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Bulletin of Experimental Biology and Medicine, Vol. 157, No. 2, June, 2014
GENERAL PATHOLOGY AND PATHOPHYSIOLOGY Role of Polymorphic Loci in HLA-Region rs2647012 and rs805288 in the Development of Non-Hodgkin’s Malignant Lymphomas in Western Siberia A. S. Weiner1,2, O. V. Berezina3, V. S. Ovchinnikov3, M. N. Surovtseva1, E. N. Voropaeva4, T. I. Pospelova3, and M. L. Filipenko1 Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 157, No. 2, pp. 139-142, February, 2014 Original article submitted February 20, 2013 The data of genome-wide association analysis suggest that human 6p21.3 chromosomal region (localization of HLA genes) contains polymorphic loci influencing the risk of developing nonHodgkin’s lymphomas. We analyzed association of rs2647012 and rs805288 loci with the risk for non-Hodgkin’s malignant lymphomas in the population of Western Siberia. Allele and genotype frequencies were determined in the group of 298 patients and in the control group including 551 individuals. Subgroups of diffuse large B-cell lymphoma (86 patients) and follicular lymphoma (25 patients) were analyzed separately. An association of rs2647012 А/А genotype with increased risk of the disease (OR=2.78, p=0.002) was detected in the subgroup of diffuse large B-cell lymphoma. Key Words: non-Hodgkin’s malignant lymphomas; HLA-region; polymorphic locus; rs2647012; rs805288 Human chromosome 6p21.3 region carries genes encoding the major histocompatibility complex (HLA genes) proteins essential for the development of anti-infective and antitumor immune response and are the most polymorphic human genes [13]. HLA class I genes (HLA-A, HLA-B, HLA-C) encode proteins expressed on the vast majority of nucleated cells. These proteins present antigens to cytotoxic CD8+ T lymphocytes [1] and cooperate with NK-cells [2]. HLA class II genes (HLA-DP, HLA-DQ, and HLA-DR) encode protein molecules expressed on antigen present1 Institute of Chemical Biology and Fundamental Medicine, Siberian Division of the Russian Academy of Sciences, Novosibirsk; 2Novosibirsk State University; 3Novosibirsk State Medical University; 4 Research Institute of Internal Medicine, Siberian Division of the Russian Academy of Sciences, Novosibirsk, Russia. Address for correspondence:
[email protected]. A. S. Weiner
ing cells (B lymphocytes, dendritic cells, monocytes, macrophages, etc.). These proteins present foreign peptides to CD4+ T-helper lymphocytes [1]. Structural changes in the coding and regulatory regions of HLA genes can impair antigen presentation (including presentation oncogenic virus antigens) and mechanisms of antitumor protection and contribute to malignant transformation [12]. Recent large association studies including four genome-wide association studies showed that 6p21.3 region contained polymorphic loci associated with the risk of non-Hodgkin’s lymphomas [3-5,7,9-12]. In particular, association of rs2647012 (G/A) locus in 6p21.32 with reduced risk of follicular lymphoma (p=4×10–12) has been demonstrated in genome-wide analysis [12] and then confirmed [4]. It has been also demonstrated [9] that rs2647012 is in close linkage
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disequilibrium (D’=1) with HLA-DRB1*15-DQA1*01DQB1*06(*) haplotype that is also associated with reduced risk of follicular lymphoma. Allele A of rs2647012 locus enhanced HLA-DQB1 gene expression in lymphoblast cell lines that may underlie its protective effect [6]. In an earlier study [5], in 6p21.33 region of other polymorphic locus, rs805288 (G/A), marker allele HLA-B*0702 had been found associated with reduced risk of follicular lymphoma (p=2×10–4). Functional effect of this locus was not studied. Since the effect of polymorphisms on the risk of disease can be modulated by ethnic factors (differences in linkage groups of genetic markers, lifestyle factors, environmental impact), results of association studies obtained on the sample of the same ethnic origin must be confirmed on samples from other population groups. In previous studies, the effect of rs2647012 and rs805288 loci was studied on samples from the U.S., Canada, Australia, and Sweden. Here we studied the associations of these polymorphic loci with the risk for non-Hodgkin’s lymphomas (NHL) in the population of Western Siberia.
MATERIALS AND METHODS The group of NHL patients (298 patients of Novosibirsk Municipal Hematological Center) included 148 (49.7%) females and 150 males (50.3 %), mean age 54.2±14.3 years. According to the WHO classification (2008), aggressive lymphomas (highly malignant), namely diffuse large cell lymphoma, Burkitt-like lymphoma, pleomorphic lymphoma, lymphoblastic lymphoma, centroblastic lymphoma, immunoblastic lymphoma, plazmoblastic lymphoma, anaplastic lymphoma, and grade 3 follicular lymphoma were diagnosed in 48% of patients. Indolent lymphomas (low-malignant), such as diffuse small cell lymphoma, prolymphocytic lymphoma, centrocytic lymphoma, mantle cell lymphoma, lymphoplasmacytic lymphoma, grade 1 and 2 follicular lymphoma, mycosis fungoides, marginal cell lymphoma, and MALT-lymphoma were detected in 52% of patients. The stage of the disease was determined according to the Ann Arbor classification (1971). The vast majority of patients suffered
from advanced grades of the disease; 76% had grade 4; 9% had grade 3; 9% had grade 2, and 5% had grade 1 NHL. The control group consisted of 551 residents of Novosibirsk (blood donors) without cancer, 346 (62.8%) females and 205 (37.2%) males, mean age 33.42±10.88 years. All the patients signed informed consent in accordance with the requirements of the Ethics Committee. DNA was isolated from the venous blood by standard procedures including isolation and lysis of blood cells, hydrolysis of proteins with proteinase K, and DNA purification by extraction of impurities with phenol-chloroform mixture and ethanol precipitation with DNA. Genotypes were determined by PCR in real time using TaqMan-competing probes. PCR was performed in a final volume of 25 μl containing 65 mM Tris-HCl (pH 8.9), 23 mM (NH4)2SO4, 3.0 mM MgCl2, 0.05% Tween-20, 0.2 mM solution of deoxynucleoside triphosphates, 0.3 μM oligonucleotide primers (Table 1), 0.3 μM TaqMan-probes (Table 1), 20-100 ng DNA and 1 U Taq-DNA polymerase. PCR was performed on a CFX96 DNA thermocycler (Bio-Rad) with initial denaturation at 96oC (3 min) followed by 48 cycles at 96oC for 8 sec and at 60oC for 40 sec. Allele and genotype frequencies in the studied samples were compared using the χ2 test. Conformity of the genotype distribution to the Hardy–Weinberg equilibrium was verified using the exact test. The differences were considered statistically significant at p70 subjects in order to reach 80% statistical power to detect such effect. Locus rs805288 also did not show the statistically significant association in the subgroups in follicular and diffuse large B-and lymphomas. It suggests that this locus do not affect the risk for above diseases in Western Siberia or its contribution to the genetic predisposition is small and can be detected only in the larger samples.
183 This work was supported by the Program “Fundamental Science to Medicine” of the Presidium of the Russian Academy of Sciences (grant No. 5.30) and Federal Target Program “Research and Scientific-Pedagogical personnel of Innovative Russia” for 2009-2013 (State Contract No. 16.740.11.0633, June 2, 2011).
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