ORIGINAL RESEARCH ARTICLE © American College of Medical Genetics and Genomics Revisiting mitochondrial diagnostic criteria in the new era of genomics Peter Witters, MD, PhD1, Ann Saada, MD, PhD2,3, Tomas Honzik, MD, PhD4, Marketa Tesarova, PhD4, Stephanie Kleinle, PhD5,RitaHorvath,MD,PhD6,AmyGoldstein,MD,PhD7 and Eva Morava, MD, PhD1,7 Purpose: Diagnosing primary mitochondrial diseases (MDs) is (7.07 ± 1.12/8 versus 5.69 ± 1.94/8). At a cutoff of 6.5/8, the challenging in clinical practice. The mitochondrial disease criteria sensitivity to diagnose patients with nDNA mutations is 72.5% (MDC) have been developed to quantify the clinical picture and with a positive predictive value of 69.5%. In the nDNA mutation evaluate the probability of an underlying MD and the need for a group, whole-exome sequencing could diagnose patients with lower muscle biopsy. In this new genetic era with next-generation scores (MDC (6.84 ± 1.51/8) compared to Sanger sequencing sequencing in routine practice, we aim to validate the diagnostic MDC (7.44 ± 1.13/8, P = 0.025)). Moreover 7/8 patients diag- value of MDC. nosed with possible MD by MDC were diagnosed by whole-exome Methods: We retrospectively studied MDC in a multicenter sequencing. cohort of genetically confirmed primary MD patients. Conclusion: MDC remain very useful in the clinical diagnosis of Results: We studied 136 patients (61 male, 91 nuclear DNA MD, in interpreting whole-exome results and deciding on the need (nDNA) mutations). Forty-five patients (33%) had probable MD for performing muscle biopsy. and 69 (51%) had definite MD according to the MDC. A muscle Genet Med advance online publication 26 October 2017 biopsy was performed in 63 patients (47%). Patients with nDNA mutations versus mitochondrial DNA mutations were Key Words: diagnosis; mitochondrial disease; mitochondrial younger (6.4 ± 9.7 versus 19.5 ± 17.3 y) and had higher MDC disease criteria; muscle biopsy; whole-exome sequencing INTRODUCTION developed to facilitate mitochondrial diagnostics.5,6 Histori- Mitochondrial disease is the most frequent, often devastating, cally, two different mitochondrial disease criteria have been neurometabolic disease, with an estimated incidence of published. In 2002 Bernier et al.7 established mitochondrial ~ 1/5,000 births.1 This is probably an underestimate as the diagnostic criteria in children based on a previously validated diagnosis is certainly not straightforward.2–4 Mutations in the adult mitochondrial score. The goal of this scoring system was same mitochondrial disease gene—which can be either to establish the complex diagnosis of clinical and biochemi- nuclear DNA (nDNA) or mitochondrial DNA (mtDNA) cally proven mitochondrial disease. The score developed by encoded—can give rise to different clinical phenotypes. On Wolf et al.6 had a different goal. It was established to help the the other hand the same clinical picture can be caused by a clinician during the diagnostic process to decide on the need myriad of genes affecting mitochondrial function, or involved for surgical muscle biopsy and enzyme complex measure- in totally different pathways, like glycosylation, neurotrans- ment. This score was further simplified as a bedside mission, or development.5 instrument for screening for a suspected mitochondrial In some patients there is a clinically recognizable syndrome disorder.5 It is this latter score that we study in the current (for instance, mitochondrial myopathy, encephalopathy, lactic manuscript. This composite score helps to quantify the acidosis, and stroke-like episodes (MELAS; MIM 540000) probability that a patient has an oxidative phosphorylation or myoclonic epilepsy associated with ragged-red fibers (OXPHOS) defect-related disease. It was also instrumental in (MERRF; MIM 545000) and the genetic analysis can be decision making on performing more invasive investigations, guided by the clinical symptoms. However, in the majority of such as a muscle biopsy. It is tailored for children in patients, the clinical phenotype is less definitive. To this whom symptoms are still evolving into a more characteristic extent, the mitochondrial disease criteria (MDC) have been phenotype.5 However, just like muscle biopsies, the MDC 1Department of Paediatrics and Metabolic Center, University Hospitals Leuven, Department of Development and Regeneration, KU Leuven, Leuven, Belgium; 2Monique and Jacques Roboh Department of Genetic Research, Hadassah-Hebrew University Medical Centre, Jerusalem Israel; 3The Department of Genetic and Metabolic Diseases, Hadassah-Hebrew University Medical Centre, Jerusalem Israel; 4Department of Pediatrics, First Faculty of Medicine, Charles University and General University Hospital, Prague, Czech Republic; 5Medical Genetics Centre, Munich, Germany; 6Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK; 7Tulane University Medical School, Hayward Genetics Center, New Orleans, Louisiana, USA. Correspondence: Peter Witters ([email protected]) or Eva Morava ([email protected]) The first three authors contributed equally to this work. The last two authors contributed equally to this work. Submitted 24 March 2017; accepted 19 June 2017; advance online publication 26 October 2017. doi:10.1038/gim.2017.125 444 Volume 20 | Number 4 | April 2018 | GENETICS in MEDICINE Revisiting mitochondrial diagnostic criteria | WITTERS et al ORIGINAL RESEARCH ARTICLE cannot differentiate between primary and secondary respira- Methods tory chain involvement.6 All participating centers used either mtDNA analysis Overall ~ 5% of the human genome is predicted to be (sequencing, restriction fragment length polymorphism, involved in mitochondria (MitoCarta8). Currently, more than mtDNA long-range polymerase chain reaction, Southern 250 genes are known to be associated with mitochondrial blot) followed by either WES or nDNA mitochondrial gene disease, but this list is growing rapidly in an era of new genetic panels or direct Sanger sequencing of nuclear genes based on techniques, such as next-generation sequencing (NGS).9–16 clinical findings. Interestingly, in patients diagnosed with a mitochondrial Data collected from electronic medical records included disease (compatible clinical signs and symptoms and a age, gender, survival, (age of death), specific clinical features, confirmed mitochondrial OXPHOS complex deficiency), the family history, biochemical testing and imaging data used for success of genetic diagnostics by NGS is between 16 and 60% obtaining the MDC (data at the time of the diagnosis5), the depending on patient selection and diagnostic pipeline genetic technique used, the genetic diagnosis, the histology, used.9–16 and OXPHOS complex measurements (if a muscle biopsy Over the past several years, the development of novel was performed). In two patients with complex II deficiency diagnostic techniques and our growing understanding of OXPHOS complex measurements on lymphoblasts were mitochondrial pathophysiology altered the clinical diagnostic performed. approach in suspected mitochondrial cases. With the The MDC were calculated adding up the scores of clinical increased availability of NGS, mainly whole-exome sequen- (muscle, central nervous system, and multisystem involve- cing (WES) or targeted panels, most diagnostic centers ment), biochemical and imaging results, and in case of biopsy changed the diagnostic strategy in suspected mitochondrial the results of histology, as described previously.5 A score of disease.17 The current approach to mitochondrial diagnostics, 1 indicates unlikely mitochondrial disorder, score 2–4: even by a low suspicion, is to rule out mtDNA mutations by possible mitochondrial disorder, score 5–7 probable mito- direct sequencing, followed by (or in parallel with) NGS panels chondrial disorder, and score ≥ 8 definite mitochondrial or WES18 looking at nDNA. Therefore, many clinicians disorder (Supplementary Data 2). practicing mitochondrial medicine question whether muscle biopsy is still the gold standard for the diagnosis of Statistics mitochondrial disease. Moreover, defects in mitochondrial See Supplementary Data 1. maintenance, fusion/fission, translation, transcription, or abnormalities in mitochondrial membrane integrity or trans- RESULTS port have taught us that some of these disorders do not always Population present with significant OXPHOS deficiencies and may present We included 136 patients (61 male, 75 female) with a mean with normal muscle biochemistry and histology results.2 age of 10.7 ± 14.1 years (median 3.75 years, range 0–68 Nevertheless, the identification of new mutations often requires years). At the time of this manuscript, 50 (37%) of these extensive research to prove its pathogenicity, and it is needless patients had died. Pathogenic nDNA variants were detected in to point out that these research costs are not included in the 91 (67%) cases. Forty-five patients (33%) had a pathogenic cost of WES or covered by any health insurance.19 mtDNA variant (n = 39) or mtDNA deletion (n = 6). In 18 WES provided us with many new genes and novel individuals with pathogenic mtDNA variants, a mutation in phenotypes, and our diagnostic strategy has changed MT-TL1 was detected. Other pathogenic mtDNA variants profoundly. One should raise the question whether the were MT-ATP6 (n = 4), MT-ND1 (n = 2), MT-ND3 (n = 1), MDC still provide us with sufficient guidance in our MT-ND5 (n = 6), MT-ND6 (n = 2), MT-RNR1 (n = 2), diagnostic choices, and if they are reliable enough to point MT-TK (n = 1),