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

揭示患有导致肺动脉高压突变的疾病的家庭成员的补偿机制

基本信息

批准号：
10460463
负责人：
Mingxia Gu
金额：
$ 24.9万
依托单位：
CINCINNATI CHILDRENS HOSP MED CTR
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-15 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10460463
关键词：
Accounting Adhesions Alagille Syndrome BIRC3 gene BMPR2 gene Bioinformatics Biological Assay Biological Sciences Blood Vessels CRISPR/Cas technology Cardiovascular Diseases Cardiovascular system Cell Adhesion Cell Survival Cell physiology Cells Cessation of life ChIP-seq Clinical Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Development Diagnosis Disease Drug Screening Endothelial Cells Endothelium Enhancers Epigenetic Process Extracellular Matrix FDA approved Family Family member Fellowship Financial compensation Foundations Functional disorder Gene Expression Gene Expression Profile Genes Genetic Diseases Genetic Polymorphism Heritability Histones Lead Light Lung MMP3 gene Marfan Syndrome Modeling Molecular Mutation Other Genetics Pathway interactions Patients Penetrance Penetration Peripheral Pharmaceutical Preparations Phase Phenotype Pulmonary Hypertension RNA Research Role Signal Pathway Signal Transduction Signaling Protein Source Symptoms System Tacrolimus Binding Protein 1A Technology Testing Transcript Vascular Diseases Vascular Proliferation Williams Syndrome austin bone morphogenetic protein receptors caveolin 1 differential expression disease-causing mutation drug use screening endothelial dysfunction epigenome genome sequencing high throughput screening improved induced pluripotent stem cell interest loss of function loss of function mutation mutation carrier novel novel therapeutic intervention novel therapeutics p38 Mitogen Activated Protein Kinase personalized medicine precision drugs preservation prevent programs pulmonary arterial hypertension pulmonary vascular remodeling right ventricular failure transcriptome transcriptome sequencing transcriptomics whole genome

项目摘要

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.

家族性肺动脉高压 (PAH) (FPAH) 是一种遗传性常染色体显性遗传疾病在大多数患者中，会导致进行性右心衰竭并在五年内死亡诊断。超过 70% 的 FPAH 发生骨形态发生蛋白受体 (BMPR) 2 单倍体不足但有趣的是，只有 20% 的突变携带者会患上临床疾病。该提案旨在揭示使用患者特异性降低 BMPR2 突变外显率导致 FPAH 的机制诱导多能干细胞衍生的内皮细胞（iPSC-EC）。了解分子机制那些没有 FPAH 的 BMPR2 突变携带者中潜在的保护性表型可能会导致新的治疗家族性和非家族性 PAH 的治疗方法，因为它们的表达均减少或 BMPR2 的功能。这些研究还掌握着了解其他遗传条件的外显率的关键与 PAH 相关，例如小窝蛋白 1 (CAV1) 突变。顾博士在AHA博士后期间进行的研究该研究项目使用了来自三组 FPAH 患者及其家庭成员的 iPSC-EC BMPR2突变但无疾病。顾博士发现了一条补偿性p-p38信号通路来自未受影响的突变携带者的 iPSC-EC 保留了粘附和存活。机制各家族中保留的 p-p38 信号传导的解释似乎有所不同。第一个目标（K99阶段）顾博士提出的研究是通过功能获得和丧失的研究来扩展上述发现推行“运营商补偿”机制。她还将确定是否修正 BMPR2 CRISPR/Cas9 技术突变恢复 FPAH iPSC-EC 中的 BMP 信号通路和 EC 功能来自三个家庭。第二个目标（K99阶段）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。 RNA-Seq 在 iPSC-EC 上进行来自所有家庭成员 (n=11)。通过比较，鉴定出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，四个感兴趣的基因已被通过 qPCR 验证。通过与 Michael Snyder 博士实验室的合作，这一目标得到了加强，以帮助将基因联系起来表达随着 ChIP-Seq 鉴定的组蛋白标记的改变和由 ChIP-Seq 调用的多态性而变化全基因组测序。第三个目标（R00 阶段）将把具体目标 1 和 2 的研究扩展到新颖的领域。与 FPAH 相关的突变，例如 Caveolin1。在第四个目标（R00阶段），顾博士将建立一个使用 iPSC-EC 作为连续细胞源进行个性化药物筛选的高通量平台。这些研究将帮助 Gu 博士启动一项研究计划，优化 iPSC 衍生血管细胞的使用，并综合“组学”技术来模拟血管疾病病理生理学并开发个性化使用生物信息学方法或高通量筛选来重新利用 FDA 批准的治疗激活保护途径的药物。

项目成果

期刊论文数量（13）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Hypoplastic left heart syndrome (HLHS): molecular pathogenesis and emerging drug targets for cardiac repair and regeneration.

DOI：
10.1080/14728222.2021.1978069
发表时间：
2021-08
期刊：
Expert opinion on therapeutic targets
影响因子：
5.8
作者：
Bejjani AT;Wary N;Gu M
通讯作者：
Gu M

Capillary cell-type specialization in the alveolus.

DOI：
10.1038/s41586-020-2822-7
发表时间：
2020-10
期刊：
Nature
影响因子：
64.8
作者：
Gillich A;Zhang F;Farmer CG;Travaglini KJ;Tan SY;Gu M;Zhou B;Feinstein JA;Krasnow MA;Metzger RJ
通讯作者：
Metzger RJ

Efficient Differentiation of Human Pluripotent Stem Cells to Endothelial Cells.