HLA-A*26-A*30 and HLA-DRB1*10 could be predictors of nasopharyngeal carcinoma risk in high-risk Tunisian families

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1 29 Journal of Oral Science, Vol. 9, No. 2, 29-29, 27 Original HLA-A*2-A* and HLA-DRB* could be predictors of nasopharyngeal carcinoma risk in high-risk Tunisian families Nehla Mokni-Baizig ), Yosr Gorgi 2), Mohamed Elghourabi ), Mouna Makhlouf 2), Hamouda Boussen ), Said Gritli ), Michèle Elmay ), Amor Gamoudi ), and Ahmed Elmay ) )Laboratory of Immunohistocytology, Salah Azaiz Cancer Institute, Tunis, Tunisia 2) Laboratory of Immunology, Charles Nicolle Hospital, Tunis, Tunisia )Department of Economic and Quantitative Methods, High School of Economic and Commercial Sciences of Tunis, Tunis, Tunisia ) Department of Otorhinolaryngology, Salah Azaiz Cancer Institute, Tunis, Tunisia ) Research Unit /UR/-7, Faculty of Medicine, University of Tunis El Manar, Tunis, Tunisia (Received September 2, 2; Accepted September 2, 2) Abstract: We investigated human leukocyte antigen (HLA) profiles for Tunisians with nasopharyngeal carcinoma (NPC), their families, and a sample of unrelated healthy Tunisians in order to identify HLA specificities associated with familial NPC. HLA-A, -B, and -DRB typing was successful for NPC patients, 72 unaffected family members, and community controls, and the chi square or Fisher exact test was used to compare allele frequencies between cases and controls. We observed a consistent protective effect of HLA-DRB* on NPC development. However, none of the NPC patients or their family members had a positive result for this HLA marker (% vs 9.2% in controls, P =.7). In addition, HLA-A*2 was probably an induction marker, as its allelic frequency was significantly higher among NPC patients than among controls (P =.) and among NPC patients than among at-risk family members (P =.7). Logistic regression analysis of the joint effect of selected HLA specificities showed that HLA-A*2 and HLA-A* Correspondence to Dr. Nehla Mokni-Baizig, Laboratory of Immunohistocytology, Salah Azaiz Cancer Institute, Bab saadoun, Tunis, Tunisia Fax: nehla.mokni@rns.tn doi.org/.2/josnusd.- DN/JST.JSTAGE/josnusd/- were co-associated and have an important effect on NPC risk. Despite the small size of our cohort, we showed that HLA-A*2-A* and HLA-DRB* might be predictive markers for NPC screening of Tunisian families with a high risk of NPC. Keywords: nasopharyngeal carcinoma; HLA typing; high-risk families; Tunisia. Introduction Nasopharyngeal carcinoma (NPC) carcinogenesis involves an interplay of genetic and environmental factors and the Epstein-Barr virus (). Existing evidence confirms an association between human leukocyte antigen (HLA) profile and NPC, and several alleles and haplotypes are associated with NPC risk (2,). We previously reported an association between a specific HLA profile and sporadic NPC in north Tunisia (). Some HLA alleles are probably directly involved in NPC pathogenesis, through modulation of viral antigen presentation, for example, but current evidence does not implicate all HLA alleles in Epstein-Barr virus immune presentation (). The influence of HLA molecules may be due to a linkage with a distinct susceptibility gene mapping at a short distance from the major histocompatibility complex (,7). Therefore, this association is likely

2 29 attributable to one mechanism or perhaps a combination of mechanisms. However, more extensive investigations, probably including HLA family studies, are required in order to clarify this issue. Familial NPC is defined as NPC occurring in two or more first-degree relatives (parents, siblings, sons, and daughters) or second-degree relatives (grandparents, uncles, aunts, nephews, and nieces). In epidemiologic studies, the excess risk for first-degree relatives with NPC was generally to times that of individuals without a family history (). Most HLA association studies investigated sporadic NPC; few have analyzed familial NPC (9). Furthermore, understanding of familial NPC is limited, particularly in North Africa, and studies have focused exclusively on epidemiologic (-) and viral approaches to the disease (-7). We attempted to characterize the HLA profiles of Tunisians with NPC, their families, and a group of unrelated healthy Tunisians, to identify HLA specificities in relation to the NPC family. Materials and Methods Recruitment of multiplex families We identified and recruited 2 NPC multiplex families with at least two related members affected with NPC for this study, which was conducted at Salah Azaiz Cancer Institute: 7 families had two NPC cases and six had three NPC cases. In most families, the related NPC cases were siblings. One family had a mother and her son with NPC, and four families had second-degree relatives with NPC (first cousins; aunt/nephew; uncle/niece). For each NPC case in a multiplex family, up to two siblings and their parents without NPC were selected. Questionnaire Eligible families were interviewed in order to obtain basic information on family members and draw an exact family pedigree. A structured questionnaire was given to the proband in each family to collect information on age, sex, ethnicity, consanguinity, and family history of NPC and other diseases among the proband s relatives for three successive generations. This study was approved by the Ethics Committee of Salah Azaiz Cancer Institute (April 2, 2), and all experiments were performed in compliance with the relevant laws and institutional guidelines and in accordance with the ethical standards of the Declaration of Helsinki. Laboratory and clinical data Data on date of NPC diagnosis, tumor histologic characteristics, and response to treatment were obtained for all familial NPC patients. Other family members underwent clinical and naso-endoscopic examinations, to confirm a healthy nasopharyngeal cavity. Biological samples DNA samples were collected from 2 NPC cases, 72 unaffected relatives, and community controls (National Blood Transfusion Center) who voluntarily participated in this study. HLA allele typing was done for NPC cases, 72 relatives, and controls. DNA extraction and HLA genotyping Genomic DNA from whole blood collected in EDTA was isolated by using a modified salting-out method (). Typing for HLA-A, -B, and -DRB was performed by using polymerase chain reaction-sequence-specific primer (PCR-SSP; One-Lambda, Canoga Park, CA, USA) and Invitrogen (Life Technologies, Grand Island, NY, USA) kits, in accordance with the manufacturers instructions. HLA-A typing was successful for 2 NPC patients, unaffected family members, and community controls. HLA-B was done for 2 NPC patients, unaffected family members, and community controls. HLA-DRB was successful in NPC patients, 72 unaffected family members, and community controls. Statistical analysis Allele frequencies of cases and controls were compared with the chi square test or Fisher exact test when the number of participants was less than five. In logistic regression analysis (MINITAB for Windows) to determine if alleles were statistically associated with NPC pathology, the joint effect of alleles with a significant difference in frequency (P <.) between the NPC case and family members was evaluated. Results HLA allele frequencies were compared among NPC patients, unaffected family members, and community controls. The results of the comparison between NPC cases and community controls are summarized in Table. Allelic frequencies were significantly higher among NPC cases for HLA-A*2 (P <.) and A* (P <.) and significantly lower for HLA-B* (P =.). The absence of HLA-DRB* among NPC patients was notable, as was the significantly lower HLA-DRB* allelic frequency (P =.7 and P =.27, respectively). In contrast, the frequency of the HLA-DRB* allele was higher in NPC patients than in community controls (P =.2).

3 29 Table HLA-Cl I and Cl II frequencies in NPC cases and healthy controls HLA-A alleles A* A*2 A* A* A*2 A*2 A*2 A*2 A*29 A* A* A*2 A* A* A* A* A* A*9 A* HLA-B alleles B*7 B* B* B* B* B* B*27 B* B* B*9 B* B* B*2 B* B* B*9 B* B* B*2 B* B* B*7 B* HLA-DRB alleles DRB* DRB* DRB* DRB*7 DRB* DRB*9 DRB* DRB* DRB*2 DRB* DRB* DRB* DRB* NPC cases (%) Unrelated healthy controls (%) Relative risk P value (n = 2) (n = ) (n = 2) (n = ) (n = ) (n = ) Chi square or Fisher exact test; statistical significance defined as P <

4 292 Table 2 HLA-Cl I and Cl II frequencies in healthy family members and community controls HLA-A alleles A* A*2 A* A* A*2 A*2 A*2 A*2 A*29 A* A* A*2 A* A* A* A* A* A*9 A* HLA-B alleles B*7 B* B* B* B* B* B*27 B* B* B*9 B* B* B*2 B* B* B*9 B* B* B*2 B* B* B* B*7 B* HLA-DRB alleles DRB* DRB* DRB* DRB*7 DRB* DRB*9 DRB* DRB* DRB*2 DRB* DRB* DRB* DRB* Related family members (%) Unrelated healthy persons (%) Relative risk P value (n = ) (n = ) (n = ) (n = 72) (n = ) (n = ) Chi square or Fisher exact test; statistical significance defined as P <

5 29 Table HLA-Cl I and Cl II frequencies in NPC cases and unaffected related family members HLA-A alleles A* A*2 A* A* A*2 A*2 A*2 A*29 A* A*2 A* A* A* HLA-B alleles B*7 B* B* B* B* B*27 B* B* B* B* B* B* B*2 B* B* HLA-DRB alleles DRB* DRB* DRB* DRB*7 DRB* DRB* DRB* DRB* DRB* DRB* NPC cases (%) Related family members (%) Relative risk P value (n = 2) (n = ) (n = 2) (n = ) (n = ) (n = 72) Chi square or Fisher exact test; statistical significance defined as P < Higher frequencies of HLA-A* and HLA-DRB* (P <.) and lower frequencies of HLA-DRB* and HLA-DRB* (P <.) were noted in a comparison of unaffected at-risk family members and community controls (Table 2). Other significant differences between these groups were the higher frequencies of HLA-A*2, HLA-B* (P =.2), and HLA-DRB* (P <.) and the lower frequencies of HLA-A*2 and HLA-A* (P <.) among at-risk family members. The results of the comparison between NPC cases and unaffected family members are summarized in Table. NPC cases had a significantly higher frequency of HLA-A* (P <.). The HLA-DRB* allele was absent in NPC cases and unaffected family members but was present in 9.2% of controls. To evaluate the joint effect of alleles associated with NPC development, alleles with significant allelic frequency differences (P <.) between NPC patients and related family members were identified, including higher frequencies for HLA-A*2 (P =.9), HLA-A*2 (P =.7), HLA-B* (P =.9), and HLA-B* (P =.9) and lower frequencies for HLA-A* (P =.) and HLA-B* (P =.9) (Table ). The results of logistic regression analysis of the joint effects of these alleles are presented in Table. HLA-A*2 and HLA-A* were significantly co-associated (P <.; A*2, z =.7, P =.7 and A*, z =.9, P =.). The values for the HLA-A*2 and HLA-A* z coefficients were positive, indicating that

6 29 Table Selected HLA alleles in NPC patients and their family members HLA-A alleles A*2 A*2 A* A* HLA-B alleles B* B* B* NPC cases (%) Family members (%) P Odds ratio (n = 2) (n = ) (.-7.72) (.7-.) (.2-.7) (.-.) (n = 2) 2 (n = ) Chi square test or Fisher exact test; statistical significance defined as P <... (.-.9).9 (.-.7) 7.2 (.-9) Table Joint effect of selective HLA alleles on NPC risk Predictor Z P Odds ratio Constant HLA-A*2 HLA-A* Constant HLA-A*2 HLA-A* Constant HLA-A*2 HLA-A*2 Constant HLA-A* HLA-A* Logistic regression (MINITAB ). Statistical significance defined as P <.. 9% confidence interval Lower Upper the presence of both alleles was associated with NPC development. As indicated by the odds ratios shown in Table, a person with an HLA-A*2 profile is.7 times as likely to develop NPC disease as unaffected members of his family, and a person with an HLA-A* profile is. times as likely to have NPC as his healthy family members. Discussion Our results suggest that the association between HLA profile and NPC in high-risk Tunisian families differs from those noted in previous studies of sporadic NPC (,9). In the present study, HLA-DRB* had a consistent protective effect against NPC development. However, none of the NPC cases or family members were positive for this HLA marker. In addition, HLA-A*2 is probably a marker of induction. Its allelic frequency was significantly higher among NPC patients than among controls and among NPC patients as compared with at-risk family members. In a previous study (), we observed consistent associations of HLA-A*2, HLA-B*, DRB*, and DRB* with NPC among Tunisian sporadic NPC cases. Thereafter, we compared associations of HLA specificities between sporadic and familial NPC cases in Tunisia. Among HLA specificities, common points between sporadic and familial NPC cases were investigated. HLA-A*2, a protective marker in sporadic NPC patients, was less frequent among familial NPC cases than among healthy controls, but the difference was not significant. In contrast, the frequency of HLA-A*2 was significantly lower among at-risk family members than among NPC cases and healthy controls. Therefore, lower HLA-A*2 frequency does not appear to be a consistent protective marker, as there was no significant difference in frequency between NPC cases and healthy controls. The frequency of HLA-DRB* was lower among NPC cases and their at-risk family members than among controls, which suggests that this HLA specificity reduces NPC risk. Furthermore, a previous study of HLA typing (2) showed that HLA-DRB* is one of the most frequent alleles in the Tunisian population (2%). However, HLA-DRB* could be a susceptible gene for NPC development because its frequency was higher in NPC cases and family members than in controls in this study. This HLA specificity was also associated with increased risk among sporadic NPC cases in our previous study ().

7 29 The frequency of DRB* is relatively high in the Tunisian population (2), among whom NPC is the most commonly diagnosed head and neck cancer (2). Thus, this allele could be a marker of induction. We evaluated the joint effect of selected HLA specificities that significantly differed between NPC cases and family members. HLA-A*2 and HLA-A*, separately and in combination, have important effects on NPC risk. Therefore, haplotypes containing these two alleles might be markers of susceptibility on the MHC region of chromosome. The suggested association might be attributable to linkage disequilibrium between these haplotypes and other NPC susceptibility genes on chromosome. However, our results contradict those of an earlier study (9), which found no consistent differences in the HLA profiles of sporadic and familial cases among a sample of Taiwanese NPC cases, except for a protective effect of HLA-A* against NPC. The HLA antigens strongly associated with NPC were HLA-A*2 and HLA-B*, although these alleles were found to have a combined effect (9). In summary, despite the small size of our cohort, our results show that, in our Tunisian sample of NPC, NPC cases from at-risk families have a specific HLA profile associated with NPC risk, including a higher frequency of HLA-A*2 and lower frequency of HLA-DRB*. However, some alleles associated with NPC were the same as those observed for sporadic and familial NPC cases, such as the higher frequency of DRB* and lower frequency of DRB*. These HLA specificities could help predict NPC risk among at-risk families. To our knowledge, this is the first study of familial NPC HLA in our country and in North Africa. However, larger-scale studies of NPC families and controls are needed in order to confirm our findings. Conflict of interest None declared. References. Hildesheim A, Wang CP (22) Genetic predisposition factors and nasopharyngeal carcinoma risk: a review of epidemiological association studies, 2-2: Rosetta Stone for NPC: genetics, viral infection, and other environmental factors. Semin Cancer Biol 22, Li X, Fasano R, Wang E, Yao KT, Marincola FM (29) HLA associations with nasopharyngeal carcinoma. Curr Mol Med 9, Lakhanpal M, Singh LC, Rahman T, Sharma J, Singh MM, Kataki AC et al. (2) Contribution of susceptibility locus at HLA class I region and environmental factors to occurrence of nasopharyngeal cancer in Northeast India. Tumour Biol, -7.. Mokni-Baizig N, Ayed K, Ayed FB, Ayed S, Sassi F, Ladgham A et al. (2) Association between HLA-A/-B antigens and -DRB alleles and nasopharyngeal carcinoma in Tunisia. Oncology, -.. Su WH, Hildesheim A, Chang YS (2) Human leukocyte antigens and Epstein-Barr virus-associated nasopharyngeal carcinoma: old associations offer new clues into the role of immunity in infection-associated cancers. Front Oncol, Tse KP, Su WH, Chang KP, Tsang NM, Yu CJ, Tang P et al. (29) Genome-wide association study reveals multiple nasopharyngeal carcinoma-associated loci within the HLA region at chromosome p2.. Am J Hum Genet, Hsu WL, Tse KP, Liang S, Chien YC, Su WH, Yu KJ et al. (22) Evaluation of human leukocyte antigen-a (HLA-A), other non-hla markers on chromosome p2 and risk of nasopharyngeal carcinoma. PLoS One 7, e277.. Chang ET, Adami HO (2) The enigmatic epidemiology of nasopharyngeal carcinoma. Cancer Epidemiol Biomarkers Prev, Yu KJ, Gao X, Chen CJ, Yang XR, Diehl SR, Goldstein A et al. (29) Association of human leukocyte antigens with nasopharyngeal carcinoma in high-risk multiplex families in Taiwan. Hum Immunol 7, Hsu WL, Yu KJ, Chien YC, Chiang CJ, Cheng YJ, Chen JY et al. (2) Familial tendency and risk of nasopharyngeal carcinoma in Taiwan: effects of covariates on risk. Am J Epidemiol 7, Ng WT, Choi CW, Lee MC, Law LY, Yau TK, Lee AW (2) Outcomes of nasopharyngeal carcinoma screening for high risk family members in Hong Kong. Fam Cancer 9, Ruan HL, Qin HD, Shugart YY, Bei JX, Luo FT, Zeng YX et al. (2) Developing genetic epidemiological models to predict risk for nasopharyngeal carcinoma in high-risk population of China. PLoS One, e2.. Ouyang PY, Su Z, Mao YP, Liang XX, Liu Q, Xie FY (2) Prognostic impact of family history in southern Chinese patients with undifferentiated nasopharyngeal carcinoma. Br J Cancer 9, Yu KJ, Hsu WL, Pfeiffer RM, Chiang CJ, Wang CP, Lou PJ et al. (2) Prognostic utility of anti-ebv antibody testing for defining NPC risk among individuals from high-risk NPC families. Clin Cancer Res 7, Qin HD, Jia WH, Zhang LL, Liu N, Zhou XX, Wang MH et al. (2) Elevated Epstein-Barr virus seroreactivity among unaffected members of families with nasopharyngeal carcinoma. J Med Virol, Chang CM, Yu KJ, Hsu WL, Major JM, Chen JY, Lou PJ et al. (22) Correlates of anti-ebv EBNA IgA positivity among unaffected relatives from nasopharyngeal carcinoma multiplex families. Br J Cancer, Baizig NM, Morand P, Seigneurin JM, Boussen H, Fourati A, Gritli S et al. (22) Complementary determination

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