Functional characterization of Adamts19, a novel gene for human heart valve defects

从全球角度来看，心脏瓣膜缺陷是人类最常见的心脏异常之一，其中相当多的人一生中需要手术干预一次。已经提出了一些机制，包括发育，遗传和后天原因，但对于大多数情况下，遗传的解释仍然缺乏。为了确定遗传原因，我们进行了两个大的遗传筛查，在单胞胎和扩展的多重家庭。我们能够在两个独立的家族中检测到ADAMTS 19的纯合缺失和纯合功能丧失突变，ADAMTS 19是心脏瓣膜缺陷的新候选基因。为了研究其在心脏瓣膜发育中的作用，我们设计了一种转基因小鼠模型，该模型重建了在人类家系中发现的Adamts 19的功能丧失（LOF）。心脏瓣膜最初通过称为内皮细胞向间充质转化（EMT）的过程形成，然后随后在幼年早期延长并成熟。在整个胚胎和出生后阶段的Adamts 19-/-小鼠的表达分析发现，在瓣膜形成后的所有心脏瓣膜的表达。高分辨率数字超声心动图显示，没有Adamts 19表达的小鼠在生命早期发展出功能障碍的主动脉瓣，这让人想起人类表型。值得注意的是，Adamts 19在瓣膜中的表达仅限于特定的心肌细胞亚群，即瓣膜间质细胞（维克），而在内皮细胞中未观察到。我们使用蛋白质组学和组织学的分析表明，Adamts 19的存在对于维持瓣膜发育及其成熟过程中的细胞外基质重塑是必要的。LOF小鼠不仅完全重现了人类表型，而且还突出了Adamts 19作为VIC的新标记物，以特异性靶向初始EMT后过程，以及作为理解人类老化瓣膜表型的重要模型。本申请的目的不仅是确定Adamts 19 LOF的表型呈现，而且还分析已知的分子遗传网络，以及在心脏瓣膜组织中蛋白质和转录组水平上以及在单细胞水平上的先前未知的分子过程，以获得对人类瓣膜疾病的更详细的理解。

On a global perspective, defects of the heart valves are one of the most common cardiac abnormalities in humans, with a considerable number of them requiring surgical intervention once in their lifetime. A number of mechanisms have been proposed including developmental, genetic and acquired causes, but for the majority of cases a genetic explanation is still lacking. To identify genetic causes, we have conducted two large genetic screens in singletons and extended multiplex families. We were able to detect a homozygous deletion and a homozygous loss of function mutation in two independent families in ADAMTS19, a novel candidate gene for valvular heart defects. To investigate its role in heart valve development, we designed a transgenic mouse model that reconstitutes the loss of function (LOF) in Adamts19 found in the human pedigrees. Cardiac valves initially form through a process called Endothelial-to-Mesenchymal Transition (EMT) then subsequently elongate and mature during early juvenile life. Expression analysis throughout embryonic and postnatal stages of Adamts19-/- mice discovered an expression in all cardiac valves after valve formation. High resolution, digital echocardiography showed that mice without Adamts19 expression develop dysfunctional aortic valves early in life, reminiscent of the human phenotype. Remarkably, the expression of Adamts19 in the valve was restricted to a specific subpopulation of cardiac cells, the valvular interstitial cells (VIC) and not observed in endothelial cells. Our analysis using proteomics and histology suggests that the presence of Adamts19 is necessary to maintain extracellular matrix remodelling during valve development and its maturation. Not only do the LOF mice fully recapitulate the human phenotype, but they also highlight Adamts19 as a novel marker for VICs to specifically target initial post-EMT processes as well as serve as an important model to understand an ageing valve phenotype in humans.The aim of this application is to characterise not only the phenotypic presentation of Adamts19 LOF, but also analyse known molecular genetic networks, as well as previously unknown molecular processes on the protein and transcriptome level in heart valve tissue and on the single cell level to gain a more detailed understanding of human valve disease.