Clinical And Experimental Study Of FCGR3B Copy Number Variations In Renal Diseases

IntroductionGreat improvement has been made in immunosuppressive therapy, the long-term graft and recipients survival rate has remained static, with only 54.4 percent of kidneys from living donors and 36.4 percent of kidneys from deceased donors functioning 10 years after transplantation. Many efforts had made to achieve an ideal result. HLA matches, pre-transplant PRA, CDC levels and recently MICA are among important factors affect acute rejection rate and hence the long time graft survival for kidney transplantation. However, it is not seldom that despite a similar well handled above factors and a standard immunosuppressive protocol, the clinical outcomes are quite different. The elucidation for the underground mechanisms may give some clues for improving the transplant outcome.Transplant relevant genes' polymorphisms was once a hot topic that may influence the transplant outcomes, e.g. vimentin, platelet-specific antigens, genes that encode various cytokines, chemokines and their receptors, and molecules of the renin-angiotensin system. Recently, a totally new type of gene polymorphisms due to copy number variations (CNVs) take scientists' eyes: deletions of the UGT2B17 gene contribute to ethnic and inter-individual differences in testosterone metabolism and risk of prostate cancer; increased copy number of the CCL3L1 gene is associated with reduced susceptibility to HIV infection and progression to AIDS. To our interest, reduced FCGR3B copy number was reported to predispose people to lupus nephritis. In the current study, we determined Fcgr3B copy number of kidney transplantation recipient and analyzed its' effect on acute rejection rate and graft survival. Part I Recipients Fcgr3B copy number variation and acute rejection, graft survival in kidney transplantationAims HLA matches, pre-transplant PRA, CDC levels and recently MICA are among important factors affect acute rejection and graft survival for kidney transplantation. Despite well handled above factors, results are not always ideal. Recently, researches on gene copy number variation and its' clinical importance exerted great interest also for nephrologist. Low Fcgr3B copy number predispose to nephritis in SLE patients. We sought to determine if Fcgr3B copy number variation influence acute rejection and graft survival in kidney transplantation.Methods We determined Fcgr3B copy number of 869 kidney transplantation recipient and 761 unrelated healthy samples with q-PCR. The association between Fcgr3B gene copy number and the acute rejection rate and graft survival were analyzed with corresponding statistical method. The median length of follow-up was 916 days, respectively.Results Fcgr3B copy number distribution of healthy East Chinese showed a similar result with a report from UK. The main frequency, i.e. one and two copy in healthy control group were 41% and 44%, respectively; while in the kidney transplant group the corresponding frequency were 55% and 34%, respectively (p<0.01). Acute rejection rate of kidney transplant recipients with 0,1,2,3 and 4 copy Fcgr3B was 50%, 16.8%, 8.0%, 16.4% and 16%, respectively (p<0.001). Compared with 2 copy recipient, the hazard ratio for risk of acute rejection of recipients with 0, 1 copy of Fcgr3B were 9.66(p<0.001), 2.12(p=0.006), respectively. In subgroup serial analyses including important factors of HLA match condition, warm ischemia time and recipient age, acute rejection rate difference proved to be more significant in the high risk categories. The cumulative kidney graft survival was different with differentFcgr3B copy (p<0.05).Conclusions Fcgr3B copy number variation was an independent factor that influencethe acute rejection and graft survival in kidney transplantation. Part II The relationship between FCGR3B copy number variations and IgA nephrology in ChineseAims IgA nephrology (IgAN) was the most common reason for Chinese patients to end stage renal disease (ESRD). Many researchers reported that the heredity was related to the onset of this disease. Our previous study found the distribution of gene FCGR3B copy number variations (CNVs) between healthy blood donors and renal recipients was significantly different. Our current study was to elucidate the relation of FCGR3B CNVs between IgAN patients and healthy blood donors, and if this gene CNVs was involved in the pathological severity of this disease.Methods 146 IgAN patients and 134 healthy blood donors were included in the study. DNA of the total 280 subjects were extracted from their peripheral blood mononuclear cells, and analyzed by real time PCR.Results The distribution of gene FCGR3B CNVs was significantly different between healthy donors and IgAN patients in Chinese (P =0.028) . One copy of this gene was easier found in IgAN patients than that of healthy blood donors. In addition, the proportion of single copy gene FCGR3B was observed higher in pathological severe IgAN patients than in pathological slightly changed subjects (P =0.021) .Conclusions CNVs of gene FCGR3B was related to IgAN in Chinese, low copy number (zero and one copy) was a risk factor for the susceptibility to this disease. Low copy of gene FCGR3B was correlated to severe pathological changes of this disease. Part III Difference of mRNA Expression and Neutrophil Phagoctyosis in different FCGR3B Gene Copy Number DonorsAims The relation between gene copy number variation and the susceptibility of diseases were verified by some research groups. Our previous study found that Fcgr3B copy number variation was an independent factor that influence the acute rejection and graft survival in kidney transplantation. Data supporting the hypothesis that low copy number of Fcgr3B gene predispose disease susceptibility of IgAN—the highest morbidity type of nephritis in China, and the possibility to the susceptibility for nephritis of progression to ESRD. The aim of this study was to elucidate the difference of mRNA expression and phagocytosis ability of neutrophil between different FCGR3B copy number renal allo-grafts recipients and healthy blood donors.Methods Extracting the total RNA of peripheral blood mononuclear cells (PBMC) from different FCGR3B copy number healthy blood donors, and screened the mRNA expression by real time PCR. Extracting the total neutrophil of peripheral blood mononuclear cells (PBMC) from different FCGR3B copy number healthy blood donors and renal allo-grafts recipients (no acute rejecters), and co-cultured with Staphylococcus aureus at 37℃, testing the phagocytosis ratios of neutrophil after 60 minutes and 120 minutes.Results mRNA expression of housekeeping gene GAPDH between 5 FCGR3B one copy healthy blood donors and 5 FCGR3B two copy healthy blood donors, verified by Student' T test, P = 0.352, that displayed no significant difference of mRNA expression in housekeeping gene between two groups. mRNA expression of gene FCGR3B between 5 one copy healthy blood donors and 5 two copy healthy blood donors, verified by Student' T test, P = 0.0001. There was no significant difference about the phagocytosis ratios of neutrophil after 60 minutes and 120 minutes, in different FCGR3B copy number healthy blood donors and renal allo-grafts recipients (no acute rejecters).Conclusions There was significant difference of mRNA expression about gene FCGR3B in peripheral blood mononuclear cells (PBMC) from different FCGR3B copy number healthy blood donors. mRNA expression from one copy gene FCGR3B donors was lower than that of two copy donors. No significant difference was observed about the phagocytosis ratios of neutrophil in different FCGR3B copy number healthy blood donors and renal allo-grafts recipients (no acute rejecters). Part IV Sequencing of Gene FCGR3B Exon 5 in Chinese Renal allograft Recipients and Healthy Donors in Zhejiang ProvinceAims Many researchers reported the relation between gene copy number variations (CNVs) including FCGR3B and susceptibilities of some human diseases. This study was to elucidate the single nucleotide polymorphism (SNP), nucleotide segments insertion or deletion of this gene between different copies.Methods 20 healthy blood donors (10 have one copy and 10 have two copies of gene FCGR3B each), 20 renal allograft recipients with one copy of FCGR3B (10 subjects suffered acute rejection after transplantation and 10 subjects did not). FCGR3B exon 5 part nucleotide sequence of genomic DNA was amplified by real time PCR using SYBR Green I method. PCR products were directly sequenced or after cloning vector by sequencing machine.Results Exon 5-204 G/A mutation was observed in 9 of 10 subjects with 1 or 2 copies of FCGR3B each. 9 of 10 renal allograft recipients with acute rejection and 8 of 10 renal recipients without rejection were observed the same mutation. No SNP was found in exon 5 coding sequence, and no nucleotide segment insertion or deletion in part nucleotide sequence of 5' un-translated region (5' UTR) of exon 5 in FCGR3B.Conclusions There was likely no high frequency mutation of SNP in exon 5 coding sequence, and no nucleotide segment insertion or deletion in part nucleotide sequence of 5' UTR in exon 5 of FCGR3B in Chinese from Zhejiang province.