Uncovering compensatory mechanisms in family members with disease causing mutations of pulmonary hypertension

家族性肺动脉高压是一种可遗传的常染色体显性遗传性疾病 在大多数患者中，这会导致进行性右心衰竭和在五年内死亡 诊断。超过70%的FPAH患者存在骨形态发生蛋白受体(BMPR)2单倍体功能不全 患者，但有趣的是，只有20%的突变携带者会患上临床疾病。这项提案旨在揭示 BMPR2突变外显率降低导致FPAH的机制 诱导多能干细胞来源的内皮细胞(IPSC-ECs)。对分子机制的理解 在那些没有FPAH的BMPR2突变携带者中潜在的保护性表型可能导致新的 家族性和非家族性PAH的治疗方法，因为它们都有表达降低或 BMPR2的功能。这些研究也掌握着理解其他遗传条件外显性的关键。 与PAH相关，如小窝蛋白1(CAV1)突变。在顾博士攻读AHA博士后期间进行的研究 联谊会利用来自三组FPAH患者及其家庭成员的IPSC-ECs BMPR2突变，但无疾病。顾博士发现了一条代偿性的p-p38信号通路，导致 从未受影响的突变载体上保存了IPSC-ECs的黏附和存活。这一机制 对保留的p-p38信号的解释在不同的家族中似乎是不同的。第一个目标(K99阶段) 顾博士建议的研究是通过功能的得失研究来扩展上述发现 推行“载体补偿”机制。她还将决定是否对BMPR2进行修正 CRISPR/Cas9技术突变恢复FPAH IPSC-EC中BMP信号通路和EC功能 都来自三个家族。第二个目标(K99阶段)将决定转录组和表观基因组如何 解释未受影响的突变携带者的保护性表型。RNA-Seq在IPSC-ECs上进行 来自所有家庭成员(n=11)。通过比较，确定了71个差异表达基因 对照和突变携带者与FPAH患者的IPSC-ECs，以及四个感兴趣的基因已被 经定量聚合酶链式反应验证。通过与迈克尔·斯奈德博士的实验室合作，帮助将基因联系起来，这一目标得到了加强 ChIP-Seq鉴定的组蛋白标记物的表达变化和由 全基因组测序。第三个目标(R00阶段)将把具体目标1和2的研究扩展到小说 与FPAH相关的突变，如小凹蛋白1。在第四个目标(R00阶段)，顾博士将建立一个 使用IPSC-ECs作为连续细胞来源的高通量个性化药物筛选平台。这些 研究将帮助顾博士启动一项研究计划，优化使用IPSC来源的血管细胞和 建立血管疾病病理生理学模型并开发个性化产品的综合“组学”技术 FDA批准的使用生物信息学方法或高通量筛查改变用途的治疗方法 激活保护通路的药物。

Pulmonary arterial hypertension (PAH) in its familial form (FPAH) is a heritable autosomal dominant disorder that, in the majority of patients, results in progressive right heart failure and death within five years of diagnosis. Bone morphogenetic protein receptor (BMPR) 2 haploinsufficiency occurs in over 70% of the FPAH patients, but intriguingly, only 20% of mutation carriers get clinical disease. This proposal aims to uncover the mechanism underlying the reduced penetrance of BMPR2 mutation in causing FPAH using patient-specific induced pluripotent stem cells derived endothelial cells (iPSC-ECs). Understanding the molecular mechanisms underlying the protective phenotype in those BMPR2 mutation carriers without FPAH could lead to novel therapeutic approaches for familial and non-familial forms of PAH as they all share reduced expression or function of BMPR2. These studies also hold the key to understanding penetrance in other genetic conditions related to PAH, such as the caveolin 1 (CAV1) mutation. Studies carried out during Dr. Gu's AHA postdoctoral fellowship utilized iPSC-ECs from three sets of FPAH patients and from their family members with the same BMPR2 mutation but without disease. Dr. Gu uncovered a compensatory p-p38 signaling pathway leading to preserved adhesion and survival of iPSC-ECs from the unaffected mutation carriers. The mechanism accounting for the preserved p-p38 signaling appears to differ among the families. The first aim (K99 phase) of Dr. Gu's proposed studies is to extend the findings described above, through gain and loss of function studies to pursue the mechanism of `carrier compensation'. She will also determine whether correction of the BMPR2 mutation by CRISPR/Cas9 technology restores BMP signaling pathways and EC functions in FPAH iPSC-EC from all three families. The second aim (K99 phase) will determine how the transcriptome and epigenome explain the protective phenotype in the unaffected mutation carriers. RNA-Seq was carried out on iPSC-ECs from all family members (n=11). Seventy-one differentially expressed genes were identified by comparing iPSC-ECs from controls and mutation carriers versus FPAH patients, and four genes of interest have been verified by qPCR. This aim is strengthened by a collaboration with Dr. Michael Snyder's lab, to help relate gene expression changes with alterations in histone marks identified by ChIP-Seq and polymorphisms called by whole genome sequencing. The third aim (R00 phase) will extend studies in Specific Aims 1 and 2 to novel mutations associated with FPAH, such as caveolin1. In the fourth aim (R00 phase), Dr. Gu will establish a high-throughput platform for personalized drug screening using iPSC-ECs as a continuous cell source. These studies will help Dr. Gu to launch a research program that optimizes the use of iPSC-derived vascular cells and integrative `omic' technologies to model vascular disease pathophysiology and to develop personalized treatments using either a bioinformatics' approach or high throughput screening to repurpose FDA approved drugs that activate the protective pathway.