Identifying all Meckel-like ciliopathy genes by established total exome capture a

描述（由申请人提供）： 通过外显子捕获和大规模测序鉴定所有Meckel样纤毛病变基因。初级纤毛-中心体复合体（CCC）蛋白质突变的鉴定定义了“视网膜-肾纤毛病”的疾病组，这是生命最初30年中肾衰竭的最常见遗传原因。视网膜-肾纤毛病变可导致肾功能衰竭、视网膜变性、肝纤维化、智力低下以及脑、骨和心脏的畸形。初级纤毛是感受细胞外部信号的通用感觉细胞器。中心体蛋白调节细胞极性和定向细胞分裂，并参与有丝分裂纺锤体。我们先前已经鉴定了9个基因（NPHP 1-NPHP 9）中的隐性突变导致视网膜-肾纤毛病变，在其发病机制中涉及Wnt信号传导、hedgehog信号传导和细胞周期调节。我们证明，错义突变导致组织维持和修复的轻度表型，并导致器官退化和过早老化（Senior-Loken综合征），而截短突变在形态发生期间导致严重的发育缺陷，并导致畸形或发育不良（Meckel综合征）。在50%的这些严重的“梅克尔样纤毛病”的致病基因仍然是未知的，我们产生的证据表明，超过30个未知的基因必须存在。最近发现的基因是罕见的，需要在单个受影响的个体中进行基因鉴定。 因此，我们采用了连续大规模测序的人类外显子组捕获技术。通过检查具有严重表型和纯合突变的个体，我们克服了外显子组捕获产生来自正常参考序列的许多变体的问题，这阻止了致病突变的鉴定。使用这种方法，我们已经确定了SDCCAG 8，PRKACA和MAP 2的突变作为Meckel样纤毛病的新原因。为了发现大多数未知的纤毛病变基因及其相关的信号传导机制，并研究它们在器官发育异常/畸形以及肾、视网膜、脑和肝的纤维化/变性中的作用，我们提出：1.使用外显子组捕获和实验室中建立的连续高通量测序鉴定Meckel样纤毛病的所有致病成分。2.使用“伤口愈合试验”进行高通量基因敲除和中心体定向不良的药物筛选。本研究采用全外显子组捕获和大规模测序的新技术，加快Meckel样纤毛病的基因鉴定。它将定义纤毛-中心体和有丝分裂纺锤体复合体的主要疾病相关信号传导途径，并阐明肾脏、视网膜、脑和肝脏中纤毛病的疾病机制，因为它们涉及i）器官发育不良和畸形的形态发生缺陷以及ii）组织维持和修复机制期间的器官变性和过早衰老。 公共卫生相关性： “纤毛病”描述了一组遗传性疾病，这些疾病是由每个受影响个体中的单个基因引起的。然而，许多不同的基因可能会导致不同个体的这种疾病，导致出生时的畸形或肾脏，眼睛，肝脏和大脑在以后的生活中退化。对引起纤毛病变的基因的鉴定产生了对器官畸形和过早衰老的疾病机制的深刻的新见解，但一直难以实现。我们帮助建立的外显子捕获和高通量测序的新技术，现在将被用于非常快速地识别和表征数十个未知的纤毛疾病基因。新的纤毛病变基因的鉴定将为许多器官的畸形和变性的疾病机制提供进一步的见解。它将允许开发这些退行性疾病的动物模型和新的治疗方法。

DESCRIPTION (provided by applicant): Identify all Meckel-like ciliopathy genes by exon capture & large-scale sequencing. Identification of mutations in proteins of the primary cilia-centrosome complex (CCC) defined the disease group of "retinal-renal ciliopathies", the most frequent genetic cause of kidney failure in the first 30 years of life. Retinal-renal ciliopathies cause kidney failure, retinal degeneration, liver fibrosis, mental retardation and malformations of brain, bone and heart. Primary cilia are universal sensory organelles that sense cell-external signals. Centrosomal proteins regulate cell polarity and oriented cell division and participate in the mitotic spindle. We have previously identified recessive mutations in 9 genes (NPHP1-NPHP9) as causing retinal- renal ciliopathies, implicating Wnt signaling, hedgehog signaling, and cell cycle regulation in their pathogeneses. We demonstrated that, missense mutations cause a mild phenotype of tissue maintenance and repair and leads to degeneration and premature aging of organs (Senior-Loken syndrome), whereas truncating mutations cause severe developmental defects during morphogenesis and lead to malformation or dysplasia (Meckel syndrome). In 50% of these severe "Meckel-like ciliopathies" the causative gene is still unknown, and we generated evidence that more than 30 unknown genes must exist. Recently identified genes were rare, necessitating gene identification in single affected individuals. We therefore adapted the technique of human exome capture with consecutive large-scale sequencing. By examining individuals with severe phenotypes and homozygous mutations, we overcame the problem that exome capture yields to many variants from normal reference sequence, which prohibits identification of the disease-causing mutation. Using this approach we already identified mutations in SDCCAG8, PRKACA and MAP2 as novel causes of Meckel-like ciliopathies. To discover most of the unknown ciliopathy genes and their related signaling mechanisms, and to study their role in organ dysplasia/malformation as well as fibrosis/degeneration of kidney, retina, brain and liver we propose to: 1. Identify all causative components of Meckel-like ciliopathies using exome capture with consecutive high-throughput sequencing as established in the lab. 2. Perform high-throughput gene knockdown and drug screening for malorientation of centrosomes using the "wound-healing assay". This study uses the new technology of total exome capture and large-scale sequencing to accelerate gene identification in Meckel-like ciliopathies. It will define major disease-relevant signaling pathways of the cilia- centrosome and mitotic spindle complexes and elucidate disease mechanisms of ciliopathies in kidney, retina, brain and liver as they pertain to i) morphogenetic defects of organ dysplasia and malformation as well as ii) organ degeneration and premature aging during mechanisms of tissue maintenance and repair. PUBLIC HEALTH RELEVANCE: "Ciliopathies" describe a group of genetic diseases that are caused by a single gene in each affected individual. However, many different genes may cause this disorder in different individuals, causing malformation at birth or degeneration in later life of kidney, eye, liver and brain. Identification of ciliopathy- causing genes has generated deep novel insights into disease mechanisms of malformation and premature aging of organs but has been difficult to accomplish. The new technique of exon capture and high-throughput sequencing, which we have helped establish, will now be utilized to very rapidly identify and characterize the dozens of unknown ciliopathy genes. Identification of novel ciliopathy genes will provide further insights into disease mechanisms of malformation and degeneration of many organs. It will allow development of animal models and novel therapeutic approaches to these degenerative diseases.