Application of massively parallel sequencing approaches in Nemaline Myopathy
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USyd Access
Type
ThesisThesis type
Doctor of PhilosophyAuthor/s
Sandaradura, SarahAbstract
Nemaline myopathy (NM) is one of the most common forms of congenital myopathy. The traditional approach to diagnosis involved skeletal muscle biopsy followed by Sanger sequencing of single genes, resulting in a genetic diagnosis in approximately 50% of patients. Use of massively ...
See moreNemaline myopathy (NM) is one of the most common forms of congenital myopathy. The traditional approach to diagnosis involved skeletal muscle biopsy followed by Sanger sequencing of single genes, resulting in a genetic diagnosis in approximately 50% of patients. Use of massively parallel sequencing (MPS) techniques such as panel testing of known disease genes, whole-exome sequencing (WES) and whole-genome sequencing (WGS) enables rapid, cost-effective analysis of large regions of the genome for potentially pathogenic variants. RNA sequencing (RNA-Seq) allows analysis of the transcriptome. The primary aim of this thesis was to explore the diagnostic utility of newly emerging massively parallel sequencing approaches for diagnosis of nemaline myopathy. In this body of work, MPS was performed on probands from 21 families with NM. A genetic diagnosis was obtained in 95% of families. RNA-Seq supported pathogenicity of extended splice variants identified on WES or panel testing and identified variants missed by other techniques. This work highlights the ongoing importance of muscle biopsy in a subset of patients, to allow analysis of histopathological features and to facilitate RNA-Seq. It establishes LMOD3 and TNNT3 as new genetic causes of NM and proves cost effectiveness of MPS in obtaining a genetic diagnosis in patients with NM. It includes collaboration on an update of the mutational spectra of NEB, collaboration on publication of another new disease gene for NM, KLHL41, and collaboration on a seminal paper on RNA-Seq. This body of work demonstrates the power of MPS techniques in genetic diagnosis of congenital myopathy, in identification of new disease genes, novel phenotypes for known disease genes and variants in known disease genes. It supports the role of targeted MPS of known neuromuscular disease genes as first line investigation of patients with clinical features of congenital myopathy, with muscle biopsy reserved as a second-line investigation.
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See moreNemaline myopathy (NM) is one of the most common forms of congenital myopathy. The traditional approach to diagnosis involved skeletal muscle biopsy followed by Sanger sequencing of single genes, resulting in a genetic diagnosis in approximately 50% of patients. Use of massively parallel sequencing (MPS) techniques such as panel testing of known disease genes, whole-exome sequencing (WES) and whole-genome sequencing (WGS) enables rapid, cost-effective analysis of large regions of the genome for potentially pathogenic variants. RNA sequencing (RNA-Seq) allows analysis of the transcriptome. The primary aim of this thesis was to explore the diagnostic utility of newly emerging massively parallel sequencing approaches for diagnosis of nemaline myopathy. In this body of work, MPS was performed on probands from 21 families with NM. A genetic diagnosis was obtained in 95% of families. RNA-Seq supported pathogenicity of extended splice variants identified on WES or panel testing and identified variants missed by other techniques. This work highlights the ongoing importance of muscle biopsy in a subset of patients, to allow analysis of histopathological features and to facilitate RNA-Seq. It establishes LMOD3 and TNNT3 as new genetic causes of NM and proves cost effectiveness of MPS in obtaining a genetic diagnosis in patients with NM. It includes collaboration on an update of the mutational spectra of NEB, collaboration on publication of another new disease gene for NM, KLHL41, and collaboration on a seminal paper on RNA-Seq. This body of work demonstrates the power of MPS techniques in genetic diagnosis of congenital myopathy, in identification of new disease genes, novel phenotypes for known disease genes and variants in known disease genes. It supports the role of targeted MPS of known neuromuscular disease genes as first line investigation of patients with clinical features of congenital myopathy, with muscle biopsy reserved as a second-line investigation.
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Date
2018-05-11Licence
The author retains copyright of this thesis. It may only be used for the purposes of research and study. It must not be used for any other purposes and may not be transmitted or shared with others without prior permission.Faculty/School
Faculty of Medicine and HealthDepartment, Discipline or Centre
Discipline of Paediatrics and Child HealthAwarding institution
The University of SydneyShare