Studying Pentalogy of Cantrell in Humans and Mice

项目一：本研究的主要目的是确定导致人类坎特雷尔五联症(POC)的基因(S)突变(S)。我们怀疑其中一个基因将是NM IIB，这是基于我们建立的小鼠模型，该模型在NM IIB中有一个点突变，并表现出与诊断为POC的人类相似的表型。然而，很可能除了NM IIB外，其他基因也可能是致病基因，因此我们计划在我们可能在POC诊断患者中发现的任何基因(S)中寻找似乎是致病基因的突变。 我们目前正在招募和收集诊断为POC的患者(及其亲属)的样本。在我们的合作者的帮助下，我们计划从其他有POC确诊病例的机构获得样本。然后，我们计划进行整个外显子基因组测序。由于已知非肌肉肌球蛋白与大量蛋白质直接或间接相互作用，我们也将寻找POC患者其他基因的任何突变。如果发现突变，我们将在体外或在小鼠模型中鉴定肌球蛋白(或其他蛋白质)的特征，以确认它是致病因素。 项目二：我们的实验室已经培育出一只NM IIB基因点突变(R709C)的小鼠，这种小鼠会患上许多先天性心脏缺陷，包括隔膜缺陷、覆盖的主动脉和瓣膜发育不全。为了调查这些异常发现，我们利用免疫组织学分析来确定细胞外和细胞外蛋白的表达模式，这些蛋白已经受到NM IIB突变的影响，因此可能位于NM IIB在心脏发育中的下游。我们正致力于建立瓣膜垫外植体培养系统，以进一步探索NM IIB在瓣膜细胞迁移、基质组织和增殖/凋亡中的作用，所有这些都对瓣膜的正常形成至关重要。我们还致力于在小鼠的心肌细胞中创建组织特异性突变，以阐明NM IIB在流出道心肌化和错位中的作用。

Project One: The major purpose of this research is to identify the mutation(s) in gene(s) that result in Pentalogy of Cantrell (POC) in humans. We suspect that one of these genes will be NM IIB, based on a mouse model that we have generated which has a point mutation in NM IIB and demonstrates a phenotype similar to humans with the diagnosis of POC. However, it is likely that other genes, in addition to NM IIB, could be causative and therefore we plan to look for mutations in any gene(s) we may find in patients with the diagnosis of POC that appear to be causative. We are currently recruiting and collecting samples from patients (and their relatives) with the diagnosis of POC. With the help of our collaborators, we plan to obtain samples from other institutions where there are identified cases of POC. We then plan to carry out whole exomic sequencing. Since nonmuscle myosins are known to directly and indirectly interact with a large number of proteins, we also will be looking for any mutations in other genes from patients with the diagnosis of POC. Should a mutation be identified, we will characterize the myosin (or other protein) in vitro or in mouse models to confirm that it is causative. Project Two: Our lab has generated a mouse with a point mutation (R709C) in NM IIB which develops a number of congenital heart defects, including septal defects, overriding aorta, and incomplete valve development. To investigate these abnormal findings, we utilized immunohistological analysis to identify expression patterns of extracellular and matricellular proteins that have been affected by the mutation in NM IIB and therefore may be downstream of NM IIB's role in heart development. We are working to create a valve cushion explant culture system to further explore the role of NM IIB in valve cell migration, matrix organization, and proliferation/apoptosis, all of which are important in proper valve formation. We are also working to create a tissue specific mutation in the cardiomyocytes of mice in order to elucidate the role of NM IIB in outflow tract myocardialization and misalignment.