Association of genetic polymorphisms in DNMT3A with the progression of gastric mucosal atrophy and susceptibility to gastric cancer in Japan

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1 ONCOLOGY LETTERS Assocition of genetic polymorphisms in DNMT3A with the progression of gstric mucosl trophy nd susceptibility to gstric cncer in Jpn WU JING 1, TOSHIMI OTSUKA 1, MASAKATSU NAKAMURA 1, NAOKO SAKURAI 1, HIKARU TAKANO 1, TASUKU HAYASHI 1, MASAFUMI OTA 1, TOMOE NOMURA 1, RANJI HAYASHI 1, TAKEO SHIMASAKI 1, TOMOMITSU TAHARA 2, TOMOYUKI SHIBATA 2 nd TOMIYASU ARISAWA 1 1 Deprtment of Gstroenterology, Knzw Medicl University, Uchind mchi, Ishikw ; 2 Deprtment of Gstroenterology, Fujit Helth University, Kutsukke cho, Toyoke , Jpn Received August 30, 2018; Accepted Jnury 2, 2019 DOI: /ol Abstrct. The im of the present study ws to investigte whether single nucleotide polymorphisms in the DNMT3A gene re ssocited with susceptibility to gstric cncer in the Jpnese popultion. The present cse control study exmined the ssocitions between single nucleotide polymorphisms (rs nd rs ) in DNMT3A nd cncer susceptibility in 343 ptients with gstric cncer nd 708 subjects without gstric mlignncies on upper gstro duodenl endoscopy. Of 708 controls, 409 were clssified into two groups histologiclly: 99 cses with nd 310 cses without gstric mucosl trophy. Overll, homozygosity for the DNMT3A rs minor llele ws significntly ssocited with reduced risk of gstric cncer (odds rtio [OR], 0.621; 95% confidence intervl [CI], ; P=0.031), especilly of the intestinl type (OR, 0.494; 95% CI, ; P=0.019). In subjects >60 yers, rs minor llele homozygosity ws significntly ssocited with gstric cncer susceptibility. Crriers of the rs minor llele hd reduced risk of severe gstric mucosl trophy (OR, 0.495; 95% CI, ; P=0.0069). In ddition, the number of minor lleles of both rs nd rs ws significntly correlted with the risk of Helicobcter pylori (HP) infection (P= nd P=0.0050, respectively). However, rs ws not ssocited with severe gstric mucosl trophy or gstric crcinogenesis. The present results suggest tht DNMT3A polymorphisms serve roles in the progression from HP infection to gstric mucosl trophy nd gstric crcinogenesis in terms of degree nd mnner. Correspondence to: Professor Tomiysu Arisw, Deprtment of Gstroenterology, Knzw Medicl University, 1 1 Digku, Uchind mchi, Ishikw , Jpn E mil: trisw@knzw med.c.jp Key words: DNMT3A, genetic polymorphism, gstric cncer, trophic gstritis, Helicobcter pylori infection Introduction The incidence of gstric cncer (GC) vries worldwide; the disese is four times more common in Jpn thn in the UK nd occurs in younger ptients (1). In Jpn, 50,000 people die of GC nnully. GC progression involves vriety of gene ltertions nd number of specific events (2), such s overexpression of oncogenes nd inctivtion of tumor suppressor genes. Helicobcter pylori (HP) infection nd consequent trophic gstritis re regrded s risk fctors for GC. Reserch hs shown tht HP infection cn cuse GC vi trophic gstritis (3,4). Severe gstric trophy (SA) nd corpus predominnt gstritis, intestinl metplsi (IM), nd dysplsi re well known predisposing fctors for GC (5,6). Previous reserch suggested tht gstric crcinogenesis involves three steps: HP infection, development of gstric precncerous conditions, nd crcinogenesis (7). However, reltively few cses of HP infection progress to GC. This discrepncy hs prompted considerble reserch exmining potentil ssocitions between genetic polymorphisms nd the risk of progression from precncerous conditions to GC. Methyltion of severl genes hs been reported in mny cncers, including GC (8). The DNA methyltrnsferse (DNMT) fmily includes three ctive mmmlin homologs: DNMT1, 3, nd 3b. DNMT3 nd DNMT3b re considered de novo enzymes tht ply criticl roles in the dynmic DNA methyltion process during embryogenesis nd pthogenesis (9). In both GC nd pr cncerous tissues, the expression of DNMTs is significntly higher thn in norml tissues (10), which suggests tht DNMT overexpression is involved in the development of gstric mucosl trophy nd subsequent tumorigenesis. Mny studies hve reported n ssocition between rs , notble polymorphism in the DNMT3A gene, nd susceptibility to vrious cncers, including GC (11,12). However, no significnt ssocition between rs nd GC susceptibility hs been reported (13,14). Thus, the influence of DNMT3A polymorphisms upon GC susceptibility remins uncler, especilly in the Jpnese popultion. In this study, we investigted potentil ssocitions between SA nd GC susceptibility nd two DNMT3A polymorphisms:

2 2 JING et l: DNMT3A POLYMORPHISMS AND GASTRIC CANCER SUSCEPTIBILITY rs C>G (in linkge with rs , rs , rs , rs , nd rs ) nd rs A>G (in linkge with rs nd rs ). Mterils nd methods Clinicl smples. All subjects were enrolled t the Endoscopy Center of Fujit Helth University Hospitl or Knzw Medicl University Hospitl between April 2005 nd Mrch The study involved 343 ptients with GC (GC group) nd 708 subjects with no evidence of gstric mlignncies (non GC group) on upper gstro duodenl endoscopy. In ddition, 409 of 708 controls in which the degree of histologic gstritis could be ssessed ccording to the updted Sydney system using biopsy specimens obtined from ntrum (15) were clssified into two groups: 99 ptients in the SA group (trophy score of 3 or metplsi score 2) nd 310 ptients in the non SA group. Dignosis of ll GCs ws mde histologiclly t the Division of Pthology of ech hospitl. Ptients with severe systemic diseses, mlignncies in other orgns, or who hd received nonsteroidl nti inflmmtory drugs, ntibiotics, or HP erdiction tretment were excluded. We judged HP infection sttus s positive when the rpid urese test, ure breth test, or histologic test ws positive. The Ethics Committees of Fujit Helth University nd Knzw Medicl University pproved the protocol, nd written informed consent ws obtined from ll prticipting subjects. Single nucleotide polymorphism selection nd detection. We selected the Tg polymorphism with high minor llele frequency (MAF) in the DNMT3A gene region. We selected rs C>G in lrge linkge disequilibrium block with Hrdy Weinberg equilibrium (HWE) P vlue >0.05 nd MAF >0.30 determined ccording to the LD TAG SNP selection dtbse ( Fig. 1). In ddition, nother SNP with high MAF (rs A>G) tht hd been investigted in previous study bsed on its ssocition with GC susceptibility (13) lso ws selected. This polymorphism is in linkge with rs (Fig. 1). Smple stocks of DNA isolted from peripherl blood were used in the study. Genotyping of DNMT3A polymorphisms ws crried out using polymerse chin rection (PCR) single strnd conformtion polymorphism (SSCP) methods, s reported previously (16,17). The rs nd rs genotypes were determined using the following primer pirs: for rs , forwrd 5' ct gct gc ggg gt cgc tgt c 3' nd reverse 5' ctc ctg gct gtg g cgg g 3'; for rs , forwrd 5' ccc ct ct gtc g tc cct ctg 3' nd reverse 5' cct tcc tg ggg c ccc ttc tt t 3'. PCR ws crried out in 20 µl rection volume contining 0.1 µg of genomic DNA. The DNA ws dentured t 95 C for 3 min, followed by 35 cycles t 96 C for 15 sec, 61 C for 30 sec, nd 72 C for 30 sec, with finl extension t 72 C for 5 min. PCR conditions for mplifiction of rs nd rs were the sme. Therefter, 2 µl of the PCR product ws dentured with 10 µl of formmide (Sigm Aldrich Co., St. Louis, MO, USA) t 90 C for 5 min. SSCP ws crried out t 18 C. We used Gene Phor DNA seprtion system with Gene Gel Excel 12.5/24 (GE Helth Cre Bio Sciences AB, Stockholm, Sweden), fter which the dentured single strnd DNA bnds were detected using DNA silver stining kit (GE Helth Cre Bio Sciences AB). Sttisticl nlysis. The HWE of ech llele ws ssessed using χ 2 test. Dt re expressed s men ± SD. Differences in men ge of ptients in ech group were evluted using the Student's t test. Differences in rtios of HP infection sttus nd mle to femle ptients were evluted using Fisher's exct test. Allele nd genotype frequencies were determined by direct counting. Differences in llele count lso were evluted using Fisher's exct test. The strength of ssocition between llele frequencies nd disese ws ssessed by clculting the odds rtio (OR) nd 95% confidence intervl (CI) by logistic regression nlysis. Adjusted ORs considered ge, gender, nd HP infection sttus. An djusted nlysis lso ws performed by logistic regression nlysis fter djustment for gender, ge, nd HP infection sttus. For ll nlyses, the level of significnce ws set t P<0.05. Anlyses were performed using Stt softwre (version 13; SttCorp LP, College Sttion, TX, USA). Results Chrcteristics of subjects nd the frequencies of genotypes. Single strnd DNAs of rs nd rs were clerly seprted by SSCP (Fig. 2). The chrcteristics of subjects in this study re summrized in Tble I. The men ge, mle to femle rtio, nd HP positivity rtio were significntly higher in the GC group thn in the non GC group. The distribution of the rs C>G genotype in the GC group ws 130CC, 181CG, nd 32GG. The distribution of the rs C>G genotype in the non GC group ws 253CC, 338CG, nd 117GG (HWE P=0.82). The rs g llele frequency in the GC nd non GC groups ws 35.7 nd 40.4%, respectively (P=0.04). In ddition, the frequency of the rs GG homozygote differed significntly between the GC nd non GC groups (P=0.0018). The distribution of the rs A>G genotype in the non GC group ws 389AA, 276AG, nd 43GG (HWE P=0.69). There ws no significnt difference in either the minor llele frequency or distribution of the rs genotype between the GC nd non GC groups. Assocition between GC susceptibility nd DNMT3A polymorphisms. Overll, ptients homozygous for the rs G llele hd significntly decresed risk for gstric crcinogenesis s determined by logistic regression nlysis fter djustment for ge, gender, nd HP infection sttus (OR, 0.621; 95% CI, ; P=0.031, Tble II). When ssessed by GC subtype, ptients who were rs GG homozygotes hd significntly decresed risk for the development of intestinl cncers (OR, 0.494; 95% CI, ; P=0.019, Tble II), wheres no significnt ssocition ws found between this genotype nd diffuse types of cncer. No significnt ssocition ws found between GC susceptibility nd rs (Tble II). Assocition between DNMT3A polymorphisms nd GC susceptibility in subjects younger or older thn 60 yers. Ptients who were rs GG homozygotes exhibited significntly decresed risk for gstric crcinogenesis by logistic regression nlysis fter djustment for ge, gender, nd HP infection sttus (OR, 0.534; 95% CI, ; P=0.024, Tble III). On the other hnd, significnt ssocition between rs nd GC susceptibility ws not seen in subjects clssified bsed on ge (whether younger or older thn 60 yers).

3 ONCOLOGY LETTERS 3 rs g llele frequency ws lso significntly different between the SA nd non SA groups (P=0.0082). In ddition, the proportion of the AA homozygotes ws significntly higher nd tht of the GG homozygotes significntly lower in the SA group thn in the non SA group (P=0.044 nd P=0.013, respectively, Tble IV). Logistic regression nlysis fter djustment for ge, gender, nd HP infection sttus indicted tht the rs CG+GG genotype ws significntly ssocited with decresed severity of gstric mucosl trophy (OR, 0.495; 95% CI, ; P=0.0069, Tble V), wheres rs ws not ssocited with mucosl trophy. Figure 1. Linkge disequilibrium of DNMT3A. This figure ws generted using snpinfo.niehs.nih.gov/snpinfo/snptg.html. Assocition between HP infection sttus nd DNMT3A polymorphisms in control subjects. The men ge nd mle to femle rtio were significntly higher in HP infected subjects thn in uninfected subjects (Tble VI). The minor llele frequency of both rs nd rs ws significntly higher in uninfected subjects thn in HP infected subjects. The proportions of ptients who were rs GG nd rs GG homozygotes were lso significntly higher in HP infected subjects thn in uninfected subjects, wheres those of the rs CC nd rs AA homozygotes were significntly lower in HP infected thn in uninfected subjects. The number of minor lleles of both rs nd rs ws significntly correlted with the frequency of HP infection (P= nd P= by ANCOVA, respectively). Discussion Figure 2. Products of polymerse chin rection SSCP nlyses using clinicl smples. Single strnd DNAs were seprted by SSCP. These dt enble determintion of the genotype. SSCP, single strnd conformtion polymorphism. Chrcteristics of subjects nd genotype frequencies in subjects in which gstric mucosl trophy ws ssessed. For 409 of 708 controls, the degree of histologic gstritis could be ssessed ccording to the updted Sydney system, nd the chrcteristics nd genotype distributions for these ptients re shown in Tble IV. In ll 409 such subjects, the genotype distribution of rs ws 165CC, 186CG, nd 58GG (HWE P=0.67), wheres tht of rs ws 249AA, 136AG, nd 24GG (HWE P=0.39). The men ge, mle to femle rtio, nd HP positivity rtio were significntly higher in the SA group thn the non SA group (Tble IV). The rs g llele frequency in the SA nd non SA groups ws 29.3 nd 39.4%, respectively (P=0.011). In ddition, the proportion of ptients who were rs GG homozygotes differed significntly between the SA nd non SA groups (P=0.0014). The Globl DNA methyltion ptterns reportedly lter the hyper methyltion of CpG islnds nd the hypo methyltion of non CpG islnds (18). The ction of de novo DNMTs, including DNMT3, is responsible for this ltertion during erly tumorigenesis (19). In gstric crcinogenesis, overexpression of DNMT3 occurs in both cncerous nd pr cncerous tissues (10). In ddition, HP infection reportedly induces berrnt DNA methyltion of CpG islnds, subsequently suppressing the function of tumor suppressor genes in the gstric mucos, nd ultimtely resulting in crcinogenesis (20,21). HP infection reportedly does not directly induce either the mrna or protein expression of DNMT1, DNMT3, or DNMT3b in the gstric mucos (22). However, the rs genetic polymorphism results in incresed trnscription of the DNMT3A gene, nd n increse in the level of DNMT3A mrna ssocited with gstric crcinogenesis (23). Thus, polymorphisms in DNMT3A re thought to ply n importnt role in gstric crcinogenesis. However, lthough mny studies hve exmined the reltionship between rs nd crcinogenesis, the potentil ssocition remins uncler. Bsed on the hypothesis tht the other polymorphism in DNMT3A is more clerly ssocited with gstric crcinogenesis, we investigted the potentil ssocitions of other polymorphisms in DNMT3A tht re not in strong linkge with rs Specificlly, we investigted two llele sites of DNMT3A (rs nd rs ). The distributions of both rs nd in our control group were in HWE (P=0.82 nd P=0.69, respectively), nd were similr to those reported in the Jpnese popultion in the HpMp dtbse (P=0.98 nd

4 4 JING et l: DNMT3A POLYMORPHISMS AND GASTRIC CANCER SUSCEPTIBILITY Tble I. Subject chrcteristics nd genotype frequencies. Chrcteristics Non GC group GC group P vlue Number of subjects Men ge ± stndrd devition (yers) 61.0± ±11.4 < Mle:femle 405: : b HP positive rte 435/ /343 < b rs C>G CC CG GG b G llele frequency 40.4% 35.7% 0.04 b rs A>G AA AG GG G llele frequency 25.6% 21.9% Student's t test, b Fisher's exct test; GC, gstric cncer; HP, Helicobcter pylori. Tble II. Assocition between DNMT3A polymorphisms nd gstric cncer. A, GG vs. CC+CG rs C>G, (n) CC CG GG OR (95% CI) P vlue Non GC (708) Reference Overll GC (343) ( ) Intestinl (195) ( ) Diffuse (133) ( ) 0.36 (Unknown) B, GG vs. AA+AG rs A>G, (n) AA AG GG OR (95% CI) P vlue Non GC (708) Reference Overll GC (343) ( ) 0.25 Intestinl (195) ( ) 0.35 Diffuse (133) ( ) 0.48 (Unknown) The number of subjects following regression nlysis with djustments for ge, gender nd HP infection sttus. OR (95% CI), odds rtio (95% confidence intervl); GC, gstric cncer; HP, Helicobcter pylori. P=0.99, respectively). We found decresed ssocition between DNMT3A homozygotes nd gstric crcinogenesis, especilly for intestinl types of cncer. However, no ssocition ws observed between rs nd susceptibility to GC. HP infection is known to cuse chronic inflmmtion, which subsequently progresses to trophic gstritis, intestinl metplsi, nd finlly, GC (15). In our present study, the rs minor llele ws ssocited with significntly lower risk of gstric mucosl trophy. These observtions suggest tht in crriers of the rs minor llele, progression to gstric mucosl trophy nd subsequent development of GC my be inhibited in the homozygotes. In ddition, stronger ssocition ws detected in older rther thn younger subjects, consistent with the expecttion tht intestinl types of GC occur s result of n extended period of chronic inflmmtion nd tissue rerrngement, including metplstic chnge. Previously, Co et l (13) reported tht rs is ssocited with higher risk of HP infection but not of gstric trophy

5 ONCOLOGY LETTERS 5 Tble III. Assocition between DNMT3A polymorphisms nd GC susceptibility in subjects younger or older thn 60 yers. A, rs Age CC CG GG GG vs. CC+CG, OR (95% CI) P vlue <60 yers Non GC (278) Reference GC (105) ( ) yers Non GC (429) Reference GC (238) ( ) B, rs Age AA AG GG GG vs. AA+AG, OR (95% CI) P vlue <60 yers Non GC (278) Reference GC (105) ( ) yers Non GC (429) Reference GC (238) ( ) 0.46 The number of subjects following logistic regression nlysis with djustment for ge, gender nd HP infection sttus. OR (95% CI), Odds rtio (95% confidence intervl); GC, gstric cncer; HP, Helicobcter pylori. Tble IV. Chrcteristics nd genotype frequencies of subjects in whom histologicl findings were evluted. Chrcteristics Totl Non SA group SA group P vlue c Number of subjects Men ge ± SD (yers) 59.9± ± ± Mle:femle 240: :142 72: b H. pylori positive rte 262/ /310 93/99 < b rs C>G CC b CG GG G llele frequency (%) b rs A>G AA b AG GG b G llele frequency (%) b Student's t test, b Fisher's exct test; c non AS group vs. SA group. SD, Stndrd devition; SA, Severe trophy; HP, Helicobcter pylori. or GC. Those uthors speculted tht this DNMT3A polymorphism fcilittes HP infection by promoting methyltion of the gene encoding MUC 1, membrne bound mucin expressed on the surfce of gstric epithelil cells tht normlly provides protective brrier ginst HP infection (24). Interestingly, our results showed tht both rs nd rs were strongly ssocited with the risk of HP infection. Specificlly, rs ws ssocited with higher HP infection risk, severe gstric mucosl trophy, nd susceptibility to GC, especilly intestinl types of cncer, wheres rs ws ssocited only with HP infection risk. Potentil ssocitions of rs with clinicl disorders hve not been reported. In ddition, there re no dt vilble regrding the influence of either polymorphism on the expression or function of DNMT3. However, we

6 6 JING et l: DNMT3A POLYMORPHISMS AND GASTRIC CANCER SUSCEPTIBILITY Tble V. Assocition between DNMT3A polymorphisms nd gstric mucosl trophy. A, rs C>G Atrophy sttus, (n) CC CG GG OR (95% CI), CG+GG vs. CC P vlue Non SA (310) Reference SA (99) ( ) B, rs A>G Atrophy sttus, (n) AA AG GG OR (95% CI), AG+GG vs. AA P vlue Non SA (310) Reference SA (99) ( ) 0.73 The number of subjects following logistic regression nlysis with djustment for ge, gender nd HP infection sttus. OR (95% CI), Odds rtio (95% confidence intervl); SA, severe trophy; HP, Helicobcter pylori. Tble VI. Assocition between HP infection sttus nd DNMT3A polymorphisms in control subjects. Vrible HP uninfected HP infected P vlue No. of subjects Men ge ± SD (yers) 59.9± ± Mle:femle 133: : b rs C>G CC b CG GG b G llele frequency (%) b rs A>G AA b AG GG b G llele frequency (%) b Student's t test, b Fisher's exct test; SD, stndrd devition; HP, Helicobcter pylori. hypothesize tht these polymorphisms ffect the expression of DNMT3 to vrying degrees, s mny studies suggest. If so, the difference in risk ssocition between rs nd rs my depend on the difference in the number of ffected genes nd/or the degree of methyltion. Of course, the expression of DNMT3 is not regulted only by DNMT3A polymorphisms. Kim et l (25) reported tht DNMT3A muttions nd llelic losses, which decrese the enzymtic ctivity of the protein product, re observed in mny solid cncers, suggesting tht bnorml expression or ccumultion of DNMT3 in cncer tissues my be due to defects in the degrdtion of mutnt products rther thn to the polymorphisms themselves. There re some clinicl limittions to our study. The first limittion is tht our subjects were ptients who visited our hospitl for either endoscopic exmintions due to specific symptoms or further follow up fter helth exmintions. Subjects reporting no symptoms re essentil for control groups. In ddition, we could not confirm whether very smll histologic neoplsi ws present in ny of the control group subjects. A second limittion is tht we ssessed histologic gstritis using biopsy smples only from the ntrum, becuse the ntrl mucos is ffected by HP infection for the longest period. Clerer results might hve been obtined if the degree of gstritis in the corpus ws ssessed t the sme time. A third limittion is tht ethnicity is n importnt fctor ffecting heterogeneity. Although different countries nd popultions hve different dietry nd lifestyle hbits (26), this retrospective reserch ws performed t single Jpnese center. In conclusion, regrding the influence of rs , our results suggest tht the risk of HP infection decreses depending on the number of minor lleles, the risk of severe gstric mucosl trophy decreses in minor llele crriers,

7 ONCOLOGY LETTERS 7 nd the risk of developing GC decreses in minor llele homozygotes. In contrst, rs is ssocited only with HP infection nd not with severe gstric mucosl trophy or gstric crcinogenesis. Thus, the dt suggest tht both DNMT3A polymorphisms prticipte in the progression from HP to gstric mucosl trophy nd ultimtely to gstric crcinogenesis in both degree nd mnner. Acknowledgements Not pplicble. Funding No funding ws received. Avilbility of dt nd mterils All dt generted or nlyzed during this study re included in this published rticle. Authors' contributions WJ nlyzed the dt nd wrote the pper. TO, MN, NS, HT, TH, MO, TN nd RH obtined the smples nd performed the experiments to obtin the dt. TS determined the genotype. TT nd ToS obtined the smples nd prticipted in the design of the study. TA ws responsible for the conception nd design of the study. All uthors hve red nd pproved the finl mnuscript. Ethics pprovl nd consent to prticipte The Ethics Committees of Fujit Helth University nd Knzw Medicl University pproved the protocol, nd written informed consent ws obtined from ll prticipting subjects. Ptient consent for publiction All ptients gve informed consent. Competing interests The uthors declre tht they hve no competing interests. References 1. Neugut AI, Hyek H nd Howe G: Epidemiology of gstric cncer. Semin Oncol 23: , Ysui W, Sentni K, Motoshit J nd Nkym H: Moleculr pthobiology of gstric cncer. Scnd J Surg 95: , Wtnbe Y, Kurt JH, Mizuno S, Muki M, Inokuchi H, Miki K, Ozs K nd Kwi K: Helicobcter pylori infection nd gstric cncer. A nested cse control study in rurl re of Jpn. Dig Dis Sci 42: , Ozs K, Kurt JH, Higshi A, Hyshi K, Inokuchi H, Miki K, Td M, Kwi K nd Wtnbe Y: Helicobcter pylori infection nd trophic gstritis: A nested cse control study in rurl town in Jpn. Dig Dis Sci 44: , Corre P: A humn model of gstric crcinogenesis. 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