In 2016, guidelines for diagnostic Next Generation Sequencing (NGS) have been published by EuroGentest in order to assist laboratories in the implementation and accreditation of NGS in a diagnostic setting. These guidelines mainly focused on Whole Exome Sequencing (WES) and targeted (gene panels) sequencing detecting small germline variants (Single Nucleotide Variants (SNVs) and insertions/deletions (indels)). Since then, Whole Genome Sequencing (WGS) has been increasingly introduced in the diagnosis of rare diseases as WGS allows the simultaneous detection of SNVs, Structural Variants (SVs) and other types of variants such as repeat expansions. The use of WGS in diagnostics warrants the re-evaluation and update of previously published guidelines. This work was jointly initiated by EuroGentest and the Horizon2020 project Solve-RD. Statements from the 2016 guidelines have been reviewed in the context of WGS and updated where necessary. The aim of these recommendations is primarily to list the points to consider for clinical (laboratory) geneticists, bioinformaticians, and (non-)geneticists, to provide technical advice, aid clinical decision-making and the reporting of the results.
INTRODUCTION: Next-generation sequencing is now used on a routine basis for molecular testing but studies on copy-number variant (CNV) detection from next-generation sequencing data are underrepresented. Utilizing an existing whole-exome sequencing (WES) dataset, we sought to investigate the contribution of rare CNVs to the genetic causality of dystonia. METHODS: The CNV read-depth analysis tool ExomeDepth was applied to the exome sequences of 953 unrelated patients with dystonia (600 with isolated dystonia and 353 with combined dystonia; 33% with additional neurological involvement). We prioritized rare CNVs that affected known disease genes and/or were known to be associated with defined microdeletion/microduplication syndromes. Pathogenicity assessment of CNVs was based on recently published standards of the American College of Medical Genetics and Genomics and the Clinical Genome Resource. RESULTS: We identified pathogenic or likely pathogenic CNVs in 14 of 953 patients (1.5%). Of the 14 different CNVs, 12 were deletions and 2 were duplications, ranging in predicted size from 124bp to 17 Mb. Within the deletion intervals, BRPF1, CHD8, DJ1, EFTUD2, FGF14, GCH1, PANK2, SGCE, UBE3A, VPS16, WARS2, and WDR45 were determined as the most clinically relevant genes. The duplications involved chromosomal regions 6q21-q22 and 15q11-q13. CNV analysis increased the diagnostic yield in the total cohort from 18.4% to 19.8%, as compared to the assessment of single-nucleotide variants and small insertions and deletions alone. CONCLUSIONS: WES-based CNV analysis in dystonia is feasible, increases the diagnostic yield, and should be combined with the assessment of single-nucleotide variants and small insertions and deletions.
- MeSH
- dospělí MeSH
- dystonické poruchy diagnóza genetika MeSH
- dystonie diagnóza genetika MeSH
- kohortové studie MeSH
- lidé MeSH
- sekvenování exomu * MeSH
- variabilita počtu kopií segmentů DNA * genetika MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Dystonia is a clinically and genetically heterogeneous condition that occurs in isolation (isolated dystonia), in combination with other movement disorders (combined dystonia), or in the context of multisymptomatic phenotypes (isolated or combined dystonia with other neurological involvement). However, our understanding of its aetiology is still incomplete. We aimed to elucidate the monogenic causes for the major clinical categories of dystonia. METHODS: For this exome-wide sequencing study, study participants were identified at 33 movement-disorder and neuropaediatric specialty centres in Austria, Czech Republic, France, Germany, Poland, Slovakia, and Switzerland. Each individual with dystonia was diagnosed in accordance with the dystonia consensus definition. Index cases were eligible for this study if they had no previous genetic diagnosis and no indication of an acquired cause of their illness. The second criterion was not applied to a subset of participants with a working clinical diagnosis of dystonic cerebral palsy. Genomic DNA was extracted from blood of participants and whole-exome sequenced. To find causative variants in known disorder-associated genes, all variants were filtered, and unreported variants were classified according to American College of Medical Genetics and Genomics guidelines. All considered variants were reviewed in expert round-table sessions to validate their clinical significance. Variants that survived filtering and interpretation procedures were defined as diagnostic variants. In the cases that went undiagnosed, candidate dystonia-causing genes were prioritised in a stepwise workflow. FINDINGS: We sequenced the exomes of 764 individuals with dystonia and 346 healthy parents who were recruited between June 1, 2015, and July 31, 2019. We identified causative or probable causative variants in 135 (19%) of 728 families, involving 78 distinct monogenic disorders. We observed a larger proportion of individuals with diagnostic variants in those with dystonia (either isolated or combined) with coexisting non-movement disorder-related neurological symptoms (100 [45%] of 222; excepting cases with evidence of perinatal brain injury) than in those with combined (19 [19%] of 98) or isolated (16 [4%] of 388) dystonia. Across all categories of dystonia, 104 (65%) of the 160 detected variants affected genes which are associated with neurodevelopmental disorders. We found diagnostic variants in 11 genes not previously linked to dystonia, and propose a predictive clinical score that could guide the implementation of exome sequencing in routine diagnostics. In cases without perinatal sentinel events, genomic alterations contributed substantively to the diagnosis of dystonic cerebral palsy. In 15 families, we delineated 12 candidate genes. These include IMPDH2, encoding a key purine biosynthetic enzyme, for which robust evidence existed for its involvement in a neurodevelopmental disorder with dystonia. We identified six variants in IMPDH2, collected from four independent cohorts, that were predicted to be deleterious de-novo variants and expected to result in deregulation of purine metabolism. INTERPRETATION: In this study, we have determined the role of monogenic variants across the range of dystonic disorders, providing guidance for the introduction of personalised care strategies and fostering follow-up pathophysiological explorations. FUNDING: Else Kröner-Fresenius-Stiftung, Technische Universität München, Helmholtz Zentrum München, Medizinische Universität Innsbruck, Charles University in Prague, Czech Ministry of Education, the Slovak Grant and Development Agency, the Slovak Research and Grant Agency.
- MeSH
- dítě MeSH
- dystonie diagnóza epidemiologie genetika MeSH
- exom genetika MeSH
- genetická variace genetika MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- novorozenec MeSH
- předškolní dítě MeSH
- rodokmen MeSH
- sekvenování exomu metody MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- multicentrická studie MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
