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.
""""""""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.
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