel or known disease genes not known to be associated with mitochondrial pathology; (4) Unresolved - patients where a single plausible genetic cause could not be identified.

Binomial confidence intervals were calculated using the Clopper-Pearson method.

Results

Clinical presentation

The clinical presentation and laboratory findings of the 53 unrelated patients are summarized in Table 1 and eTable 1. The majority (51/53, 96%, 95% CI=87-99) of the patients presented in childhood (<15 years old) and most (35/53, 66%, 95% CI=52-78) developed symptoms within the first year of life. Parental consanguinity was apparent in 24 cases, and 3 cases had an additional affected sibling. The most frequent clinical feature was muscle weakness with hypotonia (47/53, 89%, 95% CI=77-96), followed by clinical or imaging features of central neurological disease (28/53, 53%, 95% CI=39-67), cardiomyopathy (19/53, 36%, 95% CI=23-50) or abnormal liver function (9/53, 17%, 95% CI=8-30), and a combination of these symptoms were present in most cases (34/53, 64%, 95% CI=50-77). Severe lactic acidosis was observed in half of the patients (27/53, 51%, 95% CI=37-65). The presence of deafness (8/53, 15%, 95% CI=7-28), ptosis or progressive external ophthalmoplegia (PEO, 4/53, 8%, 95% CI=2-18), renal impairment (5/53, 9%, 95% CI=3-21), axonal neuropathy (3/53, 6%, 95% CI=1-16), sideroblastic anaemia (2/53, 4%, 95% CI=0-13), immune deficiency (1/53, 2%, 95% CI=0-10) and optic atrophy (2/53, 4%, 95% CI=0-13) were also noted.

DNA analysis, whole exome sequencing

The mean per base depth of coverage for the exome consensus coding sequence was 79-fold, with 88.6% of bases covered >20-fold. Coverage and depth statistics for each patient are shown in eTable 2. The results of the bioinformatic analysis are shown in eTable 3, which also includes the allele frequency data. Confirmed variants are shown in Table 1. Presumptive pathogenic variants were found in 28 of the patients (53%, 95% CI=39-67), and possible pathogenic variants in 4 patients (8%, 95% CI=2-18), giving a combined result of 32 out of 53 (60%, 95% CI=46-74). Variants of uncertain significance were found in 15 patients (28%, 95% CI=17-42), and six cases remained unresolved (11%, 95% CI=11-34).

(1). Presumptive pathogenic group

A single, novel homozygous c.1349G>C (p.*450Serext*32) RMND1 (NM_017909.3)^15,16 mutation introducing a premature stop codon was identified in 5 independent patients. In each patient, the phenotype was severe, affecting different organs but including myopathy, profound deafness and renal involvement. The 5 homozygotes were from consanguineous families of Pakistani origin. A founder effect was supported by the presence of a homogeneous haplotype flanking the mutation (Figure 1a). One other patient had different compound heterozygous mutations in RMND1. Mutations in AARS2 (NM_020745.3)¹⁷ were the second most frequently identified defect (5 patients). All presented with severe infantile cardiomyopathy, with additional muscle (3 patients) and central neurological features (2 patients) in a subgroup. Interestingly, despite being from different European ethnic backgrounds, all carried the previously reported c.1774C>T (p.Arg592Trp) mutation on at least one allele (Figure 1b).¹⁷ Mutations in MTO1 (NM_012123.3)¹⁸ were identified in 4 patients. All had muscle weakness on presentation with lactic acidosis, two had central neurological features, and unlike a previous report,¹⁸ one did not have a cardiomyopathy; two patients were homozygous for a p.Thr411Ile MTO1 mutation recently shown to cause a severe respiratory phenotype in a yeast model¹⁹ (Figure 1c). Homozygous or compound heterozygous mutations were detected in previously characterised mitochondrial translation genes, including 2 patients with EARS2 (NM_001083614.1)²⁰ mutations (one having a leukoencephalopathy and no corpus callosum),²¹ 2 patients with MTFMT (NM_139242.3) mutations,²² and one case with C12orf65 (NM_152269) mutations.²³ Single patients with a clinical presentation resembling previously described cases carried homozygous or compound heterozygous mutations in the YARS2 (NM_001040436.2),²⁴, PUS1 (NM_025215.5),²⁵ MGME1 (NM_052865.2),²⁶ ETHE1 (NM_014297.3),²⁷ ELAC2 (NM_018127.6),²⁸ and TK2 (NM_004614.3)²⁹ the latter case seen in association with severe loss of mtDNA copy number due to mutation (c.1A>G, p.Met1Val) of the initiating methionine codon. Atypical presentations included a patient with a homozygous TRMU (NM_018006.4)³⁰ mutation seen in association with heart, CNS and muscle involvement, but no liver involvement, and only a subclinical, mild anaemia in patient 23 carrying a homozygous nonsense mutation in PUS1. In addition, patient 26 had typical features of Leigh syndrome and multiple respiratory chain complex defects at the time of biopsy, but was homozygous for the p.Glu140Lys SCO2 (NM_001169111.1)³¹ founder mutation which is usually seen in association with an isolated complex IV defect and cardiomyopathy, features not present in this patient.

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.

(2). Possible pathogenic

Probable disease-causing variants were identified in novel mitochondrial disease genes in 4 patients, each predicted to affect mitochondrial protein synthesis. VARS2 (NM_001167734.1) and GARS (NM_002047.2) encode mitochondrial aminoacyl-tRNA synthetase genes. FLAD1 (NM_025207.4) encodes a key factor of the riboflavin metabolism, and PTCD1 (NM_015545.3) is a gene encoding a mitochondrially-targeted pentatricopeptide reported to be involved in mitochondrial RNA metabolism.³² In silico predictions supported a pathogenic role in each case, but given that they were only identified in single patients, further evidence is required before these variants can be considered definitively pathogenic; where familial samples were available, identified mutations were shown to segregate with disease (Table 1).

(3). Variants of unknown significance

In 15 patients we identified one or more variants in genes predicted to encode mitochondrial proteins (Table 1), where there were several plausible candidate disease genes. Most of the gene defects were detected in single patients only (except for ACSM5 (NM_017888.2) mutations in patients 34 and 36). The current lack of functional data directly linking these genes to multiple respiratory chain complex defects led to their classification as possible and not probable causative variants (Figure 2). Identification of further patients with mutations in these genes and/or functional work is required to validate these findings.

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.

(4). Unresolved

Exome sequencing did not identify any candidate pathogenic variants in 6 patients.

Discussion

In the pre-exome era, the systematic biochemical characterisation of 53 patients with multiple respiratory chain complex defects led to the underlying genetic basis in only one patient.⁵ The work presented here demonstrates the effect of whole exome sequencing in this context, which has defined the genetic aetiology in 32 of 53 patients (60%, including 28 presumptive and 4 probable causative mutations) with a confirmed biochemical defect consistent with a generalised decrease in mitochondrial translation. The detection rate was even higher in children with onset < 1 year of age (24/35, 69%). In 20 patients with prominent cardiac disease we detected the causative mutation in 15 (80%), while the detection rate was much lower in patients with liver disease (3/9, 33%). Our findings contrast with large-scale candidate gene analysis using conventional,³³ and next-generation sequencing approaches,³⁴ both of which had a lower diagnostic yield (10-13%) and by definition did not discover new potential disease genes. A more ambitious approach involving exon capture and sequencing of all predicted mitochondrial genes (the “Mitoexome”) has delivered a greater diagnostic yield (22-28%)^12,35, although in cohorts with predominantly isolated respiratory chain complex defects which are less challenging to define at the genetic level.³⁶

There are a number of reasons why our approach had a >2-fold greater diagnostic yield than previous studies, despite the known difficulty in making a molecular diagnosis in patients with multiple respiratory chain complex deficiencies. First, not being based on any prior assumptions about known candidate disease genes, the whole exome approach detected four new genes which may to be responsible for the underlying mitochondrial disorder. Further arguments supporting the pathogenic role of mutations in these new genes come from similarities in function to known mitochondrial genes. For example VARS2 and GARS encode tRNA synthase genes with products known to enter mitochondria. Recessive mutations in other mitochondrial tRNA synthetase genes responsible for charging tRNAs with different specific cognate amino acids during protein synthesis have been shown to cause identical biochemical defects and similar clinical phenotypes.^17,20,24 Compound heterozygous mutations in GARS have recently been reported in a single family with a multi-systemic mitochondrial disease with cardiomyopathy.³⁷ These findings endorse our approach, and support the pathogenic role of the GARS mutations in our patient who developed a fatal cardiomyopathy, but the lack of functional data mean that mutations in this gene should remain in the “possible pathogenic” group (Table 1). Like GARS, FLAD1 was not originally considered to encode a mitochondrial protein, but ultimately were found to encode alternatively spliced cytoplasmic and mitochondrial transcripts, and thus are plausible candidate genes.³⁸ Although we have not shown proof of the causal link, in silico predictions of the deleterious effect of the mutations in genes showing strong functional similarities with known disease genes, supports a causal association with the mutations. Secondly, the unbiased exome approach has the potential to reveal unexpected results. This was the case for three additional unreported patients who also met the selection criteria for this study. One patient presenting with neonatal myopathy, encephalopathy and lactic acidosis, with a complex I defect and a less prominent complex IV defect, and was found to have novel homozygous mutations in the complex I subunit gene NDUFS6 (c.317_320delAAAC:p.Glu106fs*21). In this context, the complex IV defect was presumably secondary, perhaps mediated through the disruption of respiratory chain super-complexes. Similarly, one child carrying the homozygous common SCO2 mutation also presented with severe combined complex I and IV defects. These results also illustrate the difficulties in interpreting respiratory chain enzyme analysis, which even in skilled hands, can misdirect a candidate gene approach. The higher diagnostic yield could, in part, reflect the growing inventory of genes known to cause mitochondrial diseases, and the relatively high proportion of familial and consanguineous cases in our study cohort.

Our findings implicate 18 different genes and further 33 possible candidates in 53 patients, underscoring the genetic heterogeneity of this group of mitochondrial disorders (Figure 2). Given this complexity, how should we approach the diagnosis in a clinical setting? Despite the relatively small number of individuals with any single mutation, our observations show the phenotypic diversity in patients with multiple respiratory chain complex defects, emerging clinical subgroups do appear to be seen in association with specific genetic defects. For example, AARS2 and MTO1 mutations were preferentially seen in association with cardiomyopathy, mutations in TRMU present with liver failure that can improve spontaneously, YARS2 and PUS1 with sideroblastic anemia and myopathy, and RMND1 with deafness, myopathy, renal involvement and a severe biochemical defect (Table 1). However, not all patients fitted neatly into these subgroups – including a patient with a TRMU mutation and normal liver function, one with a PUS1 mutation and only subclinical anaemia, and a SCO2 mutation with no cardiomyopathy. This heterogeneity is typical for mitochondrial diseases, and supports the use of next-generation sequencing early in the diagnostic approach. Although a molecular diagnosis is unlikely to lead to specific drug treatments at present, defining the genetic etiology may enable accurate genetic counselling and prenatal diagnosis, and personalized disease surveillance for genotype-specific complications.

Given that our case ascertainment was determined by clinical referral and not influenced or biased by the study team, our findings may have broader relevance. However, our study has several limitations. First, we could not identify a potential candidate mutation in only 6 patients (11%), possibly because the causative mutations lay in deep intronic regions, or may involve deletions or duplications (copy number variations), missed by the exome capture and bioinformatic analysis. Second, our work was carried out on carefully phenotyped patients defined by a biochemical defect measured in two specialist centers, and a high proportion of patients were from consanguineous families. It will be important to replicate our findings in similar patient cohorts investigated in other centers from other parts of the world, where the spectrum of nuclear gene defects may be different. The 53 patients we studied account for ~20% of the referrals to our centres with any form of biochemical defect of the respiratory chain determined by a spectrophotometric assay of enzyme activity, and the patients with multiple respiratory chain complex defects account for ~60% of patients with a biochemical defect with no known molecular diagnosis. Therefore, the role of exome sequencing in unselected patients with a clinical diagnosis of suspected multiple respiratory chain complex defects remains to be determined, and the effect of exome sequencing in patients with a general diagnosis of suspected mitochondrial disease is not clear. However, applying a whole-exome approach to a group of patients with multiple respiratory chain complex defects that are difficult to diagnose has delivered a high diagnostic yield. Curiously, most of the genes appear to be involved in intra-mitochondrial gene translation. This explains the phenotypic and biochemical overlap, but it is not at all clear why apparently subtle differences in function should lead to discrete clinical phenotypes. These discrete phenotypes may point to a few candidate genes, but in our large cohort, this only became obvious after performing this study. It is possible that next-generation sequencing will revolutionise the investigation of mitochondrial diseases, and its early application may provide a rapid diagnosis at a relatively low cost, particularly in patients with multiple respiratory chain complex defects.

Conclusions

Exome sequencing enhances the ability to identify the underlying nuclear gene mutations in patients with multiple mitochondrial respiratory chain complex defects. Additional study will be required to determine the utility of this approach compared to traditional diagnostic methods in independent patient populations.