Association Between Polymorphism At The Human IL-10RA Locus And Anti-dsDNA Antibody Rallating To Systemic Lupus Erythematosus

Systemic lupus erythematosus ( SLE) is the prototype of systemic autoimmune disease,' characterized by hypergammaglobulinemia and autoantibodies against a variety of autoantigens such as double - stran-ded DNA, which can cause lupus nephritis ( LN) through the deposi-tion of immune complex in kidneys. The treatment of SUE is uneffec-tive presently because of poor understanding to etiology that affects pa-tients. Pathogenetically, numerous genetic, hormonal, environmental and immunoregulatory factors contribute to the expression of the dis-ease.Among these, interleukin -10 (IL-10) and its receptor (IL -10 R) are related to the overproduction of pathogenic antibodies and disorder of inflammation. IL-10, firstrecongnized for its ability to in-hibit activation and effector function of T cells, monocytes, and mac-rophages, is a multifunctional cytokine. The principal routine function of IL - 10 appears to be to limit and ultimately terminate inflammatory responses. On the other hand, IL-10 relate to the balance of humoral immunity and cytoimmunity, which contribute to the pathologic process of autoimmune disease. Several studies have demonstrated that the serum levels of IL - 10 production had a strong genetic basis and were associated with SUE and its phenotype (lupus nephritis, rapidlyprogressive glomerulonephritis etc). Because SLE is a genetically complex disease with polygenic disorder and genetic heterogrneity, it is difficult to locate the gene predisposing for the disease and to repli-cate the susceptibility loci previously described. Therefore the linkage of SLE to IL -10 gene is not confirmed.In consideration of the immunologic function of IL - 10 depending on its receptor, someone have supposed that dysfunction of IL - 10 re-ceptor may be related to small rat lupus. Recently, a genome scan re-ported evidence for linkage of SLE to 11 q23 ( position of IL - 10RA coding gene). According to the above consideration, we sequenced IL - 10RA DNA from the SLE patients (from Shenyang City R. 0. C) to find novel polymorphisms in IL - 10RA gene. We also researched the relationship of IL - 10RA genotype with SLE and ds - DNA antibody via SSCP( single strand conformation polymorphism) and ELISA in or-der to confirm the linkage of SLE to the IL - 10RA gene on the chro-mosome Ilq23. Identification of the genes involved in the develop-ment of SLE will provide important insight into the development of the disease as well as the development of autoimmunity and opportunities to improve diagnosis and treatment.Materials and methodsMain reagents and instrumentPCR buffer and Taq DNA Polymerase were bought from Richo Corporation; ELISA plate, calf thymus ds - DNA antigene, enzyme mark antibody was presented from the Setaito University Japanese. Su-per - speed low temperature centrifugal machine ( Beckman Company USA) ; PCR amplifications were performed using the PE9600 ThermalCycler ( Perkin Elmer Company USA). Primers were purchased from Shanghai Sangon Corporation; The PCR - amplified products were se-quenced using ABI automated sequencer by Shanghai Genecor Compa-ny Method1. SubjectsOne hundred and sixty SLE patients from China Medical Univer-sity were divided as SLE group. Tow hundred normal blood donor were divided as control group. All SLE patients met the revised American College of Rheumatology criteria for systemic lupus erythematosus.2. MethodsBlood samples were collected in two groups, from which white blood cell and plasma were isolated. For searching novel polymor-phisms in IL - 10RA gene, ten random DNA samples were used to PCR - amplify and sequence the region of the IL - 10RA with prim-ers: Forword 5' -TCC ATA GTC CAA TCA GGT CT; Reverse 5' -GAG ACG TTG TTG GGA ATC AC. SSCP analysis was used to dis-tinguish the different types of zone electrophcresis. All of samples from two groups were analyzed via SSCP. And - dsDNA concentra-tions in plasma of SLE group were determined by ELISA.3. Statistical analysisThe x test was used to compare differences in the dist...