Molecular aspects of CRKL in heart development and human disease

摘要 人类遗传学最大的挑战之一是了解复杂疾病的遗传基础， 如先天性心脏病（CHD）。研究具有已知遗传病因的罕见疾病，如 22q11.2缺失综合征（DiGeorge/Velo-cardio-facial syndrome），可以精确定位疾病基因， 机制等大约65%的22 q11 DS患者有先天性心脏缺陷，主要影响心脏病。 心脏流出道（OFT）。这些疾病从轻度到重度不等，包括持续性动脉干， 法洛氏四联症，在严重的结束。虽然TBX 1，编码一个T-box转录因子， 染色体22q11.2上CHD的最强候选基因，具有相似心脏缺陷但非 已经鉴定了该区域内的重叠缺失。在这些非典型缺失的患者中，CRKL， 编码受体酪氨酸激酶信号传导的细胞质衔接子的基因缺失，但不缺失TBX 1。这意味着 CRKL是真正的疾病基因。尽管对TBX 1进行了广泛的研究，但CRKL研究不足 与心脏发育有关。神经嵴细胞是咽部的重要细胞群 正确对准心脏OFT所需的装置。我们建议探索神经嵴细胞的功能 通过评估目标1中的新的上游和下游途径，然后，我们将描绘出 Tbx 1和Crk 1介导的OFT的形成至关重要。Crkl-/-小鼠在E16.5时死亡，早于 预期基于仅具有OFT缺陷。我们的病理学研究发现Crkl-/-胚胎 除了对线畸形外，还存在OFT瓣膜缺陷。OFT瓣膜异常与以下高度相关： 22 q11 DS，因为9%的患者有这些缺陷。这是一种新发现的缺陷， 机制尚不清楚。基于这一点，以及心内膜特异性空 突变的胚胎，我们假设Crkl是需要在这个组织中形成衍生的OFT瓣膜通过VEGF- MAPK和其他信号通路。将在目标2中测试内皮功能。这项工作也是 临床重要性，因为现在已报告中年22 q11 DS患者突然 死因不明与众所周知的心内畸形无关这可能与 存在主动脉瓣狭窄，占22q11.2缺失患者的6.6%。连接鼠标 为了将目标1和2中的工作返回到人类患者，我们将评估现有的全外显子组序列（WES）， 我们所知道的目前最大的队列，> 1，000例CRKL相互作用突变的22 q11 DS患者 Aim 3中的网络基因。对候选基因中的非编码序列的探索性研究将在 执行。将使用来自500例22 q11 DS受试者和来自新出现的 非综合征型CHD患者的WES数据。小鼠和人类研究的协同作用将提供快速的 这些发现有助于深入了解CHD复杂的遗传病因。

ABSTRACT One of the greatest challenges in human genetics is to understand the genetic basis of complex disorders, such as congenital heart disease (CHD). Studies of rare diseases with known genetic etiologies such as the 22q11.2 deletion syndrome (DiGeorge/velo-cardio-facial syndrome), can pinpoint disease genes and mechanisms. Approximately 65% of 22q11DS patients have a congenital heart defect, mostly affecting the cardiac outflow tract (OFT). These range from mild to severe and include persistent truncus arteriosus and tetralogy of Fallot, at the severe end. Although TBX1, encoding a T-box transcription factor has been the strongest candidate gene for CHD on chromosome 22q11.2, patients with similar heart defects, but non- overlapping deletions within this region have been identified. In these patients with atypical deletions, CRKL, encoding a cytoplasmic adaptor to receptor tyrosine kinase signaling is deleted, but not TBX1. This implicates CRKL as a true disease gene. Although there have been extensive studies of TBX1, CRKL is understudied with respect to heart development. The neural crest cells are a critical cell population in the pharyngeal apparatus needed for proper alignment of the cardiac OFT. We propose to explore neural crest cell functions of Crkl by evaluating new upstream and downstream pathways in Aim 1. We will then delineate the subset that is critical for OFT development as mediated by Tbx1 and Crkl. Crkl-/- mice die at E16.5, earlier than can be expected based upon only having OFT defects. Our pathological investigation has found that Crkl-/- embryos have OFT valve defects in addition to alignment malformations. OFT valve anomalies are highly relevant to 22q11DS, as 9% of patients have these defects. This is a newly recognized defect for which the molecular mechanisms are not known. Based upon this, and the presence of similar defects in endocardial-specific null mutant embryos, we hypothesize that Crkl is required in this tissue to form derivative OFT valves via VEGF- MAPK and additional signaling pathways. Endocardial functions will be tested in Aim 2. This work is also clinically important because middle-aged 22q11DS patients have now been reported to have sudden unexplained death, unconnected to the well-known intracardiac malformations. This might be related to the presence of aortic valve stenosis, which accounts for 6.6% of 22q11.2 deletion patients. To connect the mouse work in Aims 1 and 2 back to the human patients, we will evaluate existing whole exome sequence (WES) from the current largest cohort that we are aware, of >1,000, 22q11DS patients for mutations in CRKL interaction network genes in Aim 3. Exploratory studies of non-coding sequences in the candidate genes will be performed. Validation will be done using more DNA samples from 500 22q11DS subjects and from emerging WES data on non-syndromic CHD patients. The synergy of mouse and human studies will offer rapid discoveries for a deep understanding of the complex genetic etiology of CHD.