title: Next generation sequencing for molecular diagnosis of neurological disorders using ataxias as a model.
abstract: Many neurological conditions are caused by immensely heterogeneous gene mutations. The diagnostic process is often long and complex with most patients undergoing multiple invasive and costly investigations without ever reaching a conclusive molecular diagnosis. The advent of massively parallel, next-generation sequencing promises to revolutionize genetic testing and shorten the 'diagnostic odyssey' for many of these patients. We performed a pilot study using heterogeneous ataxias as a model neurogenetic disorder to assess the introduction of next-generation sequencing into clinical practice. We captured 58 known human ataxia genes followed by Illumina Next-Generation Sequencing in 50 highly heterogeneous patients with ataxia who had been extensively investigated and were refractory to diagnosis. All cases had been tested for spinocerebellar ataxia 1-3, 6, 7 and Friedrich's ataxia and had multiple other biochemical, genetic and invasive tests. In those cases where we identified the genetic mutation, we determined the time to diagnosis. Pathogenicity was assessed using a bioinformatics pipeline and novel variants were validated using functional experiments. The overall detection rate in our heterogeneous cohort was 18% and varied from 8.3% in those with an adult onset progressive disorder to 40% in those with a childhood or adolescent onset progressive disorder. The highest detection rate was in those with an adolescent onset and a family history (75%). The majority of cases with detectable mutations had a childhood onset but most are now adults, reflecting the long delay in diagnosis. The delays were primarily related to lack of easily available clinical testing, but other factors included the presence of atypical phenotypes and the use of indirect testing. In the cases where we made an eventual diagnosis, the delay was 3-35 years (mean 18.1 years). Alignment and coverage metrics indicated that the capture and sequencing was highly efficient and the consumable cost was ∼£400 (€460 or US$620). Our pathogenicity interpretation pathway predicted 13 different mutations in eight different genes: PRKCG, TTBK2, SETX, SPTBN2, SACS, MRE11, KCNC3 and DARS2 of which nine were novel including one causing a newly described recessive ataxia syndrome. Genetic testing using targeted capture followed by next-generation sequencing was efficient, cost-effective, and enabled a molecular diagnosis in many refractory cases. A specific challenge of next-generation sequencing data is pathogenicity interpretation, but functional analysis confirmed the pathogenicity of novel variants showing that the pipeline was robust. Our results have broad implications for clinical neurology practice and the approach to diagnostic testing.
authors.affiliation: 1 Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford OX3 9DU, UK.
authors.forename: Andrea H
authors.initials: AH
authors.lastname: Németh
authors.forename: Alexandra C
authors.initials: AC
authors.lastname: Kwasniewska
authors.forename: Stefano
authors.initials: S
authors.lastname: Lise
authors.forename: Ricardo
authors.initials: R
authors.lastname: Parolin Schnekenberg
authors.forename: Esther B E
authors.initials: EB
authors.lastname: Becker
authors.forename: Katarzyna D
authors.initials: KD
authors.lastname: Bera
authors.forename: Morag E
authors.initials: ME
authors.lastname: Shanks
authors.forename: Lorna
authors.initials: L
authors.lastname: Gregory
authors.forename: David
authors.initials: D
authors.lastname: Buck
authors.forename: M
authors.initials: M
authors.lastname: Zameel Cader
authors.forename: Kevin
authors.initials: K
authors.lastname: Talbot
authors.forename: Rajith
authors.initials: R
authors.lastname: de Silva
authors.forename: Nicholas
authors.initials: N
authors.lastname: Fletcher
authors.forename: Rob
authors.initials: R
authors.lastname: Hastings
authors.forename: Sandeep
authors.initials: S
authors.lastname: Jayawant
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Morrison
authors.forename: Paul
authors.initials: P
authors.lastname: Worth
authors.forename: Malcolm
authors.initials: M
authors.lastname: Taylor
authors.forename: John
authors.initials: J
authors.lastname: Tolmie
authors.forename: Mary
authors.initials: M
authors.lastname: O'Regan
authors.forename: Ruth
authors.initials: R
authors.lastname: Valentine
authors.forename: Emily
authors.initials: E
authors.lastname: Packham
authors.forename: Julie
authors.initials: J
authors.lastname: Evans
authors.forename: Anneke
authors.initials: A
authors.lastname: Seller
authors.forename: Jiannis
authors.initials: J
authors.lastname: Ragoussis
mesh_terms: D017668:Age of Onset; D001259:Ataxia / Q000175:diagnosis / Q000235:genetics*; D005808:Genes, Recessive / Q000235:genetics; D020022:Genetic Predisposition to Disease; D005820:Genetic Testing / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D025202:Molecular Diagnostic Techniques; D009154:Mutation / Q000235:genetics*
keywords: ataxia; autosomal dominant cerebellar ataxia; autosomal recessive cerebellar ataxia; diagnosis; genetics
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