Original Article Genetic polymorphism at codon 546 of the human RAD17 contributes to the risk for esophageal squamous cell carcinoma

Transcription

1 Int J Mol Epidemiol Genet 2016;7(1): /ISSN: /IJMEG Originl Article Genetic polymorphism t codon 546 of the humn RAD17 contributes to the risk for esophgel squmous cell crcinom Yukiko Ysud 1, Akiko Ski 1, Schio Ito 1, Yuichiro Mit 1, Tkyuki Sonoym 2, Shunsuke Tnbe 3, Ysuhiro Shirkw 3, Yoshio Nomoto 4, Hiroshi Ktym 1, Kenji Shimizu 1 Deprtments of 1 Moleculr Oncology, 2 Gstroenterology nd Heptology, 3 Gstroenterologicl Surgery, Grdute School of Medicine, Dentistry nd Phrmceuticl Sciences, Okym University, Okym , Jpn; 4 Deprtment of Generl Surgery, Kwski Medicl School Kwski Hospitl, Okym , Jpn Received Jnury 29, 2016; Accepted Februry 29, 2016; Epub Mrch 23, 2016; Published Mrch 30, 2016 Abstrct: Humn RAD17, humn homolog of the Schizoscchromyces pombe cell cycle checkpoint gene RAD17, plys significnt role in ctivting checkpoint signls in response to DNA dmge. We evluted the ssocition of hrad17 Leu546Arg (rs ), missense single nucleotide polymorphism, with the risk of esophgel squmous cell crcinom in reltion to smoking nd lcohol consumption history in 154 esophgel squmous cell crcinom mle ptients nd 695 cncer-free mle controls by cse-control study conducted in Jpn. The results showed tht the hrad17 Arg/Arg genotype compred to the Leu/Leu nd Leu/Arg genotypes ws significntly ssocited with the risk of the esophgel squmous cell crcinom with n djusted odds rtios of 2.22 (95% CI: P=0.013). In strtified studies, the risk of esophgel squmous cell crcinom ws mrkedly higher in light drinkers (less thn 23 g ethnol/dy) with the Arg/Arg genotype thn in hevy drinkers (excess of 23 g ethnol/ dy) with the Arg/Arg genotype (OR=2.83, 95% CI: , P=0.04). We concluded tht the genetic vrint of hrad17 Leu546Arg polymorphism exerts significnt effect on esophgel squmous cell crcinom risk mong Jpnese men. Keywords: Esophgel squmous cell crcinom, single nucleotide polymorphism, humn RAD17, DNA dmge Introduction Esophgel cncer is one of the common cuses of cncer deth in Jpn. The gedjusted mortlity rte of mle esophgel cncer deths ws bout eight times greter thn tht of femle esophgel cncer deths. For both mle nd femle esophgel cncer ptients, the proportion of stge I t dignosis is usully smll (nerly 25%) nd the 5 yer reltive survivl rte for stge I is lmost 80% [1]. Therefore, erly detection is importnt for effective therpeutic intervention for ptients with esophgel cncer. Squmous cell crcinom nd denocrcinom re the two min histologicl subtypes of esophgel cncer, nd more thn 90% of esophgel cncer ptients re dignosed with squmous cell crcinom in Jpn [2]. Vrious risk fctors hve been ssocited with development of esophgel squmous cell crcinom (ESCC). Cigrette smoking nd lcohol drinking re the two mjor behviorl trits known to significntly increse the risk of ESCC [3-5]. On the other hnd, higher intke of fruit nd vegetble hs been shown to hve protective effect on the development of ESCC [6]. Genetic fctors lso ply n importnt role in the development of esophgel squmous cell crcinom. Polymorphisms in vrious cncerssocited genes including those involved in DNA dmge repir hve been ssocited with the risk of developing esophgel cncer [7]. Vrint lleles of genes involved in metbolism of lcohol nd crcinogens, such s, ALDH2*1*2 nd CYP1A1 show significnt correltions with the risk of esophgel cncer [8-11].

2 DNA dmge induced by norml endogenous metbolic processes or environmentl crcinogens cn led to gene muttions nd genomic instbility, which re expected to be ccelerted in cells with reduced DNA repir cpcity, lso ssocited with incresed risk of cncer. Due to similr underlying resons, polymorphism in DNA repir genes contributing vritions in DNA repir cpcity lso my be ssocited with the risk of developing ESCC [7, 12]. hrad17, humn homologue of the Schizoscchromyces pombe cell cycle checkpoint gene RAD17, is one of the importnt key regultors involved in ctivting checkpoint signls in response to DNA dmge nd incomplete DNA repliction [13, 14]. The gene is locted t chromosome 5q13.2, locus frequently deleted in humn cncers [15]. The signl trnsduction of DNA dmge checkpoints engges multiple dmge sensor proteins. Two sensor proteins recognize DNA dmge nd ctivte checkpoint signls, repliction fctor C (RFC) like clmp loder complex formed by hrad17 nd four repliction fctor C subunits nd proliferting cell nucler ntigen (PCNA)-like sliding clmp heterotrimeric complex formed by RAD9, RAD1 nd HUS1 (the complex) [16, 17]. The complex, loded by the RAD17- contining clmp loder, preferentilly binds t sites of DNA dmge nd fcilittes ATRmedited phosphoryltion nd ctivtion of Chk1 regulting S-shpe progression, G2/M rrest nd repliction fork repir [17-19]. In this pper, we report the risk of ESCC ssocited with genetic polymorphism of the hrad17 gene mong Jpnese men. Mterils nd methods Study popultion A hundred nd fifty-four Jpnese mle ptients with primry ESCC were surgiclly treted nd histologiclly confirmed t Okym University Hospitl (Okym, Jpn) between 1992 nd The clinicl nd histopthologicl clssifiction of the tumors ws defined ccording to the criteri of the UICC Tumor-Node- Metstsis Clssifiction of Mlignnt Tumors (TNM), 6th edition, As the controls, 695 Jpnese mles were enrolled nd consisted of two groups. 140 outptients without cncer were recruited from Kusk Hospitl t Okym in 2005 nd 555 helthy individuls from Junpuki Helth Cre Center t Okym in All ptients nd controls gve written informed consent. The Bioethics Committee of Okym University Medicl School pproved this study. The ge, gender, personl nd fmily medicl history, smoking nd lcohol drinking of the subjects were obtined from interviews nd medicl records. Smoking sttus ws evluted by pck-yer equivlents ([cigrettes/dy 20] [smoking yers]) nd clssified s nonsmoker, <20 pck-yers (light smokers), or 20 pck-yers (hevy smokers). Alcohol drinking ws ssessed by dily ethnol intke using the method of clculting lcohol consumption (One serving of ske contins nerly 23 g ethnol which pproximtes 2 US stndrd drinks, 1 US drink contins 14 g of ethnol) [5, 8]. The recommended dily mximum mount of lcohol ws bsed on the report of the limittion on dily ethnol intke by the Ministry of Helth, Lbor nd Welfre (20 g/dy; Helth Jpn 21, ntionl helth promotion movement) nd the result of the prospective lrge cohort study showed the risk of cncer mortlity ws lowest mong Jpnese drinkers with ethnol intke less thn 23 g/dy [5]. The subjects were clssified s non-drinker, <23 g/dy (light drinkers), or 23 g/dy (hevy drinkers). Genotype nlysis Genomic DNA ws extrcted from peripherl blood lymphocytes by the stndrd procedures using proteinse K nd phenol-chloroform. Genotypes on the polymorphisms including the hrad17 Leu546Arg (rs ) were nlyzed by SNPshot methods. Trget region were mplified for genotyping by the multiplex PCR. The PCR ws performed with finl rection volume of 10 µl contining 10 ng templte DNA, pmol of ech primer, 2.0 mm of ech dntp, 10 PCR buffer, 0.25 units of Tq DNA polymerse (TKR Bio, Kustsu, Shig, Jpn). Therml cycling ws performed with n initil denturtion t 94 C for 3 min, followed by 32 cycles t 94 C for 30 sec, 60 C for 30 sec nd 72 C for 30 sec, nd finl extension t 72 C for 7 min. The PCR products were treted with 2.0 units of exonuclese I nd shrimp lkline phosphtse to remove the unrected 59 Int J Mol Epidemiol Genet 2016;7(1):58-66

3 Tble 1. Bsic chrcteristics of cses nd controls Cses Controls P n (%) n (%) Totl Age (yers) <50 8 (5.2) 45 (6.5) (26.0) 368 (52.9) (39.6) 206 (29.6) (29.2) 76 (10.9) Medin Rnge Smoking (pck-yers) < (7.8) 196 (28.2) <20 15 (9.7) 152 (21.9) (82.5) 347 (49.9) Alcohol drinking (g of ethnol/dy) < (3.2) 196 (28.2) <23 22 (14.3) 272 (39.1) (82.5) 227 (32.7) 23 g of ethnol=nerly one serving of ske. primers nd dntps by incubting t 37 C for 90 min nd t 75 C for 15 min. First PCR primer set ws designed s: sense 5 -CAGTATC- GGGAAAATTGCCTGG-3 nd nti-sense 5 -GG- ACAGTAGAGACTCCCCCT-3. Single nucleotide primer extension rection ws performed using ABI PRISM SNPshot kit (Applied Biosystems, Foster City, CA, USA) with finl rection volume of 10 µl contining 3 µl of the purified PCR products, 20 mm of (NH 4 ) 2 SO 4, 2 µl SNPshot Redy Rection mix contining fluorescently lbeled ddntps nd DNA polymerse. The typing primer used for hrad17 Leu546Arg ws 5 -TTTTTTTTTTTTTT- TTTTTTCAAGGTATGGCAATAGCTGAGTTTGG-3. Therml cycling ws performed with n initil denturtion t 96 C for 3 min, followed by 30 cycles t 96 C for 10 sec, 50 C for 5 sec nd 60 C for 30 sec. After tretment with 1.0 unit of shrimp lkline phosphtse to remove the unincorported ddntps by incubting t 37 C for 90 min nd t 75 C for 15 min, 8.5 µl of HI-Di formmide, 0.5 µl of Genescn 120 LIZ size stndrd (Applied Biosystems) nd 1 µl of the rection mixture were combined nd dentured t 95 C for 5 min nd plced t 4 C for 2 min. The products were electrophoresed with ABI PRISM 3100 Genetic Anlyzer (Applied Biosystems) nd n- Chi squre tests were used to exmine the distribution between cncer ptients nd controls. The risk of ESCC ws estimted by odds rtio (OR) nd 95% confidence intervl (CI). Odds Rtio (OR) nd 95% confidence intervl (CI) were djusted for ge, smoking nd lcohol consumption sttus using multi- vrite logistic regression model by compring the genotypes between ptients nd controls. P vlues less thn 0.05 were considered stticlly significnt. All nlyses were performed with SPSS softwre (version 12.0, SPSS Inc., Tokyo, Jpn). Results lyzed by Gene Mpper Softwre SNPshot Anlysis (Applied Biosystems). Sttistics The llele frequency ws clculted by direct counting. Devition of the genotype frequency from Hrdy-Weinberg equilibrium ws nlyzed by the exct test. Tble 1 shows tht there were significnt differences in the distribution of ge, smoking nd lcohol consumption hbits between controls nd ESCC ptients. Most of the ptients were hevy smokers or drinkers. The never smokers nd drinkers of the controls were much lrger thn esophgel crcinom ptients. The control subjects were consisted from two sources, therefore, we ssessed the genotype distributions of polymorphism in the control subjects using the Hrdy-Weinberg equilibrium. The P vlues for the Hrdy-Weinberg equilibrium of hrad17 Leu546Arg were 0.16 nd 0.50, for these two dt-sets, respectively. Excess devition from the equilibrium in these control popultions ws not found. The llele nd genotype frequencies of hrad17 Leu546Arg mong ptients nd control subjects re shown in Tble 2. The Arg/Arg genotype compred to the Leu/Leu genotype nd 60 Int J Mol Epidemiol Genet 2016;7(1):58-66

4 Tble 2. Humn RAD17 genotype in cses nd controls Gene Genotype Cses Controls n (%) n (%) Adjusted OR (95% CI) RAD17 Leu546Arg Leu/Leu 74 (48.1) 322 (46.3) (Reference) Leu/Arg 57 (37.0) 312 (44.9) 0.67 ( ) Arg/Arg 23 (14.9) 61 (8.8) 1.85 ( ) Leu/Leu + Leu/Arg 131 (85.1) 634 (91.2) (Reference) Arg/Arg 23 (14.9) 61 (8.8) 2.22 ( ) Leu 205 (66.6) 956 (68.8) Arg 103 (33.4) 434 (31.2) Adjusted for ge, smoking, nd lcohol drinking. b P vlues, difference in genotype frequencies between cses nd controls. P b Tble 3. Effect of tobcco nd lcohol consumption on hrad17 genotype in cses nd controls RAD17 Leu546Arg Cses Controls Adjusted OR n (%) n (%) (95% CI) P Smoking (pck-yers) <20 Leu/Leu 12 (44.4) 155 (44.5) (Reference) Leu/Arg 10 (37.0) 161 (46.3) 0.67 ( ) Arg/Arg 5 (18.5) 32 (9.2) 2.12 ( ) Leu/Leu + Leu/Arg 22 (81.5) 316 (90.8) (Reference) Arg/Arg 5 (18.5) 32 (9.2) 2.58 ( ) Leu/Leu 62 (48.8) 167 (48.1) (Reference) Leu/Arg 47 (37.0) 151 (43.5) 0.71 ( ) Arg/Arg 18 (14.2) 29 (8.4) 1.78 ( ) Leu/Leu + Leu/Arg 109 (85.8) 318 (91.6) (Reference) Arg/Arg 18 (14.2) 29 (8.4) 2.07 ( ) Alcohol drinking (g of ethnol/dy) <23 Leu/Leu 13 (48.1) 219 (46.8) (Reference) Leu/Arg 8 (29.6) 205 (43.8) 0.63 ( ) Arg/Arg 6 (22.2) 44 (9.4) 2.32 ( ) Leu/Leu + Leu/Arg 21 (77.8) 424 (90.6) (Reference) Arg/Arg 6 (22.2) 44 (9.4) 2.83 ( ) Leu/Leu 61 (48.0) 103 (45.4) (Reference) Leu/Arg 49 (38.6) 107 (47.1) 0.67 ( ) Arg/Arg 17 (13.4) 17 (7.5) 1.64 ( ) Leu/Leu + Leu/Arg 110 (86.6) 210 (92.5) (Reference) Arg/Arg 17 (13.4) 17 (7.5) 1.97 ( ) Adjusted for ge, smoking, nd drinking sttus. the Leu/Arg genotype t the hrd17 Leu546Arg polymorphism in recessive genetic model ws ssocited with incresed risk of ESCC (OR=2.22, 95% CI: , p=0.01). The ssocition of the Arg/Arg genotype compred to the Leu/Leu genotype in dditive genetic model with the risk of ESCC ws not found (OR=1.85, 95% CI: , p=0.07). The difference of the llele frequencies between the cses nd controls ws not sttisticlly significnt. Next, we investigted the ssocition between the hrad17 Leu546Arg genotypes nd the risk of ESCC with strtifiction ccording to the history of smoking nd lcohol consump- 61 Int J Mol Epidemiol Genet 2016;7(1):58-66

5 Tble 4. Combined effects of tobcco nd lcohol consumption on hrad17 genotype in cses nd controls RAD17 Leu546Arg Cses Controls Adjusted OR n (%) n (%) (95% CI) P Alcohol drinking (g of ethnol/dy) <23 Smoking (pck-yers) <20 Leu/Leu 5 (55.6) 119 (46.3) (Reference) Leu/Arg 2 (22.2) 112 (43.6) 0.43 ( ) Arg/Arg 2 (22.2) 26 (10.1) 2.09 ( ) Leu/Leu + Leu/Arg 7 (77.8) 231 (89.9) (Reference) Arg/Arg 2 (22.2) 26 (10.1) 2.89 ( ) Leu/Leu 8 (44.4) 100 (47.4) (Reference) Leu/Arg 6 (33.3) 93 (44.1) 0.76 ( ) Arg/Arg 4 (22.2) 18 (8.5) 2.45 ( ) Leu/Leu + Leu/Arg 14 (77.8) 193 (91.5) (Reference) Arg/Arg 4 (22.2) 18 (8.5) 2.78 ( ) Alcohol drinking (g of ethnol/dy) 23 Smoking (pck-yers) <20 Leu/Leu 7 (38.9) 36 (39.6) (Reference) Leu/Arg 8 (44.4) 49 (53.8) 0.60 ( ) Arg/Arg 3 (16.7) 6 (6.6) 1.78 ( ) Leu/Leu + Leu/Arg 15 (83.3) 85 (93.4) (Reference) Arg/Arg 3 (16.7) 6 (6.6) 2.38 ( ) Leu/Leu 54 (49.5) 67 (49.3) (Reference) Leu/Arg 41 (37.6) 58 (42.6) 0.69 ( ) Arg/Arg 14 (12.9) 11 (8.1) 1.58 ( ) Leu/Leu + Leu/Arg 95 (87.2) 125 (91.9) (Reference) Arg/Arg 14 (12.8) 11 (8.1) 1.85 ( ) Adjusted for ge, smoking, nd drinking sttus. tion (Tble 3). Among light drinkers, including non-drinkers nd rre-drinkers, the Arg/Arg genotype compred to the Leu/Leu genotype nd the Leu/Arg genotype in recessive model ws significntly ssocited with incresed risk of ESCC (OR=2.83, 95% CI: , p=0.04). However, the ssocition between the Arg/Arg genotype mong hevy drinkers with the risk of ESCC ws not found. There ws no significnt ssocition between the Arg/Arg genotype nd the risk of ESCC in light or hevy smokers. We lso exmined the combined effects of smoking nd lcohol consumption on the genotype distribution of hrad17 Leu546Arg polymorphism (Tble 4). Among ESCC mle ptients, the combined group of hevy smokers nd hevy drinkers outnumbered the other combined group. Thus, the smple size of the groups, consisting of hevy drinkers nd light smokers, of light drinkers nd light smokers, or of light drinkers nd hevy smokers, were smll mong the ptients. In contrst to the ptients, the control subjects of light smokers nd light drinkers were much lrger. The result showed no sttisticlly significnt ssocition between ESCC risk nd the Arg/Arg genotype with ll combintions of smoking nd lcohol consumption. In ddition to ESCC, we lso exmined the ssocition between the hrad17 Leu546Arg genotypes nd the risk of vrious cncers including hed nd neck squmous cell crcinom (HNSCC), gstric denocrcinom (GC), lung denocrcinom (LAD), nd lung squmous cell crcinom (LSQ) (Tble 5). Cncer ptients were dignosed nd treted t Okym University hospitl (Okym, Jpn) nd gve written informed consent. The Bioethics Committee of Okym University Medicl School pproved this study. Gstric 62 Int J Mol Epidemiol Genet 2016;7(1):58-66

6 Tble 5. The ssocition between RAD17 Leu546Arg genotype nd GC, HNSCC, LSQ nd LAD risks Gene Genotype Cses Controls Adjusted OR n (%) n (%) (95% CI) P Gstric cncer (GC) Leu/Leu 35 (47.9) 322 (46.3) (Reference) Leu/Arg 31 (42.5) 312 (44.9) 0.80 ( ) Arg/Arg 7 (9.6) 61 (8.8) 1.00 ( ) Leu/Leu + Leu/Arg 66 (90.4) 634 (91.2) (Reference) Arg/Arg 7 (9.6) 61 (8.8) 1.11 ( ) Leu 101 (69.2) 956 (68.8) Arg 45 (30.8) 434 (31.2) 0.91 ( ) Hed nd neck squmous cell crcinom (HNSCC) Leu/Leu 41 (55.4) 322 (46.3) (Reference) Leu/Arg 27 (36.5) 312 (44.9) 0.71 ( ) Arg/Arg 6 (8.1) 61 (8.8) 0.73 ( ) Leu/Leu + Leu/Arg 68 (91.9) 634 (91.2) (Reference) Arg/Arg 6 (8.1) 61 (8.8) 0.86 ( ) Leu 109 (73.6) 956 (68.8) Arg 39 (26.4) 434 (31.2) 0.79 ( ) Lung cncer squmous cell crcinom (LSQ) Leu/Leu 59 (47.2) 322 (46.3) (Reference) Leu/Arg 50 (40.0) 312 (44.9) 0.92 ( ) Arg/Arg 16 (12.8) 61 (8.8) 1.38 ( ) Leu/Leu + Leu/Arg 109 (87.2) 634 (91.2) (Reference) Arg/Arg 16 (12.8) 61 (8.8) 1.43 ( ) Leu 168 (67.2) 956 (68.8) Arg 82 (32.8) 434 (31.2) 1.08 ( ) Lung cncer deno crcinom (LAD) Leu/Leu 97 (46.0) 322 (46.3) (Reference) Leu/Arg 92 (43.6) 312 (44.9) 0.99 ( ) Arg/Arg 22 (10.4) 61 (8.8) 1.19 ( ) Leu/Leu + Leu/Arg 189 (89.6) 634 (91.2) (Reference) Arg/Arg 22 (10.4) 61 (8.8) 1.20 ( ) Leu 286 (67.8) 956 (68.8) Arg 136 (32.2) 434 (31.2) 1.05 ( ) LSQ, LAD nd HNSCC were djusted for ge nd smoking sttus. GC ws djusted for ge, smoking, nd drinking sttus. cncer cohort ws djusted for ge, smoking hbit nd lcohol consumption. Hed nd neck squmous crcinom nd lung cncers were djusted for ge nd smoking hbit becuse no lcohol consumption dt ws vilble. There ws no significnt ssocition between hrad17 Leu546Arg polymorphism nd these other cncers exmined. Discussion In this cse-control study, we nlyzed ssocition between the codon 564 polymorphism cusing mino-cid substitution of leucine to rginine, missense SNP in hrad17 gene, nd ESCC risk. We found tht the hrad17 Leu564Arg vrint ws ssocited with the occurrence of esophgel squmous cell cncer in the recessive genetic model. To our knowledge, this is the first report describing tht the hrd17 polymorphism hs n ssocition with the risk of ESCC. Humn RAD17 is humn homologue of Schizoscchromyces pombe cell cycle checkpoint gene RAD17 [14]. The protein encoded by hrad17 gene is required for cell cycle rrest nd DNA dmge repir in response to DNA 63 Int J Mol Epidemiol Genet 2016;7(1):58-66

7 dmge nd incomplete DNA repliction. The phosphoryltion of hrad17 protein by ATR is required for cell cycle G2 phse rrest with CHK1 ctivtion induced by DNA dmge [18, 19]. HRAD17 protein hs two importnt regions, P loop (Wlker A motif) which is criticl for nucleotide binding of ATPses nd C terminus contining two SQ motifs (Ser635 nd Ser645) tht re strong trgets for ATP kinse. The phosphoryltion on both SQ sites is essentil for hrad17 function fcilitting interction with the RAD9-HUS1-RAD1 complex nd is implicted in the ctivtion of the G2/M checkpoint [18, 19]. Repliction fctor C is clmp loder plying importnt roles in DNA metbolism by loding the clmps in response to DNA dmge. HRAD17 forms repliction fctor C like clmp loder complex with the four smll RFC subunits nd lods the RAD9-HUS1-RAD1 clmp complex, PCNA-like sliding clmp, onto dmged DNA site in n ATP-dependent ctivtion process. The RAD9-HUS1-RAD1 complex fcilittes ATR medited phosphoryltion nd ctivtion of CHK1 which regultes S-shpe progression, G2/M rrest, nd repliction fork repir [16]. The hrad17 codon 546 polymorphism is locted ner SQ motif phosphorylted by ATR. The mino cid substitutions from leucine to rginine t codon 546 my hve effect on the ATR-dependent phosphoryltion t SQ site in response to DNA dmge. A recent study showed tht loss of hrad17 expression occurred frequently in hed nd neck squmous cell crcinom [20]. The expression of hrad17 mrna in tumor tissue decresed compred with norml tissue of hed nd neck squmous cell crcinom ptients. Although low expression of hrad17 in ESCC hs not been reported, strong correltion between excess smoking nd lcohol drinking nd hed nd neck squmous cell crcinom hs been found, s is the cse for ESCC. Therefore, it is likely tht down regultion of hrad17 my lso be contributed to the development of ESCC. The dt showed tht Arg/Arg homozygosity hs significnt ssocition with the risk of ESCC mong mle ptients compred to Leu/ Leu homozygosity nd Leu/Arg heterozygosity mong light drinkers. Previous studies observed tht excessive lcohol drinking incresed the risk of ESCC mong Jpnese men who hd deficient phenotype for ldehyde dehydrogense-2 (ALDH2) [8-10]. However, we could not find the ssocition between the hrad17 Leu546Arg polymorphism nd the risk of ESCC mong mle ptients with history of hevy drinking. Our result lso indicted tht there ws no significnt ssocition between the hrad17 Leu546Arg polymorphism nd the risk of other cncers including hed nd neck squmous cell crcinom, gstric cncer, lung squmous cell crcinom nd lung denocrcinom. Recently, RAD17 homolog isofom1 hs been reported to be down-regulted in ethnol-treted humn embryonic crcinom cell-derived embryoid bodies [21]. Chronic nd hevy exposure of esophgel squmous cell to lcohol my led to down-regulted hrad17 gene trnscription. Consequently, llele independent trnscriptionl downregultion of the gene rther thn the genetic vrint hrad17 Leu546Arg my be ssocited with the risk of ESCC mong hevy drinkers. The expression level of hrad17 is different for ech type of cncer, lower expression in hed nd neck squmous cell crcinom [20] nd overexpression in colon crcinom, non-smll cell lung crcinom nd brest cncer [15, 22-24]. In fission yest, the reduction of yest colony growth nd slower progression through cell cycle were observed by expression of hrad17 in S. pombe rd17 deleted strin [25]. These reports suggest tht hrad17 plys n importnt role in regultion of tumor growth. Our cse-control study hs severl limittions including smll smple size nd indequte djustment for vrious confounding fctors. The smll smple size my limit the sttisticl power of our study, prticulrly for the subgroup nlysis. The number of femle ptients with ESCC is smller thn mle ptients, so we could not investigte the effect of hrad17 polymorphism mong femle ESCC ptients. The difference in smoking nd lcohol drinking sttus between ESCC mle ptients nd cncerfree controls ws sttisticlly significnt. Therefore, we could not perform more detiled strtifiction of ptients of non-smoker nd non-drinker. Although our study hs these limi- 64 Int J Mol Epidemiol Genet 2016;7(1):58-66

8 ttions, we identified the ssocition between hrad17 polymorphism nd the high risk group of ESCC. In conclusion, we found tht homozygosity of vrint llele of hrad17 is significntly ssocited with ESCC risk in Jpnese men, in prticulr, mong moderte lcohol drinkers. Lrger studies on the hrad17 polymorphisms re wrrnted to confirm our results. Acknowledgements This work ws supported by Grnt-in-Aid from the Ministry of Eduction, Culture, Sports, Science nd Technology (grnt no ), nd prtilly supported by the Smoking Reserch Foundtion (K. S.). We pprecite Drs. Ouchid M., Knzki T., nd Koide N. (Okym University School of Medicine), Kusk Y. (Kusk Hospitl, Bizen, Okym), Hmnishi S. nd Senoh E. (Junpuki Helth Mintennce Center, Okym) for their generous help in this study. Disclosure of conflict of interest None. Address correspondence to: Hiroshi Ktym nd Kenji Shimizu, Deprtment of Moleculr Oncology, Okym University Grdute School of Medicine, Dentistry nd Phrmceuticl Sciences, 2-5-1, Shikt-Cho, Kit-Ku, Okym , Jpn. Tel: ; Fx: ; E-mil: (HK), (KS) References [1] Foundtion for Promotion of Cncer Reserch: Cncer Sttistics in Jpn Edited by Kym T, Sobue T, Ktnob K, Tsukum H, Mikmi H nd Kiti A: Editoril Bord of the Cncer Sttistics in Jpn. Tokyo, 2010, pp [2] Ishiguro S, Sszuki S, Inoue M, Kurhshi N, Iwski M nd Tsugne S. Effect of lcohol consumption, cigrette smoking nd flushing response on esophgel cncer risk: popultion-bsed cohort study (JPHC study). Cncer Lett 2009; 275: [3] Oze I, Mtsuo K, Ito H, Wki K, Ngt C, Mizoue T, Tnk K, Tsuji I, Tmkoshi A, Sszuki S, Inoue M nd Tsugne S. Cigrette smoking nd esophgel cncer risk: n evlution bsed on systemtic review of epidemiologic evidence mong the Jpnese popultion. Jpn J Clin Oncol 2012; 42: [4] Oze I, Mtsuo K, Wki K, Ngt C, Mizoue T, Tnk K, Tsuji I, Sszuki S, Inoue M nd Tsugne S. Alcohol drinking nd esophgel cncer risk: n evlution bsed on systemtic review of epidemiologic evidence mong the Jpnese popultion. Jpn J Clin Oncol 2011; 41: [5] Lin Y, Kikuchi S, Tmkoshi A, Wki K, Kwmur T, Iso H, Ogimoto I, Ygyu K, Obt Y nd Ishibshi T. Alcohol consumption nd mortlity mong middle-ged nd elderly Jpnese men nd women. Ann Epidemiol 2005; 15: [6] Ymji T, Inoue M, Sszuki S, Iwski M, Kurhshi N, Shimzu T nd Tsugne S. Fruit nd vegetble consumption nd squmous cell crcinom of the esophgus in Jpn: the JPHC study. Int J Cncer 2008; 123: [7] Hiym T, Yoshihr M, Tnk S nd Chym K. Genetic polymorphisms nd esophgel cncer risk. Int J Cncer 2007; 121: [8] Yokoym A, Kto H, Yokoym T, Tsujink T, Muto M, Omori T, Hned T, Kumgi Y, Igki H, Yokoym M, Wtnbe H, Fukud H nd Yoshimizu H. Genetic polymorphisms of lcohol nd ldehyde dehydrogenses nd glutthione S-trnsferse M1 nd drinking, smoking, nd diet in Jpnese men with esophgel squmous cell crcinom. Crcinogenesis 2002; 23: [9] Yokoym T, Yokoym A, Kto H, Tsujink T, Muto M, Omori T, Hned T, Kumgi Y, Igki H, Yokoym M, Wtnbe H nd Yoshimizu H. Alcohol flushing, lcohol nd ldehyde dehydrogense genotypes, nd risk for esophgel squmous cell crcinom in Jpnese men. Cncer Epidemiol Biomrkers Prev 2003; 12: [10] Yokoym A, Kto H, Yokoym T, Igki H, Tsujink T, Muto M, Omori T, Kumgi Y, Yokoym M nd Wtnbe H. Esophgel squmous cell crcinom nd ldehyde dehydrogense-2 genotypes in Jpnese femles. Alcohol Clin Exp Res 2006; 30: [11] Yokoym T, Yokoym A, Kumgi Y, Omori T, Kto H, Igki H, Tsujink T, Muto M, Yokoym M nd Wtnbe H. Helth risk pprisl models for mss screening of esophgel cncer in Jpnese men. Cncer Epidemiol Biomrkers Prev 2008; 17: [12] Pn J, Lin J, Izzo JG, Liu Y, Xing J, Hung M, Ajni JA nd Wu X. Genetic susceptibility to esophgel cncer: the role of the nucleotide excision repir pthwy. Crcinogenesis 2009; 30: Int J Mol Epidemiol Genet 2016;7(1):58-66

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