Mapping And Studying Susceptibility Gene To Lupus Nephritis

Systemic lupus erythematosus ( SLE ) is characterized by multiorgan pathology and autoantibodies agaist a variety of autoantigens such as double-stranded DNA, intrcellular ribonuclear proteins and membrane phospholipids. The most severe pathological concequences of SLE result from the deposition of immune complexes on basement membranes, leading to the development of glomerulone-phritis(GN)and culminating in kidney failure. SLE susceptibility has strong genetic basis, SLE susceptibility appears to be inherited in a polygenetic fashion involving both environmental and genetic factors, we set up New Zealand black (NZB)xNew Zealand white ( NZW) FlxNZB backcross mice model and used polymorphic microsatlite marker quantitive trait locous(QTL) analysis and amplified fragment length polymorphism to map the susceptibility genes for Lupus Nephritis ( LN) in New Zealand mice and study the polymorphism of human Csf3 gene and to see if there was relationship between the polymorphism of human Csf3 gene and SLE/LN.MATERIALS(NZB x NZW) F1 mice, originally obtained from the Shizuoka Laboratory Animal Center, were maintained in the animal facility of Chinese medical University ( shenyang, China). Backcross mice were obtained by crossing female (NZB x NZW)F1 mice with male NZB mice. All these animals were housed in the same room and fed an identical diet. Because of female predominance of SLE, only female mice were used in the present study.215 with SLE,who were being follow up in then Department of nephrology , Chinese medical university were studied. All patients were Northern Chinese,andfulfilled the classification criteria of American College of Rheumatology. The control group consist of 459 healthy people ,78 other non-sle autoimmune patients and 91 primary kidney disease patients. All of them were Northern Chinese . The clinical characteristics of the patients were obtained by reviewing their medical records. When sufficient information was not available, such patients were excluded from the paticular analysis.METHODSDNA was extracted from the mouse tail tissues. Select microsatlite markers that had polymorphism between NZB and NZW. For quantitative trait loci ( QTL) analysis, severity of renal disease was scored from grade 0 to grade 6, according to the amount of urinary albumin. Linkages of particular microsatellite loci with development of proteinuria was estimated using a computer package program of MAPMAKER/EXP and MAPMAKER/QTL to identify chromosomal locations of QTL. The likelihood ratio statistic ( base-10 likelihood of the odds (LOD) score) of 3=3. 3 was used as the threshold for statistically significant linkage.SUE patients DNA was extracted from whole blood using standard molecular techniques. Finding the DNA sequence of Csf3 gene on internet and design the primer according to it ( NT-010755 ). Single-strand DNA fragments in the gel were visualized by silver staining.RESULTS1. According to the outcome of QTLanalysis LN were linked to microsatlite markerD11Mit263 on chromosom 11 andD17Mit61 on chromosom 11 in NZW. There is Csf3 near D11Mit263 and there were Tnfa and H-2 genes near D17Mit61 . It is suggested that these genes proboboly were the candidate genes for LN.2. Comparing backcross progeny DYlMit61 and DllMit263 genotypes, we found in both group the cumulative incidence of proteinuria of B/W genotypes was greater than that of W/W genotypes,that meaned the candidate genes camefromNZW.3. The backcross progeny was separated into four groups, according to combinations of D17Mit61 andD11Mit263 genotypes, it is conceivable that the mice that has D17Mit61 NZB/NZWand NZB/NZW D11Mit263 genotypes stimus-ly has a higher incidence of proteinuria than that has only D17Mit61 NZB/NZW or NZB/NZW DllMit263 genotypes . It means susceptibility genesof LN can promote each other.4. Among the 12 productions of Csf3 gene segements ,4 can be found having single nucleotide polymorphism with the techneque of SSCP. They are Csfi (394) Csf3 (1205) Csf3 (CDS) and Csf3 (1869).5. The...