Analysis Of HLA Gene And Haplotype Polymorphisms Of ESRD Patients In Chinese South Han Population

End-stage renal failure (End-stage renal disease, ESRD) is a chronic progressive renal damage caused by a variety of reasons, leading to a dramatic drop in the kidney which can not maintain its basic functions. Its clinical performances appear to the derangement of metabolites, water, electrolyte, acid-base balance, also known as uremia. The continued progress of various kidney diseases finally can lead to renal failure. According to the countries, the incidence and prevalence rate of chronic renal failure was on the rise. The total number of patients with chronic renal failure had reached 13.63 million in the U.S, the prevalence rate being 9900/100 million.Our country reported the incidence of end-stage renal failure was up to 100/100 million. As we all know, chronic renal failure is a serious harm to people's health, which have caused a heavy financial burden to the families and society. But its pathogenesis is still unclear.Now the current interpretation of CRF is high perfusion, pressure, filtration of the glomerular, high pressure glomerular capsule, and high metabolic theory of compensatory hypertrophy in renal tubular.Other than the above,glomerular and renal interstitial changes, tubular cells and renal interstitial infiltration of immune cells to produce a large number of inflammatory cytokines, are also involved in the process of deterioration of renal function.Human leukocyte antigen system is the human major histocompatibility complex (Major histocompatibitily complex, MHC), which encodes HLA and is the most complex of inmunocomplex. HLA gene region is divided into three categories.HLA-Ⅰgenes, including the classic A, B, C sites, the atypical E, F, G sites, were own with the function of identifying self and nonself in the immune system, so playing an important role in the immune systems against viruses and tumors;Ⅱgenes, including DP, DQ, DR locus in the classic area, DN, DM, DO site in atypical area, encoded its main products which are in charge of handling, processing, and providiing antigens;Ⅲgenes, include the genes encoding C2, Bf, C4A, C4B complement components and MIC, LTA, TNF, LTB, HSP70 etc, of which the latter expressed protein with inflammatory response.Discovered by Dausset in 1958, HLA has become one of the most active areas of research in the medical profession, eventually leding to the edge of a new discipline-the rapid development of immunogenetics. With the gradual deepening study, the role of HLA in human physiology and pathology major was found more and more important, not only in transplant rejection, immune response of regulation and genetic control, but also in recognizing and limiting each other of immune cells. Most of kidney disease is immune-mediated inflammatory reaction. There were many factors involved in immune inflammation, such as NK cells, B cells, T cells, platelets, granulocytes, etc. The receptor on the cell membrane of these immune cells need to be bond with HLA gene as the ligand, urging the immune inflammatory response. Some scholars had found there were the deposition of complement C4 in mesangial area of ESRD patients. The complement C4 was encoded by HLAⅢgene. These all indicate that the HLA plays an important role in the occurrence and development of kidney diseases. Researching on the association between HLA and ESRD, we could found the susceptible and protective genes associated with ESRD.The work was useful for kidney transplant patients and had the clinical meanings. In recent years, some foreign transplant centers had work more on the research that HLA played the immune genetic role in the progress of ESRD, founding the possiblely susceptible and protective HLA gene associated with ESRD. Rogus JJ proposed HLA susceptible genes play an important role in the pathogenesis of the existence of type 1 diabetic nephropathy complicated with end-stage renal failure, and reported susceptible genes may be located on HLA region of sixth chromosome through family research. CRISPIM JC reported 105 Brazilian patients awaiting kidney transplantation with end-stage renal failure were with significantly high antigen frequency of HLA-A78, DR11, low antigen frequency of HLA-B14, suggesting that HLA-A78, DR11 may be susceptible genes to ESRD in Brazilian patients with kidney disease. KARAHAN GE reported 587 ESRD patients with different the original disease expressed HLA-B58, DR3 with high antigen frequencies. And he proposed that the determination of susceptible genes were helpful for patients with renal disease to delay the progression of ESRD and for ESRD pratients to prevent the recurrence of the primary disease after transplantation.There is no domestic information about the large sample research on the association between HLA and ESRD. The paper was to research on the HLA typing data of renal transplant patients with end-stage renal failure and normal healthy people as subjects, using sequence specific primer polymerase chain reaction (polymerase chain reaction sequence specific primer, PCR-SSP) assay. Anlysis of the expression of the HLA-A, B, DR gene and haplotype polymorphism in the two group,we could known that how HLA played the immune and genetic role in the progress of the ESRD.Chapter 1 Analysis of HLA gene polymorphism in patients with end-stage renal failureObjective:Authors analyse the expression of HLA-A,B and DRB1 alleles of ESRD patients awaiting kidney transplantation in Chinese south Han population in order to discuss HLA polymorphism as the role of immune and genetic factors in the pathogenesis of ESRD.Methods:DNA was extracted by Salting-out method. Poly-merase chain reaction-sequence specific primer (PCR-SSP) were used to analyze the type of HLA-A, B and DRB1 alleles of 3911 ESRD patients and control group. HLA phenotype frequency (phenotype frequencies, PF)=observed positive number of HLA gene (n)/the number of ESRD patients. HLA gene frequency (gene frequencies, GF) GF=1-(1-PF)1/2.The gene frequency of two groups and genotype frequencies were compared by x2 test.The OR value were calculated by SPSS 13.0 software. P was corrected as Pc value (P value was multiplied by the detected antigen number of a loci).Results:1.19 HLA-A locus genes was detected in ESRD patients,17 ones detected in the control group. HLA-A*25 and A*80 were detected in ESRD patients more than the control group. The phenotype frequency of HLA-A30 (Pc＜0.00001) was significantly reduced in ESRD patients group, no gene with significantly higher frequency in the goup.2.41 HLA-B locus genes was detected in ESRD patients.38 ones were detected in the control group. HLA-B*28, B*42, B*72, B*78 and B*73 were only detected in the ESRD group.HLA-B*47 and B*64 were only detected in the control group. The phenotype frequency of HLA-B75 (Pc<0.00001,OR=1.479, 95%CI=1.315-1.664) was significantly higher in the ESRD patients, being no the gene with significantly lower frequency in the group compared with the control group.3.14 HLA-DRB1 locus genes were detected in ESRD patients,13 ones detected in control group. The gene HLA-DR*18 was detected in ESRD patients more than the control group. The phenotype frequency of the gene DR*04 (Pc<0.00001,OR= 1.294,95%CI=1.172-1.428), DR*17 (Pc<0.00001,OR=2.639,95%CI=2.240-3.110) were significantly higher,that of the DR*08 (Pc＜0.00001, OR=0.697, 95%CI=0.615-0.791)being significantly reduced in the ESRD patient group.Conclusion:1. Susceptible and protective HLA genes associated with ESRD were different in different regions and races.2. Although the phenotype frequency of HLA-A*30 were significantly different between the ESRD group and the control group, but its frequency was low in both groups. Considering the clinical appliance, we do not think there is association between A30 and ESRD.3. HLA-B75, DR4 and DR17 may be the susceptible genes to ESRD in patients of Han people in south China waiting for kidney transplantion, while the expression of HLA-DR8 in patients with renal diseases may delay the occurrence of ESRD. 4. The research of the susceptible and protective HLA gene associated with ESRD were helpful for kidney transplant patients to choose a suitable donor to improve the survival time of patients after transplantation and long-term effect of transplantation.Chapter2 Analysis of the susceptibility of HLA haplotypes which contain susceptible genes to ESRD.Objective:HLA typing data of patients waiting for renal transplant were analyzed compared with that of the healthy people, founding that HLA-B75, DR4, DR17 were the susceptible genes to ESRD (end-stage renal disease).Based on this, the susceptibitity of the HLA haplotype containing susceptible genes to ESRD were analysed.Methods:Population genetics software Arlequin was used to calculate frequencies of the two loci HLA haplotype in ESRD patients and control groups, x2 test was applied to test whether the frequencies of HLA haplotypes were with statistical difference. Then the odds ratio (odds ratio, OR) of these haplotypes were caculated by the formla Woolf. P value was calibrated as Pc value (P value was multiplied by the number of two loci HLA haplotype).Results:1. ESRD patients group expressed 11 susceptible gene HLA-B*75-A loci haplotypes, the control group expressed 7 ones. The haplotype A* 33-B*75, A*66-B*75, A*03-B*75, A*29-B*75, A*34-B*75, A*68-B*75 were only expressed in ESRD patients. The haplotype A30-B75, A25-B75 was only expressed in the control group. After comparing haplotype frequencie of the two groups, there was no significant difference.2 ESRD patient group expressed 27 susceptible gene HLA-DR* 04-B loci haplotypes. The haplotype B*44-DR*04, B*37-DR*04, B*57-DR*04, B* 81-DR*04, B*28-DR*04 (frequency of 0.166%,0.111%,0.061%,0.051%, 0.013%).were only expressed in ESRD patients.The control group expressed 25 ones. The haplotype B*63-DR*04, B*49-DR*04, B*42-DR*04 (all of frequencies were 0.040%) were only expressed in the control group. Compared two groups, the frequencies of these haplotypes were significantly increased(Pc<0.05)in ESRD patient group.They were the B*13-DR*04(1.149%vs0.043%),B*46-DR*04 (1.952%vs0.758%),B*58-DR*04(0.957%vs0.138%),B*75-DR*04(1.354% vs0.450%).3.ESRD patients group expressed 31 the susceptible gene HLA-DR*17-B loci haplotypes.The control group expressed 16 ones.The haplotype B*13-DR*17,B* 62-DR*17,B*39-DR*17,B*61-DR*17,B*48-DR*17,B*56-DR*17,B* 07-DR*17,B*45-DR*17,B*76-DR*17,B*18-DR*17,B*77-DR*17,B* 72-DR*17,B*78-DR*17,B*63-DR*17,B*42-DR*17 were expressed more in ESRD group than control group.Compared the haplotype frequencies of the two groups,there was no haplotypes with significantly higher frequency in the ESRD group.Conclusion:1.There were the different susceptible and protective HLA haplotypes associated with ESRD in different regions and races.2.The frequencies of haplotype B*13-DR*04,B*46-DR*04,B*58-DR* 04,B*75-DR*04 were significantly increased in the ESRD patient group.The haplotypes B*75-DR*04(Pc<0.05,OR=3.101,95%CI=1.664-5.779)consisted of independent susceptible gene B75,DR4,with a significantly susceptible collection, suggesting that it was the susceptible haplotypes associated with the end-stage renal failure.