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

家族性肺动脉高压（FPAH）是一种可遗传的常染色体显性遗传疾病 在大多数患者中，导致进行性右心衰竭和五年内死亡。 诊断.骨形态发生蛋白受体（BMPR）2单倍不足发生在超过70%的FPAH中。 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 使用患者特异性免疫组织化学方法降低BMPR 2突变导致FPAH的潜在机制 诱导多能干细胞衍生的内皮细胞（iPSC-EC）。了解分子机制 在那些没有FPAH的BMPR 2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，因为它们都具有降低的表达或 BMPR 2的功能这些研究也掌握了理解其他遗传条件下的突变的关键 与PAH相关，如caveolin 1（CAV 1）突变。顾博士在AHA博士后期间进行的研究 奖学金利用了来自三组FPAH患者及其家庭成员的iPSC-EC，并进行了相同的研究 BMPR 2突变但无疾病。Gu博士发现了一种代偿性p-p38信号通路， 保留了未受影响的突变携带者的iPSC-EC的粘附和存活。机制 对保留的p-p38信号传导的解释似乎在家族中不同。第一个目标（K99阶段） 博士Gu的研究计划是通过功能获得和丧失的研究来扩展上述发现 推行“承运人补偿”机制。她还将决定是否纠正BMPR 2 CRISPR/Cas9技术突变恢复FPAH iPSC-EC中的BMP信号通路和EC功能 三个家族的人第二个目标（K99期）将确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。在iPSC-EC上进行RNA测序 所有家庭成员（n=11）。通过比较，筛选出71个差异表达基因， 来自对照和突变携带者与FPAH患者的iPSC-EC，以及四种感兴趣的基因已经被 通过qPCR验证。与迈克尔·斯奈德博士实验室的合作加强了这一目标， ChIP-Seq鉴定的组蛋白标志物的改变和ChIP-Seq鉴定的多态性的表达变化， 全基因组测序第三个目标（R 00阶段）将把具体目标1和2的研究扩展到新的 与FPAH相关的突变，如小窝蛋白1。在第四个目标（R 00阶段），顾博士将建立一个 使用iPSC-EC作为连续细胞来源进行个性化药物筛选的高通量平台。这些 这些研究将帮助顾博士启动一项研究计划，优化iPSC衍生血管细胞的使用， 综合“组学”技术，以模拟血管疾病病理生理学并开发个性化的 使用生物信息学方法或高通量筛选的治疗方法， 激活保护途径的药物。

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.