Genetic analysis of the ciliopathies ARPKD and Meckel syndrome

描述（由申请人提供）：在过去的几年中，由于纤毛相关基因的突变，多囊肾病（pkd）已经明确为纤毛病。在这个建议中，我们将研究两种明显隐性遗传PKD，常染色体隐性PKD （ARPKD）和Meckel综合征（MKS）。在ARPKD中，这种疾病主要局限于肾脏和肝脏，而MKS是一种致命的综合征型PKD，通常也会发现中枢神经系统和数字缺陷。由于pkpkd突变，ARPKD被认为是一种遗传同质性疾病，尽管对具有ARPKD样表型的大量人群的研究发现PKHD1突变仅在大约一半的个体中发生。MKS在遗传上更复杂，有11个基因被鉴定出来，其他位点的等位基因也被认为起作用；可能的寡生遗传。虽然从表面上看，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PKD （ADPKD）基因PKD1的亚形态突变已被证明可引起arpkd样疾病和外显子富集，NGS显示MKS患者中存在PKHD1和相关PKHDL1等位基因。本研究的目的是采用尖端的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重要性，以及纤毛病基因之间的上位效应的强度。在第一个目标中，将通过纤毛病和其他纤毛基因的外显子富集和下一代测序，以及适当的全外显子组分析，共分析约100个MKS和约200个arpkd样家族。变异将被生物信息学评估，并在Aim 2细胞系统和模式生物秀丽隐杆线虫和斑马鱼中评估最有希望的变异的意义。在MKS和一些arpkd样家族中发现的表型是由多个基因的等位基因引起的，这一概念将在Aim 3中使用线虫、斑马鱼和杂交的纤毛病小鼠模型进行测试。最终目的是通过开发敲入/敲出小鼠模型来表征与所有转录本破坏相关的表达和表型，探索PKHD1平行基因和疑似纤毛基因PKHDL1的作用。这些研究将为arpkd样疾病和MKS的病因提供更清晰的观点，并阐明这些“简单”遗传疾病的真正复杂性。

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.