Genetic basis of congenital nephrotic syndrome and generation of an animal model

Sammanfattning: Congenital nephrotic syndrome, NPHS1, is a disease highly enriched in Finland with incidence of 1:82oo births.The disease is characterized by massive proteinuria, go % of which is albumin, starting in utero, large placenta, which weighs over 25 % of the child's birth weight, and manifestation of proteinuria soon after birth. Historically, NPHS1 children have died before the age Of 2, but an aggressive nutritional therapy together with kidney transplantation can today save the life of the child. Irregular dilation of proximal and distal tubules and irregular fusion of podocyte foot processes are the most typical findings from histological and ultrastructural studies, respectively. The homogenous population structure of Finns, with the presence of a founder effect and substantial isolation, was utilized in attempts to clone the disease causing gene using linkage disequilibrium. Linkage disequilibrium is an efficient tool for positional cloning and can be used to narrow down the critical chromosomal region into which the disease gene is first linked. Haplotype sharing analysis of genetic markers, which were found to be in linkage disequilibrium in the critical region of the disease gene 19q13.1, was used in specific prenatal diagnosis Of NPHS1. Four main categories of haplotypes in Finnish NPHS1 patients were found, prenatal diagnosis being 95 % accurate in the Finnish families with history Of NPHS1. The 150 kb chromosomal region containing the NPHS1 gene was sequenced entirely and the sequence data was subsequently subjected to computer-based exon prediction. Over 100 potential exons and at least ten novel genes were found in this region, four of which were subjected to mutation search. One of the genes bore mutations, two of which comprise over go % of the Finnish NPHS1 chromosomes either as homozygote or compound heterozygote. The more common mutationFinmajor-is a deletion in exon 2 and results in a frameshift, which generates a stop codon within the same exon. The second mutation-Fin minor -is a nonsense mutation in exon 26. The protein product of the NPHS1 gene, named nephrin, comprises 1241 residues and is, based on its predicted domain structure, a member of the immunoglobulin superfamily. The gene was first shown by Northern hybridization analysis to be expressed solely in human embryonic and adult kidney and then by in situ analysis in human embryonic kidney in the periphery of mature and developing glomeruli of the kidney. The primary sequence of mouse and rat nephrin homologs were obtained from CDNA Clones. The sequence identity between rat and mouse proteins was about 93 %, both rodent protein sequences showing about 83 % identity to overlapping human protein regions. The predicted domain structures of nephrin of these species are similar to that of human. The in situ analysis of mouse nephrin showed that at the embryonic day 11 the cranial tubules of the mesonephric kidney with podocyte- like structures were strongly positive. Expression was seen in metanephros from E13 in late s-shaped bodies in presumptive podocytes. Expression was also seen at Ell in the hindbrain area continuing dorsally in the mantle layer neurons of the spinal cord. From E13 to E17, signal was detected in the neuroepithelium of the cerebellum primordium at the roof of the fourth ventricle. The epithelial cells of the developing choroid plexus remained negative at all stages. A mouse model with targeted inactivation of the nephrin gene was generated in order to study the expression pattern in more detail with the beta-galactosidase gene inserted in frame to nephrin exon 1. The different genotypes were born in expected ratios, and inactivation of the nephrin gene was confirmed with Western analyses of extracted kidney proteins. The null allele animals, however, became edemic soon after birth and died within 24 hours. Protein analysis of the urine showed extensive proteinuria, most of the protein being albumin. Histological and ultrastuctural findings in the kidney were similar to human NPHS1, thus confirming that a mouse model Of NPHS1 was generated. The expression of beta-galactosidase was observed, in addition to kidney, transiently in the developing spinal cord and pancreas, in main olfactory bulb glomeruli, in hippocampal dentate gyrus, and in cerebellum. In newborn mice, nephrin was localized to radial glial cells. Histological analysis of newborn mice brain revealed no substantial abnormalities in the cerebellum.