An ENU-induced Point Mutation on Chromosome Eight Causes a Nephronophthisis-like Kidney Phenotype

Ji, Ae-Ri
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University of Guelph

The forward genetics ENU mutagenesis and phenotyping program at MRC Harwell is designed to produce and screen recessive mutant mouse pedigrees. We examined the histopathology of six selected pedigrees based on their clinical phenotypes. Homozygotes from pedigree MPC232 showed growth retardation, smaller organ size, increased serum BUN and creatinine, and renal dysplasia and hepatic oval cell/biliary hyperplasia as determined by histopathology. The phenotype was consistent and robust at 4-5 weeks of age leading to renal failure. The renal histopathology was characterized by tubular ectasia at the corticomedullary junction and increased interstitial mesenchyme. Considering the renal phenotype, a defect of mesenchymal-epithelial transition (MET) during renal development or a cystic kidney disease was suspected. An embryonic and early postnatal development study confirmed no developmental defect between embryonic day 15.5 and postnatal day 11. In kidneys of homozygous mice at 4-5 weeks of age, ciliary defect in tubules, epithelial-mesenchymal transition in some tubules, interstitial fibrosis, and interstitial infiltrates of inflammatory cells was verified and validated by Masson’s trichrome staining, immunoblotting, and immunohistochemistry. The temporal and histological features of this renal dysplasia resemble human nephronophthisis. Genetic analysis was performed to identify the causative mutations. Candidate mutations were narrowed down to 4 loci on chromosome 8 using linkage analysis, Illumina genome sequencing, and comparative genomic analysis of G3 littermate mice. Additional genetic investigation using Sanger sequencing and SNaPshot (single nucleotide polymorphism genotyping) further narrowed the candidate mutations down to two A to T mutations, at 8:104324177 (intron variant of Cmtm2b) and at 8:105912712 (missense variant of Pskh1). Little is known about either of these genes. However, a mutation in Pskh1 was recently found in mice with ciliopathies, and a protein interaction between PSKH1 and CDC6 has been proposed in humans. MPC232 homozygous mice showed a significant decrease in PSKH1 and CDC6 expression, and an increase in E-cadherin and β-catenin. Further, augmented cytoplasm-only β-catenin suggested that disrupted β-catenin-related pathways mechanistically underlie the phenotype of the MPC232 pedigree. In summary, we characterized a mouse model of nephronophthisis and we propose a potential novel role of Pskh1 in its pathogenesis.

ciliopathy, Pskh1, Cmtm2b, nephronophthisis, cystic kidney disease, ENU, point mutation