Targeted next-generation sequencing of the ATP7B gene for molecular diagnosis of Wilson disease.

OBJECTIVES In recent years, next-generation sequencing (NGS) technologies, which enable high throughput sample processing at relatively lower costs, are adopted in both research and clinical settings. A multiplex PCR-based NGS assay to identify mutations in the ATP7B gene for routine molecular diagnosis of Wilson disease was evaluated in comparison with the gold standard direct Sanger sequencing. DESIGN AND METHODS Five multiplex PCRs to amplify the partial promoter, 5' untranslated and the entire coding regions of the ATP7B gene were designed. Indexed paired-end libraries were generated from the pooled amplicons using Nextera XT DNA Sample Preparation Kit and subjected to NGS on the MiSeq platform. DNA from the peripheral blood of 12 patients with Wilson disease, 2 B-lymphocyte cell lines and 3 external quality assurance samples were sequenced by the MiSeq and Sanger sequencing. RESULTS Complete coverage was achieved across the targeted bases without any drop-out sequences. The observed read depth in a single run with 20 samples was >100X. Comparison of the NGS results against Sanger sequencing data on a panel of clinical specimens, cell lines and European Molecular Genetics Quality Networks (EMQN) quality assurance samples showed 100% concordance in identifying pathogenic mutations. CONCLUSION With the capability of generating relatively higher throughput in a short time period, the NGS assay is a viable alternative to Sanger sequencing for detecting ATP7B mutations causally linked to Wilson disease in the clinical diagnostic laboratory.

[1]  T. Gilliam,et al.  Characterization of the Wilson disease gene encoding a P-type copper transporting ATPase: genomic organization, alternative splicing, and structure/function predictions. , 1994, Human molecular genetics.

[2]  Maido Remm,et al.  Enhancements and modifications of primer design program Primer3 , 2007, Bioinform..

[3]  B. Faircloth,et al.  Primer3—new capabilities and interfaces , 2012, Nucleic acids research.

[4]  D. Cox,et al.  Sequence variation database for the Wilson disease copper transporter, ATP7B , 2007, Human mutation.

[5]  L. Kádasi,et al.  Detection of His1069Gln mutation in Wilson disease by bidirectional PCR amplification of specific alleles (BI-PASA) test. , 2007, General physiology and biophysics.

[6]  L. Kozák,et al.  Mutation analysis of the ATP7B gene and genotype/phenotype correlation in 227 patients with Wilson disease. , 2005, Molecular genetics and metabolism.

[7]  Lin-fu Zhou,et al.  Wilson disease: identification of two novel mutations and clinical correlation in Eastern Chinese patients. , 2007, World journal of gastroenterology.

[8]  D. Kim,et al.  New novel mutation of the ATP7B gene in a family with Wilson disease , 2012, Journal of the Neurological Sciences.

[9]  F. Tsai,et al.  Development of a high-resolution melting method for the screening of Wilson disease-related ATP7B gene mutations. , 2010, Clinica chimica acta; international journal of clinical chemistry.

[10]  G. Mortier,et al.  Novel pathogenic COL11A1/COL11A2 variants in Stickler syndrome detected by targeted NGS and exome sequencing. , 2014, Molecular genetics and metabolism.

[11]  D. Cox,et al.  The Wilson disease gene: spectrum of mutations and their consequences , 1995, Nature Genetics.

[12]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[13]  M. Schilsky,et al.  Diagnosis and treatment of Wilson disease: An update , 2008, Hepatology.

[14]  S. Henikoff,et al.  Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm , 2009, Nature Protocols.

[15]  S. Tam,et al.  Rapid diagnosis of Wilson disease by a 28-mutation panel: real-time amplification refractory mutation system in diagnosing acute Wilsonian liver failure. , 2008, Clinica chimica acta; international journal of clinical chemistry.

[16]  Wei Zhao,et al.  Exome sequencing identifies novel compound heterozygous mutations in SPG11 that cause autosomal recessive hereditary spastic paraplegia , 2013, Journal of the Neurological Sciences.

[17]  H. Shiraishi,et al.  Pilot study of screening for Wilson disease using dried blood spots obtained from children seen at outpatient clinics , 1999, Journal of Inherited Metabolic Disease.

[18]  S. Hahn,et al.  Clinical molecular diagnosis of Wilson disease. , 2011, Seminars in liver disease.

[19]  Joshua L. Deignan,et al.  ACMG clinical laboratory standards for next-generation sequencing , 2013, Genetics in Medicine.

[20]  DNA sequencing research group: 2006 general survey of DNA sequencing facilities. , 2007, Journal of biomolecular techniques : JBT.

[21]  P. Bork,et al.  A method and server for predicting damaging missense mutations , 2010, Nature Methods.

[22]  P. Ott,et al.  Homozygosity for a gross partial gene deletion of the C‐terminal end of ATP7B in a Wilson patient with hepatic and no neurological manifestations , 2005, American journal of medical genetics. Part A.

[23]  Dong Hwan Lee,et al.  Pilot study of mass screening for Wilson's disease in Korea. , 2002, Molecular genetics and metabolism.

[24]  T. Meitinger,et al.  DHPLC mutation analysis of the hereditary nonpolyposis colon cancer (HNPCC) genes hMLH1 and hMSH2. , 2001, Journal of biochemical and biophysical methods.

[25]  S. Antonarakis,et al.  Mutation nomenclature extensions and suggestions to describe complex mutations: A discussion , 2000 .

[26]  W. Stremmel,et al.  EASL Clinical Practice Guidelines: Wilson's disease. , 2012, Journal of hepatology.

[27]  Xiang-min Xu,et al.  Mutation analysis of 73 southern Chinese Wilson's disease patients: identification of 10 novel mutations and its clinical correlation , 2011, Journal of Human Genetics.

[28]  Helga Thorvaldsdóttir,et al.  Integrative Genomics Viewer , 2011, Nature Biotechnology.

[29]  G. Zang,et al.  Clinical and molecular characterization of Wilson's disease in China: identification of 14 novel mutations , 2011, BMC Medical Genetics.

[30]  Sheng Lin,et al.  Mutational analysis of ATP7B in north Chinese patients with Wilson disease , 2012, Journal of Human Genetics.

[31]  F. Parant,et al.  Molecular analysis of Wilson patients: direct sequencing and MLPA analysis in the ATP7B gene and Atox1 and COMMD1 gene analysis. , 2012, Journal of trace elements in medicine and biology : organ of the Society for Minerals and Trace Elements.