A Study Of Acting Mechanism Of FAM40A Gene In Faminial FSGS

Background:According to foreign reports FSGS(focal and segmental glomerulosclerosis) accounts for 12-35% of adult primary nephrotic syndrome, FSGS, including primary and secondary two. Familial FSGS accounted for about 8.9% in primary FSGS, mainly due to mutations,including two genetic methods autosomal dominant and recessive, and different races have different genetic mutations. Familial FSGS compared with sporadic FSGS, no significant difference in early clinical manifestations, easily missed; but earlier onset familial FSGS, response to treatment is relatively weak, and easily moved to end-stage renal disease.In clinical work, our group found a family four generations of 36 people, 11 showed FSGS by biopsy pathology, and they does not belong to any known familial FSGS by genetic diagnosis. Objective:1.We determine pathogenic genes by genetic linkage analysis and whole exon sequencing; 2. We explore the pathogenesis by microarray; 3. We validate pathogenesis by knockout animal models Methods:Fisrt:the analysis of Genetic way and mutation genes:1.a newly discovered family of FSGS were pedigree analysis;2. using genetic linkage analysis and full exon scanning for possible mutations in genes.Second:Microarray gene expression profiling fam40 a effect and mechanism in mouse podocytes.1. Gene expression profiling microarray analysis to understand the interaction between genes and fam40a;2, the use of qPCR, western blotting validate microarray results.Third Build a fam40 a conditional knock out mice model to research the pathogenic role of fam40 a. Experimental design as follows:1. the use of homologous recombination in mouse ES cells and ES cell technology blastocyst injection technology, to obtain chimeric male mice.2. The chimeric male mice mating with C57 female mice to obtaine flox heterozygous mice.3.flox heterozygous mice mating with EIIAcre mice to obtain systemic knockout fam40a(+/-) mice.4 We collect heterozygous, wild-type mice hematuria and renal tissue specimens to observe fam40 a pathogenic situation; the use of qPCR, western blotting to detect changes of fam40 a and laminin in mouse kidney. Result:One, pedigree analysis and genetic scan1. By pedigree analysis,we found that mode of inheritance in this familial FSGS is autosomal dominant;2. We found that the causative genes is FAM40 A bu genetic linkage analysis and whole exon scan.Second, the cell model parts:1. Microarray Results: Compared with the control group, the expression of fam40 a in the interference group was down regulation, the gene expression of lamb1, lamb2 which encode laminin was down regulation;2.qPCR, western blotting Results: Compared with the control group, the interference group lamb1, lamb2 reduced expression; we found that the expression of laminin in the interference group was down regulations by Western blotting.Third, the animal model portion:1. successfully constructed flox heterozygous mice;2. successfully constructed fam40 a heterozygotes;3. Clinical indicators: ①24 hour urine protein increased;②serum tests: blood urea nitrogen and creatinine are in the normal range.4. pathological indicators: ① light microscope: no significant pathological changes ② electron microscopy: heterozygous Group: basement membrane thickening, wrinkled, podocyte fusion segments; ③ Immunohistochemistry: compared with the wild-type group, heterozygous group kidney fam40 a staining is lighter, laminin Traveling along the GBM, stained is lighter.5,Fam40 a and laminin detection in the wild-type control group and model group renal tissue: ① real time PCR show: compared with wild-type mice heterozygous,mRNA expression of fam40 a, lamb1 and lamb2 decline;②western blotting Show: compared with wild-type, expression of fam40 a and laminin protein in the heterozygous mice kidney tissue is lower; Conclusion:By pedigree analysis and genetic testing, we found that the genetic approach in this pedigree is autosomal genetic profiling,and the mutant gene is FAM40 A. at 8 weeks after birth the fam40 a homozygous model(fam40a +/-) mice showed significant proteinuria, and electron microscopy showed foot process fusion, GBM thickening and shrinkage. In fam40 a heterozygous mice and interference fam40 a podocytes the expressing of laminin was affected.Above all: FAM40 A is very important to maintain glomerular filtration function. Its changes will cause in proteinuria, podocytes and basement membrane disease; FAM40 A may influence podocyte adhesion to the basement membrane by affecting the expression of laminin, eventually leading to glomerular filtration membrane dysfunction.