RESEARCH COMMUNICATION. Interactions Between MTHFR C677T - A1298C Variants and Folic Acid Deficiency Affect Breast Cancer Risk in a Chinese Population

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1 OI: Intertions etween MTHFR 77T - nd Foli id efiieny in rest ners in hin RSRH OMMUNITION Intertions etween MTHFR 77T - Vrints nd Foli id efiieny ffet rest ner Risk in hinese Popultion Xi-Yu Wu 1,2, Jun Ni 2, Wei-Jing Xu 2, To Zhou 2, Xu Wng 1,2 * strt kground: Our ojetive ws to evlute the MTHFR 77T- polymorphisms in ptients with rest ner nd in individuls with no history of ner, to ompre the levels of geneti dmge nd poptosis under foli id (F) defiieny etween ptients nd ontrols, nd to ssess ssoitions with rest ner. Methods: Geneti dmge ws mrked y mironuleted inuleted ells (MNN) nd poptosis ws estimted y ytokinesis-lok mironuleus ssy (MN). PR-RFLP moleulr nlysis ws rried out. Results: The results showed signifint ssoitions etween the MTHFR 77TT or the omined MTHFR 77T- nd rest ner risk (OR = 2.51, I =.5 to 7.7, p =.; OR =.11, I =.7 to 21., p <.1). The MNN from the omined MTHFR 77T- ws higher nd the poptosis ws lower thn tht of the single vrints (p <.5). t to nmol /L F, the MNN in ses with the genotype ws higher thn ontrols (p <.5), wheres no signifint differene in poptosis ws found etween the ses nd ontrols fter exluding the geneti kground. onlusions: ssoitions etween the omined MTHFR 77T- polymorphism nd rest ner re possile from this study. dose of nmol/l F ould enhne poptosis in ses with MTHFR 77T-. rest ner individuls with the genotype my e more sensitive to the genotoxi effets of F defiieny thn ontrols. Keywords: Foli id - omined MTHFR 77T- - rest ner - geneti dmge - poptosis sin Pifi J ner Prev, 1, Introdution Folte is n importnt mironutrient found in green vegetles, fruits nd ens. The syntheti form tht is dded to foods nd found in supplements is known s foli id (F). F plys n essentil role in severl omplex metoli pthwys inluding N synthesis nd pthwys tht re involved in the methionine metoli pthwy, whih is ruil for N methyltion (Feneh et l., 21; ihholzer et l., 2; Mzz et l., 21; oukert et l., 211). F levels re determined y oth dietry F intke nd F metolism. F defiieny indues defiient methyltion of deoxyuridyli id (dump) to deoxythymidyli id (dtmp) leding to uril misinorportion (Feneh, 21; Mzz et l., 21) nd N glol hypomethyltion (Rmpersud et l., 2); this not only leds to point muttions, the genertion of single- nd doule-strnded N reks, hromosome rekge, mironuleus formtion nd norml poptosis (verson et l., 1; Feneh et l., 21; Feneh et l., 22; ihholzer et l., 2; Yng et l., 2; Mzz et l., 21; oukert et l., 211), ut lso my lso e ssoited with neuploidies of the inrement hromosomes 17, 21 nd (Wng et l., 2; Ruosri et l., 2; Thoms et l., 2; Ni et l., 21). Low dietry F intke lso results in n interruption of the N repir pility (Wei et l., 2). The mjority of prospetive nd se-ontrolled studies showed tht low levels of serum F, red ell F onentrtion nd low dietry F intke re inversely orrelted with olon ner, rest ner, denom nd other ners t severl sites (Kim et l., 21; Wng et l., 2; Lin et l., 21; Johnson et l., 211). isturnes in the F metoli pthwy re ssoited with wide rnge of onditions, nd vrints from genes involved in this pthwy my ply role in the genesis of these onditions. t lest different enzymes re involved in this omplex pthwy inluding methylenetetrhydrofolte redutse (MTHFR), methionine synthse, methionine synthse redutse, nd thymidylte synthse (Kim et l., 1; Suleeporn et l., 21). efets or polymorphi enzymes my lter the iovilility of F nd influene ner suseptiility (Mruti et l., 2). MTHFR is responsile for the vilility of methyl groups for iologil 1 Shool of Life Sienes, Yunnn University, 2 Shool of Life Sienes, The ngineering Reserh enter of Sustinle evelopment nd Utiliztion of iomss nergy, Ministry of dution, Yunnn Norml University, Kunming, hin *For orrespondene: wngxu@fudn.edu.n sin Pifi Journl of ner Prevention, Vol 1, 2 21

2 Xi-Yu Wu et l methyltion retions nd tlyzes the onversion of 5, 1-methylenetetrhydrofolte to 5-methyltetrhydrofolte, whih is o-sustrte for homoysteine remethyltion to methionine (hillon et l., 2); muttions of MTHFR will lter the tivity of the enzyme. The esthrterized polymorphism of MTHFR is the 77T trnsition. This muttion uses n lnine to vline sustitution nd redues the enzyme s tlyti tivity (Thoms et l., 2; Rodrigues et l., 21). n dditionl polymorphism, MTHFR, results in the hnge from glutmi id to n lnine residue nd dereses the enzyme s tivity (Weiserg et l., 1; Promthet et l., 21). Redued MTHFR tivity inreses the 5, 1-methylenetetrhydrofolte onentrtion, wheres it dereses the 5-methyltetrhydrofolte onentrtion. Suh sitution is expeted to fvor the synthesis of dtmp over methyltion of pg, nd to minimize oth the uril inorportion into N nd lso the hromosoml reks used y uril. Two ommon polymorphisms hve een reported tht re linked to n inresed risk of wide rnge of dverse helth onditions (suh s irth defets, slow growth, nemi, weight loss, digestive disorders, some ehviorl issues, rdiovsulr disese nd severl ners) (Weiserg et l., 1; Hung et l., 2; Thoms et l., 2; hillon et l., 2; Promthet et l., 21; Rodrigues et l., 21). pidemiologil studies indite tht the 77TT genotype is ssoited with lower risk of ute lymphoyti leukemi (Skiol et l., 1) nd olon rinom (Promthet et l., 21), espeilly in lowrisk sujets who onsume diet high in folte (rott et l., 21). In ontrst, under F defiieny, the 77TT vrint genotype hs een ssoited with n inresed risk of ner t vrious sites (rest, gstri, ervil nd prostte) (eily et l., 2; Zoodsm et l., 25; istulfi et l., 21). Previous studies showed tht vrition of the F onentrtion within the physiologil rnge ( to 2 nmol/l) signifintly influened the genomi instility nd ytotoxiity in ultured humn lymphoytes with the 77T polymorphism (Wu et l., 2). However, the extent of the geneti dmge nd the levels of poptosis under F defiieny etween rest ner ses nd nonner ontrol ses with the omined MTHFR polymorphisms of 77T nd hve not een eluidted. In the present work, we investigted nd ompred the effets of F defiieny etween rest ner ses nd non-ner ontrols on the levels of geneti dmge nd poptosis of humn lymphoytes with omined MTHFR 77T- polymorphisms. Mterils nd Methods Study design pprovl for the present study ws otined from the Ntionl Nturl Sienes Foundtion of hin (NSF) nd the Yunnn Sientifi nd Tehnologil ommittee. The peripherl lood from 75 femle rest ner ptients nd 75 ontrol femle volunteers who did not reeive ny vitmin supplements ws utilized for the study fter otining informed onsent. 22 sin Pifi Journl of ner Prevention, Vol 1, 2 ll of the ses were pthologilly dignosed femles in the Seond ffilited Hospitl of Kunming Medil ollege, Yunnn, hin. ligile ses were newly dignosed rest-ner femles tht hd not yet reeived hemotherpy nd did not hve ny previous history of ny ner. Genotype ssys Peripherl lood smples were olleted from the ner nd ontrol groups, nd genomi N ws extrted from whole lood smples y the hydroxyenzene nd hloroform method (Song et l., 1). N frgments ontining the MTHFR nuleotides 77 nd were mplified ording to the onditions desried y P. Yi et l. (22). The PR primers to mplify the 77-frgment re 5 TTGGGTGGGG nd 5 GGTGGTGTGT, nd the primers for the -ontining segment re 5 GGTGGGG nd 5 GGTGT. The PR onditions were one yle of, min; irles of, 5 s, 5, 5 s, nd 72, 5 s, nd finl yle of 72, 1 min. PR produts with nuleotides 77 nd were sujeted to HinF1 nd MoII digestion, respetively, followed y eletrophoresis using 2% grose gel. The PR produts were vlidted y sequening nlysis. Mutgenesis ws performed to generte ll nine possile omintions of the polymorphi vrints: (wild-type), (heterozygous mutnt t ), (homozygous mutnt t ), (heterozygous mutnt t 77), (heterozygous mutnt t oth 77 nd ), (heterozygous mutnt t 77 nd homozygous mutnt t ), (homozygous mutnt t 77), (homozygous mutnt t 77 nd heterozygous mutnt t ) nd (homozygous mutnt t oth 77 nd ). Lymphoyte ulture, viility nd ell growth ssys h individul donted 5 ml of peripherl lood y venipunture fter n overnight fst. The donted lood ws olleted in heprinized tues. The lymphoytes were isolted using lymphoyte seprtion medium (Shnghi Hujing ioteh ompny, hin). The lymphoyte ultures were prepred t onentrtion of.5 1 ells / ml in 1 ml of RPMI 1 medium ontining vrying onentrtions of F (,,, nd 2 nmol/l). ll of the other onstituents of the RPMI 1 medium were s previously desried, nd ll were purhsed from Gio Medi, merin. Five perent dilyzed fetl lf serum (FS, P the ell ulture ompny, merin), 1 ku/l interleukin 2 (Shenzhen Xinjier Phrmeutil, hin), 2 mmol/l L-glutmine (Sigm), 1 U/ml peniillin G nd 1 mg/ml streptomyin (eijing Xisi iotehnology, hin) were dded to the medium. The lymphoytes were ultured t onentrtion of.5 1 ells / ml in 5 ml volumes fter stimultion with phytohemgglutinin (5 mg/ml) (PH; eixing ioteh ompny, hin), nd ultures were inuted t 7 nd 5% O 2 in humidified inutor. fter dys, the ell numer nd viility were determined using hemoytometer nd trypn lue exlusion, respetively.

3 OI: Intertions etween MTHFR 77T - nd Foli id efiieny in rest ners in hin The ultures were ontinued in.7 ml fresh medium nd. ml onditioned medium from the previous -dy ulture t onentrtion of.5 1 vile ells/ml. The omponents of the fresh medium were the sme s ove ut without PH. The medium hnge ws repeted t dys post-ph tretment, nd finl vile ell ount ws mesured on dy. ytokinesis-lok mironuleus (MN) ssys ight dys fter PH tretment, ytohlsin (.5 μg/ml; Sigm) ws dded to eh tue, nd ells were hrvested 2 h lter onto mirosope slides using ytoentrifuge (Shndon Southern Produts, heshire, UK). The slides were then ir-dried, fixed with ml eh methnol:eti id nd old methnol for 1 min nd stined with 5% Giems for 5 min. ll of the slides were oded prior to soring. Soring ws performed y single individul using n Olympus light mirosope t 1 mgnifition under oil immersion. oded slides were sored for the frequeny of mironuleted inuleted (MNN) ells, mironuleted mononuleted (MNed mono) ells, nuleoplsmi ridges (NPs) ells nd nuler uds (NUs) ells, poptoti ells nd neroti ells. The soring riteri followed the ytokinesis-lok mironuleus (MN) ssy estlished y Feneh (Feneh, 2). In this study, the geneti dmge ws mesured y the frequeny MNN. Sttistil nlysis Genotypi frequenies were ssessed y the X 2 test. The frequenies of MNN nd poptosis for the vrious ultures were ompred using one-wy. The men vlues of the frequenies of MNN nd poptosis for the ontrol nd rest ner groups with different genotypes under vrying F onditions were ompred using the Newmn Keuls post-test (two-tiled). The differenes in sensitivity to F defiieny etween rest ner nd ontrol groups were determined using differene of differene nlysis y sutrting the pooled oserved frequenies of reltive iomrkers t 2 nmol/l F from those oserved t to nmol/l F for eh individul (ndrew et l., 21). The differenes for these results were then ompred etween the groups using two-tiled Student s t-test. The nlyses were onduted using SPSS (Sttistil Pkge for the Soil Sienes) (Version.) nd Prism. softwre (GrphPd, Sn iego, ). Results Tle 1. istriution of MTHFR 77T nd Polymorphisms Genotype ses (%) s (%) lleli frequeny: χ 2 P T of 77, of N = 75 N = 75 ses (%) s (%) MTHFR77 75(1.) 75(1.) >.5 2(2.7) 7(.).71 >.5 T () 2(2.7).11 >.5 TT 1(17.) (.) 2.5 >.5 MTHFR 75(1.) 75(1.) >.5 7(.) 2(5.). >.5 2(2.7) 2(7.). >.5 (.) 5(.7). >.5 Genotypi frequenies were ssessed y the X 2 test Tle 2. The Frequeny of omined MTHFR 77T- Polymorphisms MTHFR MTHFR ses (%) s (%) χ 2 P 77 2 (.) 27 (.). >.5 (1.7) 7 (.).7 >.5 1 (1.) (.) 1.27 >.5 T 1 (1.) 11 (1.7).55 >.5 T 17 (22.7) 1 (25.).1 >.5 T (.) 2 (2.7).27 >.5 TT (5.) (5.) >.5 TT 7 (.) 2 (2.7) 2.55 >.5 TT 2 (2.7) (.) - - Genotypi frequenies were ssessed y the X 2 test Tle. Odd Rtions (ORs) nd 5% I for MTHFR Polymorphisms with Risk of rest ner Genotype No. ses (%) No. ontrols (%) OR(5% I)* p 77 2(2.7) 7(.) 1. (Referene) 77T () 2(2.7) 1.(.55 to 2.1). 77TT 1(17.) (.) 2.51(.5 to 7.5). 7(.) 2(5.) 1. (Referene) 2(2.7) 2(7.) 1.1(. to 2.5). (.) 5(.7) 1.1(. to.).5 omined genotype 77-2(.) 27(.) 1. (Referene) 77- (1.7) 7(.) 1.(.2 to.27). 77-1(1.) (.) 1.(. to.2).1 77T- 1(1.) 11(1.7) 1.7(. to 2.).2 77T- 17(22.7) 1(25.) 1.5(.5 to 2.). 77T- (.) 2(2.7) 1.7(.27 to 11.7).17 77TT- (5.) (5.) 1.17(.2 to 5.2).51 77TT- 7(.) 2(2.7).11(.7 to 21.).1 77TT- 2(2.7) (.) - - *OR, odds rtio; I, onfidene intervl sttus, ORs were further djusted for ge t enrollment ontrol group were insignifint (Tle 1-2). No Frequenies of omined MTHFR 77T- genotype ws deteted in the ontrol group.the genotype genotypes, T nd frequenies of the 77T polymorphism The distriutions of the MTHFR polymorphisms in were respetively %, % nd 1% for ptients, nd se nd ontrol groups did not devite signifintly %, % nd 1% in ontrols. There were no signifint from the Hrdy Weinerg equilirium. The frequenies differenes etween groups (p=.2). of 77 nd 77T lleles were. nd.7 in se Multiple logisti regression tests were pplied to group nd.71 nd.2 in ontrol group, respetively, evlute the effet of vriles on disese progression while the frequenies of nd lleles were (genotypes) (Tle ). MTHFR 77TT (OR = 2.51; I=.71 nd.2 in se group nd.7 nd.2 in ontrol.5 to 7.7; p =.) nd MTHFR 77TT- (OR= group, respetively. The differenes in frequenies of.11; I =.7 to 21.; p <.1) were preditors of rest 77T nd genotype etween the se group nd ner. sin Pifi Journl of ner Prevention, Vol 1, 2 221

4 Xi-Yu Wu et l Genotype F (nmol/l) p Totl Totl Genotype F (nmol/l) p Totl Totl 2 22 <.1 <.1~.1 poptosis /5 ells <.1~.1.7±. 5.±1. 5.2±2..± ±1.7.2±. <.1~.1.5± ±1. 5.5±1.5.7±..±..2±. <.1~.1.± ±1..±..1±.5.±.25.2±.7 <.1~.5.7±.2 5.±..1±..22± ±.7.±. <.1~.5 2.2±..±..2±1.1 2.±.5 2.2±. 2.1±.7 <.1~.5.2±..±1..±.2 2.7± ±.57 2.±.5 <.1~.5.2±1.1.51±. 5.2±. 2.7±.7 2.5±.1 2.7±. <.1~.5 2.±.5.±1.1.57±1. 1.5±.2 1.7±.2 1.7±.7 <.1 1.5± ±.5 2.5±.7 1.1±. 1.±.1 1.2±.21 <.1.5±1.1.2± ± ±. 2.±. 2.±.21 <.1 <.1 MNN /1 N ells <.1 < ± ± ±.7.±2.7 d.7±1. d.7±1.1 d <.1 1.5± ±1. 11.±.7.1±.7 d.±.2 d.±1. d <.1 1.2±1..51± ±.75.±1.2 d.1±1. d.5±1. d <.1 2.7±2. 1.1± ±2.1.±.7 d.±2.2 d.±1.1 d < ± ±1.17.5±1.2.7±. d.7±.77 d.7±2.2 d 2.±. 17.5±..1±.1.5±.1 d.2±. d.±2.2 d 2.± ±2.2.2±1.1.±1.22 d.±1.1 d.7±1. d 2.± ±2. 1.5±.1 1.±1.1 d 1.±.7 d 1.±1. d 25.7±. 22.2±1.1.7±1.7.7±. d.57±.2 d.5±.2 d 21.2± ±2.2.2±2.1.±1. d.7±1. d.1±1.7 d Tle. The Frequenies of poptosis nd MNN in se Group under Vrious F onentrtions. Vlues re mens ± S; n = 2 for ; n = for ; n = 1 for ; n = 1 for ; n = 17 for ; n = for ; n = for ; n = 7 for ; n = 2 for. rs mrked with different letters differ from eh other in the sme type in different medium; Repeted mesures one-wy of dt 2 22 <.1 <.1~.1 poptosis /5 ells <.1~.1 <.1~.1.7±.2.21±1.22.5±1.2.72±.75.75±..±. <.1~.1.±..2±1.55.1±1.2.5±.2.±1.7.17±. <.1~.5.±1..2±1..±1.2.1±.7.2±.2.5±.2 <.1~.5.5± ±1.2 7.±..2±.5.±.7.2±. <.1~.5 2.±..1±1.1.± ± ± ±1.11 <.1~.5.21±.7.±..±.1.1±.2.±1.2.±.7 <.1.1±. 5.±1..5±1..27±.2.±.71.1±. <.1.±.7.±.2.5±.7 2.1±. 2.5±. 2.7±1.2 <.1.21± ±1.2.±1..52±..±1..±1. <.1 <.1 <.1 MNN /1 N ells <.1 < ± ± ±.7.±2.7 d.7±1. d.7±1.1 d <.1 1.5± ±1. 11.±.7.1±.7 d.±.2 d.±1. d 1.2±1..51± ±.75.±1.2 d.1±1. d.5±1. d 2.7±2. 1.1± ±2.1.±.7 d.±2.2 d.±1.1 d 22.2± ±1.17.5±1.2.7±. d.7±.77 d.7±2.2 d 2.±. 17.5±..1±.1.5±.1 d.2±. d.±2.2 d 2.± ±2.2.2±1.1.±1.22 d.±1.1 d.7±1. d 2.± ±2. 1.5±.1 1.±1.1 d 1.±.7 d 1.±1. d 21.2± ±2.2.2±2.1.±1. d.7±1. d.1±1.7 d Tle 5. The Frequenies of poptosis nd MNN in Group under Vrious F onentrtions. Vlues re mens ± S; n = 27 for ; n = 7 for ; n = for ; n = 11 for ; n = 1 for 77T /; n = 2 for ; n = for ; n = 2 for ; n = for. rs mrked with different letters differ from eh other in the sme type in different medium; Repeted mesures one-wy of dt MNN nd poptosis in different F onentrtion The frequeny of MNN signifintly deresed with inresing F onentrtion from to or 2 nmol/l F (p <.5 to.1), nd the frequeny of poptosis inresed with inresing F onentrtion from to or nmol/l. The frequeny of poptosis ws likewise redued when the F onentrtion inresed from to or 2 nmol/l in ll tested groups (p <.5 to.1). There ws no signifint differene etween the deteted iomrkers etween nd 2 nmol/l F in ll of the groups (Tle, 5). Frequenies of MNN in MTHFR 77T- omined genotypes in ner ses The omined genotypes of 77, 77T with ny lleles nd of 77TT with showed the lowest frequenies of MNN under ny onentrtion of F, nd no signifint differene ws deteted. The genotype of 77TT with either or ( nd ) showed signifint inreses in the frequenies of MNN regrdless of the F onentrtion reltive to the frequenies of MNN from the genotypes 77 with ny lleles nd to or (p <.5 to.1). The differene of the frequenies of MNN 222 sin Pifi Journl of ner Prevention, Vol 1, 2 MNed N ells / 1 N ells 2 1 p<.5 MNed N ells / 1 N ells 2 1 Figure 1. The Frequenies of MNN in the se nd Group with the omined MTHFR 77T- Genotypes. () nm F () nm F () nm F () nm F () 2 nm F MNed N ells / 1 N ells MNed N ells / 1 N ells 2 1 p<.1~.5 MNed N ells / 1 N ells 2 1 MNed N ells / 1 N ells p<.1~ Figure 2. The Frequenies 5.of MNN in the Group with 5. the omined MTHFR 77T Genotypes. () nm F () nm F () nm F () nm F () 2 nm F in lymphoytes 25.with the 77T, 77TT genotype with, (,,. nd ) ws not signifint t onentrtions of to 2 nmol/l 2.7 F, ut or ws signifintly lower thn the or genotypes t to nmol/l in the ner group (p <.5 to.1) (Figure 1). Newly dignosed without tretment p<.1~.5 p<.1~.5 MNed N ells / 1 N ells MNed N ells / 1 N ells 2 1 p<.1~ MNed N ells / 1 N ells 1.1 Newly dignosed with tretment 2 1 p<.1~.5 2. Persistene or reurrene p<.1~.5 MNed N ells / 1 N ells 2 1 p<.1~.5 p<.1~.5 Frequenies of MNN mong MTHFR 77T- genotypes in ontrols The 77, 77T with ny lleles nd genotypes hd the lowest frequenies of MNN, nd there ws no signifint differene etween them. No individuls were found in the ontrol group. The MNN levels from the genotype were signifintly inresed reltive to the MNN levels from the 77 nd 77T genotypes with ny lleles (p <.5 to.1), regrdless of F onentrtion hnge (Figure 2). Frequenies of poptosis mong MTHFR 77T- genotypes in ner ses The 77 with ny lleles nd 77T with genotypes showed higher frequenies of poptosis in lymphoytes thn other genotypes (p <.5 to.1), nd there were no signifint differenes in the levels of poptosis etween them t ny onentrtion of F. The 77TT with or ( nd ) genotypes hd lower frequenies of poptosis thn the 77 with ny lleles nd genotypes t ny F onentrtion (p <.5 to.1). The frequenies of 25. Remission

5 poptoti ells per 1 ells p<.1~.5 poptoti ells per 1 ells poptoti ells per 1 ells OI: Intertions etween MTHFR 77T - nd Foli id efiieny in rest ners in hin p<.5 p<.1~.5 Figure. The Frequeny of poptosis in the se Group with the omined MTHFR 77T- Genotypes. () nm F () nm F () nm F () nm F () 2 nm F poptoti ells per 1 ells p<.5 poptoti ells per 1 ells poptoti ells per 1 ells poptosis of 77T heterozygote with either the or ( nd ) genotypes were signifintly lower thn from the 77 with ny lleles nd the genotypes in ll of the F onentrtion groups (p <.1); however, the frequeny of poptosis ws inresed when F ws inresed to nmol/l (p <.5). The 77TT or 77 T with ( nd T) nd 77 with or ( nd ) genotypes did not show signifint differenes in the levels of poptosis reltive to in the ner se group (Figure ). poptosis mong MTHFR 77T- genotypes in ontrols The genotype 77 with ny lleles (, nd ) hd higher levels of poptosis thn other genotypes (p <.1) nd did not hve signifint differenes in the level of poptosis reltive to eh other. The omintion of the 77TT nd 77T with ny lleles (,, nd ) showed signifint derese in the levels of poptosis reltive to the 77 with ny llelegenotypes t to nmol/l of F (p<.5 to.1), nd inresed signifintly s F ws inresed to over nmol / L (p<.5) (Figure ). omprison of MNN/poptosis frequeny etween ner ses nd ontrols differene of differenes nlysis showed tht poptoti ells per 1 ells p<.1~.5 poptoti ells per 1 ells p<.1~.5 p<.5 Figure. The Frequenies of poptosis in the Group with the omined MTHFR 77T- Genotypes. () nm F() nm F () nm F () nm F () 2 nm F p<.5 poptoti ells per 1 ells poptoti ells per 1 ells p<.1~.5 p<.5 Redutionin MNed Ns per 1 N ells with Redutionin MNed Ns per 1 N ells with - - se se p=.1 *** nm F se p>.5 nm F se p<.1 nm F se nm F se nm F nm F se se Figure 5. omprison of the MNN Frequenies etween the se nd Groups with the omined MTHFR 77T- Genotypes fter the ifferene of ifferenes nlysis under F efiieny. The results re shown s olumn nd r of results for eh individul. () genotype () genotype () genotype () genotype () genotype (F) genotype (G) genotype (H) genotype Redution in poptoti ells per 1 ells with Redution in poptoti ells per 1 ells with - - se se p>.5 nm F se nm F se p>.5 nm F se nm F se nm F se se Redution in poptoti ells per 1 ells with nm F MNN levels in individuls with the 77T with mutted lleles from the ner group were signifintly higher thn those in the respetive ontrol group t nmol/l F (p <.1 to.1), wheres the MNN levels in the ner se group shring the 77TT with genotype were signifintly higher t to nmol/l F (p <.5) thn in the ontrols (Figure 5). There were no signifint differenes in the ourrene of poptosis mong individuls with ny genotype etween the ner se nd ontrol groups s determined using differene of differenes nlysis y sutrting the pooled vlues of the iomrker t 2 nmol/l F from those oserved t to nmol/l F for eh individul (Figure ). isussion Redutionin MNed Ns per 1 N ells with Redutionin MNed Ns per 1 N ells with Redution in poptoti ells per 1 ells with - F se se nm F se p=. ** nm F F se se p>.5 nm F se p<.1 se p>.5 nm F se nm F se nm F nm F nm F se p>.5 se nm F se nm F se se nm F se nm F se Reports studying the ssoition of folte metolism nd hnges in MTHFR tivity with rest ner re onfliting t ll time (mpell et l., 22; Rodrigues et l., 21). Therefore, the present report sin Pifi Journl of ner Prevention, Vol 1, 2 22 Redution in poptoti ells per 1 ells with Redution in poptoti ells per 1 ells with Redutionin MNed Ns per 1 N ells with Redutionin MNed Ns per 1 N ells with - - se se G nm F se p>.5 nm F se p>.5 nm F se nm F se p>.5 nm F se nm F se Redutionin MNed Ns per 1 N ells with Redutionin MNed Ns per 1 N ells with - se - H se nm F se p>.5 nm F se p<.5 p=.11 nm F * p=. * Figure. omprison of the poptosis Frequenies etween the se nd Groups with the omined MTHFR 77T- Genotypes fter the ifferene of ifferenes nlysis under F efiieny. The results re shown s olumn nd r of results for eh individul. () genotype () genotype () genotype () genotype () genotype (F) genotype (G) genotype (H) genotype - - G se se nm F se nm F se p>.5 nm F se nm F se nm F se nm F se Redution in poptoti ells per 1 ells with Redution in poptoti ells per 1 ells with - - H se se se nm F p=.25 * se p>.5 nm F se nm F se p>.5 nm F se nm F se nm F se nm F se nm F se nm F se

6 Xi-Yu Wu et l studied the reltionship etween omined MTHFR polymorphisms nd geneti dmge nd poptosis under folte defiieny, nd ompred the differenes in response to folte defiieny etween rest ner nd nonner popultions with omined. However, the results must e onsidered with some ution euse the ulture onditions my not predit preisely wht hppens in vivo. In this study, we found tht superfluous supplementtion of F (more thn or nmol/l) deresed the frequeny of poptosis. In ontrst, MNN exhiited strong negtive orreltion with foli id onentrtion ut plteued t nmol/l ( versus 2 nmol/l; p >.5). Tken together, these results suggest tht F defiieny my inrese the risk of ner y induing n imlne in N preursors, leding to modified N synthesis nd repir, nd onentrtion of nmol/l F is required to minimize ytotoxiity nd geneti dmge. hnges of MTHFR tivity my tilt the lne of one ron metolism in fvor of N synthesis t the expense of methyl supply for methyltion retions; suoptiml methyl supply n led to errnt N methyltion, whih hs een ssoited with rest ner etiology. The 77T polymorphism is in exon, whih is within the N-terminl tlyti domin of the enzyme, nd the polymorphism is in exon 7, whih is within the -terminl regultory domin. The more dynmi effet of 77T is due to its lotion within the tlyti region. The polymorphism my ffet enzyme regultion possily y influening S-denosylmethionine, whih is n llosteri inhiitor of MTHFR nd is known to ind in the -terminl region (Yi et l., 22; Promthet et l., 21). onsequently, effets on MTHFR tivity my eome signifint when n individul rries oth muttions. The study found tht the tivity for the g polymorphism ws pproximtely 5% of tht of the ontrols nd the tivity for the 77gT muttion ws pproximtely % of tht of the ontrols. However, individuls tht were heterozygous for oth the g nd 77gT muttion hd somewht lower tivities in lymphoytes (5% to % of ontrols) thn single 77gT heterozygotes (Weiserg et l., 1). Low enzyme tivity my redue the pity of N methyltion nd possily redue uril misinorportion into N (Weiserg et l., 1; Skiol et l., 1; rott et l., 21; Hung et l., 2; Thoms et l., 2; hillon et l., 2; Promthet et l., 21; Rodrigues et l., 21). One puzzling result of the present study is tht there ws no trend towrd redution of MNN levels in single mutnt () ells ompred with wild-type () ells. dditionlly, there ws n inresed frequeny of MNN in the homozygous mutnt t oth 77 nd () ompred with homozygous mutnt t 77 () ells within the ner se group nd in homozygous mutnt t 77 nd heterozygous mutnt t () ompred with homozygous mutnt t 77 () ells within oth groups. This is ounterintuitive euse uril in N is positively orrelted with MNN in other studies using this system (rott et l., 21). However, MNN my originte not only from hromosome rekge used y uril in 22 sin Pifi Journl of ner Prevention, Vol 1, 2 N, ut lso from hromosoml loss events tht my e used y hypomethyltion of N (Friso et l., 22). One study reported tht the 77TT with mutted lous genotype inreses N hypomethyltion in lymphoytes in vivo (mpell et l., 22), nd N hypomethyltion in lymphoytes in vitro or in vivo inreses the loss of hromosomes 1, nd 1, whih re then inluded in MNN (Guttenh et l., 1; Xu et l., 1). Therefore, the mrginl inrese in MNN levels in 77TT with mutted lous ells my e due to inresed hromosoml loss events; thus, omined muttions ould enhne the instility of the genome in the hinese popultion. In ordne with the results disussed ove, poptosis levels were lower in the 77TT with thn the 77TT with genotype nd lower in the 77T with genotype thn the 77T with genotype t to nmol / L F in ner ses, ut the lower poptosis level in the 77TT with genotype reltive to the 77TT with genotype nd in 77T with genotype reltive to the 77T with genotype ws not signifint in the ontrol group. To the est of our knowledge, this is the first report showing tht spontneous poptosis in rest ner ptients is ssoited with or my even depend on s. It lso found tht the spontneous poptosis rtes fter 2 h were signifintly inresed in hroni lymphoyti leukemi ptients with the MTHFR 77 genotype versus. MTHFR 77T nd MTHFR 77TT genotypes (1, 5 nd 25%, respetively, p =.5). dditionlly, the spontneous poptosis rte ws signifintly deresed in the genotype vs. genotype (2 nd %, respetively, p =.) (istulfi et l., 21). It reported on the diversion of oneron units towrd nuleotide synthesis t the expense of N methyltion in young women with the 77TT genotype fter 7 weeks of folte restrition (Quinlivn et l., 25), whih suggested tht the derese in poptosis ws relted to N synthesis. Menwhile, the study found tht poptosis ws oservly lower in the 77TT with genotype thn 77TT with genotype nd lower in the 77T with genotype thn the 77T with genotype t to nmol/l F in ner ses, whih suggested tht the omined muttion of MTHFR my e more likely to influene poptosis proesses in the ner ses group with F defiienies. Our dt provide some evidene suggesting tht the hypomethyltion of N might derese poptosis proesses in some ner ptients; this result ws in greement with those of eltiki et l. (2) nd Xio et l. (2). In tht of my study, poptosis did not pper to e relted to the omined MTHFR polymorphisms in the ontrol group. lthough poptosis ws lower in 77T with ells thn in 77T with ells in the ontrol group, the mgnitude of this differene ws smll, nd it is unler if this is physiologilly relevnt differene. possile wekness of our study is tht we did not mesure N methyltion diretly. It is known tht mlignnies derived from rpidly proliferting tissues re expeted to hve n inresed requirement for N synthesis, nd ptients with suh

7 OI: Intertions etween MTHFR 77T - nd Foli id efiieny in rest ners in hin mlignnies ould, therefore, e even more endngered y folte defiieny nd the resulting N dmge. y the differene of differenes nlysis, MNN from the genotype t to nmol/l F nd the 77T with mutted lous genotype t nmol/l F in ner ses is signifintly higher thn in ontrols, nd in individuls reeiving dequte F, there were no signifint differenes in sensitivities to the genomi dmge etween the ner ses nd ontrols in ll of the genotypes. This finding seems to suggest tht the rest ner ptients with the omined muttion of 77TT with genotype my e more sensitive to F defiieny thn ontrols, nd the effet of the resulting genomi dmge in the ner se groups y MTHFR polymorphisms seems to e dependent on folte sttus. dditionlly, for the ourrene of poptosis mong individuls with ny genotype, our dt showed no signifint differenes etween ner ses nd ontrols, whih suggests tht women with the omined muttion of MTHFR 77T- genotypes hve signifintly inresed odds of developing rest ner. In summry, our study hs shown tht the omined mutnts of MTHFR 77T- hve signifint effet on inresing geneti dmge nd deresing poptosis levels ompred with single vrints in rest ner ptients. rest ner ptients with the omined muttion of MTHFR 77T- my e more sensitive thn ontrols to F defiieny tht n use the indution of genomi instility. n ssoition ws found etween MTHFR 77T nd omined MTHFR 77T- polymorphism nd rest ner in our series. Unfortuntely, there ws no 77TT with genotype ontrol in the study, so we nnot ompre the differene on geneti dmge nd poptosis in rest ner ses nd non-ner ontrols with the 77TT with genotype. knowledgements We re grteful to the volunteers for their prtiiption in the study. This work is supported y the Ntionl Nturl Siene Foundtion of hin (No. 7, 1) nd the Nturl Siene Foundtion of Yunnn Provine (No.2Z, 21). The uthor(s) delre tht they hve no ompeting interests. Referenes ndrew JV, ougls G (21). nlysing ontrolled trils with seline nd follow up mesurements. rit Med J, 2, 1-. eily J, Ingrm, Hhnel R, Rossi (2). Redued rest ner risk with inresing serum folte in se-ontrol study of the 77T genotype of the methylenetetrhydrofolte redutse gene. ur J ner,, 5-. istulfi G, Vndette, Mtsui S, Smirgli J (21). Mild folte defiieny indues geneti nd epigeneti instility nd phenotype hnges in prostte ner ells. 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