Autosomal recessive polycystic kidney disease (ARPKD) and Meckel syndrome (MKS) are examples of simple and complex recessive forms of PKD, respectively. ARPKD is considered a monogenic disease, due to PKHD1 mutations, while 16 genes, most commonly TMEM67, cause MKS. They are representatives of a larger group of diseases that result in PKD due to defects in the functioning of primary cilia, ciliopathies. During the previous funding period, increasingly complex mutation screening protocols from Sanger sequencing, through ciliopathy gene panels, to whole exome sequencing (WES) were employed to analyze patients. Further genetic and allelic heterogeneity was found for both diseases, along with evidence of oligogenic inheritance. Mutation screening will continue here employing WES and sophisticated in silico, including 3D modeling approaches, to score variants of uncertain significance (VUS) (Aim 1). Preliminary explorations were made by cellular assays to measure surface localization and protein maturation of the TMEM67 membrane protein, meckelin, with some mutations found to cause folding defects, which were rescued in an enhanced protein-folding environment, lower temperature. Here, cellular assays will be employed for meckelin and the Pkhd1 protein, fibrocystin (FPC), to assess the pathogenicity, penetrance and rescue ability of VUS (Aim 2). The value of potential chemical chaperones to rescue the maturation/localization of folding mutants will be tested employing the cellular systems. Mouse models mimicking the adult ciliopathy phenotypes associated with TMEM67 mutations, such as Joubert syndrome (JBTS), will be generated by engineering two homozygous, hypomorphic, folding mutations employing the CRISPR/Cas9 gene editing method (Aim 3). The adult phenotype will be characterized and chaperone treatment tested. Another discovery during the previous funding period was the greatly enhanced renal phenotype when a Pkhd1 null model (Pkhd1LSL/LSL) and the autosomal dominant PKD (ADPKD) gene, Pkd1, hypomorphic model, Pkd1RC/RC were interbred. While both models alone had a mild renal cystic phenotype, the digenic animals had explosive PKD and early lethality. Here, the pathogenesis associated with the loss of Pkhd1 to make the digenic model will be analyzed by RNA seq and cilia analysis (Aim 4). Since functional Pkhd1 can be reactivated, as a tagged gene/protein (Pkhd1PK) from the Pkhd1LSL model, we will determine if somatic reactivation of Pkhd1 alters the course of the PKD (Aim 4). Using a global expressing Cre, Pkhd1 will be activated at P3 and P14 and the resulting phenotype and expression profile analyzed. This is an important preliminary experiment to determine the phenotypic value of somatic repair of Pkhd1 in this disorder. Overall this study will contribute major insight into the etiology and pathogenicity of recessive forms of PKD, and provide clues for therapeutics.
This study of recessive forms of polycystic kidney disease is designed to better understand causes both in terms of the gene involved and the specific mutation. Mutations that result in protein folding and localization defects will be tested for the ability to be rescue by drug treatment. Also, with an eye to future treatment options, the value of genetically reactivating the disease gene in the cystic kidney will be evaluated.
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