Akkafa, F.

Dilmec, F.

Ozgonul, A.

Uzunkoy, A.

van Kuilenburg, A.B.P.

English

Colorectal cancer (CRC) is the leading cause of cancer death among human around the world. The vitamin D receptor gene (VDR) is a member of the nuclear receptor super family, which is expressed in the tissue of gastrointestinal tract, known to modulate the rate of cell proliferation. We aimed to investigate the genotype and allele frequencies and association of the VDR gene: c.1025-49G&gt;T (ApaIG&gt;T) and c.1056T&gt;C (TaqIT&gt;C) polymorphisms with CRC in Turkish patients. Fifty-six patients with CRC and 169 healthy individuals were enrolled to study, and their DNA was isolated. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was used to detect the frequency of c.1025-49G&gt;T and c.1056T&gt;C polymorphisms. The prevalence of the c.1025-49G&gt;T and c.1056T&gt;C alleles and the genotype frequencies in patients with CRC was similar to that in the normal population. The investigated polymorphisms in the VDR gene do not represent I significant risk factor for CRC in our populatio

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UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl)UvA-DARE (Digital Academic Repository)Determination of ApaI and TaqI Polymorphisms of VDR Gene in a Group ofTurkish Patients with Colorectal CancerDilmec, F.; Ozgonul, A.; Akkafa, F.; Uzunkoy, A.; van Kuilenburg, A.B.P.Publication date2009Document VersionFinal published versionPublished inUluslararasÿ¸ Hematologi-Onkoloji Dergisi : UHOD = International Journal of Hematology andOncologyLink to publicationCitation for published version (APA):Dilmec, F., Ozgonul, A., Akkafa, F., Uzunkoy, A., & van Kuilenburg, A. B. P. (2009).Determination of ApaI and TaqI Polymorphisms of VDR Gene in a Group of Turkish Patientswith Colorectal Cancer. Uluslararasÿ¸ Hematologi-Onkoloji Dergisi : UHOD = InternationalJournal of Hematology and Oncology, 19(1), 18-22. http://www.uhod.org/pdf/PDF_345.pdfGeneral rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s)and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an opencontent license (like Creative Commons).Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, pleaselet the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the materialinaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letterto: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. Youwill be contacted as soon as possible.Download date:09 Mar 202318 UHOD Say› / Number: 1    Cilt / Volume: 19   Y›l / Year: 2009Determination of ApaI and TaqI Polymorphismsof VDR Gene in a Group of Turkish Patients with Colorectal CancerFuat DİLMEÇ1, Abdullah ÖZGÖNÜL2, Feridun AKKAFA1, Ali UZUNKOY2, André B.P. van KUILENBURG31 Harran University, Faculty of Medicine, Department of Medical Biology2 Harran University, Faculty of Medicine, Department of General Surgery, ŞANLIURFA3 University of Amsterdam, Emma Children’s Hospital and Department of Clinical Chemistry, Academic Medical Center, AMSTERDAMABSTRACTColorectal cancer (CRC) is the leading cause of cancer death among human around the world. The vitamin D recep-tor gene (VDR) is a member of the nuclear receptor super family, which is expressed in the tissue of gastrointestinaltract, known to modulate the rate of cell proliferation. We aimed to investigate the genotype and allele frequencies andassociation of the VDR gene: c.1025-49G>T (ApaIG>T) and c.1056T>C (TaqIT>C) polymorphisms with CRC inTurkish patients. Fifty-six patients with CRC and 169 healthy individuals were enrolled to study, and their DNA wasisolated. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was used todetect the frequency of c.1025-49G>T and c.1056T>C polymorphisms. The prevalence of the c.1025-49G>T andc.1056T>C alleles and the genotype frequencies in patients with CRC was similar to that in the normal population.The investigated polymorphisms in the VDR gene do not represent a significant risk factor for CRC in our popula-tion.Key Words: VDR gene, Colorectal cancer, Polymorphism, PCR, RFLPÖZETKolorektal Kanserli Bir Grup Türk Hastasında VDR Geni ApaI ve TaqI Polimorfizmlerinin BelirlenmesiKolorektal kanser (KRK), Dünyada insanlar arasında kanserden ölümün önde gelen sebebidir. Vitamin D reseptör(VDR) geni, mide bağırsak dokusunda ifadesini bulan ve hücre bölünme oranını düzenleyen çekirdeğe ait reseptörbüyük ailesinin bir üyesidir. Türk hastalarında KRK ile VDR geni: c.1025-49G>T (ApaIG>T) ve c.1056T>C(TaqIT>C) polimorfizmlerinin ilişkisini, genotip ve allel oranlarını araştırmayı amaçladık. KRK’li 56 hasta ve 169sağlıklı birey çalışmaya dahil edildi ve bunların DNA’sı izole edildi. c.1025-49G>T ve c.1056T>C polimorfizmlerininbelirlenmesinde, Polimeraz zincir reaksiyonu-restriksiyon parça uzunluk polimorfizm (PCR-RFLP) tekniği kullanıldı.KRK’li hastalarda c.1025-49G>T ve c.1056T>C allel ve genotip frekanslarının yaygınlığı, normal popülasyondakideğerlerle benzerdir. VDR geninde araştırılan polimorfizmleri, toplumumuzda KRK için anlamlı bir risk faktörüolduğunu göstermiyor.Anahtar Kelimeler: VDR geni, Kolorektal kanser, Polimorfizm, PCR, RFLPULUSLARARASı HEMATOLOJI-ONKOLOJI DERGISI ARTICLE / MAKALE International Journal of Hematology and OncologyINTRODUCTIONColorectal cancer (CRC) is the third most commontumor and the fourth most common cancer-relatedcause of death in both genders worldwide. CRC in-cidence and mortality vary markedly between eth-nicities in different populations. The vitamin D re-ceptor gene (VDR) may be an important modulatorof the risk of CRC, either independently or in con-junction with vitamin D, and calcium intake.1Polymorphisms in the VDR gene (OMIM 601769)may influence the expression or function of the vi-tamin D (1,25-dihydroxyvitamin D3) receptor.2The VDR gene TaqI polymorphism is associatedwith numerous diseases, such as diabetes mellitustype 1 (T1DM)3, and psoriasis4; while not associ-ated with osteoporosis5, T2DM6, breast cancer7, andprostate cancer8. In the same way, some studies in-dicate that the genotypes of ApaI polymorphism isa significant association with prostate cancer8, Gra-ves’ disease9, and Epithelial Ovarian Cancer10; whi-le not association with osteoporosis 5, (T1DM)3,T2DM 6.Molecular variants of the VDR gene may be relatedto the development of colon cancer.1,11,12 The VDRTru9I polymorphism may be associated with lowerrisk for colorectal adenoma, particularly in interac-tion with various risk factors, but not with calciumor vitamin D in a USA population.13 The homozy-gous variant genotypes (CC) are associated with adecreased risk of squamous cell carcinoma of thehead and neck (SCCHN) compared to the commonTaqI TT genotypes.14Some studies demonstrate that there is no associati-on of ApaI, TaqI, BsmI, FokI, and Tru9I polymorp-hisms of the VDR gene with patients with colorec-tal adenoma, compared with healthy controls in aUSA and Russian populations.15-19 Whereas, the ot-her study shows that the ApaI TT genotype of theVDR gene are associated with an increased risk forrenal cell carcinoma, while the other genotypes(TaqI CC and BsmI TT) are no associated with thiscarcinoma.20The purpose of this study was to investigate the as-sociation of the VDR gene c.1025-49G>T andc.1056T>C polymorphisms with CRC in a Turkishpopulation.MATERIAL AND METHODSIndividuals and DNA IsolationFifty-six patients with CRC (30 females and 26 ma-les: average age: 56±2), who were diagnosed at theSurgery Unit in Harran University and 169 healthycontrols (79 females, mean age 57±3; and 90 males,mean age 56±3), who did not have any disease, we-re investigated in this study. Blood was taken fromthese individuals, and genomic DNA was extractedfrom nuclear cells by using a standard salting outprocedure, as described by Miller et al.21PCR-RFLPThe VDR gene NM001017535: c.1025-49G>T(ApaIG>T or g.59979G>T) and c.1056T>C (Ta-qIT>C or g.60058T>C) polymorphic sites were in-vestigated by a touchdown polymerase chain reac-tion (PCR) method modified by us, and restrictionfragment length polymorphisms (RFLP) technique.The PCR reactions were performed in a 10-ml ofreaction volume, including 1xPCR buffer, 2 mMMgCl2, 0.2 mM each deoxynucleotide triphospha-te (dNTPs, Fermentas, St. Leon-Rot, Germany), 0.2µM of each primer (A:5’-CAGAGCATGGA-CAGGGAGCAAG-3’, B:5’-GCAACTCCTCATGGCTGAGGTCTCA-3’)22 (BioBasic Inc, Ontario,Canada), 40 ng of DNA, and 0.3 unit of Taq DNAPolymerase (Fermentas). The touchdown PCRprogram was performed at 94˚C for 3 min (initialdenaturation), 10 cycles, with 94˚C for 30s, 72-62˚C for 30s (decreasing 1˚C per cycle), 72˚C for30s, and 20 cycles, with 94˚C for 30s, 62˚C for 30s,72˚C 30s; and 72˚C for 5 min (final extension). The PCR products (5 µl) of VDR gene were sepa-rately restricted in a 20 µl reaction volume for twohours with 1.5 Units of ApaI at 37˚C and TaqI (Fer-mentas, St. Leon-Rot, Germany) at 65˚C, respecti-vely. The digested PCR product was separated on2% agarose gel, and analyzed using Alpha-ImagerGel System (AlphaInnotech, San Leandro, Califor-nia USA). The ApaI product; G allele yielded frag-ments of a 528-bp, 217-bp (wild type), and T alleleyielded 745-bp (mutant) (Figure 1); and TaqI T al-lele yielded fragments of 494-bp, 251-bp (wildtype), and C allele yielded 293, 251, 201-bp (mu-tant) (Figure 2).19UHOD Say› / Number: 1    Cilt / Volume: 19   Y›l / Year: 2009Statistical AnalysisGenotype and allele frequencies of c.1025-49G>Tand c.1056T>C polymorphic sites of the VDR genewere tested for Hardy-Weinberg equilibrium byusing chi-square test. Besides, genotype and allelefrequencies observed in this study were analyzedwith Fisher’s exact test by using the SPSS statisticsprogram. Statistical significance was defined as p <0.05. The odds ratio (OR) was calculated to measu-re the strength of the association observed (Table 1).EthicsThe institutional review board approved the study,and written informed consents were obtained fromall patients. The study complied with the HelsinkiDeclaration.RESULTSThe c.1025-49G>T and c.1056T>C polymorphismsin the VDR gene were investigated by PCR-RFLPanalysis in CRC patients and healthy subjects. Thefrequencies of the alleles and the genotypes were inHardy-Weinberg equilibrium among the patientsand the controls. There was no significant association between theVDR gene c.1025-49 TT (mutant) and c.1056 CC(mutant) genotypes and CRC patients, comparedwith healthy control group (Table 1). In addition,we could not reveal any effect of the VDR genec.1025-49 T (OR: 0.815, 95%CI: 0.529-1.254) andc.1056 C (OR: 0.673, 95%CI: 0.419-1.082) alleleson the risk of CRC (Table 1).DISCUSSIONHigher serum 25-OH vitamin D levels are associ-ated with decreased colorectal adenoma risk, andvitamin D has been shown to reduce proliferationand increase differentiation in human colon cancercells.19 The VDR polymorphisms are important fac-tors in the assessment of colorectal cancer risk, pos-sibly indicating vitamin D and calcium as preventi-ve measures.13 Vitamin D is a potential agent for theprevention of colorectal cancer possibly throughmechanisms mediated by the VDR.9, 15, 16 These fin-dings led to the question of whether variants in theVDR gene influence an individual’s susceptibilityto colorectal cancer development. Polymorphismsin the VDR gene may influence the expression orfunctioning of the vitamin D (1,25-hydroxyvitaminD3) receptor protein.2In our study, there was no the association of thec.1025-49G>T and c.1056T>C polymorphisms ofthe VDR gene in patients with CRC and healthycontrols in a Turkish population. These findings in-dicated that the VDR gene c.1025-49 T allele wasnot a risk for CRC patients (55.4%), compared withhealthy control (60.4%). Similarly, c.1025-49 TTgenotype frequency in CRC patients (30.4%) wasUHOD Say› / Number: 1    Cilt / Volume: 19   Y›l / Year: 200920Figure 2. The TaqI restriction profile of the VDRgene. Lane M: GeneRuler DNA ladder (50-1000bp,Fermentas); lane 1: undigested PCR product; lane 2:TT genotype (homozygous, wild type); lane 3: TCgenotype (heterozygous); lane 4: CC genotype(homozygous, mutant).Figure 1. The ApaI restriction profile of the VDRgene. Lane M: GeneRuler DNA ladder (50-1000bp,Fermentas); lane 1: undigested PCR product; lane 2:TT genotype (homozygous, mutant); lane 3: GTgenotype (heterozygous); lane 4: GG genotype(homozygous, wild type).not significant difference from healthy group(36.1%). On the other hand, the present study fin-dings demonstrated that the frequency of c.1056 Callele of VDR gene allele frequency was not signi-ficant association between CRC patients (26.8%)and healthy control (35.2%). Furthermore, althoughthe c.1056 CC genotype frequency of the VDR ge-ne in patients with CRC (7.1%) was lower than inhealthy subjects (11.2%), this difference was notsignificant (p=0.456) (Table 1). The VDR multiplepolymorphisms have been examined for the associ-ation with CRC in several populations. In this man-ner, our results corresponded with those reported inthe literature for various populations. Recent studi-es demonstrate that there is no association betweenthe VDR c.1056 TT (37.7%), TC (46.9%) and CC(15.3%) polymorphism in 3’UTR of VDR gene andpatients with colorectal adenoma, compared withhealthy controls (38.9%, 45.6%, and 15.5%, res-pectively) in a USA population.15-17 Whereas, theexpression of the nuclear vitamin D receptor(VDR), which is involved in regulating cell growthand proliferation, may contribute to the develop-ment of CRC.2 The finding that genetic variation inthe vitamin D pathway contributes to risk supportsa role for vitamin D in colon cancer etiology.12 TheTT genotype at the c.1025-49 site of the VDR genemay be a risk of incidence and poor prognosis fac-tor for renal cell carcinoma (RCC) in the Japanesepopulation.20Two case-control studies report a decreased risk ofcolorectal neoplasia in participants with the c.1056CC genotype compared with those with TC or TTgenotype either overall 4 or in conjunction with lowcalcium or vitamin D intake.3In conclusion, we have not found any evidence thatdifferences in the oncogenic properties of the VDRgene c.1025-49 T and c.1056 C alleles could confera genetic predisposition to colorectal carcinogene-sis. We proposed for detect whether the sequencedifferences in the VDR gene are susceptibility vari-ants for CRC, additional studies in different popu-lations are required in a large study group.AcknowledgementWe thank to the staff of the General Surgery Unit ofHarran University Hospital for collecting bloodsamples from patients with CRC.21UHOD Say› / Number: 1    Cilt / Volume: 19   Y›l / Year: 2009Table 1. Statistical analysis of SNPs in patients with CRC vs. control groupSNP CRC patients Healthy controls X2 OR (95% CI) p-valuesGenotype/Allele (n:56) (n:169)c.1025-49G>TGG 11 (19.6%) 26 (15.4%) 0.555 1.344  (0.616-2.934) 0.533GT 28 (50.0%) 82 (48.5%) 0.037 1.061  (0.580-1.942) 0.878TT 17 (30.4%) 61 (36.1%) 0.611 0.772  (0.403-1.479) 0.611G 50 (44.6%) 184 (39.6%) 0.869 1.228  (0.797-1.890) 0.376T 62 (55.4%) 204 (60.4%) 0.869 0.815  (0.529-1.254) 0.376c.1056T>CTT 30 (53.6%) 69 (40.8%) 2.772 1.672  (0.910-3.072) 0.120TC 22 (39.3%) 81 (47.9%) 1.266 0.703  (0.380-1.301) 0.282CC 4 (7.1%) 19 (11.2%) 0.770 0.607  (0.197-1.868) 0.456T 82 (73.2%) 219 (64.8%) 2.694 1.485  (0.925-2.386) 0.106C 30 (26.8%) 119 (35.2%) 2.694 0.673  (0.419-1.082) 0.106Abbreviations: X2 = Chi-square, OR = Odds ratio, CI = Confidence interval, SNP = Single Nucleotide PolymorphismREFERENCES1. Slattery ML, Neuhausen SL, Hoffman M, et al.Dietary calcium, vitamin D, VDR genotypes andcolorectal cancer. Int J Cancer 111: 750-756,2004.2. Park K, Woo M, Nam J, Kim JC. Start codonpolymorphisms in the vitamin D receptor andcolorectal cancer risk. Cancer Lett 237: 199-206, 2006.3. Skrabic V, Zemunik T, Situm M, Terzic J. Vita-min D receptor polymorphism and susceptibilityto type 1 diabetes in the Dalmatian population.Diabetes Res Clin Pract 59: 31-35, 2003.4. Halsall JA, Osborne JE, Pringle JH, HutchinsonPE. Vitamin D receptor gene polymorphisms,particularly the novel A-1012G promoter poly-morphism, are associated with vitamin D3 res-ponsiveness and non-familial susceptibility inpsoriasis. Pharmacogenet Genomics 15: 349-55,2005.5. Langdahl BL, Gravholt CH, Brixen K, EriksenEF. Polymorphisms in the vitamin D receptorgene and bone mass, bone turnover and osteopo-rotic fractures. Eur J Clin Invest 30: 608-617,2000.6. Oh JY, Barrett-Connor E. Association betweenvitamin D receptor polymorphism and type 2 di-abetes or metabolic syndrome in community-dwelling older adults: The Rancho BernardoStudy. Metabolism 51: 356-359, 2001.7. Buyru N, Tezol A, Yosunkaya-Fenerci E, DalayN. Vitamin D receptor gene polymorphisms inbreast cancer. Exp Mol Med 35: 550-555, 2004.8. Onen IH, Ekmekci A, Eroglu M, et al. Associati-on of genetic polymorphisms in vitamin D re-ceptor gene and susceptibility to sporadic pros-tate cancer. Exp Biol Med (Maywood) 233:1608-14, 2008.9. Ban Y, Taniyama M, Ban Y. Vitamin D receptorgene polymorphism is associated with Graves'disease in the Japanese population. J Clin En-docrinol Metab 85: 4639-4643, 2000. 10. Lurie G, Wilkens LR, Thompson PJ, et al. Vita-min D receptor gene polymorphisms and epithe-lial ovarian cancer risk. Cancer Epidemiol Bi-omarkers Prev 16: 2566-71, 2007.11. Slattery ML, Yakumo K, Hoffman M, Neuha-usen S. Variants of the VDR gene and risk of co-lon cancer (United States). Cancer Causes Cont-rol 12: 359-364, 2001. 12. Wong HL, Seow A, Arakawa K, et al. Vitamin Dreceptor start codon polymorphism and colorec-tal cancer risk: effect modification by dietarycalcium and fat in Singapore Chinese. Carcino-genesis 24: 1091-1095, 2003.13. Gong YL, Xie DW, Deng ZL, et al. Vitamin Dreceptor gene Tru9I polymorphism and risk forincidental sporadic colorectal adenomas. WorldJ Gastroenterol 11: 4794-4799, 2005.14. Liu Z, Calderon JI, Zhang Z, et al. Polymorp-hisms of vitamin D receptor gene protect againstthe risk of head and neck cancer. PharmacogenetGenomics 15: 159-165, 2005.15. Peters U, McGlynn KA, Chatterjee N, et al. Vi-tamin D, calcium, and vitamin D receptor poly-morphism in colorectal adenomas. Cancer Epi-demiol Biomarkers Prev 10: 1267-1274, 2000.16. Peters U, Hayes RB, Chatterjee N, et al. Circu-lating vitamin D metabolites, polymorphism invitamin D receptor, and colorectal adenoma risk.Cancer Epidemiol Biomarkers Prev 13: 546-552, 2004.17. Grau MV, Baron JA, Sandler RS, et al. VitaminD, calcium supplementation, and colorectal ade-nomas: Results of a randomized trial. J NatlCancer Inst 95: 1765-1771, 2003.18. Zhou W, Heist RS, Liu G, et al. Polymorphismsof vitamin D receptor and survival in early-stagenon-small cell lung cancer patients. Cancer Epi-demiol Biomarkers Prev 15: 2239-2245, 2006.19. Flügge J, Krusekopf S, Goldammer M, et al. Vi-tamin D receptor haplotypes protect against de-velopment of colorectal cancer. Eur J Clin Phar-macol 63: 997-1005, 2007.20. Obara W, Suzuki Y, Kato K, et al. Vitamin D re-ceptor gene polymorphisms are associated withincreased risk and progression of renal cell car-cinoma in a Japanese population. Int J Urol 14:483-487, 2007.21. Miller SA, Dykes DD, Polesky HF. A simplesalting out procedure for extracting DNA fromhuman nucleated cells. Nucleic Acids Res 16:1215, 1998.22. Mitra S, Desai M, Ikram KM. Vitamin D recep-tor gene polymorphisms and bone mineral den-sity in postmenopausal Indian women. Maturitas55: 27-35, 2006.CorrespondenceDr. Fuat DİLMEÇHarran Üniversitesi, Tıp Fakültesi Tıbbi Biyoloji Bölümü 63300   Yenişehir -  ŞANLIURFAFax: (0.414) 313 96 15e-mail: fdilmec@yahoo.com.trfdilmec@harran.edu.trUHOD Say› / Number: 1    Cilt / Volume: 19   Y›l / Year: 200922

Determination of ApaI and TaqI Polymorphisms of VDR Gene in a Group of Turkish Patients with Colorectal Cancer

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Determination of ApaI and TaqI Polymorphisms of VDR Gene in a Group of Turkish Patients with Colorectal Cancer

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