HLA Class II Allele and Haplotype Frequencies in Iranian Patients with Acute Myelogenous Leukemia and Control Group

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1 ORIGINAL ARTICLE Iran J Allergy Asthma Immunol September 2006; 5(3): HLA Class II Allele and Haplotype Frequencies in Iranian Patients with Acute Myelogenous Leukemia and Group Abdolfattah Sarafnejad 1, Farideh Khosravi 2, Kamran Alimoghadam 3, Saied Dianat 2, Bita Ansaripour 2, Batoul Moradi 2, Shahin Dorkhosh 2, and Aliakbar Amirzargar 2 1 Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2 Department of Immunology, Medical School, Tehran University of Medical Sciences, Tehran, Iran 3 Department of Hematology, Oncology and BMT, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran Received: 12 June 2006; Accepted: 6 July 2006 ABSTRACT Previous studies have demonstrated some significant differences in HLA allele frequencies in leukemic patients and normal subjects. We have analyzed HLA class II and haplotypes in 60 Iranian patients with acute myelogenous leukemia () and 180 unrelated normal subjects. Blood samples were collected after obtaining informed consents. From the patients and control DNA extraction and HLA typing were performed using PCR-SSP method. Significant positive association with the disease was found for HLA-DRB1*11 allele (35% vs. 24.7%, p=0.033). Two including HLA-DRB4 and DQB1*0303 were found to be significantly decreased in patients compared to controls. Regarding haplotype analysis, no significant association was found between case and control groups. It is suggested that HLA-DRB1*11 allele plays as a presumptive predisposing factor while the HLA-DRB4 and DQB1*0303 are suggested as protective genetic factors against acute myelogenous leukemia. Larger studies are needed to confirm and establish the role of these associations with acute myelogenous leukemia. Key words: Acute Myelogenous Leukemia; HLA class II; Polymorphism INTRODUCTION Leukemia is classified into few categories according to their origin and pathogenesis. Acute myelogenous leukemia () accounting for less than 1% of all cancers and 34% of all types of leukemia affects humans with a peak incidence between 15 and 39 years of life, but it is also observed in children. 1 Corresponding Author: Aliakbra Amirzargar, PhD; Department of Immunology, Immunogenetic laboratory, Medical school, Tehran University of Medical Sciences, Tehran, Iran. Tel: (+98 21) , Fax: (+98 21) , Amirzargar_ali@yahoo.com Its heterogeneity caused by complex stages of differentiation of myeloid cells is distinctive from Chronic Myelogenous Leukemia, which is mainly characterized by the presence of Philadelphia chromosome formation. 2 More than one mutation is required as a genetic susceptibility to as is true for other human malignancies. Several genetic translocations occurring in have been reported such as t(15;17), t(4;11) and t(12;21) encoding PML- RARα, MLL-AF4 and ETV6-RUNX1, respectively. 3 Mutated genes introduce two effects to human white cells including proliferation and/or survival of hematopoietic progenitors and impairment of hematopoietic differentiation. 4 Copyright 2006, IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY. All rights reserved. 115

2 A. Sarafnejad, et al. Several environmental risk factors for the development of include benzene, radiation, 5,6 alkylating agents exposure and history of chemotherapy for other cancers. 7 Moreover, some of the subtypes rarely transform from the myelodysplastic syndromes (MDS). 8 There are several reports indicating the influence of inflammatory processes in the pathogenesis of MDS. Also, there is much evidence of autoimmune phenomena and role of inflammatory cytokines such as tumor necrosis factor α (TNF-α). 9,10,11 The first HLA study in human leukemia in 1967 showed an increase of HLA-A2 allele frequency in ALL. 12 Seremetis et al. 13 have reported the strongest association between leukemia and HLA using a monoclonal antibody specific for the HVR3 epitope of HLA-DR53 and (Pv< ). In another study by Duncan Gowans in 150 patients from UK no significant association with HLA-DRB1 was reported. 14 Recently, in a Chinese study on the association of HLA class I with, HLA- A*01 and -B*37 were more frequent while HLA-A*33 and B*51 were less frequent in patients comparing to the controls. 15 In the present study, we have analyzed HLA-DRB, -DQA1 and -DQB1 and haplotypes frequency in patients and a normal unrelated control group. In addition we compared allele frequencies between case and control groups. PATIENTS AND METHODS Sampling and DNA Extraction A group of sixty patients with referred to Hematology, Oncology and BMT center of Shariati Hospital a referral center for hematological and leukemic disorders in Tehran, took part in this study. A hundred and eighty normal blood donor subjects as control group were randomly selected from healthy blood donors admitted to Iranian Blood Transfusion Organization (IBTO). patients had been diagnosed on the combination of morphologic, immunophenotyping and cytogenetic studies. Informed consent was obtained from all patients and control subjects participating in this study. Genomic DNA was extracted from 10 ml peripheral blood in EDTA vacutainers by modified salting out method. 16 HLA- DRB, DQA1 and DQB1 typing were performed by polymerase chain reaction based on sequence specific primers (PCR-SSP), following Olerup and Zetterquist method. 17 HLA-DRB, DQA1 and DQB1 PCR-SSP kits were supplied by Biotest (Heidelberg, Germany). TAQ DNA polymerase was purchased from Roche (Basel, Switzerland). The PCR reactions were carried out in 10 µl volumes. Samples were amplified in Techne genius thermal cyclers, after initial denaturation at 94 C for two minutes, followed by 10 cycles of 94 C denaturation for 10 seconds, 65 C annealing and extension for 60 seconds, and finally 20 cycles of 94 C denaturation for 10 seconds, 61 C annealing for 50 seconds and 72 C extension for 30 seconds. After amplification, PCR products were run on an agarose gel, and then result was interpreted for specific bands using a UV trans-illuminator. The haplotypes were calculated according to Iranian population specific linkage disequilibrium pattern among HLA-DRB, - DQA, and -DQB. 18 Statistical Analysis The differences in allele and haplotype frequencies of HLA among studied groups were analyzed using the Chi-square test after Yates correction. 19 Each allele frequency in patients was compared against the same allele in controls. The odds ratio (OR) and its 95% confidence intervals (CI) were calculated and a P- value of 0.05 or less was considered to be significant using InStat version 3.06 (GraphPad Software Inc, CA, 2003). RESULTS The distribution of HLA class II and haplotypes in patients and control subjects are presented in Tables 1-4. On analysis of distribution, significant differences were obtained. Significant positive association of HLA-DRB1*11 allele with is reported (35% vs. 24.7%, p =0.033). The HLA-DRB4 allele was observed in patients with significantly lower frequency compared with controls (11.6% vs. 21.1%, p=0.021). The HLA-DQB1*0303 allele was never observed in patients while its frequency in control subjects was 4.2% (p=0.028). In patients, HLA-DRB1*07 and -DQ1*0201 allele frequencies were lower than in control group with statistically quite insignificant (p=0.058). 116/ IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY Vol. 5, No. 3, September 2006

3 HLA class II and Acute Myelogenous Leukemia Table 1. HLA-DRB allele frequencies in Iranian patients and controls. DRB / / DRB3 DRB4 DRB5 11(9.2) 17(14.2) 10(8.3) 42(35) 63(52.5) 14(11.6) 26(21.6) 24(6.6) 47(13.1) 32(8.8) 33(9.2) 36(10) 8(2.2) 7(1.9) 12(3.3) 89(24.7) 3(0.8) 21(5.8) 10(2.7) 4(1.1) 18(5) 177(49.1) 76(21.1) 50(13.8) : not significant; OR: Odds Ratio OR: 0.39 ( ) OR: 1.64( ) OR: 0.49( ) OR: 1.71( ) Table 2. HLA-DQA1 allele frequencies in Iranian patients and controls DQA (12.5) 20(16.6) 7(5.8) 4(3.3) 52(43.4) 35(9.7) 49(13.6) 43(11.9) 34(9.4) 36(10) 38(10.5) 6(1.6) 130(33.3) : not significant; OR: Odds Ratio OR: 0.39( ) HLA-DRB5 frequency was higher in patients (21.6%) than in controls (13.8%), but the difference is not statistically significant (p=0.059). Haplotype analysis showed no significant result in frequencies of haplotypes in patients and controls. Table 3. HLA-DQB1 allele frequencies in Iranian patients and controls DQB (13.3) 42(35) 7(5.8) 26(21.6) 14(11.6) 12(10) 68(18.8) 1(0.3) 102(28.3) 18(5) 1 2(0.5) 1(0.3) 6(1.6) 66(18.3) 9(2.5) 25(6.9) 37(10.3) 10(2.7) : not significant; OR: Odds Ratio OR: 0.09( ) Table 4. HLA Class II haplotype frequencies in Iranian patients and controls DRB/DQA1/DQB1 haplotypes 0101/0101/ /0104/ /01021/ /0103/ /01021/ /05011/ /05/ /03011/ /0103/ /0104/05 07/0201/ /0401/ /0104/0501 : not significant 3(2.5) 12(10) 39(32.4) 3(2.5) 20(5.5) 17(4.7) 23(6.4) 32(8.8) 81(22.4) 21(5.8) 17(4.7) 30(8.3) 4(1.1) 8(2.2) Vol. 5, No. 3, September 2006 IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY /117

4 A. Sarafnejad, et al. DISCUSSION Mouse studies have shown the role of MHC class II in virus-induced leukemia. 20 The only mechanism of much evidence is immune response related to MHC. The first human study in association with certain HLA gene with leukemia showed an increase of HLA-A2 allele frequency in ALL in However, the mechanism of such association has not been obviously identified. Only few studies have been reported in the case of. HLA-DR53 allele has been suggested by Seremetis et al 13 to be associated with susceptibility to. In another study by Gowans et al in 150 patients from UK, no significant association with HLA- DRB1 was found. 14 Accordingly, we have investigated HLA-DRB,-DQA1,-DQB1 and haplotypes frequency in patients with and a normal control group. The only significant positive association with against control group was HLA- DRB1*11(35% vs. 24.7, p=0.033). HLA-DRB4 allele was significantly less frequent in patients compared to controls (11.6% vs. 21.1%, p=0.021). HLA-DQB1*0303 was never found in any patients, suggesting to be a protective factor against. Two other including HLA-DRB1*07 and -DQA1*0201 are negatively associated with however they are not statistically significant (p=0.058). Reporting some significant HLA allele association with, future direction would be toward the role of these in the disease in search of an immunemediated basis beside the known genetic mutations observed in this disease. Genetic association studies in different diseases will help the physicians for prediction of disease and the patient prognosis. One of the strongest HLA allele association studies is HLA-B27 association in ankylosing spondilitis that is useful in differential diagnosis of this disease. Genetic association studies in leukemic disorders are also helpful in clarification of pathogenesis as well as prediction or treatment of leukemic disorders of poor prognosis. In conclusion, it is suggested that the presence of HLA-DRB1*11 allele increases the risk of while HLA-DRB4 and DQB1*0303 are thought to be protective genetic factors. Larger studies are required to investigate more extensively the role of every specific HLA allele in the development of this disease. REFERENCES 1. O'Brien K, Cokkinides V, Jemal A, Cardinez CJ, Murray T, Samuels A, et al. Cancer statistics for Hispanics, 2003.CA Cancer J Clin 2003; 53(4): Kurzrock R, Kantarjian HM, Druker BJ, Talpaz M. Philadelphia chromosome-positive leukemias: from basic mechanisms to molecular therapeutics. Ann Intern Med 2003; 138(10): McKenzie SB. Advances in understanding the biology and genetics of acute myelocytic leukemia. Clin Lab Sci 2005; 18(1): Gilliland DG, Jordan CT, Felix CA. The Molecular Basis of Leukemia. Hematology 2004; 1: Sandler DP. Epidemiology of acute myelogenous leukemia. Semin Oncol 1987; 14(4): Byrd JC, Lawrence D, Arthur DC, Pettenati MJ, Tantravahi R, Qumsiyeh M, et al. Patients with isolated trisomy 8 in acute myeloid leukemia are not cured with cytarabinebased chemotherapy: results from Cancer and Leukemia Group B Clin Cancer Res 1998; 4(5): Pedersen-Bjergaard J, Philip P, Larsen SO, Andersson M, Daugaard G, Ersboll J, et al. Therapy-related myelodysplasia and acute myeloid leukemia. Cytogenetic characteristics of 115 consecutive cases and risk in seven cohorts of patients treated intensively for malignant diseases in the Copenhagen series. Leukemia 1993; 7(12): Germing U, Gattermann N, Aivado M, Hildebrandt B, Aul, C. Two types of acquired idiopathic sideroblastic anaemia (AISA): a time-tested distinction. Br J Haematol 2000; 108: Okamoto T, Okada M, Mori A, Saheki K, Takatsuka H, Wada H, et al. Correlation between immunological abnormalities and prognosis in myelodysplastic syndrome patients.int J Hematol 1997; 66(3): Symeonidis A, Kourakli A, Katevas P, Perraki M, Tiniakou M, Matsouka P, et al. Immune function parameters at diagnosis in patients with myelodysplastic syndromes: correlation with the FAB classification and prognosis. Eur J Haematol 1991; 47(4): Shetty V, Mundle S, Alvi S, Showel M, Broady-Robinson L, Dar S, et al. Measurement of apoptosis, proliferation and three cytokines in 46 patients with myelodysplastic syndromes. Leuk Res 1996; 20(11-12): Dausset J. The major histocompatibility complex in man. Science 1981; 213(4515): Seremetis S, Cuttner J, Winchester R. Definition of a possible genetic basis for susceptibility to acute 118/ IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY Vol. 5, No. 3, September 2006

5 HLA class II and Acute Myelogenous Leukemia myelogenous leukemia associated with the presence of a polymorphic Ia epitope. J Clin Invest 1985; 76(4): Gowans D, O'Sullivan A, Rollinson S, Roddam P, Groves M, Fegan C, et al. Allele and haplotype frequency at human leucocyte antigen class I/II and immunomodulatory cytokine loci in patients with myelodysplasia and acute myeloid leukaemia in search of an autoimmune aetiology. Br J Haematol 2002; 117(3): Li D, Xi B, Liu HY, Yu Y. Association of gene HLAclass I with leukemia. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2005; 13(4): Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells.nucleic Acids Res 1988; 16(3): Emtestam L, Zetterquist H, Olerup O. HLA-DR, -DQ and -DP in nickel, chromium, and/or cobalt-sensitive individuals: genomic analysis based on restriction fragment length polymorphisms.j Invest Dermatol 1993; 100(3): Amirzargar A, Mytilineos J, Farjadian S, Doroudchi M, Scherer S, Opelz G, et al. Human leukocyte antigen class II allele frequencies and haplotype association in Iranian normal population. Hum Immunol 2001; 62(11): Haldane Jbs. The estimation and significance of the logarithm of a ratio of frequency. Ann Hum Genet 1956; 20: Vasmel WL, Zijlstra M, Radaszkiewicz T, Leupers CJ, de Goede RE, Melief CJ. Major histocompatibility complex class II-regulated immunity to murine leukemia virus protects against early T- but not late B-cell lymphomas. J virol 1988; 62(9): Vol. 5, No. 3, September 2006 IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY /119