Heart failure is a leading cause of death and indication for cardiac transplantation in the US. Non- ischemic idiopathic dilated cardiomyopathy (iDCM ), an important cause for heart failure, can arise from mutation in many different genes. Sequence analyses of iDCM genes often yield data with uncertain clinical significance due to a lack of knowledge of the full genetic architecture of iDCM and the significance of different gene variants. In this proposal we will sequence and analyze 105 candidate genes in three iDCM populations to define pathogenic genes and variants. We will harness cardiomyocytes differentiated from iPS cells (iPS-CM) to functionally validate gene variant classification and to explore the transcriptional responses to iDCM mutations. Through these studies we aim to a) Determine the allelic spectrum, including loss of function and missense variants that cause iDCM; b) Determine if the genetic architecture of iDCM varies in patients with different ancestries and c) Determine if iDCM genes perturb cardiac biology by altering shared or distinct molecules and pathways. These studies are predicated on successes achieved during the prior funding period. Under the aegis of this grant we demonstrated that truncating titin (TTNtv) cause ~13% unselected iDCM, 22% end- stage iDCM, and 25% severe, familial iDCM. Loss-of-function variants in ~20 other iDCM genes contribute to an additional 10% of the burden of iDCM. We will extend these studies to characterize iDCM genes and variants in patients with non-EU ancestry. Our sequencing data also revealed vast numbers of nonsynonymous variants that encode missense alleles. We will improve our interpretation of these rare missense alleles by harnessing robust bioinformatic strategies that incorporate large sequencing datasets to predict deleterious missense iDCM variants; these will be validated through functional analyses. To improve mechanistic understanding of iDCM we will capitalize on our success in unbiased transcriptional profiling of human and mouse iDCM tissues and iPS-CM that carry iDCM mutations. By defining the pathways and mechanisms that lead to iDCM we hope to identify therapeutic targets that can attenuate progression of disease to heart failure.
Our specific aims are to: 1. Expand the genetic architecture of iDCM by studying patients with EU and non-EU ancestry 2. Construct iPS-CM with iDCM gene variants and assess pathogenicity by functional analyses 3. Produce and analyze selected iDCM gene mutations in vivo
; Dilated cardiomyopathy, a heart disease in which the chambers of the heart are enlarged and the heart's ability to pump blood is significantly diminished, leads to heart failure, the leading cause of death in the United States. Although 30% of unexplained dilated cardiomyopathy is inherited, only ~1/3 of this heritability is understood. We propose to use multiple methods including next generation sequencing and induced pluripotent stem cells to further define the genetic causes of dilated cardiomyopathy and to define the mechanisms by which gene mutation leads to disease.
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| Sharma, Arun; Toepfer, Christopher N; Schmid, Manuel et al. (2018) Differentiation and Contractile Analysis of GFP-Sarcomere Reporter hiPSC-Cardiomyocytes. Curr Protoc Hum Genet 96:21.12.1-21.12.12 |
| Sharma, Arun; Toepfer, Christopher N; Ward, Tarsha et al. (2018) CRISPR/Cas9-Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells. Curr Protoc Hum Genet 96:21.11.1-21.11.20 |
| Garfinkel, Amanda C; Seidman, Jonathan G; Seidman, Christine E (2018) Genetic Pathogenesis of Hypertrophic and Dilated Cardiomyopathy. Heart Fail Clin 14:139-146 |
| Sharma, Arun; Mücke, Michael; Seidman, Christine E (2018) Human Induced Pluripotent Stem Cell Production and Expansion from Blood using a Non-Integrating Viral Reprogramming Vector. Curr Protoc Mol Biol 122:e58 |
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| Ito, Kaoru; Patel, Parth N; Gorham, Joshua M et al. (2017) Identification of pathogenic gene mutations in LMNA and MYBPC3 that alter RNA splicing. Proc Natl Acad Sci U S A 114:7689-7694 |
| Alamo, Lorenzo; Ware, James S; Pinto, Antonio et al. (2017) Effects of myosin variants on interacting-heads motif explain distinct hypertrophic and dilated cardiomyopathy phenotypes. Elife 6: |
| Schafer, Sebastian; de Marvao, Antonio; Adami, Eleonora et al. (2017) Titin-truncating variants affect heart function in disease cohorts and the general population. Nat Genet 49:46-53 |
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