Variations in the bitterness perception-related genes TAS2R38 and CA6 modify the risk for colorectal cancer in Koreans

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1 /, 2017, Vol. 8, (No. 13), pp: Vritions in the itterness pereption-relte genes TAS2R38 n CA6 moify the risk for oloretl ner in Korens Jeong-Hw Choi 1, Jeonghee Lee 1, Je Hwn Oh 2, Hee Jin Chng 2, De Kyung Sohn 2, Aesun Shin 3, Jeongseon Kim 1 1 Moleulr Epiemiology Brnh, Division of Cner Epiemiology n Prevention, Ntionl Cner Center, Ilsnong-gu, Goyng-si, Gyeonggi-o, 10408, Kore 2 Center for Coloretl Cner, Ntionl Cner Center Hospitl, Ntionl Cner Center, Ilsnong-gu, Goyng-si, Gyeonggi-o, 10408, Kore 3 Deprtment of Preventive Meiine, Seoul Ntionl University College of Meiine, Jongno-gu, Seoul, 03080, Kore Corresponene to: Jeongseon Kim, emil: jskim@n.re.kr Aesun Shin, emil: shinesun@snu..kr Keywors: itterness pereption, CA6, oloretl ner, ietry intke, TAS2R38 Reeive: Novemer 08, 2016 Aepte: Ferury 07, 2017 Pulishe: Ferury 19, 2017 ABSTRACT Reserh Pper Bitterness pereption is known to e n importnt ftor in iniviuls ietry ehviors n is lso ssoite with the sensing of nutritious/noxious moleules for susequent metoli responses in multiple orgns. Therefore, the geneti vrition in itterness sensing my e ssoite with iet-relte iseses, inluing oloretl ner (CRC). We investigte the influene of vritions in the itterness-sensing genes tste reeptor type 2 memer 38 (TAS2R38) n roni nhyrse 6 (CA6) on the onsumption of foo, too n lohol n the risk of CRC in Korens. The stuy popultion onsiste of 681 ses n 1361 ontrols, n their intke of vegetles, fruits, fier, ft-foo n sweets ws nlyze. The genotypes for TAS2R38 A49P, V262A n I296V n CA6 rs A/G were ssesse using the MssArry tehnique. Our finings suggeste tht the TAS2R38 iplotype, CA6 rs n their omine genotype h negligile influene on ietry n lohol intke. The omine TAS2R38-CA6 AVI/AVI-AA genotype ws ssoite with higher too onsumption thn the other genotypes in CRC ses only. However, the geneti vritions were signifint risk ftor for CRC. The TAS2R38 AVI/AVI iplotype n CA6 G llele were ssoite with reue risk of CRC. Moreover, when the omine genotypes of the sujets were nlyze, possessing oth the vrint iplotype/vrint llele (AVI/AVI+G*) ws ssoite with greter reution in the risk of CRC (juste OR = 0.49; 95%CI: ). In summry, vritions in the itterness pereption genes TAS2R38 n CA6 i not influene the exmine foo intke in Korens. However, those geneti vrints were eisive moifying ftor of CRC suseptiility. INTRODUCTION Coloretl ner (CRC) hs een mjor helth onern in the Western hemisphere n is urrently n emerging issue in Kore. Although the reent mortlity rte of CRC seems to hve stilize or eline, the gejuste inienes of CRC for men n women were 45.6 n 24.4 per 100,000 in 2013, respetively, mking CRC the thir most ommon type of ner in Kore [1]. Eviene suggests tht in ition to ommon environmentl ftors, inluing nutrition n ietry intke, n iniviul s geneti kgroun is ritil omponent of CRC etiology [2]. Therefore, geneti vrints in tste pereption, espeilly itterness sensing n its influene on ietry intke, re onsiere signifint risk ftors for CRC suseptiility. Beuse itterness is key eterminnt in the rejetion/ eptne of foo prouts, genetilly moulte sensitivity to itterness intensity my le to n iniviul s ifferentil intke of ietry n onsumer goos, whih my further e linke to the risk of iet-relte iseses [3]

2 The pereption of the itter thioynte (N-C=S) moiety s teste with 6-n-propylthiouril (PROP) n phenylthiormie (PTC) hs een stuie extensively s humn tsting trit with respet to ietry ehvior n helth outomes [3]. The vriility of PROP/PTC itterness senstion mong iniviuls is known to e ssoite with the tste reeptor type 2 memer 38 (TAS2R38, T2R38) gene, n its hplotype onsists of three ommon missense vritions (A49P, V262A n I296V). Iniviuls with the PAV hplotype re sensitive to the itterness of PROP/PTC (tster), wheres those who possess the AVI/AVI hplotype re not (non-tster) [4]. The TAS2R38 iplotype ws therefore oserve to e ssoite with n iniviul s ifferentil intke of itter tsting everges, ruiferous vegetles (whih re high in gluosinoltes ontining thioure moieties), fruits n loo folte onentrtions [3, 5 8]. Furthermore, the TAS2R38 iplotype hs een inepenently ssoite with the risk of gstrointestinl ner regrless of moifitions in ietry foo intke [9 11]. The sensing of hrmful moleules y the gusttory T2R38 my initite susequent protetive responses, suh s the neutrliztion or expulsion of rinogeni moleules from the limentry trk. Therefore, the ifferentil T2R38 tivity my moify the risk of CRC inepenent of ietry intke [12]. This iet-inepenent isese risk-moifying effet of T2R is lso supporte y eviene from extr-gstrointestinl trt tissues. In ells in the upper respirtory system, T2R38 respons to quorum-sensing moleules serete y grm-negtive teri n is ssoite with the tivtion of nitri oxie proution n irwy infetion [13]. T2R reeptors re lso expresse on the thyroi, whih is the enter of enorine metolism, n regulte thyroyte funtion n triioothyronine n thyroxine proution [14]. These finings suggest tht TAS2R38 geneti vrints my e geneti mrker for orgn or metoli funtion s well s itterness sensing [10, 11]. However, some ontroversy still exists: the TAS2R38 hplotype oes not ompletely esrie the ifferentil intensity of itterness n ietry intke mong iniviuls, n the ssoition etween the TAS2R38 iplotype, ietry onsumption n CRC risk lso iffers mong ethniities [11, 15]. These finings suggest tht other geneti omponents my nee to e onsiere to etter unerstn the mehnism of itterness pereption n the relte suseptiility to CRC. Croni nhyrse VI (CAVI, gustin, CA6), zin-ontining slivry protein, is known to e involve in PROP-meite itterness senstion [16 18]. CA fmily proteins ply entrl role in ph regultion n eletrolyte lne, n CAVI is known to e trophi ftor for the growth n evelopment of tste us n itterness senstion [19, 20]. One sequene vrition in CAVI (rs , A/G, S90G) ws reently reporte to moify protein funtion. Iniviuls with vrint G llele showe ifferentil fungiform ppille ensity n morphology, n the vrint G llele moifie the intensity of itterness oth on its own n in onert with the TAS2R38 hplotype [16]. The vrint CAVI protein lso showe n ssoition with the risk of ries n other orl helth prmeters [21, 22]. Moreover, some CA isozymes re thought to e ssoite with unregulte ell prolifertion n mlignny invsion [23, 24]. Consiering these regultory roles of CAVI in ellulr homeostsis n itterness senstion, vrint CAVI my e ritil moifying ftor for ietry intke n gstrointestinl ysfuntions [25]. However, the epiemiologil eviene for the moifying effet of vrint CAVI in ietry intke n its pthologil role in gstrointestinl iseses hs not een fully investigte. This stuy exmine whether the itter tste reeptor proteins T2R38 n CAVI influene the ietry, lohol n too onsumption of Korens. The role of geneti vritions in TAS2R38 n CA6 in the evelopment of CRC ws lso investigte. RESULTS Generl hrteristis of the stuy popultion The generl hrteristis of the stuy sujets showe signifint ifferenes epening on CRC phenotype (Tle 1). CRC ws more likely to evelop in former rinkers n in those with lower oy mss inex n lower eution n inome levels. Ptients with CRC were lso less likely to regulrly exerise n were more likely to live lone n to hve fmily history of CRC. These ifferenes re known to moify the risk of CRC; therefore, suh vriles were inlue in the susequent nlyses s potentil onfouners. Lstly, the ietry zin intke ws etermine euse CAVI is zinepenent metlloprotein. Sine the ontrols n CRC ptients exhiite signifint ifferenes in ietry zin, the ietry zin intke ws juste for in the sttistil moels of CA6 geneti vrition. However, there ws no signifint ifferene in the ietry zin intke mong the CA6 genotypes (t not shown). Distriution of TAS2R38 n CA6 geneti vritions n the omine genotype Tle 2 presents the istriution of the TAS2R38 iplotype, the CA6 rs genotype n their omine genotype. All geneti vrints were in Hry- Weinerg equilirium (P > 0.05). The three geneti vrints in TAS2R38 (A49P, V262A n I296V) were highly orrelte eh other (r 2 > 0.99). A totl of 6 hplotypes were present in the urrent Koren popultion. The most frequently oserve hplotypes were PAV n AVI, n their omintions (PAV/PAV, PAV/AVI n AVI/ AVI) esrie the mjority of the TAS2R38 iplotypes (99.7%). Four other hplotypes (AAV, AVV, PVI n PVV) n iplotypes (AAV/AVI, PAV/AVV, PVI/AVI n 21254

3 Tle 1: Desriptive t of the stuy popultion y oloretl ner phenotype Totl Cses Controls p Numer of prtiipnts (%) 2,042 (100) 681 (33.4) 1361 (66.7) Sex (%) Mle 1,390 (68.1) 463 (68.0) 927 (68.1) Femle 652 (31.9) 218 (32.0) 434 (31.9) Age (men, yer) 56.1 ± ± ± Boy mss inex (kg/m 2 ) < 25 1,374 (67.3) 466 (68.4) 908 (66.7) (32.7) 215 (31.6) 453 (33.3) Too smoking (%) Never 909 (44.5) 308 (45.2) 601 (44.2) Former 742 (36.3) 228 (33.5) 514 (37.8) Current 391 (19.15) 145 (21.3) 246 (18.1) Alohol rinking (%) Never 619 (30.3) 208 (30.5) 411 (30.2) Former 220 (10.8) 97 (14.2) 123 (9.0) Current 1,203 (58.9) 376 (55.2) 827 (60.76) Regulr exerise (%) <.001 Yes 1,029 (50.4) 217 (31.9) 812 (59.6) No 1,006 (49.3) 646 (68.1) 542 (39.8) Missing 7 (0.3) - 7 (0.5) First-egree fmily history of oloretl ner (%) Yes 127 (6.2) 53 (7.8) 74 (5.4) No 1,915 (93.8) 628 (92.2) 1,287 (94.6) Mritl sttus (%) <.001 Mrrie or with prtner 1,800 (88.2) 576 (84.6) 1,224 (89.9) Single 171 (8.4) 82 (12.0) 89 (6.5) Never mrrie 65 (3.2) 21 (3.1) 44 (3.2) Missing 6 (0.3) 2 (0.3) 4 (0.3) Eution level (%) <.001 Mile shool grute or less 439 (21.5) 249 (36.6) 190 (14.0) High shool grute 855 (41.9) 261 (38.3) 594 (43.6) College grute or more 739 (36.2) 171 (25.1) 568 (41.7) Missing 9 (0.4) - 9 (0.7) Househol inome (10,000 won/month) <.001 < (8.6) 91 (13.4) 85 (6.3) (18.3) 143 (21.0) 230 (16.9) (43.2) 301 (44.2) 582 (42.8) > (28.0) 146 (21.4) 424 (31.2) Missing 40 (2.0) - 40 (2.9) Dietry zin intke (mg/y) 8.4 ± ± ± 1.6 <.001 P-vlues present the ifferene etween ses n ontrols. Age n zin intke were exmine using Stuent s t-tests; other vriles were ssesse using hi-squre nlyses. Numers in prentheses re perentges; other t re presente s the mens ± stnr evition

4 Tle 2: Distriution of the TAS2R38 iplotype, CA6 rs genotype n the omine genotype TAS2R38 iplotype Totl (n = 2,042) Cse (n = 681) Control (n = 1,361) p hisq PAV/PAV 726 (35.6) 251 (36.9) 475 (34.9) PAV/AVI 981 (48.0) 337 (49.5) 644 (47.3) AVI/AVI 329 (16.1) 92 (13.5) 237 (17.4) AAV/AVI 3 (0.15) - 3 (0.2) PAV/AVV 1 (0.05) 1 (0.2) - PVI/AVI 1 (0.05) - 1 (0.1) PVV/AVI 1 (0.05) - 1 (0.1) PAV/PAV+PAV/AVI 1,707 (83.6) 588 (86.5) 1,119 (82.2) CA6 rs AA 353 (17.3) 142 (20.9) 211 (15.5) GA 957 (46.9) 299 (43.9) 658 (48.4) GG 732 (35.9) 240 (35.2) 492 (36.2) GA + GG 1,689 (82.8) 539 (79.1) 1,150 (84.6) Comine genotype (TAS2R38+CA6) e PAV/*+AA 288 (14.2) 119 (17.5) 169 (12.5) PAV/*+G* 1,419 (69.7) 469 (69.0) 950 (70.1) AVI/AVI+AA 65 (3.1) 23 (3.3) 42 (3.0) AVI/AVI+G* 264 (13.0) 69 (10.2) 195 (14.4) n inites numers of sujets. P-vlues from hi-squre tests. Comprison for the istriution of the reessive moel (PAV/PAV+PAV/AVI versus AVI/AVI) y oloretl ner phenotype. Comprison for the istriution of the ominnt moel (AA versus GA+GG) y oloretl ner phenotype. e Comine genotype of reessive moels for the TAS2R38 iplotype n ominnt moel for CA6 rs , PAV/*+G*=[TAS2R38 (PAV/PAV+PAV/AVI)+CA6 (GA+GG)], AVI/AVI+G*=[TAS2R38 (AVI/AVI) + CA6 (GA+GG)], PAV/*+AA=[TAS2R38 (PAV/PAV+PAV/AVI)+CA6 (AA)] n AVI/AVI+AA=[TAS2R38 (AVI/AVI)+CA6 (AA)]. PVV/AVI) were lso ompute for the urrent popultion. However, ue to the rrity of these iplotypes (0.3%, n = 6), these sujets were exlue from the susequent sttistil investigtion. The hi-squre test showe tht the istriutions of the three iplotypes (PAV/PAV, PAV/AVI n AVI/AVI, o-ominnt moel) were not ssoite with CRC outome. However, when the sujets were lssifie se on the presene of the PAV hplotype (PAV/* vs. the AVI/AVI, reessive moel), the istriution of iplotypes iffere y CRC phenotype (p hisq = 0.022). The istriution of the CA6 rs genotype is lso shown in Tle 2. The G llele ws more frequent in the entire n ontrol popultion thn the A llele, with frequenies of 0.59 n 0.60, respetively. However, eviene hs suggeste tht the sustitution of enine to gunine les to the struturl ltertion of the CAVI protein, therey reuing oth the protein tivity n itterness intensity [26, 27]. Sine the tster hplotype PAV is onsiere the ominnt hplotype for TAS2R38 n to etter estimte the effet of reue itterness sensitivity on ietry intke n isese risk, we onsiere enine (tster llele) s the referene llele for this stuy. A hi-squre evlution revele tht the istriution of the CA6 rs genotype iffere etween CRC ses n ontrols (p hisq = 0.009). This genotype-crc ssoition ws lso retine when the sujets were groupe oring to the presene of the vrint G llele (p hisq = 0.003, AA vs. G*). The numers of groups or sugroups with limite numers of sujets oul osure the ssoitions etween geneti vrints n phenotypi outomes. Therefore, to estlish the TAS2R38-CA6 omine genotype, the results of hi-squre tests were pplie. Sujets were lssifie se on the presene of tster PAV/* (reessive moel) n AA (ominnt moel). Four omintions (PAV/*+AA, PAV/*+G*, AVI/AVI+AA, AVI/AVI+G*) were evient in the urrent stuy sujets, n the istriutions of these omintions iffere istintively y CRC phenotype (p hisq = 0.002). This 21256

5 omine genotype ws therefore pplie for susequent sttistil nlyses (the istriutions of ll omine genotypes re presente in Supplementry Tle 1). Geneti vritions n ietry intke, lohol n too onsumption The effet of TAS2R38 n CA6 geneti vrints n their omine genotype on the intke of totl energy, ll vegetles, ruiferous vegetles, rk green vegetles, ll fruits, itrus fruits, fier, ft-foo, sweets, lohol n too were exmine, n the results re presente in Tles 3, 4 n 5. Tles 3 n 4 present the men onsumption of the evlute vriles for eh of the TAS2R38 n CA6 geneti vrints in ll sujets s well s in CRC ses n ontrols, respetively. Generlize liner moels n Stuent s t-tests, however, revele no signifint ifferenes in the vriles mong the genotypes either in the presene or sene of onfouners in ny of the exmine groups of sujets. The only mrginl ssoition ws oserve in reessive moel etween the TAS2R38 iplotype n too intke in CRC ses (p = 0.046). When the omine genotype of TAS2R38 n CA6 ws nlyze, the results gin showe tht the geneti vrints h no meningful influene on the intke of the exmine foos or lohol in either ll sujets or the ontrols (Tle 5). However, mong the CRC ses, the omine genotypes were ssoite with ifferentil too intke when onfouning ftors were onsiere (p = 0.024). Former/urrent smoking ses with the AVI/ AVI ± AA genotype tene to e ssoite with higher ily too use thn the other genotypes, lthough the ifferenes etween genotypes were not lerly preite y Tukey s test (Figure 1). The ssoition etween itter tste geneti vrints n the risk for oloretl ner The ssoitions etween the TAS2R38 iplotype, the CA6 rs genotype n CRC suseptiility re presente in Tle 6. Our logisti regression moels provie ler eviene tht geneti vritions in itterness pereption moify the risk for CRC inepenently, without ny moifitions in ietry intke. Hving the TAS2R38 AVI/AVI iplotype erese the risk of CRC y pproximtely 25% ompre to hving the PAV hplotype [juste os rtio (OR) = 0.74, 95% onfiene intervl (CI): ]. CA6 rs ws lso preite to moify the risk of CRC: sujets with the G llele h lower risk of eveloping CRC thn those with the AA genotype (juste OR = 0.71, 95% CI: ). When TAS2R38 n CA6 vritions were oth tken into ount, the omine genotype retine the protetive effet of eh vrint llele/iplotype ginst CRC suseptiility (Tle 6). Sujets with the TAS2R38 PAV/* iplotype n the CA6 vrint G llele were less likely to hve CRC (juste OR = 0.71, 95% CI: ), ompre to those with the referene PAV/* n AA genotype. This protetive effet ppere to e greter when sujets possesse oth the vrint iplotype n the vrint llele (juste OR for AVI/AVI+G* = 0.49, 95% CI: ). Sujets with the AVI/AVI iplotype n the AA genotype lso showe reue risk of CRC, though this result ws not sttistilly signifint. DISCUSSION Geneti vritions in tste pereption mehnisms hve een known to moify humn ietry ehvior n helth outomes. The urrent stuy exmine whether geneti vrints relte to itterness sensing re ssoite with the intke of ietry n onsumer goos n the suseptiility to CRC in Korens. Our finings suggeste tht the itterness-relte geneti vrints i not influene the intke of the exmine foos. However, these geneti vrints seeme to e ssoite with the risk of CRC vi other potentil rinogeni mehnisms n not y moifying ietry onsumption. Experimentl stuies hve shown tht geneti vrints in TAS2R38 n CA6 re responsile for itter tste sensitivity, TAS2R38 vrints hve minly een investigte to etermine their moifying effets on ietry intke. To the est of our knowlege, this is the first stuy to use n epiemiologil pproh to exmine whether CAVI is ssoite with the onsumption of ietry foos n onsumer goos. However, the finings suggeste tht the CA6 rs genotype i not le to ny ifferentil intke of ietry n onsumer goos. Furthermore, the omintion of the CA6 rs genotype n TAS2R38 vrition lso showe n miguous effet on the ietry n lohol intke of Korens. A similrly negligile influene of itterness geneti vrints on foo intke ws oserve in our erlier stuy on gstri ner n TAS2R38; in tht stuy, the geneti vritions h no signifint influene on the popultion s ietry intke [11]. Though ruiferous vegetles re mjor vegetle soure for Korens (48% of the totl vegetle intke), these foos re generlly onsume s pikle/slte ishes or s kimhi with multiple types of oniments, inluing slt, ginger, pepper, grli, hili n fish sue, whih ll hve strong spiity. The use of nturl n rtifiil oniments (e.g., monosoium glutmte) my msk the ntive itter flvor of ruiferous vegetles, n onsequently, geneti vrition in itterness sensing might not influene the intke of itter-tsting foo. Aitionlly, the istriution of the omine TAS2R38-CA6 vrition my e ssoite with the miniml ifferene in the ietry intke etween genotypes. TAS2R38 n CA6 resie on ifferent hromosomes n their vritions re 21257

6 inepenent (P = 0.221, hi-squre test, Supplementry Tle 1). However, mjority of Korens (69.7%) possess oth the tster TAS2R38 PAV n the non-tster CA6 G llele. Hving oth vrints my neutrlize the ontrsting effet of eh llele on itterness sensitivity, thus inresing the tolerne to itter-tsting foos. Therefore, no ler ifferenes in ietry intke my e oserve mong genotypes. Nonetheless, we shoul not unerestimte the effets of itterness-relte geneti vrints on ietry n onsumer goos intke: in our t set, ses with TAS2R38 AVI/AVI n the omine AVI/AVI-AA genotype tene to onsume more too thn other genotypes. It is iffiult to etermine whether the presene of the vrint llele ws the min effetor of the high too onsumption, s the ssoition etween TAS2R38 AVI/AVI n too intke ws mrginl. Furthermore, only smll numers of former/ urrent smoking ses h the omine genotype (n = 10), n the effets of other onfouning ftors eyon geneti omponents my exist (the genotypetoo intke ssoition ws only preite y the sttistil moel fter justing for onfouners). Despite these limittions, this eviene suggests tht geneti moultion of itterness intensity oul influene n iniviul s intke of ietry n onsumer goos y interting with soio-eonomi hrteristis n helth ehvior n, s result, my potentilly ontriute to the risk of CRC [28]. Aitionl stuies re require to estlish lerer role for tste-relte geneti vritions in the ietry n onsumer goos intke of Korens. Though the effets of geneti vrints on ietry intke were miniml, the TAS2R38 n CA6 geneti vritions were ssoite with CRC outomes on their own. This result supports the erlier finings tht itterness sensing-relte vritions in TAS2R38 n CA6 oul e geneti mrkers of gstrointestinl funtion n isese [10, 15, 25]. When the effet of eh geneti vrition on the risk of CRC ws evlute, the TAS2R38 AVI/AVI iplotype erese the risk of CRC pproximtely 30% ompre to the PAV hplotype. Beuse the AVI hplotype ws ssoite with reue itterness intensity n n inrese risk of CRC in Czeh-Germn popultion, the vrint AVI/AVI iplotype hs een suggeste to e potentil iomrker for impire gstrointestinl funtion [10]. However, nother experimentl stuy prove tht the AVI/AVI iplotype is not simply funtionl mrker for the impire T2R38 vrint protein, s expression of the homozygous AVI trnsript ws etete, n iniviuls with the AVI/ AVI iplotype were shown to respon to other itter ompouns [29]. Aoringly, it oul e hypothesize tht lthough the AVI vrint rely respons to thioure ligns, the struturl perturtion of the AVI vrint protein my enhne the sensing of other unknown rinogeni moleules. Therefore, the AVI vrint protein my hve vntges in terms of signl trnsmission involve in the neutrliztion n expulsion of those unknown rinogens from the intestine, thus reuing the risk of CRC. A similr protetive effet of the AVI/AVI iplotype ws lso evient in nother stuy: in Jpnese- Amerins, the AVI/AVI iplotype tene to e protetive ginst CRC, lthough the power of the sttistil moel ws limite [15]. The ifferentil genotype istriution n the potentil rinogens in ifferent ietry ultures n surrouning environments my hve le to suh ontrsting ssoition etween the TAS2R38 genotype n CRC mong ifferent ethni groups. Figure 1: The onsumption of too igrettes for the TAS2R38-CA6 genotypes y oloretl ner phenotype. Eh r presents men ± stnr evition. The men onsumption ws estimte mong urrent/former smokers. Numers in rkets represent the numers of sujets. P-vlues re from generlize liner moels juste for sex, ge, oy mss inex, lohol rinking, regulr exerise, fmily history of oloretl ner, mritl sttus, eution level, househol inome n ietry zin intke. No signifint ifferene etween pir of genotypes ws estimte y Tukey s tests

7 Tle 3: Men onsumption of selete foos, lohol n too for the TAS2R38 iplotype in ll sujets n oloretl ner ses n ontrols (men ± stnr evition) TAS2R38 All sujets (n = 2036) Energy (kl/y) Vegetles Fruits All Cruiferous Drk green All Citrus Fier Ft-foo Sweets Alohol Too (igrettes/ y) PAV/PAV (n = 726) ± ± ± ± ± ± ± ± ± ± ± 9.5 PAV/AVI (n = 981) ± ± ± ± ± ± ± ± ± ± ± 9.0 AVI/AVI (n = 329) ± ± ± ± ± ± ± ± ± ± ± p ttest e Controls (n = 1,356) PAV/PAV (n = 475) ± ± ± ± ± ± ± ± ± ± ± 8.1 PAV/AVI (n = 644) ± ± ± ± ± ± ± ± ± ± ± 8.1 AVI/AVI (n = 237) ± ± ± ± ± ± ± ± ± ± ± p ttest Cses (n = 680) PAV/PAV (n = 251) ± ± ± ± ± ± ± ± ± ± ± 11.5 PAV/AVI (n = 337) ± ± ± ± ± ± ± ± ± ± ± 10.3 AVI/AVI (n = 92) ± ± ± ± ± ± ± ± ± ± ± p ttest Sujets with the AAV/AVI (n = 3); PAV/AVV (n = 1), PVI/AVI (n = 1), n PVV/AVI (n = 1) iplotypes were exlue from the sttistil nlyses ue to the limite numers. The men onsumption ws estimte mong urrent/former rinkers n smokers. P-vlues for rue generlize liner moels ompre the intke of selete foos, lohol n too mong groups. P-vlues for generlize liner moels juste for sex, ge, oy mss inex, smoking, lohol rinking, regulr exerise, fmily history of oloretl ner, mritl sttus, eution level n househol inome s pproprite. e P-vlues from Stuent s t-tests of the omprison etween genotypes of the reessive moel for TAS2R38 (PAV/PAV+PAV/AVI versus AVI/AVI). The pthogeni role of CAVI hs een intensively investigte in reltion to entl helth euse the CAVI protein is minly serete y the slivry glns. However, humns swllow lrge quntities of CAVI every y, n the physiologil roles n ssoitions of CAV1 with upper limentry trt iseses hve een previously oserve [20, 25]. The urrent stuy lso revels tht CAVI my e linke to vrious gstrointestinl iseses, inluing oloretl mlignnies. In silio nlyses preite tht the rs vrint G llele les to ritil struturl moifition of CAVI tht limits the ining of zin suh tht the GG vrint protein my hve reue effiy in tlyzing CO 2 + H 2 O HCO 3 + H + [26]. Beuse this vrint protein my le to inresingly ii onitions in the igestive orgns, iniviuls with the GG genotype re thought to e vulnerle to entl n ulertive iseses. However, this rtionle runs ounter to the urrent finings, s iniviuls with the G llele h erese risk of CRC. Severl hypotheses my e propose to explin the ontritory outomes mong the stuies. Although exessive iity is onsiere risk ftor for limentry isorers, gstri iity is eisive protetive rrier from foo- or wter-orne toxi moleules [30]. Gstri iity is lso responsile for the omposition of the miroiome in the verterte intestinl system, n the optiml gstri iity my vry epening on ietry hits [30]. The potentilly higher iity of oy fluis, inluing sliv n gstri juie, my enefit the limentry trt y onferring protetion ginst potentilly rinogeni ompouns tht Korens re expose to, therey potentilly reuing the risk of CRC. Aitionlly, the iity of the gstri environment is ritil for the stility/slvging of folte vitmers. In ii gstri juie (ph 3.5), 5-methyltetrhyfrofolte (the iotive form of folte) is more stle thn t higher ph [31]. Moreover, the improve iovilility of 5-methyltetrhyfrofolte my e ssoite with the suffiient provision of folte in the synthesis of pyrimiine n thymiylte, whih oul e involve in ntirinogeni mehnisms. Finlly, CAVI is likely linke to the immune response, whih my e ssoite with rinogeni etiology. In murine CAVI-efiient moel, silene CAVI expression resulte in the perturtion of lymphoi folliles in the intestinl Peyer s pthes n 21259

8 Tle 4: Men onsumption of selete foos, lohol n too for the CA6 rs genotype in ll sujets n oloretl ner ses n ontrols (men ± stnr evition) CA6 rs All sujets (n = 2042) Energy (kl/y) Vegetles Fruits Fier All Cruiferous Drk green All Citrus Ft-foo Sweets Alohol Too (igrettes/y) AA (n = 353) ± ± ± ± ± ± ± ± ± ± ± 10.7 GA (n = 957) ± ± ± ± ± ± ± ± ± ± ± 8.7 GG (n = 732) ± ± ± ± ± ± ± ± ± ± ± p ttest Controls (n = 1,361) AA (n = 211) ± ± ± ± ± ± ± ± ± ± ± 6.8 GA (n = 658) ± ± ± ± ± ± ± ± ± ± ± 8.2 GG (n = 492) ± ± ± ± ± ± ± ± ± ± ± p ttest Cses (n = 681) AA (n = 142) ± ± ± ± ± ± ± ± ± ± ± 14.2 GA (n = 299) ± ± ± ± ± ± ± ± ± ± ± 9.4 GG (n = 240) ± ± ± ± ± ± ± ± ± ± ± p ttest The men onsumption ws estimte mong urrent/former rinkers n smokers. P-vlues for rue generlize liner moels ompring the intke of selete foos, lohol n too mong groups. P-vlues for generlize liner moels juste for sex, ge, oy mss inex, smoking, lohol rinking, regulr exerise, fmily history of oloretl ner, mritl sttus, eution level n househol inome s pproprite. P-vlues from Stuent s t-tests ompring etween genotypes of the ominnt moel (AA versus GG+GA). the up- n own-regultion of 127 genes, whih were mostly involve in toli proesses in the uoenum [18]. Vrint CAVI my le to the perturtion of the immune system n relte rinogeni mehnisms n my, therefore, e linke to the risk of CRC. Cner evelopment n progression re multiftoril proesses, n the role of CAVI in these proesses hs rely een explore. It ws therefore iffiult to eluite the preise mehnisti reltionship etween the vrint CAVI protein n the risk of CRC in this stuy. However, the urrent finings n the mehnisms speulte ove oul imply tht vrint CAVI plys pthologil role in CRC etiology. This hypothesis shoul e teste in future investigtions. Sine T2R38 n CAVI show funtionl overlp in itterness pereption, the role of eh protein n their omine effets in itterness sensing hve een of prtiulr interest in mny stuies. Eviene hs suggeste tht the TAS2R38 PAV hplotype is ssoite with the pereption of higher onentrtions of PROP, wheres the CA6 A llele is relevnt to sensing lower PROP onentrtions [16]. However, susequent stuy reporte tht the vrint CAVI ws only ssoite with fungiform ppill ensity, wheres T2R38 moifie itterness sensing [32]. In ition to those oservtions regring itterness sensing, the present stuy s more eviene for the omine effet of oth T2R38 n CAVI in isese etiology. The omine genotype onferre the sme protetive effet of single vrint llele, n hving oth the vrint llele n the vrint hplotype resulte in n itive reution in the risk of CRC. Unlike the PROP tste intensity stuy, the urrent oservtionl stuy i not llow us to preit how either T2R38 or CAVI inepenently or oopertively moify the risk of CRC. The moifying effet of the omine TAS2R38-CA6 genotype on the risk of CRC my rise inepenently euse these two proteins re minly involve in ifferent physiologil metolisms (signl trnsution n mintining homeostsis). However, oth proteins re ommonly responsive to thioure moieties n re involve in energy metolism n oy homeostsis [33]. Aitionlly, T2R38 n CAVI re ftors in the regultion of innte immunity, whih is ritil for the evelopment n progression of CRC [34]. Consiering our finings tht the protetive effet of the omine genotype ginst CRC inrese with the 21260

9 Tle 5: Men onsumption of selete foos, lohol n too for the omine TAS2R38 n CA6 genotype in ll sujets n oloretl ner ses n ontrols (men ± stnr evition) TAS2R38+CA6 All sujets (n = 2036) Energy (kl/y) numers of vrint lleles n hplotypes, the presene of mutully Supplementry roles or potentil mehnisti linkge etween T2R38 n CAVI in CRC etiology (s well s itterness sensing) nnot e isounte. More investigtions with lrger popultion sizes n ifferent experimentl pprohes ime t verifying the unerpinning mehnism etween T2R38 n CAVI re require. This stuy provies new epiemiologil eviene for the role of T2R38 n CAVI in the ietry intke of n CRC onset in Korens. However, the finings must e interprete with ution euse some potentil limittions my exist. First, this reserh employe se-ontrol stuy esign; therefore, it my e ffete y seletion or rell is in sujet reruitment n t olletion. Seon, the vlite self-reporte FFQ pplie in the urrent stuy my hror potentil systemti n rnom mesurement issues in ietry evlution. Thir, the ontrols were enrolle on volunteer sis from mong All Vegetles Cruiferous Drk green Fruits Fier Ft-foo Sweets Alohol Too (igrettes/ y) PAV/*+AA (n = 288) ± ± ± ± ± ± ± ± ± ± ± 10.1 PAV/*+G* (n = 1,419) ± ± ± ± ± ± ± ± ± ± ± 9.1 AVI/AVI+AA (n = 65) ± ± ± ± ± ± ± ± ± ± ± 13.4 AVI/AVI+G* (n = 264) ± ± ± ± ± ± ± ± ± ± ± e Controls (n = 1,356) PAV/* + AA (n = 169) ± ± ± ± ± ± ± ± ± ± ± 7.2 PAV/* + G/* (n = 950) ± ± ± ± ± ± ± ± ± ± ± 8.2 AVI/AVI + AA (n = 42) ± ± ± ± ± ± ± ± ± ± ± 5.1 AVI/AVI + G/* (n = 195) All ± ± ± ± ± ± ± ± ± ± ± Cses (n = 680) PAV/* + AA (n = 119) ± ± ± ± ± ± ± ± ± ± ± 12.9 PAV/* + G/* (n = 469) ± ± ± ± ± ± ± ± ± ± ± 10.3 AVI/AVI + AA (n = 23) ± ± ± ± ± ± ± ± ± ± ± 18.9 AVI/AVI + G/* (n = 69) ± ± ± ± ± ± ± ± ± ± ± Sujets with the AAV/AVI (n = 3), PAV/AVV (n = 1), PVI/AVI (n = 1), PVV/AVI (n = 1) iplotype were exlue from the sttistil nlyses ue to the limite numers. Comine genotype for the reessive n ominnt moels of the TAS2R38 n CA6 geneti vrints, PAV/*=PAV/PAV+PAV/ AVI, G* = GG + GA. The men onsumption ws estimte mong urrent/former rinkers n smokers. P-vlues for rue generlize liner moels ompring the intke of selete foos, lohol n too mong groups. e P-vlues for generlize liner moels juste for sex, ge, oy mss inex, smoking, lohol rinking, regulr exerise, fmily history of oloretl ner, mritl sttus, eution level n househol inome s pproprite. Citrus prtiipnts of helth sreening exmintion. These ontrols my e more onerne with helthier lifestyle tht is ssoite with reue risk of CRC, though we trie to minimize suh ifferenes y ressing onfouning ftors in the sttistil moels. Lstly, we exmine the intke of vrious types of foos n onsumer goos s well s the geneti vrints known to ruilly moify the tivity of T2R38 n CAVI. However, other unexmine foos n polymorphisms linke to, or retine in, those genes my ontriute to the ietry intke n CRC outomes. In onlusion, TAS2R38 n CA6 geneti vrints in itterness pereption i not influene the ietry intke of Korens. However, TAS2R38 n CA6 geneti vrints were moifying ftors of CRC suseptiility. This my inite tht the itterness sensing reeptors T2R38 n CAVI re involve in oloretl rinogenesis n tht their geneti vritions re potentil iomrkers for gstrointestinl funtion

10 Tle 6: The ssoitions for the TAS2R38 iplotype, CA6 rs genotype n their omine genotype with risk for oloretl ner TAS2R38 iplotype OR rue (95%CI) OR juste (95%CI) p p tren PAV/PAV 1.00 (Referene) 1.00 (Referene) PAV/AVI 0.99 ( ) 0.94 ( ) AVI/AVI 0.74 ( ) 0.70 ( ) PAV/* vs. AVI/AVI e,f 0.74 ( ) 0.74 ( ) CA6 rs AA 1.00 (Referene) 1.00 (Referene) GA 0.68 ( ) 0.69 ( ) GG 0.73 ( ) 0.73 ( ) AA vs. G* g 0.70 ( ) 0.71 ( ) Comine genotype (TAS2R38+CA6) h PAV/*+AA 1.00 (Referene) 1.00 (Referene) PAV/*+G* 0.70 ( ) 0.71 ( ) AVI/AVI+AA 0.78 ( ) 0.73 ( ) AVI/AVI+G* 0.50 ( ) 0.49 ( ) <.001 Crue os rtios (OR) from the logisti regression moels for geneti vrints n risk for oloretl ner. 95% onfiene intervl (CI). OR juste for sex, ge, oy mss inex, smoking, lohol rinking, regulr exerise, fmily history of oloretl ner, mritl sttus, eution level, househol inome n ietry zin intke where pproprite. P-vlues for tren. e vs.=versus. f PAV/*=PAV/PAV+PAV/AVI. g G*=GG+GA. h Comine genotype for the reessive n ominnt moels of the TAS2R38 n CA6 geneti vrints. MATERIALS AND METHODS Sujet reruitment n t olletion This stuy ws prt of CRC reserh onute in Ntionl Cner Center (NCC), Kore, from Otoer 2007 to Deemer The etils of sujet reruitment n t olletion proeures were esrie previously [35]. Briefly, ses were efine s ptients who unerwent surgery for CRC or were histologilly ignose with CRC t the Center for Coloretl Cner, NCC. Controls were enrolle mong visitors for helth sreening exmintion ( enefit progrm of the Ntionl Helth Insurne Coopertion) t the Center for Cner Prevention n Detetion, NCC. A totl of 1,070 oloretl ner ptients n 14,201 ontrols volunteere for the stuy. However, iniviuls with inomplete esriptive n foo frequeny questionnire (FFQ) t, unknown energy intke, or those with loo smples tht were missing or oul not e ollete were exlue from the stuy (see Figure 2 for the etils of the sujet seletion proeure). Among the remining sujets, 701 ses n 1,402 ontrols were selete for the stuy t 1:2 frequeny n mthe y sex n 5-yer ge group, n their genotypes were etermine. Finlly, 681 ses n 1,361 ontrols with qulifie genotypi t were nlyze for the stuy. Prior to the ommenement of the stuy, ll stuy proeures were pprove y the ethil ommittee of NCC (NCCNCS n NCC ) n the tul stuy ws rrie out following pprove protools. Dietry intke nlyses Prtiipnts were requeste to omplete the vlite FFQ [36]. The FFQ presente three portion sizes n nine egrees of frequeny for eh foo item, n prtiipnts were ske to esrie their foo intke over the lst 12 months. Dietry intke ws nlyze using CAN-PRO 4.0 (Computer Aie Nutritionl Anlysis Progrm, The Koren Nutrition Soiety, Seoul, Kore). The mjor fous of the present stuy (the effet of geneti vrints on ietry intke) ws evlute from the following ten groups of foo n nutrients: totl 21262

11 energy, ll vegetles, ruiferous vegetles, rk green vegetles, ll fruits, itrus fruits, fier, ft-foo, sweets n zin. Aitionlly, sujets ily onsumption of lohol n too prouts (igrettes/y) were lso etermine using struture questionnire (see Supplementry Tle 2 for the investigte foo items n loholi everges in eh tegory). Genotyping n geneti t nlyses Genotypes of TAS2R38 (A49P, V262A n I296V) n CA6 (rs ) were ssesse using the Agenio MssArry iplex gol ssy (Sn Diego, CA, USA). The primry t were nlyze using Agenio TYPER version 4.0, n the rw results were only epte s qulifie genotype if the ll rte for eh lous ws over 95%. The funmentl geneti nlyses were onute using Hploview (Version 4.2). The iplotypes of TAS2R38 were ompute using FAMHAP softwre [37]. Sttistil nlyses The generl hrteristis of the stuy popultion were ompre se on CRC phenotype using Stuent s t-tests n hi-squre tests. Differenes in the istriution of TAS2R38 iplotypes n CA6 genotypes were evlute using hi-squre tests. All ietry t were nlyze fter justing for the totl energy intke using Willet s resiul metho [38] n log-trnsformtion. Anlyses of vrine were employe to evlute the ifferene in foo n onsumer goos intke etween genotypes n iplotypes in the presene or sene of potentil onfouning ftors. Tukey s tests were employe for post ho omprisons. The logisti regression moels were estlishe to preit the ssoition etween risk of CRC n geneti vritions n enote s OR with 95% CI. All sttistil nlyses were performe using SAS version 9.3 (SAS Institute In., Cry, NC, USA). A two-sie P-vlue of less thn 0.05 ws onsiere sttistilly signifint. Figure 2: Simplifie flow hrt for the seletion of stuy sujets. CRC = oloretl ner; NCC = Ntionl Cner Center; FFQ = foo frequeny questionnire

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