Genetic analysis of the ciliopathies ARPKD and Meckel syndrome

描述(申请人提供)：在过去的几年里，由于纤毛相关基因的突变，多囊肾病(PKD)是一种纤毛疾病。在这个方案中，我们将研究两个明显的隐性遗传性PKD，常染色体隐性遗传性PKD(ARPKD)和Meckel综合征(MKS)。在ARPKD中，疾病主要局限于肾脏和肝脏，而MKS是一种致命的综合征性PKD，通常也会发现中枢神经系统和手指缺陷。ARPKD被认为是一种由PKHD1突变引起的遗传同质性疾病，尽管对具有ARPKD样表型的大量人群的研究发现，只有大约一半的人存在PKHD1突变。MKS在遗传上更为复杂，有11个基因被发现，其他基因座的等位基因也被认为起到了作用；可能是寡基因遗传。尽管从表面上看，这些疾病看起来非常不同，但最近的出版物和我们的初步数据表明，基因重叠。常染色体显性遗传性PKD(ADPKD)基因PKD1亚型突变可导致ARPKD样病和外显子丰富，NGS在MKS患者中显示PKHD1及相关的PKHDL1等位基因。这项研究的目的是使用尖端遗传学方法来了解这两种疾病的全部遗传负荷，同时将利用动物模型来确定检测到的变异的意义，以及纤毛病变基因之间的上位性效应的强度。在第一个目标中，总共大约100个MKS和大约200个ARPKD样家系将通过外显子丰富和下一代纤毛疾病和其他纤毛发生的测序进行分析，如果合适的话，还将进行完整的外显子分析。将在AIM 2细胞系统和模式生物秀丽线虫和斑马鱼中对变异进行生物信息学评估，以评估最有希望的变异的重要性。在MKS和一些类似ARPKD的家系中发现的表型是由于多个基因上的等位基因，上位性起核心作用的概念，将在目标3中使用线虫、斑马鱼和纤毛病小鼠模型的杂交进行测试。最终目的将通过建立敲入/敲除小鼠模型来研究PKHD1准同源基因和可疑纤毛基因PKHDL1的作用，以表征与所有转录本中断相关的表达和表型。总之，这些研究将为ARPKD样疾病和MKS的病因学提供更清晰的观点，并阐明这些“简单”遗传病的真正复杂性。

DESCRIPTION (provided by applicant): In the last few years it has become clear that polycystic kidney diseases (PKDs) are ciliopathies, due to mutations in cilia related genes. In this proposal we will study two apparently recessively inherited PKDs, autosomal recessive PKD (ARPKD) and Meckel syndrome (MKS). In ARPKD, the disease is largely restricted to the kidney and liver, while MKS is a lethal, syndromic PKD in which central nervous system and digital defects are typically also found. ARPKD is considered a genetically homogenous disorder due to PKHD1 mutation, although studies of large populations with an ARPKD-like phenotype have found PKHD1 mutations in only about one half of individuals. MKS is genetically more complex with 11 genes identified and alleles at other loci also thought to play a role; possible oligogenic inheritance. Although on the surface these diseases appear very different, recent publications and our preliminary data suggest genetic overlap. Hypomorphic mutations in the autosomal dominant PKD (ADPKD) gene, PKD1, have been shown to cause an ARPKD-like disease and exon enrichment and NGS has shown PKHD1, and the related PKHDL1, alleles in MKS patients. The purpose of this study is to employ cutting-edge genetic methods to understand the full genetic load in these two disorders, while animal models will be utilized to determine the significance of detected variants, plus the strength of epistatic effects between ciliopathy genes. In the first aim a total of ~100 MKS and ~200 ARPKD-like families, that are genetically unresolved, will be analyzed by exon enrichment and next-generation sequencing of ciliopathy and other ciliogenes, plus, when appropriate, whole exome analysis. Variants will be assessed bioinformatically and in Aim 2 cellular systems and the model organisms C. elegans and zebrafish employed to assess the significance of the most promising variants. The concept that the phenotype found in MKS and some ARPKD-like families is due to alleles at more than one gene, that epistasis plays a central role, will be tested in Aim 3 employing C. elegans, zebrafish and interbreeding of mouse models of ciliopathies. The final aim will explore the role of the PKHD1 paralog and suspected ciliogene, PKHDL1, by developing knock- in/out mouse models to characterize expression and the phenotype associated with disruption of all transcripts. Together these studies will provide a much clearer view of the etiology of ARPKD-like disease and MKS and clarify the true complexity of these "simple" genetic diseases.