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Assessment of a Targeted Gene Panel for Identification of Genes Associated with Movement Disorders

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Supplementary Online Content Montaut S, Tranchant C, Drouot N, et al; French Parkinson’s and Movement Disorders Consortium. Assessment of a targeted gene panel for identification of genes associated with movement disorders. JAMA Neurol. Published online June 18, 2018. doi:10.1001/jamaneurol.2018.1478 eMethods. Supplemental methods. eTable 1. Name, phenotype and inheritance of the genes included in the panel. eTable 2. Probable pathogenic variants identified in a cohort of 23 patients with cerebellar ataxia using WES analysis. eTable 3. Negative cases in a cohort of 23 patients with cerebellar ataxia studied using WES analysis. eTable 4. Variants of unknown significance (VUSs) identified in the cohort. eFigure 1. Examples of pedigrees of cases with identified causative variants. eFigure 2. Pedigrees suggesting mendelian inheritance in negative cases. eFigure 3. Examples of pedigrees of cases with identified VUSs. eResults. Supplemental results. This supplementary material has been provided by the authors to give readers additional information about their work. © 2018 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/02/2021 eMethods. Supplemental methods Patients selection In the multicentric, prospective study, patients were selected from 25 French, 1 Luxembourg and 1 Algerian tertiary MDs centers between September 2014 and July 2016. Inclusion criteria were patients (1) who had developed one or several chronic MDs (2) with an age of onset below 40 years and/or presence of a family history of MDs. Patients suffering from essential tremor, tic or Gilles de la Tourette syndrome, pure cerebellar ataxia or with clinical/paraclinical findings suggestive of an acquired cause were excluded. Most of the patients underwent common biochemical testing (copper, ceruloplasmin), brain imaging and most common relevant genetic testing (such as Wilson’s disease or Huntington’s disease) before their inclusion. Demographic, clinical and paraclinical data were collected, as well as family history and a family tree. For all patients, a written informed consent for genetic testing was obtained, either from adult probands or from the legal representative in case of minors and the study was approved by the local ethics committee. Targeted genes and capture design An exhaustive literature review was performed in order to design a targeted genes panel made up of 127 genes known to be either candidates, or clearly involved in MDs (Supplementary table 1). Some genes included were considered as candidate genes since the pathogenicity of their mutations was not clear 6, since they were suspected to act as risk factors 2, or because they are classically responsible for a different phenotype than MDs 7. The total size of targeted regions, all exons, 50-bp flanking sequences of target exons (RefSeq database, hg19 assembly) encompassed 753 kb (kilobases). © 2018 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/02/2021 Library preparation, targeted capture and sequencing Genomic DNA samples were extracted from peripheral blood samples, prepared and controlled following standard procedures in the Genetics and Cellular and Molecular Biology Institute (IGBMC, Illkirch, France). The capture design was performed with SeqCap EZ Designs following the manufacturer’s instructions (Roche, Madison, WI). DNAs (1µg) were sheared mechanically using Covaris E210 (duty cycle: 10%; intensity: 5; cycles per burst: 200; time: 340s) (Woburn, MA). Sequencing and multiplexing adaptors were added simultaneously on 900 ng of sheared DNA using the KAPA Library Preparation Kit (KAPA Biosystems, Wilmington, MA) and the NimbleGen™ SeqCap Adapter Kits A and B (Roche, Madison, WI). After amplification and quality assessments, in-solution targeted capture and post-hybridization amplification were performed on 1µg of multiplexed DNA-prepped libraries using the NimbleGen™ SeqCap EZ Accessory Kit v2 and Pure Capture Bead Kit. Steps of washing, purification and elution were performed using Agencourt SPRI® AMPure XP (Beckman Coulter, Brea, CA). Finally, paired-end sequencing was performed on a NextSeq550 platform (Illumina, Inc., San Diego, CA), multiplexing up to 24 samples per sequencing lane. Bioinformatic pipeline Read mapping and variant calling were performed following standard procedures. Variant filtering was performed using Polyweb, a computer interface developed by the Université Paris Descartes which collects variant-specific information to rank them according to their predicted pathogenicity using data bases (Exome Variant Server (EVS), Exome Aggregation Consortium (ExAC), Single Nucleotide Polymorphism database (dbSNP)) and prediction tools (Sorting Intolerant from Tolerant (SIFT), Polymorphism Phenotyping (PolyPhen)). It © 2018 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/02/2021 also enabled evaluation of the sequencing quality through the assessment of coverage and depth. Variant ranking After using Polyweb filters to exclude a substantial amount of benign polymorphisms, several candidate variants remained, requiring additional filtering strategies. The common polymorphic variants that were frequently observed in the general population were excluded using NGS population data bases (1000 Genomes Project, EVS, ExAC). In addition, repositories of known disease-causing variants (dbSNP, ClinVar, OMIM) were used. Moreover, prediction tools based on the analysis of the evolutionary conservation of the nucleotide/amino acid targeted by the variant or on the prediction of the functional damage caused by the mutated amino acid were used. Literature (e.g. PubMed, OMIM) was also used to identify patients/families with similar phenotype carrying the same or other mutations in the same gene, functional studies or to assess biological relevance. The above-mentioned approaches aimed at reducing the number of variants of unknown significance (VUS) identified, for whom available evidence is not conclusive to claim pathogenicity or to exclude pathogenicity. Exonic copy number variants (CNVs) detection pipeline Putative heterozygous/homozygous/hemizygous structural variants or exonic CNVs were highlighted using a method based on a double normalization of the average depth-of- coverage, comparing the number of reads in the index sample with other regions in the same sample and with the same region in the other samples. © 2018 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/02/2021 Mutation validation All candidate mutations were discussed collegially between geneticists and clinical neurologists before validation. For the proof-of-principle experiment, identified variations were systematically validated by Sanger sequencing in the 48 first patients. Sanger sequencing has also been performed punctually, in particular to validate variants in susceptibility or candidate genes, or CNVs. Moreover, three patients with molecularly confirmed MD diagnosis, identified prior to this study by Sanger sequencing, were included as control. In the event of VUS identification, its involvement in the patient’s phenotype was investigated by specific imaging or biochemical tests if available or by segregation study within the family of the patient. Mutations were considered as causative if they fulfilled the following criteria: convincing sequencing quality (high number of reads) occurring in genes which are consistent with the phenotype, predicted to be deleterious by several algorithms, very rare, or absent in population databases. Whole-exome sequencing Whole-exome sequencing was performed on DNA extracted from whole blood by exon capture with the Agilent SureSelect kit and high-throughput sequencing with an Illumina HiSeq2500 sequencer (IGBMC sequencing platform) for 13 individuals (WHA#, NLDT#, ALG72) and at the Centre National de Génotypage, Institut de Génomique, CEA, France for 10 individuals (ATX#). © 2018 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/02/2021 eTable 1. Name, phenotype and inheritance of the genes included in the panel. Phenotype Name refSeq Disease Inheritance MIM Number Dementia, Lewy body 127750 AD SNCA including Rep1 NM_001146055 Parkinson disease 1 168601 AD promoter region Parkinson disease 4 605543 AD LRRK2 NM_198578 {Parkinson disease 8} 607060 AD 608013, 230800, Gaucher disease AR 230900, GBA NM_001005742 231000, 231005 {Lewy body dementia, susceptibility to} 127750 AD {Parkinson disease, late-onset, susceptibility to} 168600 AD PINK1 NM_032409 Parkinson disease 6, early onset 605909 AR DJ-1/PARK7 NM_007262 Parkinson disease 7, autosomal recessive early-onset 606324 AR ?Neurodegeneration with optic atrophy, childhood 615491 AR UCHL1 NM_004181 onset ?{Parkinson disease 5, susceptibility to} 613643 AD GIGYF2 NM_001103146 {Parkinson disease 11} 607688 AD parkin/PARK2 NM_004562 Parkinson disease, juvenile, type 2 600116 AR 3-methylglutaconic aciduria, type VIII 617248 AR HTRA2 NM_013247 {Parkinson disease 13} 610297 AD VPS35 NM_018206 {Parkinson disease 17} 614203 AD EIF4G1 NM_001194947 {Parkinson disease 18} 614251 AD Parkinson disease 19a, juvenile-onset 615528 AR DNAJC6 NM_001256864 Parkinson disease 19b, early-onset 615528 AR Reclassified - Variant of unknown significance DNAJC13 NM_015268 AD Parkinson disease 21 - AD SYNJ1/synaptojanin NM_003895 Parkinson disease 20, early-onset 615530 AR 1 RAB7L1 NM_001135662 {Parkinson disease
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  • Carrier Detection for Tay-Sachs Disease: a Model for Genetic Disease Prevention

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    Carrier detection for Tay-Sachs disease: a model for genetic disease prevention Irene De Biase, MD PhD Assistant Professor of Pathology, University of Utah Assistant Medical Director, Biochemical Genetics and Supplemental Newborn Screening, ARUP Laboratories Conflict of Interest . None to declare Learning objectives . Review the clinical characteristics and the biochemical features of Tay-Sachs Disease . Describe the population-based screening for Tay-Sachs disease and its impact on disease incidence . Explore the unique challenges in carrier testing for Tay-Sachs disease Cherry red spot Warren Tay British ophthalmologist In 1881, he described the cherry red spot on the retina of a one-year old child with mental and physical retardation NOVEL collection University of Utah Bernard Sachs Jewish-American neurologist In 1896, observed the extreme swelling of neurons in autopsy tissue of affected children Also noticed the disease seemed to be of Jewish origin TSD is a lysosomal storage disease . The underlying biochemical defect is the profound deficiency of the lysosomal hydrolase b-hexosaminidase A . HexA is necessary for the break-down of the ganglioside GM2, a component of the plasma membrane Okada et al. Science 1969; 165:698-700 Degradation of glycosphingolipids GM2 Generalized Gangliosidosis GA2 Sandhoff Disease Tay-Sachs Disease Fabry Disease Metachromatic leukodystrophy Gaucher Disease Krabbe Disease Essentials of Glycobiology Second Edition b-hexosaminidase isoforms: HexA and HexB GM2 Generalized Gangliosidosis GA2 Tay-Sachs Disease HexB: ββ Sandhoff Disease GM2 HexA: αβ activator Fabry Disease GM2 activator Metachromatic leukodystrophy Gaucher Disease Krabbe Disease Essentials of Glycobiology Second Edition Three gene system required for HexA activity 1. 2.