Elucidating Genotype-Phenotype Relationship of Polygenic Dilated Cardiomyopathies

项目摘要 扩张型心肌病（DCM）是一种遗传性心脏病，发病率高达1：250。 虽然一般认为这是一种单基因遗传的疾病，但最近的遗传研究表明， 复杂的多基因原因。多基因心肌病的功能概况知之甚少， 阻碍了有效治疗的发展。干细胞技术的最新进展，如患者- 诱导多能干细胞（iPSC）和基因组编辑，提供了前所未有的机会 来研究这种疾病的表型。心肌细胞将从取自患有以下疾病的患者的iPSC分化： 两个DCM相关突变并测序，以了解可能与 疾病易感性该分析将通过使用CRISPR来标准化以创建同基因对照，并且还 插入与这些患者相关的健康个体中的突变。我们将利用iPSC 与组织工程相结合的平台，以开发由心脏特异性 细胞我们将进行单细胞RNA测序，以测试细胞间的相互作用和细胞间的串扰。 细胞我们还将进行功能测定，如钙处理和收缩性。CRISPR/dCas 9系统 将使我们能够识别出适合药物靶向的基因。父R 01补助金的首要目标是 使用iPSC平台了解多基因DCM的潜在机制。建议多样性 通过研究不同种族的患者并考虑这些因素如何影响 疾病严重程度。非裔美国人和西班牙裔患者在健康研究中的代表性不足， 因此更容易受到疾病管理不善的影响。卡洛斯维拉医生会把我们的20人小组 通过来源于这些少数群体的六个iPSC系对这些个体进行研究，并执行所提出的一组 实验

PROJECT SUMMARY Dilated cardiomyopathy (DCM) is a genetic cardiac disorder with high morbidity and an incidence of 1:250. Although generally thought of as a disease with monogenic heritability, recent genetic studies suggest complex polygenic causes. Functional profiles of polygenic cardiomyopathies are poorly understood and this hampers effective therapeutics development. Recent advances in stem cell technology such as patient- derived induced pluripotent stem cells (iPSCs) and genome-editing, provides an unprecedented opportunity to study such disease phenotypes. Cardiomyocytes will be differentiated from iPSCs taken from patients with two DCM related mutations and sequenced to understand transcriptome differences that may correlate with disease susceptibility. The analysis will be normalized by using CRISPR to create isogenic controls and also insert the mutations in healthy individuals that are related to these patients. We will then utilize the iPSC platform coupled with tissue engineering to develop engineered heart tissues composed of cardiac specific cells. We will perform single cell RNA sequencing to test the cell-cell interactions and crosstalk between the cells. We will also perform functional assays like calcium-handling and contractility. CRISPR/dCas9 systems will allow us to identify genes suitable for drug targeting. The overarching goal of the parent R01 grant is to understand underlying mechanisms of polygenic DCM using the iPSC platform. The proposed diversity supplement extends this work by studying patients of different ethnicities and considering how this factors in disease severity. African-American and Hispanic patients are underrepresented in health studies, and therefore more vulnerable to poor disease management. Dr. Carlos Vera will expand our cohort of 20 individuals by six iPSC lines derived from these minority populations and perform the proposed set of experiments.