The Use of Genetic Analyses and Functional Assays for the Interpretation of Rare Variants in Pediatric Heart Disease
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The use of genetic analyses and functional assays for the interpretation of rare variants in pediatric heart disease A dissertation submitted to the Division of Graduate Studies and Research, University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Molecular Genetics by Jeffrey A. Schubert Bachelor of Science, Mount St. Joseph University, 2012 Committee Chair: Stephanie M. Ware, M.D., Ph.D. Edmund Choi, Ph.D. Benjamin Landis, M.D. Anil Menon, Ph.D. David Wieczorek, Ph.D. Molecular Genetics, Biochemistry, and Microbiology Graduate Program College of Medicine, University of Cincinnati Cincinnati, Ohio, USA, 2018 ABSTRACT The use of next generation technologies such as whole exome sequencing (WES) has paved the way for discovering novel causes of Mendelian diseases. This has been demonstrated in pediatric heart diseases, including cardiomyopathy (CM) and familial thoracic aortic aneurysm (TAA). Each of these conditions carries a high risk of a serious cardiac event, including sudden heart failure or aortic rupture, which are often fatal. Patients with either disease can be asymptomatic before presenting with these events, which necessitates early diagnosis. Though there are many known genetic causes of disease for both conditions, there is still room for discovery of novel pathogenic genes and variants, as many patients have an undefined genetic diagnosis. WES covers the protein-coding portion of the genome, which yields a massive amount of data, though it comprises only 1% of the genome. Sorting and filtering sequencing information to identify (sometimes) a single base pair change responsible for the patient phenotype is challenging. Further, interpreting identified candidate variants must be done according to strict standards, which makes it difficult to definitively say whether a coding change is pathogenic or benign. This thesis uses a combined approach of genetic analyses via WES, and functional assessment of variants via in vitro assays to aid interpretation of pathogenicity for variants identified in CM and TAA patients. Much of the work described in the following chapters focuses on the gene filamin C (FLNC). FLNC is an important structural protein for the maintenance of sarcomeres in skeletal and cardiac muscle cells, and FLNC variants are associated with either skeletal myopathy or cardiomyopathy, but not both. Within, I describe two novel FLNC variants identified by WES in two families with restrictive CM, and determine both are pathogenic by using genetic and ii functional evidence. Further, I define the rate of rare, clinically significant FLNC variants in a large pediatric CM cohort, which mirrored previously reported values (2-4%). To investigate disease mechanisms associated with FLNC, I used RNA-sequencing to identify specific patterns of gene expression and affected pathways in Flnc-deficient mouse skeletal and cardiac muscle cells, as it is unknown why individual FLNC variants affect only one tissue type. This analysis revealed divergently affected mitotic pathways between the two cell types, and highlighted other affected cellular processes, including downregulation of the contractile apparatus in cardiomyocytes. Further, I sought to define the molecular signatures of three FLNC variants causing three types of CM (hypertrophic, dilated, and restrictive) by knocking-in each into human stem cells via CRISPR/Cas9, followed by cardiomyocyte differentiation. However, as I did not produce successful knock-ins, I instead describe my efforts to optimize this approach. Finally, I used WES to screen a broad panel of genes in a cohort of TAA patients with previous inconclusive genetic testing. I found clinically significant variants in 6/10 patients, including two likely pathogenic variants in two families. This highlighted the utility of using WES as an initial approach over single gene or panel testing; WES also could reduce costs considering the additive expense of iterative testing. iii iv ACKNOWLEDGEMENTS: There are many people and even a couple animals worthy of thanks and acknowledgement for their support throughout my dissertation research. First and foremost I would like to thank my committee members for their guiding wisdom the past four years—their insightful comments and suggestions helped teach me to think like a scientist, and (perhaps to their chagrin due to its length), played a large part in molding the thesis into its final product. Notably, I’d like to thank my advisor Stephanie Ware for her calm demeanor and steadfast support in the face of numerous unpredictable mishaps and unforeseen problems. Every time I came to meet with her, confidence shaken, questioning myself and the chaotic universe responsible for these problems, I left with a sense of relief and a re-kindled belief that those difficulties were indeed surmountable. I would also like to thank the many members of the Ware Lab, past and present, for their continuous support and comraderie. Whether the situation called for sharing a laugh or sharing a question, this group was always available, and for that I’m truly thankful. Finally I’d like to thank my friends and family for their support and well wishes throughout. I certainly wouldn’t have been in this position without the love and support of my parents, for whom I am eternally grateful. Also the past five years would have been profoundly more difficult, less entertaining, and rewarding without the constant support of my loving wife Leah (certainly editing the thesis would have been much more difficult, too). And to not leave out the animals, thanks to our cats Node and BooBoo for sitting with me at the computer while I wrote and only sometimes getting in the way. The following work is dedicated with love to Mom and Aunt Ma Sœur. v Table of Contents ABSTRACT .................................................................................................................................... ii CHAPTER 1: Introduction ............................................................................................................. 1 1.1 CARDIOMYOPATHY CLINICAL SPECTRUM ............................................................... 1 1.1.1 Classification of cardiomyopathies ................................................................................ 1 1.1.2 Hypertrophic cardiomyopathy ........................................................................................ 2 1.1.3 Dilated cardiomyopathy ................................................................................................. 3 1.1.4 Restrictive cardiomyopathy ............................................................................................ 5 1.1.5 Other inherited cardiomyopathies .................................................................................. 6 1.2 GENETICS OF CARDIOMYOPATHY .............................................................................. 8 1.2.1 Introduction .................................................................................................................... 8 1.2.2 Known genes and genetic testing for HCM.................................................................... 9 1.2.3 Known genes and genetic testing for DCM.................................................................. 10 1.2.4 Known genes and genetic testing for RCM .................................................................. 11 1.3 USE OF WHOLE EXOME SEQUENCING AS A METHODOLOGY TO IDENTIFY NOVEL GENETIC CAUSES OF DISEASE ........................................................................... 12 1.3.1 Next generation sequencing and The Pediatric Cardiomyopathy Genes Study ........... 12 1.3.2 Challenges and limitations of WES data analysis ........................................................ 15 1.3.3 The VUS problem ......................................................................................................... 16 1.4 FILAMIN C AS AN IMPORTANT PROTEIN IN MUSCLE CELLS .............................. 18 1.4.1 Filamins and their structure .......................................................................................... 18 1.4.2 Functions of filamins .................................................................................................... 20 1.4.3 FLNC and its roles in cardiac and skeletal muscle ....................................................... 26 1.5 MUTATIONS IN FLNC CAUSE SKELETAL AND CADIAC MUSCLE DISEASE ..... 33 1.5.1 Distal and myofibrillar myopathies .............................................................................. 33 1.5.2 FLNC mutations causing distal and myofibrillar myopathies ...................................... 34 1.5.3 Potential pathomechanisms for FLNC-related skeletal myopathies ............................. 35 1.5.4 Cardiomyopathy causing FLNC mutations .................................................................. 36 1.6 CLINICAL SPECTRUM AND GENETICS OF THORACIC AORTIC ANEURYSM ... 38 1.6.1 Classifying thoracic aortic aneurysm ........................................................................... 38 1.6.2 Genetics of syndromic and non-syndromic familial TAA ........................................... 40 1.7 SUMMARY OF HYPOTHESES AND MAJOR FINDINGS ............................................ 44 1.8 FIGURES AND TABLES ............................................................................................... 47 vi Figure 1.8.1 Filamin protein structure ................................................................................... 47 Table 1.8.2: