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

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

Expanding the disease phenotype of ADSSL1-associated myopathy in non-Korean patients

Adenylosuccinate synthase (ADSSL1) is a muscle specific enzyme involved in the purine nucleotide cycle and responsible for the conversion of inosine monophosphate to adenosine monophosphate. Since 2016, when mutations in the ADSSL1 gene were first described to be associated with an adult onset distal myopathy, nine patients with compound heterozygous variants in the ADSSL1 gene, all of Korean origin, have been identified. Here we report a novel ADSSL1 mutation and describe two sporadic cases of Turkish and Indian origin. Many of the clinical features of both patients and muscle histopathology and muscle MRI findings, were in accordance with previously reported findings in the adult onset distal myopathy individuals. However, one of our patients presented with progressive, proximally pronounced weakness, severe muscle atrophy and early contractures. Thus, mutations in ADSSL1 have to be considered in patients with both distal and proximal muscle weakness and across various ethnicities. (C) 2020 Published by Elsevier B.V

Prominent scapulae mimicking an inherited myopathy expands the phenotype of CHD7-related disease

CHD7 variants are a well-established cause of CHARGE syndrome, a disabling multi-system malformation disorder that is often associated with deafness, visual impairment and intellectual disability. Less severe forms of CHD7-related disease are known to exist, but the full spectrum of phenotypes remains uncertain. We identified a de novo missense variant in CHD7 in a family presenting with musculoskeletal abnormalities as the main manifestation of CHD7-related disease, representing a new phenotype. The proband presented with prominent scapulae, mild shoulder girdle weakness and only subtle dysmorphic features. Investigation revealed hypoplasia of the trapezius and sternocleidomastoid muscles and semicircular canal defects, but he did not fulfill diagnostic criteria for CHARGE syndrome. Although the shoulders are often sloping and anteverted in CHARGE syndrome, the underlying neuromuscular cause has never been investigated. This report expands the phenotypes associated with CHD7 mutations to include a musculoskeletal presentation, with hypoplasia of the shoulder and neck muscles. CHD7 should be considered in patients presenting in childhood with stable scapular winging, particularly if accompanied by dysmorphic features and balance difficulties

Diagnosis and etiology of congenital muscular dystrophy: We are halfway there.

Objective: To evaluate the diagnostic outcomes in a large cohort of congenital muscular dystrophy (CMD) patients using traditional and next generation sequencing (NGS) technologies. Methods: A total of 123 CMD patients were investigated using the traditional approaches of histology, immunohistochemical analysis of muscle biopsy, and candidate gene sequencing. Undiagnosed patients available for further testing were investigated using NGS. Results: Muscle biopsy and immunohistochemical analysis found deficiencies of laminin a2, a-dystroglycan, or collagen VI in 50% of patients. Candidate gene sequencing and chromosomal microarray established a genetic diagnosis in 32% (39 of 123). Of 85 patients presenting in the past 20 years, 28 of 51 who lacked a confirmed genetic diagnosis (55%) consented to NGS studies, leading to confirmed diagnoses in a further 11 patients. Using the combination of approaches, a confirmed genetic diagnosis was achieved in 51% (43 of 85). The diagnoses within the cohort were heterogeneous. Forty-five of 59 probands with confirmed or probable diagnoses had variants in genes known to cause CMD (76%), and 11 of 59 (19%) had variants in genes associated with congenital myopathies, reflecting overlapping features of these conditions. One patient had a congenital myasthenic syndrome, and 2 had microdeletions. Within the cohort, 5 patients had variants in novel (PIGY and GMPPB) or recently published genes (GFPT1 and MICU1), and 7 had variants in TTN or RYR1, large genes that are technically difficult to Sanger sequence. Interpretation: These data support NGS as a first-line tool for genetic evaluation of patients with a clinical phenotype suggestive of CMD, with muscle biopsy reserved as a second-tier investigation.Gina L. O, Grady, Monkol Lek, Shireen R. Lamande, Leigh Waddell, Emily C. Oates, Jaya Punetha, Roula Ghaoui, Sarah A. Sandaradura, Heather Best, Simranpreet Kaur, Mark Davis, Nigel G. Laing, Francesco Muntoni, Eric Hoffman, Daniel G. MacArthur, Nigel F. Clarke, Sandra Cooper, and Kathryn Nort

Patient care standards for primary mitochondrial disease in Australia: an Australian adaptation of the Mitochondrial Medicine Society recommendations

This document provides consensus-based recommendations for general physicians and primary care physicians who diagnose and manage patients with mitochondrial diseases (MD). It builds on previous international guidelines, with particular emphasis on clinical management in the Australian setting. This statement was prepared by a working group of medical practitioners, nurses and allied health professionals with clinical expertise and experience in managing Australian patients with MD. As new treatments and management plans emerge, these consensus-based recommendations will continue to evolve, but current standards of care are summarised in this document

Prominent scapulae mimicking an inherited myopathy expands the phenotype of CHD7-related disease

CHD7 variants are a well-established cause of CHARGE syndrome, a disabling multi-system malformation disorder that is often associated with deafness, visual impairment and intellectual disability. Less severe forms of CHD7-related disease are known to exist, but the full spectrum of phenotypes remains uncertain. We identified a de novo missense variant in CHD7 in a family presenting with musculoskeletal abnormalities as the main manifestation of CHD7-related disease, representing a new phenotype. The proband presented with prominent scapulae, mild shoulder girdle weakness and only subtle dysmorphic features. Investigation revealed hypoplasia of the trapezius and sternocleidomastoid muscles and semicircular canal defects, but he did not fulfill diagnostic criteria for CHARGE syndrome. Although the shoulders are often sloping and anteverted in CHARGE syndrome, the underlying neuromuscular cause has never been investigated. This report expands the phenotypes associated with CHD7 mutations to include a musculoskeletal presentation, with hypoplasia of the shoulder and neck muscles. CHD7 should be considered in patients presenting in childhood with stable scapular winging, particularly if accompanied by dysmorphic features and balance difficulties

Improving genetic diagnosis in Mendelian disease with transcriptome sequencing

Exome and whole-genome sequencing are becoming increasingly routine approaches in Mendelian disease diagnosis. Despite their success, the current diagnostic rate for genomic analyses across a variety of rare diseases is approximately 25 to 50%. We explore the utility of transcriptome sequencing [RNA sequencing (RNA-seq)] as a complementary diagnostic tool in a cohort of 50 patients with genetically undiagnosed rare muscle disorders. We describe an integrated approach to analyze patient muscle RNA-seq, leveraging an analysis framework focused on the detection of transcript-level changes that are unique to the patient compared to more than 180 control skeletal muscle samples. We demonstrate the power of RNA-seq to validate candidate splice-disrupting mutations and to identify splice-altering variants in both exonic and deep intronic regions, yielding an overall diagnosis rate of 35%. We also report the discovery of a highly recurrent de novo intronic mutation in COL6A1 that results in a dominantly acting splice-gain event, disrupting the critical glycine repeat motif of the triple helical domain. We identify this pathogenic variant in a total of 27 genetically unsolved patients in an external collagen VI-like dystrophy cohort, thus explaining approximately 25% of patients clinically suggestive of having collagen VI dystrophy in whom prior genetic analysis is negative. Overall, this study represents a large systematic application of transcriptome sequencing to rare disease diagnosis and highlights its utility for the detection and interpretation of variants missed by current standard diagnostic approachesThis project was supported by funding from the Broad Institute’s BroadIgnite and Broadnext10 programs. B.B.C. is supported by the NIH GM096911 training grant. T.T. is supported by the Academy of Finland, the Finnish Cultural Foundation, the Orion-Farmos Research Foundation, and the Emil Aaltonen Foundation. M.L. is supported by the Australian NHMRC (National Health and Medical Research Council) CJ Martin Fellowship, the Australian American Association Sir Keith Murdoch Fellowship, and a Muscular Dystrophy Association/American Association of Neuromuscular and Electrodiagnostic Medicine (MDA/AANEM) development grant. L.B.W., S.A.S., N.G.L., N.F.C., K.N.N., and E.C.O. are supported by the NHMRC of Australia (1080587, 1075451, 1002147, 1113531, 1022707, 1031893, and 1090428). K.J.K. is supported by a National Institute of General Medical Sciences (NIGMS) fellowship grant (F32GM115208). A.H.O.-L. is supported by an NIGMS fellowship grant (4T32GM007748). A.H.B. is supported by the NIH R01 HD075802 and R01 AR044345 and by MDA383249 from the Muscular Dystrophy Association. P.B.K., E.E., and H.K.M. are supported by NIH R01NS080929. Funding relevant to this research includes fellowship support of S.T.C. and a project grant supporting an Australian-wide program about gene discovery in inherited neuromuscular disorders performed in collaboration with D.G.M. [NHMRC APP1048816 (2013–2017) and NHMRC APP1080587 (2015–2019)]. The Broad CMG was funded by the National Human Genome Research Institute (NHGRI), the National Eye Institute, and the National Heart, Lung, and Blood Institute (NHLBI) grant UM1 HG008900 to D.G.M. and H. Rehm. The GTEx project was supported by the Common Fund of the Office of the Director of the NIH (http://commonfund.nih.gov/GTEx). Additional funds were provided by the National Cancer Institute (NCI), NHGRI, NHLBI, National Institute on Drug Abuse (NIDA), National Institute of Mental Health (NIMH), and National Institute of Neurological Disorders and Stroke (NINDS). Donors were enrolled at Biospecimen Source Sites that were funded by NCI/Science Applications International Corporation (SAIC)–Frederick Inc. (SAIC-F) subcontracts to the National Disease Research Interchange (10XS170) and the Roswell Park Cancer Institute (10XS171). The Laboratory, Data Analysis, and Coordinating Center (LDACC) was funded through a contract (HHSN268201000029C) to the Broad Institute Inc. Biorepository operations were funded through an SAIC-F subcontract to the Van Andel Institute (10ST1035). Additional data repository and project management were provided by SAIC-F (HHSN261200800001E). The Brain Bank was supported by a supplement to the University of Miami grant DA00622