Mutation screening in 86 known X-linked mental retardation genes by droplet-based multiplex PCR and massive parallel sequencing

The HUGO Journal - 2010
Hao Hu1, Klaus Wrogemann1,2, Vera Kalscheuer1, Andreas Tzschach3, Hugues Richard1, Stefan A. Haas3, Corinna Menzel1, Melanie Bienek1, Guy Froyen4, Martine Raynaud5, Hans Van Bokhoven6, Jamel Chelly7, Hilger Ropers1, Wei Chen3,8
1Max Planck Institute for Molecular Genetics, Berlin, Germany
2Department of Biochemistry & Medical Genetics,, University of Manitoba, Winnipeg, Canada
3Max-Planck-Institute for Molecular Genetics, Berlin, Germany
4Human Genome Laboratory, Centre for Human Genetics, VIB, K.U.Leuven, Leuven, Belgium
5INSERM, U930; Centre Hospitalier Régional Universitaire de Tours, Service de Genetique, Tours, France
6Department of Human Genetics, Radboud University Medical Centre, Nijmegen, The Netherlands
7Faculté de Médecine Cochin, INSERM 129-ICGM, Paris, France
8Max-Delbrück-Centrum für Molekulare Medizin, Berlin Institute for Medical Systems Biology, Berlin, Germany

Tóm tắt

Massive parallel sequencing has revolutionized the search for pathogenic variants in the human genome, but for routine diagnosis, re-sequencing of the complete human genome in a large cohort of patients is still far too expensive. Recently, novel genome partitioning methods have been developed that allow to target re-sequencing to specific genomic compartments, but practical experience with these methods is still limited. In this study, we have combined a novel droplet-based multiplex PCR method and next generation sequencing to screen patients with X-linked mental retardation (XLMR) for mutations in 86 previously identified XLMR genes. In total, affected males from 24 large XLMR families were analyzed, including three in whom the mutations were already known. Amplicons corresponding to functionally relevant regions of these genes were sequenced on an Illumina/Solexa Genome Analyzer II platform. Highly specific and uniform enrichment was achieved: on average, 67.9% unambiguously mapped reads were derived from amplicons, and for 88.5% of the targeted bases, the sequencing depth was sufficient to reliably detect variations. Potentially disease-causing sequence variants were identified in 10 out of 24 patients, including the three mutations that were already known, and all of these could be confirmed by Sanger sequencing. The robust performance of this approach demonstrates the general utility of droplet-based multiplex PCR for parallel mutation screening in hundreds of genes, which is a prerequisite for the diagnosis of mental retardation and other disorders that may be due to defects of a wide variety of genes.

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Tài liệu tham khảo

Albert TJ, Molla MN et al (2007) Direct selection of human genomic loci by microarray hybridization. Nat Methods 4(11):903–905

Bau S, Schracke N et al (2009) Targeted next-generation sequencing by specific capture of multiple genomic loci using low-volume microfluidic DNA arrays. Anal Bioanal Chem 393(1):171–175

de Brouwer AP, Yntema HG et al (2007) Mutation frequencies of X-linked mental retardation genes in families from the EuroMRX consortium. Hum Mutat 28(2):207–208

Edwards MC, Gibbs RA (1994) Multiplex PCR: advantages, development, and applications. PCR Methods Appl 3(4):S65–S75

Gnirke A, Melnikov A et al (2009) Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat Biotechnol 27(2):182–189

Hodges E, Xuan Z et al (2007) Genome-wide in situ exon capture for selective resequencing. Nat Genet 39(12):1522–1527

Jensen LR, Amende M et al (2005) Mutations in the JARID1C gene, which is involved in transcriptional regulation and chromatin remodeling, cause X-linked mental retardation. Am J Hum Genet 76(2):227–236

Li R, Li Y et al (2008) SOAP: short oligonucleotide alignment program. Bioinformatics 24(5):713–714

Mardis ER (2008) Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet 9:387–402

Mercer TR, Dinger ME et al (2008) Specific expression of long noncoding RNAs in the mouse brain. Proc Natl Acad Sci USA 105(2):716–721

Meuzelaar LS, Lancaster O et al (2007) MegaPlex PCR: a strategy for multiplex amplification. Nat Methods 4(10):835–837

Ng SB, Turner EH et al (2009) Targeted capture and massively parallel sequencing of 12 human exomes. Nature 461(7261):272–276

Okou DT, Steinberg KM et al (2007) Microarray-based genomic selection for high-throughput resequencing. Nat Methods 4(11):907–909

Olson M (2007) Enrichment of super-sized resequencing targets from the human genome. Nat Methods 4(11):891–892

Porreca GJ, Zhang K et al (2007) Multiplex amplification of large sets of human exons. Nat Methods 4(11):931–936

Ropers HH (2007) New perspectives for the elucidation of genetic disorders. Am J Hum Genet 81(2):199–207

Ropers HH (2008) Genetics of intellectual disability. Curr Opin Genet Dev 18(3):241–250

Rosenberg EH, Almeida LS et al (2004) High prevalence of SLC6A8 deficiency in X-linked mental retardation. Am J Hum Genet 75(1):97–105

Shendure J, Ji H (2008) Next-generation DNA sequencing. Nat Biotechnol 26(10):1135–1145

Sunyaev S, Ramensky V et al (2001) Prediction of deleterious human alleles. Hum Mol Genet 10(6):591–597

Tabolacci E, Pomponi MG et al (2006) A truncating mutation in the IL1RAPL1 gene is responsible for X-linked mental retardation in the MRX21 family. Am J Med Genet A 140(5):482–487

Tarpey PS, Smith R et al (2009) A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation. Nat Genet 41(5):535–543

Tewhey R, Warner JB et al (2009) Microdroplet-based PCR enrichment for large-scale targeted sequencing. Nat Biotechnol 27(11):998–999

Turner EH, Lee C et al (2009) Massively parallel exon capture and library-free resequencing across 16 genomes. Nat Methods 6(5):315–316

Varley KE, Mitra RD (2008) Nested Patch PCR enables highly multiplexed mutation discovery in candidate genes. Genome Res 18(11):1844–1850

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