Genetic Diagnosis of Duchenne and Becker Muscular Dystrophy
Background Duchenne muscular dystrophy or Becker muscular dystrophy might be a suitable candidate disease for application of next-generation sequencing in the genetic diagnosis because the complex mutational spectrum and the large size of the dystrophin gene require two or more analytical methods and have a high cost. The authors tested whether large deletions/duplications or small mutations, such as point mutations or short insertions/deletions of the dystrophin gene, could be predicted accurately in a single platform using next-generation sequencing technology.
Methods A custom solution-based target enrichment kit was designed to capture whole genomic regions of the dystrophin gene and other muscular-dystrophy-related genes. A multiplexing strategy, wherein four differently bar-coded samples were captured and sequenced together in a single lane of the Illumina Genome Analyser, was applied. The study subjects were 25 patients: 16 with deficient dystrophin expression without a large deletion/duplication and 9 with a known large deletion/duplication.
Results Nearly 100% of the exonic region of the dystrophin gene was covered by at least eight reads with a mean read depth of 107. Pathogenic small mutations were identified in 15 of the 16 patients without a large deletion/duplication. Using these 16 patients as the standard, the authors' method accurately predicted the deleted or duplicated exons in the 9 patients with known mutations. Inclusion of non-coding regions and paired-end sequence analysis enabled accurate identification by increasing the read depth and providing information about the breakpoint junction.
Conclusions The current method has an advantage for the genetic diagnosis of Duchenne muscular dystrophy and Becker muscular dystrophy wherein a comprehensive mutational search may be feasible using a single platform.
Duchenne muscular dystrophy (DMD; MIM #310200) and Becker muscular dystrophy (BMD; MIM #3003376) are the most common forms of childhood muscular dystrophy affecting from 1 in 3500 to 1 in 6000 male births. Genetic testing of the dystrophin gene (DMD; Xp21.2) is now the initial method for confirming the diagnosis, although a muscle biopsy might be considered if rapid and reliable genetic testing is unavailable. However, full characterisation of the mutational spectrum is necessary for genetic counselling, prenatal diagnosis and selecting the patients eligible for future mutation-specific treatments. Although the proportion of mutations differs slightly between studies, possibly reflecting bias in cohort selection and application of different molecular diagnostic methods, the mutational spectrum can be approximated as follows: a large deletion in about 60% of patients, a large duplication in about 10% of patients and small mutations confined mostly to coding exons in about 30% of patients. To date, no genetic testing has been developed to cover this whole mutational spectrum in a single platform. In most laboratories, methods for detecting large deletions/duplications and methods for detecting small mutations are conducted separately. In addition, the large size of the dystrophin gene requires considerable effort, cost and time for direct sequencing using the Sanger method.
Massively parallel or next-generation sequencing (NGS) technologies have become essential tools for searching for new human disease genes, most of which were identified by either whole-exome sequencing or targeted sequencing of the regions identified by linkage analyses. NGS technology is also useful for molecular diagnosis of certain diseases where laborious sequencing efforts are required because of the large gene size or the presence of multiple causative genes in single disease entities. A few proof-of-concept studies have been published recently in this field, although further optimisation and validation are required. Besides small mutations including point mutations and short insertions/deletions (indels) spanning several base pairs, large deletions/duplications can also be identified by NGS technologies through read depth estimation. Because NGS provides a comprehensive mutation search from large deletions/duplications to small mutations in a single platform, DMD/BMD is a suitable candidate disease for testing whether NGS can be applied for molecular diagnosis.
To demonstrate whether large deletions/duplications and small mutations of DMD/BMD can be predicted accurately in a single NGS platform, we analysed the entire dystrophin gene regions of 25 patients with DMD or BMD using solution capture with bar-code multiplexing and massively parallel sequencing. First, the pathogenic mutations were searched in 16 patients with pathologically proven DMD/BMD who were negative for large deletions/duplications. Next, we showed how large deletion/duplication mutations could be detected in the same platform by analysing the sequencing data of nine patients with DMD/BMD with known deletion/duplication mutations.
Abstract and Introduction
Abstract
Background Duchenne muscular dystrophy or Becker muscular dystrophy might be a suitable candidate disease for application of next-generation sequencing in the genetic diagnosis because the complex mutational spectrum and the large size of the dystrophin gene require two or more analytical methods and have a high cost. The authors tested whether large deletions/duplications or small mutations, such as point mutations or short insertions/deletions of the dystrophin gene, could be predicted accurately in a single platform using next-generation sequencing technology.
Methods A custom solution-based target enrichment kit was designed to capture whole genomic regions of the dystrophin gene and other muscular-dystrophy-related genes. A multiplexing strategy, wherein four differently bar-coded samples were captured and sequenced together in a single lane of the Illumina Genome Analyser, was applied. The study subjects were 25 patients: 16 with deficient dystrophin expression without a large deletion/duplication and 9 with a known large deletion/duplication.
Results Nearly 100% of the exonic region of the dystrophin gene was covered by at least eight reads with a mean read depth of 107. Pathogenic small mutations were identified in 15 of the 16 patients without a large deletion/duplication. Using these 16 patients as the standard, the authors' method accurately predicted the deleted or duplicated exons in the 9 patients with known mutations. Inclusion of non-coding regions and paired-end sequence analysis enabled accurate identification by increasing the read depth and providing information about the breakpoint junction.
Conclusions The current method has an advantage for the genetic diagnosis of Duchenne muscular dystrophy and Becker muscular dystrophy wherein a comprehensive mutational search may be feasible using a single platform.
Introduction
Duchenne muscular dystrophy (DMD; MIM #310200) and Becker muscular dystrophy (BMD; MIM #3003376) are the most common forms of childhood muscular dystrophy affecting from 1 in 3500 to 1 in 6000 male births. Genetic testing of the dystrophin gene (DMD; Xp21.2) is now the initial method for confirming the diagnosis, although a muscle biopsy might be considered if rapid and reliable genetic testing is unavailable. However, full characterisation of the mutational spectrum is necessary for genetic counselling, prenatal diagnosis and selecting the patients eligible for future mutation-specific treatments. Although the proportion of mutations differs slightly between studies, possibly reflecting bias in cohort selection and application of different molecular diagnostic methods, the mutational spectrum can be approximated as follows: a large deletion in about 60% of patients, a large duplication in about 10% of patients and small mutations confined mostly to coding exons in about 30% of patients. To date, no genetic testing has been developed to cover this whole mutational spectrum in a single platform. In most laboratories, methods for detecting large deletions/duplications and methods for detecting small mutations are conducted separately. In addition, the large size of the dystrophin gene requires considerable effort, cost and time for direct sequencing using the Sanger method.
Massively parallel or next-generation sequencing (NGS) technologies have become essential tools for searching for new human disease genes, most of which were identified by either whole-exome sequencing or targeted sequencing of the regions identified by linkage analyses. NGS technology is also useful for molecular diagnosis of certain diseases where laborious sequencing efforts are required because of the large gene size or the presence of multiple causative genes in single disease entities. A few proof-of-concept studies have been published recently in this field, although further optimisation and validation are required. Besides small mutations including point mutations and short insertions/deletions (indels) spanning several base pairs, large deletions/duplications can also be identified by NGS technologies through read depth estimation. Because NGS provides a comprehensive mutation search from large deletions/duplications to small mutations in a single platform, DMD/BMD is a suitable candidate disease for testing whether NGS can be applied for molecular diagnosis.
To demonstrate whether large deletions/duplications and small mutations of DMD/BMD can be predicted accurately in a single NGS platform, we analysed the entire dystrophin gene regions of 25 patients with DMD or BMD using solution capture with bar-code multiplexing and massively parallel sequencing. First, the pathogenic mutations were searched in 16 patients with pathologically proven DMD/BMD who were negative for large deletions/duplications. Next, we showed how large deletion/duplication mutations could be detected in the same platform by analysing the sequencing data of nine patients with DMD/BMD with known deletion/duplication mutations.
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