Panel-Based Nuclear and Mitochondrial Next-Generation Sequencing Outcomes of an Ethnically Diverse Pediatric Patient Cohort with Mitochondrial Disease
2019; Elsevier BV; Volume: 21; Issue: 3 Linguagem: Inglês
10.1016/j.jmoldx.2019.02.002
ISSN1943-7811
AutoresMaryke Schoonen, Izelle Smuts, Roan Louw, Joanna L. Elson, Etresia van Dyk, Lindi-Maryn Jonck, Richard J. Rodenburg, Francois H. van der Westhuizen,
Tópico(s)ATP Synthase and ATPases Research
ResumoMitochondrial disease (MD) is a group of rare inherited disorders with clinical heterogeneous phenotypes. Recent advances in next-generation sequencing (NGS) allow for rapid genetic diagnostics in patients who experience MD, resulting in significant strides in determining its etiology. This, however, has not been the case in many patient populations. We report on a molecular diagnostic study using mitochondrial DNA and targeted nuclear DNA (nDNA) NGS of an extensive cohort of predominantly sub-Saharan African pediatric patients with clinical and biochemically defined MD. Patients in this novel cohort presented mostly with muscle involvement (73%). Of the original 212 patients, a muscle respiratory chain deficiency was identified in 127 cases. Genetic analyses were conducted for these 127 cases based on biochemical deficiencies, for both mitochondrial (n = 123) and nDNA using panel-based NGS (n = 86). As a pilot investigation, whole-exome sequencing was performed in a subset of African patients (n = 8). These analyses resulted in the identification of a previously reported pathogenic mitochondrial DNA variant and seven pathogenic or likely pathogenic nDNA variants (ETFDH, SURF1, COQ6, RYR1, STAC3, ALAS2, and TRIOBP), most of which were identified via whole-exome sequencing. This study contributes to knowledge of MD etiology in an understudied, ethnically diverse population; highlights inconsistencies in genotype-phenotype correlations; and proposes future directions for diagnostic approaches in such patient populations. Mitochondrial disease (MD) is a group of rare inherited disorders with clinical heterogeneous phenotypes. Recent advances in next-generation sequencing (NGS) allow for rapid genetic diagnostics in patients who experience MD, resulting in significant strides in determining its etiology. This, however, has not been the case in many patient populations. We report on a molecular diagnostic study using mitochondrial DNA and targeted nuclear DNA (nDNA) NGS of an extensive cohort of predominantly sub-Saharan African pediatric patients with clinical and biochemically defined MD. Patients in this novel cohort presented mostly with muscle involvement (73%). Of the original 212 patients, a muscle respiratory chain deficiency was identified in 127 cases. Genetic analyses were conducted for these 127 cases based on biochemical deficiencies, for both mitochondrial (n = 123) and nDNA using panel-based NGS (n = 86). As a pilot investigation, whole-exome sequencing was performed in a subset of African patients (n = 8). These analyses resulted in the identification of a previously reported pathogenic mitochondrial DNA variant and seven pathogenic or likely pathogenic nDNA variants (ETFDH, SURF1, COQ6, RYR1, STAC3, ALAS2, and TRIOBP), most of which were identified via whole-exome sequencing. This study contributes to knowledge of MD etiology in an understudied, ethnically diverse population; highlights inconsistencies in genotype-phenotype correlations; and proposes future directions for diagnostic approaches in such patient populations. CME Accreditation Statement: This activity ("JMD 2019 CME Program in Molecular Diagnostics") has been planned and implemented in accordance with the accreditation requirements and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity ("JMD 2019 CME Program in Molecular Diagnostics") for a maximum of 18.0 AMA PRA Category 1 Credit(s)™. Physicians should claim only credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity ("JMD 2019 CME Program in Molecular Diagnostics") has been planned and implemented in accordance with the accreditation requirements and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity ("JMD 2019 CME Program in Molecular Diagnostics") for a maximum of 18.0 AMA PRA Category 1 Credit(s)™. Physicians should claim only credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Mitochondria are ubiquitous in the human body and serve mainly as the energy-producing organelle via oxidative phosphorylation (OXPHOS). This metabolic pathway comprises five protein complexes (CI to CV), consisting of a total of 92 structural subunits encoded by both mitochondrial DNA (mtDNA) and nuclear DNA genes.1Chinnery P.F. Hudson G. Mitochondrial genetics.Br Med Bull. 2013; 106: 135-159Crossref PubMed Scopus (227) Google Scholar, 2Anderson S. Bankier A.T. Barrell B.G. De Bruijn M. Coulson A.R. Drouin J. Eperon I. Nierlich D. Roe B.A. Sanger F. Sequence and organization of the human mitochondrial genome.Nature. 1981; 290: 457-465Crossref PubMed Scopus (7631) Google Scholar Underlying genetic mutations generate disruptions within this system, which manifest clinically, often affecting multiple highly energy-demanding organs simultaneously. The heterogeneous class of clinical phenotypes associated with such mutations is referred to as mitochondrial disease (MD).3Gorman G.S. Chinnery P.F. DiMauro S. Hirano M. Koga Y. McFarland R. Suomalainen A. Thorburn D.R. Zeviani M. Turnbull D.M. Mitochondrial diseases.Nat Rev Dis Primers. 2016; 2: 16080Crossref PubMed Scopus (693) Google Scholar Many genes (at least 289), extending beyond the structural OXPHOS genes, have been identified as being involved in MD.4Wortmann S.B. Mayr J.A. Nuoffer J.M. Prokisch H. Sperl W. A guideline for the diagnosis of pediatric mitochondrial disease: the value of muscle and skin biopsies in the genetics era.Neuropediatrics. 2017; 48: 309-314Crossref PubMed Scopus (42) Google Scholar Traditionally, MDs are diagnosed through extensive clinical evaluation, including biochemical tissue analysis, followed by genetic screening for selected mutations. The current suite of next-generation sequencing (NGS) options available for MD diagnosis, and for heterogeneous disease diagnosis in general, includes targeted panel sequencing, unbiased whole-exome sequencing (WES), and whole-genome sequencing; each has particular advantages, disadvantages, and considerations. Recent publications advocate for a genetics first diagnostic approach, with the promise of eliminating the need for functional and biochemical analyses in most diagnoses.5Wortmann S.B. Koolen D.A. Smeitink J.A. van den Heuvel L. Rodenburg R.J. Whole exome sequencing of suspected mitochondrial patients in clinical practice.J Inherit Metab Dis. 2015; 38: 437-443Crossref PubMed Scopus (148) Google Scholar, 6Craven L. Alston C.L. Taylor R.W. Turnbull D.M. Recent advances in mitochondrial disease.Annu Rev Genomics Hum Genet. 2017; 18: 257-275Crossref PubMed Scopus (166) Google Scholar This raises some concerns for understudied ethnically diverse populations in developing countries, where relatively little progress has been made toward understanding the genetic etiology of MD and where the genotype-phenotype correlations are poorly understood and are inconsistent with those for non-African populations (as an example). To address these limitations, we and others have undertaken various clinical, biochemical, and genetic studies on MDs in the South African population, one of the few developing countries to do so (Supplemental Figure S1).7Smuts I. Louw R. Du Toit H. Klopper B. Mienie L.J. van der Westhuizen F.H. An overview of a cohort of South African patients with mitochondrial disorders.J Inherit Metab Dis. 2010; 33: 95-104Crossref PubMed Scopus (18) Google Scholar, 8van der Walt E.M. Smuts I. Taylor R.W. Elson J.L. Turnbull D.M. Louw R. van Der Westhuizen F.H. Characterization of mtDNA variation in a cohort of South African paediatric patients with mitochondrial disease.Eur J Hum Genet. 2012; 20: 650-656Crossref PubMed Scopus (28) Google Scholar, 9Reinecke C.J. Koekemoer G. Van der Westhuizen F.H. Louw R. Lindeque J.Z. Mienie L.J. Smuts I. Metabolomics of urinary organic acids in respiratory chain deficiencies in children.Metabolomics. 2012; 8: 264-283Crossref Scopus (51) Google Scholar, 10Meldau S. De Lacy R. Riordan G. Goddard E. Pillay K. Fieggen K. Marais D. Van der Watt G. Identification of a single MPV17 nonsense-associated altered splice variant in 24 South African infants with mitochondrial neurohepatopathy.Clin Genet. 2018; 93: 1093-1096Crossref PubMed Scopus (10) Google Scholar, 11Van der Watt G. Owen E.P. Berman P. Meldau S. Watermeyer N. Olpin S.E. Manning N.J. Baumgarten I. Leisegang F. Henderson H. Glutaric aciduria type 1 in South Africa: high incidence of glutaryl-CoA dehydrogenase deficiency in black South Africans.Mol Genet Metab. 2010; 101: 178-182Crossref PubMed Scopus (32) Google Scholar To date, a traditional diagnostic trajectory of extensive clinical evaluation and functional biochemical diagnosis of referred patients, followed by screening for known, common mutations, has been followed.12Van der Westhuizen F.H. Sinxadi P.Z. Dandara C. Smuts I. Riordan G. Meldau S. Malik A.N. Sweeney M.G. Tsai Y. Towers G.W. Understanding the implications of mitochondrial DNA variation in the health of black southern African populations: the 2014 workshop.Hum Mutat. 2015; 36: 569-571Crossref PubMed Scopus (16) Google Scholar Currently, published patient data, and public genetic and disease databases from predominantly non-African populations, are used as reference because of the limited (specific) information available on African MD etiology. To address these diagnostic challenges for MD in an understudied ethnically diverse population, we report on the outcome of an NGS approach when targeting reported nuclear and mtDNA-encoded genes involved in MD. This approach was investigated in a predominantly African cohort of 212 South African pediatric patients selected on the basis of clinical and muscle respiratory chain (RC) enzymology data; this approach would be considered prudent in a diagnostic setting. We highlight the contrasting outcome of a WES approach in a small subset of this patient cohort and, with specific consideration of the genotype-phenotype correlation suggested by selected cases, propose future diagnostic directions that should be considered for similar understudied population groups. Since 2006, >6000 patients with neurologic symptoms have been referred to the Steve Biko Academic Hospital (a state-funded institution in Pretoria, South Africa) and clinically evaluated, according to an MD criteria scoring system first set forth by Wolf and Smeitink13Wolf N.I. Smeitink J.A. Mitochondrial disorders: a proposal for consensus diagnostic criteria in infants and children.Neurology. 2002; 59: 1402-1405Crossref PubMed Scopus (189) Google Scholar (2002) and subsequently refined by Smuts et al.7Smuts I. Louw R. Du Toit H. Klopper B. Mienie L.J. van der Westhuizen F.H. An overview of a cohort of South African patients with mitochondrial disorders.J Inherit Metab Dis. 2010; 33: 95-104Crossref PubMed Scopus (18) Google Scholar Currently, this cohort consists of 212 pediatric patients who manifested clinically with MD signs or symptoms from as early as the neonatal period. This cohort originated from the northern provinces of South Africa and is predominantly African (64%), with an equal number of males and females. Urine and muscle (vastus lateralis) samples were collected from the entire cohort for subsequent biochemical and molecular genetic investigations. Limited availability of samples from parents and patients prevented segregation analysis. This study was approved by the Ethics Committees of the University of Pretoria (Pretoria, South Africa; number 91/98 and amendments) and the North-West University (Potchefstroom, South Africa; number NWU-00170-13-A1). Muscle RC enzyme analyses for CI to CIV [Enzyme Commission number (EC) 1.6.5.3, EC 1.3.5.1, EC 1.10.2.2, and EC 1.9.3.1, respectively] and CII + CIII were performed and normalized against citrate synthase (EC, 2.3.3.1) activity for all 212 patients. Enzymes were analyzed in 600 × g homogenates prepared from frozen muscle samples, as previously described.7Smuts I. Louw R. Du Toit H. Klopper B. Mienie L.J. van der Westhuizen F.H. An overview of a cohort of South African patients with mitochondrial disorders.J Inherit Metab Dis. 2010; 33: 95-104Crossref PubMed Scopus (18) Google Scholar, 14Smith P.E. Krohn R.I. Hermanson G. Mallia A. Gartner F. Provenzano M. Fujimoto E. Goeke N. Olson B. Klenk D. Measurement of protein using bicinchoninic acid.Anal Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18634) Google Scholar Other biochemical analyses performed included urine metabolic analysis7Smuts I. Louw R. Du Toit H. Klopper B. Mienie L.J. van der Westhuizen F.H. An overview of a cohort of South African patients with mitochondrial disorders.J Inherit Metab Dis. 2010; 33: 95-104Crossref PubMed Scopus (18) Google Scholar, 9Reinecke C.J. Koekemoer G. Van der Westhuizen F.H. Louw R. Lindeque J.Z. Mienie L.J. Smuts I. Metabolomics of urinary organic acids in respiratory chain deficiencies in children.Metabolomics. 2012; 8: 264-283Crossref Scopus (51) Google Scholar, 15Venter L. Lindeque Z. van Rensburg P.J. Van der Westhuizen F. Smuts I. Louw R. Untargeted urine metabolomics reveals a biosignature for muscle respiratory chain deficiencies.Metabolomics. 2015; 11: 111-121Crossref Scopus (22) Google Scholar and muscle coenzyme Q10 (CoQ10) analysis16Louw R. Smuts I. Wilsenach K.-L. Jonck L.-M. Schoonen M. van der Westhuizen F.H. The dilemma of diagnosing coenzyme Q10 deficiency in muscle.Mol Genet Metab. 2018; 125: 38-43Crossref PubMed Scopus (9) Google Scholar in muscle samples. In total, 127 patients were identified to have an RC deficiency. Genomic DNA preparation from muscle homogenate was performed using a standard protocol, as previously described.8van der Walt E.M. Smuts I. Taylor R.W. Elson J.L. Turnbull D.M. Louw R. van Der Westhuizen F.H. Characterization of mtDNA variation in a cohort of South African paediatric patients with mitochondrial disease.Eur J Hum Genet. 2012; 20: 650-656Crossref PubMed Scopus (28) Google Scholar The Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA) was used for quantification of genomic DNA. The complete mitochondrial genome was sequenced in 123 patients, all of whom had a known combined clinical and biochemical MD profile. Two chemistries were used: 71 patient samples were sequenced using the 454 GS-FLX platform (Roche, Basel, Switzerland), and 52 patient samples were sequenced using the Ion PGM platform (Thermo Fisher Scientific). The 454 GS-FLX sequencing, including library preparation and enrichment, was done according to procedures described by van der Walt et al.8van der Walt E.M. Smuts I. Taylor R.W. Elson J.L. Turnbull D.M. Louw R. van Der Westhuizen F.H. Characterization of mtDNA variation in a cohort of South African paediatric patients with mitochondrial disease.Eur J Hum Genet. 2012; 20: 650-656Crossref PubMed Scopus (28) Google Scholar The Ion PGM sequencing, including library preparation and enrichment, was done according to the manufacturer's protocol for Ion Torrent platform. Four samples could not be sequenced because of insufficient sample quantity and quality. For nuclear gene investigations, Ion Torrent amplicon panel sequencing was performed on 86 patients, all of whom had a known combined clinical and biochemical MD profile. Patients were included for sequencing based on their MD criteria scores for clinical presentations and the severity of their biochemical deficiencies [ie, patients who had enzyme activity lower than the reference range when expressed against at least two of three enzyme markers (citrate synthase, CII, or CIV) were considered].7Smuts I. Louw R. Du Toit H. Klopper B. Mienie L.J. van der Westhuizen F.H. An overview of a cohort of South African patients with mitochondrial disorders.J Inherit Metab Dis. 2010; 33: 95-104Crossref PubMed Scopus (18) Google Scholar Genes to be included in sequencing panels were selected on the basis of their mitochondrial RC involvement, either direct or indirect (Supplemental Tables S1, S2, and S3). Three custom gene panels consisting of 136 genes in total were designed and are briefly described. Panel 1 consisted of 78 targeted genes associated with CI deficiency (HaloPlex Target Enrichment System; Agilent Technologies, Santa Clara, CA) (Supplemental Table S1). This customized panel had a target region size of 360,091 kbp and 99.6% targeted coverage. In total, 30 patients were sequenced using panel 1. Panel 2, from Ion AmpliSeq Custom DNA Panels (Thermo Fisher Scientific), had 78 targeted genes associated with CI to IV deficiency (CI, 38 genes; CII, 6 genes; CIII, 10 genes; and CIV, 24 genes), with a total target region size of 157,834 kbp and 98.2% targeted coverage (Supplemental Table S2). In total, 48 patients were sequenced using panel 2, of which 5 patients overlapped with panel 1 and 10 patients overlapped with panel 3. Panel 3, from Ion AmpliSeq, targeted 18 genes known to be involved with primary and secondary CoQ10 deficiency (Supplemental Table S3). The design size was 61 kbp with a targeted coverage of 98%. In total, 26 patients were sequenced in panel 3 (6 patients overlapping with panel 1). The entire coding region of each gene, including flanking regions of introns-exons, was sequenced using the Ion PGM platform, as per manufacturers' protocol (HaloPlex, catalog number G9912C, and AmpliSeq, catalog number 4480441). The selected genes and panels were not African population specific, as the underlying genetic cause for MD is mostly unclear in African populations. As an initial comparison on the outcomes of a panel versus the WES approach, WES was performed on a subset of eight randomly selected African (haplogroup L) cases in whom no strong candidate disease-causing variants had been identified by initial mtDNA and/or nuclear panel sequencing. WES was performed at the Central Analytical Facilities, Stellenbosch University (Stellenbosch, South Africa), using the Ion Proton sequencer (Thermo Fisher Scientific), according to the manufacturer's protocol for the Ion Torrent platform. An approximate 95% on-target coverage was achieved, with an average depth coverage of approximately 140×. Mitochondrial DNA sequences were aligned against the human mitochondrial revised Cambridge Reference Sequence (NC_012920 gi:251831106). Haplogroup assignment, and variant identification and annotation, was performed using mtDNA-Server version 1.20.0,17Weissensteiner H. Forer L. Fuchsberger C. Schopf B. Kloss-Brandstatter A. Specht G. Kronenberg F. Schonherr S. mtDNA-Server: next-generation sequencing data analysis of human mitochondrial DNA in the cloud.Nucleic Acids Res. 2016; 44: W64-W69Crossref PubMed Scopus (90) Google Scholar MitoMap, and MitoMaster.18Lott M.T. Leipzig J.N. Derbeneva O. Xie H.M. Chalkia D. Sarmady M. Procaccio V. Wallace D.C. mtDNA variation and analysis using MITOMAP and MITOMASTER.Curr Protoc Bioinform. 2013; : 1.23.1-1.23.26Crossref PubMed Scopus (320) Google Scholar Homoplasmic and heteroplasmic (levels >30%) nonsynonymous variants were further evaluated on the basis of their allele frequency reported in GenBank and appearance on Phylotree,19Benson D.A. Cavanaugh M. Clark K. Karsch-Mizrachi I. Lipman D.J. Ostell J. Sayers E.W. GenBank.Nucleic Acids Res. 2012; 41: D36-D42Crossref PubMed Scopus (2072) Google Scholar, 20Van Oven M. PhyloTree Build 17: growing the human mitochondrial DNA tree.Forensic Sci Int. 2015; 5: e392-e394Scopus (136) Google Scholar and those with a population allele frequency 0.5 suggests a probable damaging impact on protein function, with scores between 0.75 and 1.0 indicating such functional damage on a protein/amino acid with high confidence. Mitochondrial-tRNA variants were individually evaluated using MitoTIP25Sonney S. Leipzig J. Lott M.T. Zhang S. Procaccio V. Wallace D.C. Sondheimer N. Predicting the pathogenicity of novel variants in mitochondrial tRNA with MitoTIP.PLoS Comput Biol. 2017; 13: e1005867Crossref PubMed Scopus (62) Google Scholar and classified according to a scoring system first set forth by McFarland et al22McFarland R. Elson J.L. Taylor R.W. Howell N. Turnbull D.M. Assigning pathogenicity to mitochondrial tRNA mutations: when "definitely maybe" is not good enough.Trends Genet. 2004; 20: 591-596Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar and subsequently refined by Yarham et al.26Yarham J.W. Al-Dosary M. Blakely E.L. Alston C.L. Taylor R.W. Elson J.L. McFarland R. A comparative analysis approach to determining the pathogenicity of mitochondrial tRNA mutations.Hum Mutat. 2011; 32: 1319-1325Crossref PubMed Scopus (152) Google Scholar According to Yarham et al,26Yarham J.W. Al-Dosary M. Blakely E.L. Alston C.L. Taylor R.W. Elson J.L. McFarland R. A comparative analysis approach to determining the pathogenicity of mitochondrial tRNA mutations.Hum Mutat. 2011; 32: 1319-1325Crossref PubMed Scopus (152) Google Scholar a low score ( 10, with substantial evidence from functional tests, weighs more toward a pathogenic classification. Variants were also evaluated using the guidelines put forth by the American College of Medical Genetics and Genomics, where possible (notably for mtDNA variants).27Richards S. Aziz N. Bale S. Bick D. Das S. Gastier-Foster J. Grody W.W. Hegde M. Lyon E. Spector E. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.Genet Med. 2015; 17: 405Abstract Full Text Full Text PDF PubMed Scopus (14880) Google Scholar Raw sequencing files, obtained from the Ion PGM, were analyzed with the use of Torrent Suite version 5.0.2 (Thermo Fisher Scientific). The sequence files were aligned against Genome Reference Consortium Human Build 37 (hg19), followed by coverage analysis and variant calling using the coverageAnalysis and variantCaller plugins version 5.0 from the Torrent Suite, respectively. The variant calling format files were further annotated using the offline Variant Effect Predictor version 81 from Ensembl (https://uswest.ensembl.org/info/docs/tools/vep/index.html, last accessed July 10, 2018),28McLaren W. Gil L. Hunt S.E. Riat H.S. Ritchie G.R. Thormann A. Flicek P. Cunningham F. The ensembl variant effect predictor.Genome Biol. 2016; 17: 122Crossref PubMed Scopus (2966) Google Scholar followed by variant mining using GEMINI version 20.29Paila U. Chapman B.A. Kirchner R. Quinlan A.R. GEMINI: integrative exploration of genetic variation and genome annotations.PLoS Comput Biol. 2013; 9: e1003153Crossref PubMed Scopus (276) Google Scholar The output text files generated using GEMINI contained information on both novel and reported variants. Most notably, they detailed whether a variant had previously been reported as pathogenic. Further variant filtering was done using population databases, such as Exome Aggregation Consortium and gnomAD30Karczewski K.J. Weisburd B. Thomas B. Solomonson M. Ruderfer D.M. Kavanagh D. Hamamsy T. Lek M. Samocha K.E. Cummings B.B. The ExAC browser: displaying reference data information from over 60 000 exomes.Nucleic Acids Res. 2016; 45: D840-D845Crossref PubMed Scopus (393) Google Scholar (specifically for African allele frequencies); disease-specific databases, such as ClinVar and Online Mendelian Inheritance in Man (OMIM); and sequence databases, such as the National Center for Biotechnology Information Genome and RefSeqGene. As supporting evidence, the missense variants of interest were cautiously evaluated using various in silico predictive algorithms (SIFT, Polyphen-2, and CADD).31Ng P.C. Henikoff S.J. Predicting amino acid changes that affect protein function.Nucleic Acids Res. 2003; 31: 3812-3814Crossref PubMed Scopus (4109) Google Scholar, 32Adzhubei I. Jordan D.M. Sunyaev S.R. Predicting functional effect of human missense mutations using PolyPhen-2.Curr Protoc Hum Genet. 2013; 76: 7.20.21-7.20.41Google Scholar, 33Kircher M. Witten D.M. Jain P. O'roak B.J. Cooper G.M. Shendure J. A general framework for estimating the relative pathogenicity of human genetic variants.Nat Genet. 2014; 46: 310Crossref PubMed Scopus (3717) Google Scholar These algorithms, however, have been shown to have low sensitivity, specificity, and accuracy.34Ernst C. Hahnen E. Engel C. Nothnagel M. Weber J. Schmutzler R.K. Hauke J. Performance of in silico prediction tools for the classification of rare BRCA1/2 missense variants in clinical diagnostics.BMC Med Genomics. 2018; 11: 35Crossref PubMed Scopus (61) Google Scholar Candidate variants of interest were evaluated using American College of Medical Genetics and Genomics guidelines, and are classified as pathogenic, likely pathogenic, variants of uncertain significance, likely benign, or benign. From the >6000 neurologic patients referred for clinical assessment, 212 patients (113 males and 99 females) presented with mitochondrial phenotypes, forming the cohort that is described herein. These clinically defined MD patients were comprehensively evaluated and were predominantly of African ancestry (n = 130) (Figure 1A). Age of onset was as early as the first year of life, including the neonatal period (n = 139) (Figure 1B). The most common clinical finding was muscle involvement, which manifested in 73% (n = 155) of the cohort. Cardiac involvement and deafness were the least observed, at 5% (n = 10) and 8% (n = 17), respectively (Figure 1C). An RC enzyme complex deficiency was identified in 127 cases (64 males and 63 females); 64 cases had isolated deficiency, and the remaining 63 had a combined deficiency (Figure 2A). CI deficiency, either isolated (n = 43) or combined (n = 32), was most prevalent, as is frequently reported,35McFarland R. Kirby D.M. Fowler K.J. Ohtake A. Ryan M.T. Amor D.J. Fletcher J.M. Dixon J.W. Collins F.A. Turnbull D.M. De novo mutations in the mitochondrial ND3 gene as a cause of infantile mitochondrial encephalopathy and complex I deficiency.Ann Neurol. 2004; 55: 58-64Crossref PubMed Scopus (148) Google Scholar, 36Kirby D.M. Salemi R. Sugiana C. Ohtake A. Parry L. Bell K.M. Kirk E.P. Boneh A. Taylor R.W. Dahl H.-H.M. 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