- •DYSF gene analysis is time-consuming and laborious by conventional sequencing method.
- •We set up a next-generation sequencing-based screening method for dysferlinopathy.
- •This method is accurate and efficient for genetic diagnosis of dysferlinopathy.
Dysferlinopathy comprises a group of autosomal recessive muscular dystrophies caused by mutations in the DYSF gene. Due to the large size of the gene and its lack of mutational hot spots, analysis of the DYSF gene is time-consuming and laborious using conventional sequencing methods. By next-generation sequencing (NGS), DYSF gene analysis has previously been validated through its incorporation in multi-gene panels or exome analyses. However, individual validation of NGS approaches for DYSF gene has not been performed. Here, we established and validated a hybridization capture-based target-enrichment followed by next-generation sequencing to detect mutations in patients with dysferlinopathy. With this approach, mean depth of coverage was approximately 450 fold and almost all (99.3%) of the targeted region had sequence coverage greater than 20 fold. When this approach was tested on samples from patients with known DYSF mutations, all known mutations were correctly retrieved. Using this method on 32 consecutive patient samples with dysferlinopathy, at least two pathogenic variants were detected in 28 (87.5%) samples and at least one pathogenic variant was identified in all samples. Our results suggested that the NGS-based screening method could facilitate efficient and accurate genetic diagnosis of dysferlinopathy.
To read this article in full you will need to make a payment
Purchase one-time access:Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
One-time access price info
- For academic or personal research use, select 'Academic and Personal'
- For corporate R&D use, select 'Corporate R&D Professionals'
Subscribe:Subscribe to Neuromuscular Disorders
Already a print subscriber? Claim online access
Already an online subscriber? Sign in
Register: Create an account
Institutional Access: Sign in to ScienceDirect
- A gene related to Caenorhabditis elegans spermatogenesis factor fer-1 is mutated in limb-girdle muscular dystrophy type 2B.Nat Genet. 1998; 20: 37-42
- Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular dystrophy.Nat Genet. 1998; 20: 31-36
- Phenotypic study in 40 patients with dysferlin gene mutations: high frequency of atypical phenotypes.Arch Neurol. 2007; 64: 1176-1182
- Identical dysferlin mutation in limb-girdle muscular dystrophy type 2B and distal myopathy.Neurology. 2000; 55: 1931-1933
- Identical mutation in patients with limb girdle muscular dystrophy type 2B or Miyoshi myopathy suggests a role for modifier gene(s).Hum Mol Genet. 1999; 8: 871-877
- Intracellular accumulation and reduced sarcolemmal expression of dysferlin in limb–girdle muscular dystrophies.Ann Neurol. 2000; 48: 902-912
- Protein and gene analyses of dysferlinopathy in a large group of Japanese muscular dystrophy patients.J Neurol Sci. 2003; 211: 23-28
- Disease-targeted sequencing: a cornerstone in the clinic.Nat Rev Genet. 2013; 14: 295-300
- Comprehensive mutation analysis for congenital muscular dystrophy: a clinical PCR-based enrichment and next-generation sequencing panel.PLoS ONE. 2013; 8 (e53083)
- Exome sequencing as a second-tier diagnostic approach for clinically suspected dysferlinopathy patients.Muscle Nerve. 2014; 50: 1007-1010
- An analysis of exome sequencing for diagnostic testing of the genes associated with muscle disease and spastic paraplegia.Hum Mutat. 2012; 33: 614-626
- Next generation sequencing for molecular diagnosis of neuromuscular diseases.Acta Neuropathol. 2012; 124: 273-283
- Two common mutations (p.Gln832X and c.663+1G>C) account for about a third of the DYSF mutations in Korean patients with dysferlinopathy.Neuromuscul Disord. 2012; 22: 505-510
- Heterogeneous characteristics of Korean patients with dysferlinopathy.J Korean Med Sci. 2012; 27: 423-429
- Fast and accurate long-read alignment with Burrows-Wheeler transform.Bioinformatics. 2010; 26: 589-595
- The sequence alignment/map format and SAMtools.Bioinformatics. 2009; 25: 2078-2079
- The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data.Genome Res. 2010; 20: 1297-1303
- A framework for variation discovery and genotyping using next-generation DNA sequencing data.Nat Genet. 2011; 43: 491-498
- From FastQ data to high-confidence variant calls: the genome analysis toolkit best practices pipeline.Curr Protoc Bioinformatics. 2013; 43: 11.10.1-33
- ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data.Nucleic Acids Res. 2010; 38 (e164)
- Predicting functional effect of human missense mutations using PolyPhen-2.Curr Protoc Hum Genet. 2013; Chapter 7 (Unit7 20)https://doi.org/10.1002/0471142905.hg0720s76
- MutationTaster evaluates disease-causing potential of sequence alterations.Nat Methods. 2010; 7: 575-576
- SIFT web server: predicting effects of amino acid substitutions on proteins.Nucleic Acids Res. 2012; 40: W452-W457
- Copy number variation detection and genotyping from exome sequence data.Genome Res. 2012; 22: 1525-1532
- LOVD v.2.0: the next generation in gene variant databases.Hum Mutat. 2011; 32: 557-563
- Muscle pathology in dysferlin deficiency.Neuropathol Appl Neurobiol. 2002; 28: 461-470
- Muscular dystrophy with marked dysferlin deficiency is consistently caused by primary dysferlin gene mutations.Eur J Hum Genet. 2011; 19: 974-980
- Analysis of the DYSF mutational spectrum in a large cohort of patients.Hum Mutat. 2009; 30: E345-E375
- Limb-girdle muscular dystrophy in the United States.J Neuropathol Exp Neurol. 2006; 65: 995-1003
- Efficient bypass of mutations in dysferlin deficient patient cells by antisense-induced exon skipping.Hum Mutat. 2010; 31: 136-142
- Validation of comparative genomic hybridization arrays for the detection of genomic rearrangements of the calpain-3 and dysferlin genes.Clin Genet. 2012; 81: 99-101
- Identification of different genomic deletions and one duplication in the dysferlin gene using multiplex ligation-dependent probe amplification and genomic quantitative PCR.Genet Test Mol Biomarkers. 2009; 13: 439-442
- MotorPlex provides accurate variant detection across large muscle genes both in single myopathic patients and in pools of DNA samples.Acta Neuropathol Commun. 2014; 2: 100
Published online: March 17, 2015
Accepted: March 11, 2015
Received in revised form: February 16, 2015
Received: December 4, 2014
© 2015 Elsevier B.V. Published by Elsevier Inc. All rights reserved.