Indian Journal of Nephrology Indian J Nephrol 2001;11: 88-97

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1 88 Indian Journal of Nephrology Indian J Nephrol 2001;11: ARTICLE HLA gene and haplotype frequency in renal transplant recipients and donors of Uttar Pradesh (North India) S Agrawal, AK Singh, RK Sharma Deptt. of Genetics and Nephrology Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow Abstract Previous studies have shown that there is a need for population data to calculate the HLA antigen and haplotype frequencies that may help in creating the national registries and organ-sharing network for a particular population and country. This study is the first report from India to find out distribution of HLA A, B, DR antigen and haplotype frequencies in live related renal transplant recipients and donors from the state of Uttar Pradesh, North India. Study population consists of live related renal transplant recipients and donors. HLA class I and class II typing was done using microlymphocytotoxicity assay (NIH protocol). Standard statistical methods were used to calculate the HLA phenotype, genotype, haplotype frequencies and genetic distances. HLA antigen and haplotype frequencies were analysed in renal transplant recipients and live related donors. It has been seen that the HLA-A and B antigen frequencies were not significantly different in two groups. At DR locus DR16 was totally absent in the recipient population but was present among the donors. Two and three locus haplotype analysis was also carried out in the renal transplant recipients and donors to find out the most common haplotypes. It has been seen that two locus haplotypes like HLA-A2-B7; A3-B51; A11-DR1; B5-DR7 and B12-DR1 were found to be common among recipients and donors of Uttar Pradesh (North India) population at HLA- A&B; A&DR and B&DR locus. Threelocus haplotype analysis revealed that there were only four haplotypes i.e. A1-B27- DR4, A11-B13-DR11, A19-B62-DR5, and A24-B57-DR7 were common in both donors and recipients. We have compared our results with other world and Indian populations and have observed unique pattern of HLA phenotypes and haplotypes, which suggests that it is important to create a national donor registry and organ-sharing network for Indian subcontinent. Introduction Renal transplant has become the standard care for the fatal renal diseases and the success of such transplantations correlates with the degree of HLA compatibility between recipients and donors. However, to find out matched donor and recipient, is a difficult task as HLA locus is highly polygenic and polymorphic in nature. Address for Correspondence: Dr. Suraksha Agrawal, Addl Professor Dept of Medical Genetics Sanjay Gandhi Postgraduate Institute of Medical Sciences Rae Bareli Road,Lucknow (UP) suraksha@sgpgi.ac.in Human leukocyte antigens are encoded by major histocompatability complex (MHC) located on short arm of chromosome six. HLA molecule binds and presents peptide to T lymphocytes in cell mediated immune response and plays a key role in shaping the T cell repertoire and is also associated with allograft rejection. HLA antigens are inherited in a co-dominant manner from parents to the offsprings. In the live related transplants there are only 25% chances that two sibs would be hundred percent identical and 50% chances would be that they will share one haplotype and 25% would be that they will not share any of the haplotype. When matched donor is not found in a family then search may be performed to find out unrelated donors. Therefore, the information about frequencies with which a particular HLA haplotype occurs in a population is

2 Indian J Nephrol 2001;11: important. This information is required to calculate the probability to find out unrelated donors from a particular ethnic group 1,2. In the present study we have analyzed the allele and haplotype frequencies of HLA-A, B, and DR antigens in the live related renal transplant recipients and donors of Uttar Pradesh and have also compared our results with the available data on Indian populations and other world population. Material and Methods We have analyzed the HLA-A, B and DR phenotypes of 382 renal transplant recipients and their prospective live related donors referred to our center from Serological HLA typing for class I and II antigens was done by complement dependent cytotoxic (CDC) method. (NIH protocol). Both class I and class II antisera for microlymphocytotoxicity assay were obtained from Pelfreeze and One Lambda. For each specificity 3 antisera were used we have taken into consideration both broad and split antigens. For gene frequency analysis broad antigens are independent of the split antigens therefore, there is a difference when the sum of split antigens is calculated. Allele frequencies 6 were obtained by direct counting. The Hardy Weinberg equilibrium analysis was performed for the observed genotypes to assess the inbreeding status of these populations. Chi-square test was used to determine whether there was a significant deviation from Hardy Weinberg equilibrium. The haplotype frequencies and linkage disequilibrium were computed using 2 X 2 contingency tables according to the formula of Mattiuz et al (1970) 3. Allele frequencies were used to measure differences between populations and to compare our results with other populations. Nei s genetic distance was computed between each pair of populations. Observation Allele frequency distribution of HLA A, B & DR antigen in renal transplant recipients and donors is shown in tables 1 3. It is evident from the data that the allele frequencies of various antigens at HLA- A, B, & DR locus for broad and split antigens did not differ significantly in recipients and donors (p > 0.05). Goodness of fit test was applied (X 2 analysis) to determine, whether the distribution of observed frequency in recipients and donor population were in genetic equilibrium or not. It was noted that the observed frequencies do not deviate significantly from the expected frequencies among two groups, therefore, both the study groups are in genetic equilibrium. Two locus (HLA A&B, A&DR, and B&DR) and three locus (HLA-A, B&DR) extended haplotype analysis was done for both renal transplant recipients and donors. HLA gene and haplotype in transplant 89 Most common two locus HLA-A&B, A&DR and B&DR and three locus HLA-A, B&DR haplotypes are shown in table 4, 5, 6, 7. From tables 4, 5 and 6, it is observed that the common haplotypes found in both recipients and donors were A2-B7, A3-B51, A11-DR1, B5-DR7 and B12-DR1. The occurrence of some of these common haplotypes between recipients and donors were because the incidence of these antigens were almost equal in both the groups. From the three-locus haplotype analysis (Table 7) it has been noted that A1-B27-DR4 A11-B13-DR11, A19-B62- DR5 and A24-B57-DR7 were only common haplotypes in the recipients and donors. Discussion Our center performs live related renal transplants. Most of the time donor and recipient are either siblings (35.78%), parents vs offspring (47.34%), offspring vs parents (9.24%). Less than ten percent are spousal donors. Only (1.15%) are other donors like cousins or uncles. In the present study an attempt has been made to see whether there is any difference between recipient and donor at individual antigen basis. Our results show that phenotypic and genotypic frequency of HLA A, B, and DR antigens do not differ from one another in donors and recipients. Both donor and recipient population are found to be in the genetic equilibrium and obey the null hypothesis. HLA antigen frequencies of the present study have been compared with other Indian population s i.e. North Indians from Delhi and South Indians from Tamil Nadu. It has been observed that antigen frequencies do not differ significantly from North Indians from Delhi except at certain loci like HLA B35, DR2 and DR8 6. The allele frequency of B35 and DR2 is extremely reduced in our population as compared to North Indians from Delhi, whereas DR8 is significantly raised. Our study sample is different from South Indian population at A11 and B35 antigens 7. Frequency of antigen A11 and B35 is reduced in our population as compared to South Indians. The antigen frequencies were compared with donors of other ethnic groups (i.e. African Americans, Caucasians, Orientals, and Hispanics) from United Nation of Organ Sharing (UNOS) renal registry data at HLA-A, B and DR loci. Our population differs significantly from the other ethnic groups. The differences were maximum with Orientals followed by Hispanics and Caucasians (Table 8). Two and three locus haplotype analysis was carried out and it has been seen that HLA-A2-B7; A3B51; A11-DR1; B5 - DR7 and B12-DR1 at two-locus haplotype were found to be common haplotypes among recipients and donors of UP population. Three locus haplotype analysis

3 90 Indian Journal of Nephrology Indian J Nephrol 2001;11: revealed that some of the haplotypes, viz. A1-B27-DR4, A11-B13-DR11, A19-B62-DR5 and A24-B57-DR7 were found more commonly while other haplotypes were rare and occurred with low frequencies. The number of different haplotypes observed i.e. for which the frequencies have been estimated may be the function of the sample size, the distribution of haplotype frequencies and also the estimation process. The number of haplotypes theoretically possible in a population of infinite size is equal to the product of the number of alleles occurring at each locus. The size of the groups studied here was too small to observe all theoretically possible haplotypes because of the rarity of antigen frequencies and further the haplotypes to haplotypes. In our study the number of different haplotypes estimated, given as percentage of the number of theoretically possible haplotypes were less than 50% for A-B, B-DR and A-B-DR haplotypes (i.e. 43.7%, 44.7% and 31%) while the number of A-DR was 60.65%. Similar results were also found in all the groups of the UNOS renal transplant registry data where the population is bigger but not too large (Zachary et al. 1996) 2. The number of haplotypes increase with increasing population size, the relationship between size of the population and the number of haplotypes was not linear but rather appeared to approach the maximum asymptotically. In an analysis of the national marrow donor program (NMDP) registry data the number of A- B and A-B-DR haplotypes detected were 66% and 64% respectively, of the number of haplotypes, theoretically possible. However, these percentages were for combined population of different races and may over estimate those seen in individual racial groups. Haplotypes may be absent from a sample either because the size of the sample is too small to accommodate all possible haplotypes, or the fact that haplotypes do not exist at all in the population. This data suggests that it is unlikely that all the possible haplotypes that may occur in a population can be detected. The differences between patients and the donors of the same race are in the linkage disequilibrium. However, the population sizes are not sufficiently large to eliminate the possibility that these differences are due to chance. The data presented in this study for the HLA allele and haplotype frequencies of North Indians from UP is the first report for renal transplant recipients and donor from this part of the country. While the accuracy of the frequency estimates diminishes for every rare haplotypes, comparison with similar data from other sources indicate that this data are overall reasonable estimates of the haplotype frequencies that may be applied for various purposes, such as predictive modelling in transplantation. UNOS renal transplant registry demonstrates that the large disparity exists between African-American and Caucasoid in the HLA frequencies. If phenotypically identical cadaver renal transplant is required then large registries are to be created for end stage renal disease patients. Certain haplotypes particularly two loci and three loci are common to some of the racial groups which reduces the registry size. However, this does not hold true for our population, as Indian population does not appear to share the phenotypes with other racial and ethnic groups within the country and also with the world population. Hence, there is a need to compare haplotype frequencies among the populations from various geographical regions of India. This would be important for creating a national registry and organ-sharing network for Indian populations both for Indians living in India and also abroad. References 1. Lefell MS, Steinberg Bias WB, Machan CH and Zachary AA, The distribution of antigens and phenotyes among donor and patients in the UNOS registry. Transplantation 1994; 10: Zachary AA, Steinberg AG, Bias WB, and Lefell MS, The frequencies of HLA alleles and haplotypes and their distribution among the donors and renal transplant patients in the UNOS registry. Transplantation 1996; 62: Mattiuz PL, Inde D, Piazza A, Ceppellini R, Bodmer WF; New approaches to the population genetics and segregation analysis of HLA system In : Histocompatability testing; Terasaki PI, Munksgaard, Copenhagen; 1970; Nei M: Genetic distance between populations. American Naturalist 1972; 106: Piazza A. Haplotype and linkage disequilibrium from three locus phenotype; Histocompatablitity Testing; 1975; Copenhegan, Munkgaard; pp Mehra NK, Taneja V, Kailash S, Raizada N, Vaidya MC; Distribution of HLA antigen in samples of North Indian Hindu population; Tissue antigen 1986; 27: Pitchappan RM, Kakkanaiah VN, Rajshekhar, Arulraj N, Muttukkaruppan VR; HLA antigens in south India: I Major group of Tamilnadu. Tissue Antigen 1984; 24: 1906

4 Indian J Nephrol 2001;11: HLA gene and haplotype in transplant 91 Table. 1: Allele frequencies of HLA A locus in renal Tx recipients and donors of different populations PATIENT DONOR ANTIGEN U P AFA CAU HIS U P NI SI AFA CAU HIS A UP = Uttar Pradesh, AFA = African-Americans, CAU = Caucasians, HIS= Hispanics, NI = North Indians (Delhi), SI = South Indians

5 92 Indian Journal of Nephrology Indian J Nephrol 2001;11: Table : 2A Allele frequencies of HLA B locus in renal Tx recipients and donors PATIENT DONOR ANTIGEN UP AFA CAU HIS UP NI SI AFA CAU HIS

6 Indian J Nephrol 2001;11: HLA gene and haplotype in transplant 93 Table : 2B Allele frequencies of HLA - B locus in renal Tx recipients and donors PATIENT DONOR ANTIGEN UP AFA CAU HIS UP NI SI AFA CAU HIS B UP = Uttar Pradesh, AFA = African-Americans, CAU = Caucasians, HIS= Hispanics, NI = North Indians (Delhi), SI = South Indians

7 94 Indian Journal of Nephrology Indian J Nephrol 2001;11: Table: 3. Allele frequencies of HLA DR locus in renal Tx recipients and donors of different populations PATIENT DONOR ANTIGEN U P AFA CAU HIS U P NI SI AFA CAU HIS Blank UP = Uttar Pradesh, AFA = African-Americans, CAU = Caucasians, HIS= Hispanics, NI = North Indians, SI = South Indians

8 Indian J Nephrol 2001;11: Table: 4 : Most common HLA-A&B Haplotypes of renal transplant recipients and donors of UP (HF > 15/ 10000, t >2.0) Patients Donors A Ag B Ag H F A Ag B Ag H F Table 5 : Most common HLA-A&DR haplotypes of renal Tx recipients and donors of UP (HF > 15/10000 t >2.0) Patients Donors A Ag DR Ag H F A Ag DR Ag H F HF = Haplotype HLA gene and haplotype in transplant 95 HF = Haplotype frequency (%) (HF < 5% not listed).

9 96 Indian Journal of Nephrology Indian J Nephrol 2001;11: Table 6 : Most common HLA-B&DR haplotypes of renal transplant recipients and donors of UP (HF> 15 / t >2.0) Patients Donors B-Ag DR Ag H F B Ag DR Ag H F Table 7 : Most common HLA-A, B & DR haplotypes of renal transplant recipients and donors of UP (HF >15 / t >2.0) Patient Donors A Ag B Ag DR Ag H F A Ag B Ag DR Ag H F Table 8 : Statistical evaluation of comparison of antigen distribution between renal transplant recipients of UP and other ethnic groups A Locus B Locus DR Locus χ2 p value χ2 p value χ2 p value Recipients of UP < < < vs Caucasian recipients Recipients of UP < < < vs Hispanic recipients Recipients of UP < < < vs Oriental recipients

10 Indian J Nephrol 2001;11: HLA gene and haplotype in transplant Table 9 : Genetic distances between various groups based on HLA- A frequencies Groups Genetic Distances Recipients of UP vs donors of UP Recipients of UP vs Caucasian recipients Donors of UP vs Caucasian donors Recipients of UP vs Hispanic recipients Donors of UP vs Hispanics donors Recipients of UP vs Oriental recipients