The application of whole exome sequencing for the diagnosis of limb-girdle muscular dystrophies

Abstract

The limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of inherited muscle disorders characterized by progressive weakness and wasting that primarily affects the proximal muscle of the upper and lower extremities. By definition, disease onset occurs after the age of 2 years and varies from early childhood to adulthood with a spectrum of clinical severity. The LGMDs typically show dystrophic changes on muscle biopsy. To date, 33 different genetic forms of LGMD have been described and are grouped according to inheritance pattern; LGMD1 refers to dominantly inherited forms whilst LGMD2 refers to recessively inherited forms. As the genetic basis of the various subtypes within LGMD have been identified, it has become clear that the clinical presentations and histopathology overlap, and predicting the genetic form of LGMD based on clinical examination or histological appearances alone is difficult. In Australia, the rate of genetic diagnosis of LGMD remains low at approximately 24%, despite traditional approaches of histopathological findings to triage genetic screening of known LGMD genes. The aim of my PhD project was to improve the genetic diagnoses of LGMDs by translating whole-exome sequencing (WES) to clinical practice. I initially ascertained LGMD families retrospectively through the Institute for Neuroscience and Muscle Research Biospecimen Bank (INMR) between 2006 and 2014 to determine the frequency of the LGMD subtypes in Australia. By collecting this data, I determined that 65% of patients have remained undiagnosed at our centre despite previous extensive investigations. At the outset of my PhD project, and in collaboration with Associate Professor Daniel MacArthur at the Broad Institute of Harvard, WES was the NGS technology available to screen my LGMD families and also provided the opportunity to identify new disease genes. 60 LGMD families were initially recruited for WES but with ongoing collaboration with Australian and New Zealand Neurologist colleagues, a total of 90 LGMD families were enrolled for the study. I also investigated whether the WES platform provides adequate coverage of known LGMD-related genes by performing Neurogenetic Subexomic Supercapture (NSES)(also referred to as targeted neuromuscular disease gene panel) on all undiagnosed LGMDs after WES. NSES did not identify any variants missed by WES. Using NGS, I achieved 45% diagnostic rate for this patient cohort who remained undiagnosed after extensive investigations (biochemistry and candidate gene sequencing). Inclusion of family members increased the diagnostic efficacy of WES, with a diagnostic rate of 60% for “trios” (an affected proband with both parents) versus 40% for single probands. Common causes of phenotypic overlap with LGMD were due to mutations in genes associated with congenital muscular dystrophy and Bethlem myopathy. The most common causes of LGMD in an Australasian population were due to the recessive LGMD genes, specifically FKRP, DYSF and CAPN3. During the course of this work, I also expanded the clinical phenotypes associated with known myopathy genes specifically discussing the following genes; GMPPB, HSPB8 and TOR1AIP1 (attached publications), ACTA1 and LAMA2. My research demonstrates the effectiveness of WES for genetic diagnosis of the highly heterogeneous LGMDs. In addition, my results stress the importance of accurate clinical examination and histopathological data for interpretation of WES, with many diagnoses requiring follow-up review and ancillary investigations of biopsy specimens, or further imaging or serum samples. The marked success of WES and other NGS approaches (NSES, Neuromuscular Gene Panel) challenge the current diagnostic algorithm for the diagnosis of patients with neuromuscular disorders. By incorporating NGS into clinical practice, a muscle biopsy becomes a secondary investigation and should be requested for cases that have remained undiagnosed or as a follow up investigation after NGS. NGS is time- and cost-effective relative to traditional diagnostics approaches and will significantly reduce the long diagnostic odyssey for many families. WES approaches will also enhance gene discovery, a vital step in the process of understanding the genetic and biological mechanisms of inherited diseases. I hope that the discoveries presented in this thesis will be incorporated into clinical practice and the future diagnostic, and also guide clinicians in their investigation of patients with neuromuscular disorders. My study provides strong evidence supporting the diagnostic efficacy of NGS approaches. However, ascribing likely pathogenicity of identified variants remains challenging for many patients, in particular, non-essential splice site variants and missense variants not previously linked to disease. Thus, education of referring clinicians about NGS and its limitations is essential. There is also a need to develop guidelines relating to the use of NGS technology in clinical practice including ‘gate-keeping’ for NGS, as to who can request the test, how, and when it should be implemented relative to current diagnostic practices. These guidelines should also address the ethical, legal and social implications of NGS technology. Future work should focus on providing evidence of the cost-effectiveness of NGS, so it can be implemented into clinical practice