Molecular aspects of CRKL in heart development and human disease

摘要 人类遗传学中最大的挑战之一是理解复杂疾病的遗传基础， 例如先天性心脏病(CHD)。对具有已知遗传病因的罕见疾病的研究，如 22q11.2缺失综合征(DiGeorge/velo心脏面部综合征)，可精确定位疾病基因和 机械装置。大约65%的22q11DS患者有先天性心脏缺陷，主要影响 心脏流出道(OFT)。这些症状从轻微到严重不等，包括持续性动脉干和 法洛四联症，最严重的结局。尽管编码T-box转录因子的TBX1一直是 位于染色体22q11.2上的CHD最强候选基因，有类似的心脏缺陷，但不是 该区域内的重叠缺失已被发现。在这些非典型缺失的患者中，CRKL， 编码受体酪氨酸激酶信号的细胞质适配器被删除，但不是TBX1。这意味着 CRKL是一个真正的致病基因。尽管已经有了广泛的研究，但对CRKL的研究还很少 在心脏发育方面。神经脊细胞是咽部的一个重要细胞群。 心脏OFT正确对准所需的设备。我们建议探索神经脊细胞的功能 通过评估目标1中的新的上行和下行路径。然后，我们将描述 在Tbx1和Crk1介导的OFT发生中起关键作用。Crkl-/-小鼠在E16.5岁死亡，比可能的死亡时间更早 基于只有OFT缺陷的预期。我们的病理检查发现，Crk1-/-胚胎 除对齐畸形外，还存在OFT阀门缺陷。软性瓣膜异常与 22q11DS，因为9%的患者有这些缺陷。这是一种新发现的缺陷，分子 机制尚不清楚。根据这一点，心内膜特异性零点存在类似的缺陷 突变的胚胎，我们假设CRK1在这个组织中需要通过血管内皮生长因子形成衍生的OFT瓣膜。 MAPK和其他信号通路。心内膜功能将在目标2中进行测试。这项工作也是 临床上很重要，因为现在有报道称中年22q11ds患者患有突发性 不明原因的死亡，与众所周知的心脏内畸形无关。这可能与 存在主动脉瓣狭窄，占22q11.2缺失患者的6.6%。连接鼠标的步骤 在目标1和2中的工作回到人类患者，我们将评估现有的完整外显子序列(WES)从 目前我们所知的CRKL相互作用突变的最大队列为&gt；1,000，22q11DS患者 目标3.候选基因中非编码序列的探索性研究 已执行。验证将使用来自500名22q11DS受试者和Emerging的更多DNA样本进行 非综合征型冠心病患者的WES资料。老鼠和人类研究的协同作用将提供快速 这些发现有助于深入了解冠心病的复杂遗传病因。

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.