research article the vitamin d receptor (vdr) gene...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 295791 6 pageshttpdxdoiorg1011552013295791

Research ArticleThe Vitamin D Receptor (VDR) Gene Polymorphisms inTurkish Brain Cancer Patients

Bahar ToptaG1 Ali Metin Kafadar2 Canan Cacina1 Saime Turan1 Leman Melis Yurdum1

Nihal YiLitbaGJ1 Muhammed OLuz Goumlkccedile1 Uumlmit Zeybek1 and Ilhan YaylJm1

1 Department of Molecular Medicine The Institute of Experimental Medicine Istanbul University Vakıf Gureba CaddesiCapa 34390 Istanbul Turkey

2Department of Neurosurgery Cerrahpasa Faculty of Medicine Istanbul University Istanbul Turkey

Correspondence should be addressed to Ilhan Yaylım ilhanyaylimgmailcom

Received 25 September 2012 Revised 30 January 2013 Accepted 25 March 2013

Academic Editor Daniel Pra

Copyright copy 2013 Bahar Toptas et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Objective It has been stated that brain cancers are an increasingly serious issue in many parts of the world The aim of our studywas to determine a possible relationship between Vitamin D receptor (VDR) gene polymorphisms and the risk of glioma andmeningiomaMethodsWe investigated the VDRTaq-I andVDR Fok-I gene polymorphisms in 100 brain cancer patients (including44 meningioma cases and 56 glioma cases) and 122 age-matched healthy control subjectsThis study was performed by polymerasechain reaction-based restriction fragment length polymorphism (RF LP)Results VDRFok-I ff genotypewas significantly increasedin meningioma patients (159) compared with controls (25) and carriers of Fok-I ff genotype had a 647-fold increased riskfor meningioma cases There was no significant difference between patients and controls for VDR Taq-I genotypes and allelesConclusions We suggest that VDR Fok-I genotypes might affect the development of meningioma

1 Introduction

The term of brain cancer is a heterogeneous group of neo-plasms which occurs in intracranial tissue and the meningesThe incidence of malignant brain tumors is rare and approx-imately 2 of all cancers in adults [1 2] According to theresult of several studies the frequencies of brain tumors are ofdifferent rates according to countries worldwide In additionthese differences can be observed among different ethnicgroups in the same country [1] Primary brain tumors areclassified according to histopathological features like variousother cancer types into the followingmajor histologic groupstumors of neuroepithelial tissue including astrocytoma glio-blastoma oligodendroglioma and ependymoma tumors ofmeninges including meningioma and hemangioblastomaand tumors of cellular region including pituitary tumors andcraniopharyngioma [3] Glioblastoma and meningioma aretwo of the most common and most malignant primary braintumors Gliomas are responsible for approximately 80 andmeningioma is responsible for nearly 13ndash26 of all primarymalignant brain tumors [4ndash6] Primary brain tumors are a

very heterogeneous group of diseases that are still not fullyunderstood in their pathological mechanism Despite allthese developments the contribution of genetic factors in thedevelopment of brain tumors is still not fully understoodUnderstanding of the genetic risk factors is very importantfor choosing the most suitable cancer treatment [6 7]

Vitamin D is a steroid hormone that regulates severalendocrine functions and cell functions such as proliferationand differentiation [8 9] The 125(OH)

2D is one of potent

regulators of cell growth and differentiation which is aneffect on cell death tumor invasion and angiogenesis whichmakes it a candidate compound for cancer regulation [9] Itis known that vitamin D inhibits malignant cell proliferationand differentiation in several tissues such as breast colonskin and brain [10] The nuclear functions of vitamin Drequire binding to the vitamin D receptor (VDR) [11] Thevitamin D receptor (VDR) is involved in multiple pathwayssuch as insulin-like growth factor (IGF) signaling it also hasa role in the inflammation and estrogen-related pathwaysthat may be related to the prognosis of cancer [12 13] Thegene encoding the VDR is located on the chromosome

2 BioMed Research International

12q12-14 [14] Various studies suggested the effect of VDRgene polymorphisms in the development of several typesof carcinoma such as breast prostate and colon carcinoma[9 15ndash17] One of the VDR gene polymorphisms is the Fok-I (rs2228570) polymorphism which is located in exon 2 andresults in an alternative transcription initiation site leadingto alter the activity of VDR protein The polymorphic Fok-Isite in exon 2 results in different translation initiation regiondue to thymine (T) to cytosine (C) substitution The one ofthe most well known of these polymorphisms such as Taq-I(rs731236) which are located in exons 9 and a TC nucleotidesubstitution (ATT to ATC) leading to a synonymous changeat codon 352 (isoleucine) [18] It has been reported that VDRslocalized in neuronal and glial cells affect the metabolismof brain cells and change the expression of VDR [18 19]It has been reported that the potential effect of vitamin Don the treatment of cancer was first identified in myeloidleukemic cells [20] Synthetic vitaminD analogues are amongthe preferred options in the treatment of central nervoussystem tumors In addition phase II clinical studies havecorrelated with positive effects of vitamin D therapy onglioblastoma cells [21ndash26]The increased vitamin D synthesisin glioma cells after treatment can regulate cell proliferation[19] However the consist of this effect is required in VDRgene expression [20 25] Several studies have determinedthe contribution of VDR polymorphisms in various types ofcancer The aim of this study was to evaluate the associationbetween polymorphisms in the VDR Fok-I and Taq-I and therisk of brain cancer in Turkish patients

2 Materials and Methods

Subject Selection We investigated the VDR Taq-I and VDRFok-I gene polymorphisms in 100 brain cancer patients(including 44 meningioma cases and 56 glioma cases) and122 age-matched healthy control subjects who were in thefollow-up Cerrahpasa Faculty of Medicine Department ofNeurosurgery in IstanbulUniversityThemean ages of gliomaand meningioma patients and control group were 4475 plusmn1563 5026 plusmn 1268 and 4722 plusmn 1063 years respectively

The specimens were taken after obtaining informed con-sent and the study was conducted prospectivelyTheMedicalEthics Committee of Istanbul Medical Faculty approval wasobtained for the study The protocol followed was consistentwith the World Medical Association Declaration of Helsinki(Ethical Principles for Medical Research Involving HumanSubjects)

21 Polymorphism Analysis Blood specimens were collectedin tubes containing EDTA and DNA samples were extractedfrom the whole blood by a salting out procedure (Miller etal 1988) [27] Genotyping was performed by the polymerasechain reaction (PCR) and restriction fragment length poly-morphism (RF LP) For Taq-I polymorphism the followingprimers were used to amplify the VDR gene 51015840-CAG AGCATG GAC AGG GAG CAA G-31015840 51015840-GCA ACT CCT CATGGGCTGAGGTCTCA-31015840 For detection of the Taq-I RFLP50ndash100 ng genomic DNA was amplified with 1x polymerase

chain reaction (PCR) buffer 3mM MgCl2 02mM of each

dNTP 02mM of each primer and Taq polymerase in a 50120583Lreaction volume The PCR conditions were as follows Initialdenaturation step of 94∘C for 4min followed by 5 cycles of94∘C for 45 sec 64∘C for 60 sec and 72∘C for 2min and afurther 25 cycles of 94∘C for 30 sec 64∘C for 30 sec and 72∘Cfor 45 sec PCR products were digested with TaqI restrictionenzyme at 65∘C electrophoresed on 2 agarose gels andstained with ethidium bromide Genotypes were determinedas TT (490 245 bp) Tt (490 290 245 and 205 bp) or tt (290245 and 205 bp) for Taq-I polymorphism [28] The primers(MBI Fermentas Lithuania) for Fok-I polymorphism were51015840-GAT GCC AGC TGG CCC TGG CAC TG-31015840 51015840-ATGGAA ACA CCT TGC TTC TTC TCC CTC-31015840 The DNAtemplate was amplified by PCR using 3mM MgCl

2 02mM

of each dNTP 025mM of each primer and Taq polymerase(MBI Fermentas Lithuania) in a 50 120583L final volumeThePCRconditions involved an initial denaturation of 4min at 94∘Cand followed by 30 cycles of 94∘C for 1min annealing at 60∘Cfor 1min and extension at 72∘C for 1min A final extensionstep at 72∘C for 4min was also studied PCR productswere digested with FokI restriction enzyme (MBI FermentasLithuania) at 37∘C for 3 h followed by electrophoresis in a 2agarose gelThe FF genotype (homozygote of common allele)shows only one band of 272 bp in agarose gelThe ff genotype(homozygote of infrequent allele) generates two fragmentsof 198 and 74 bp The heterozygote displays three fragments(272 198 and 74 bp) [29]

22 Statistical Analysis Statistical analyses were performedusing the SPSS software package (revision 16 SPSS IncChicago IL USA) Data are expressed as means + SD Dif-ferences in the distribution of genotypes or alleles betweencases and controls were tested using the chi-square statisticOdds ratios (ORs) and 95 confidence intervals (95 CI)were calculated to estimate the risk of glioma and menin-gioma Values of119875 lt 005were considered statistically signif-icant Haplotype frequencies1198631015840 and 1199032 were calculated usingHaploview 40 programme

3 Results

The analysis included 100 brain cancer (including 44 menin-gioma cases 56 glioma cases and 122 healthy controlsTable 1 shows the clinical characteristics of the study groupsGenotype and allele frequencies ofmeningioma cases gliomacases and controls are shown in Table 2 The risks ofmeningioma and glioma associated with VDR genotypes areshown in Table 3 There was a significant difference in thedistribution of VDR Fok-I genotypes inmeningioma patientsbut no significant difference was detected in glioma patients(119875 gt 005) (Table 2) VDR Fok-I ff genotype was significantlyincreased in meningioma patients (159) compared withcontrols (25) and carriers of Fok-I ff genotype had anincreased risk for meningioma cases (119875 = 0004) (1205942 1033OR 6470 95 CI 1749ndash23926) The VDR Taq-I genotypefrequencies for meningioma glioma and control cases werenot significantly different (119875 gt 005) In addition vitamin D

BioMed Research International 3

Table 1 General demographic informations and parameters of patients and control groups

Parameters Meningioma cases (119899 = 44) Glioma (119899 = 56) Controls (119899 = 122)Age (years) (mean plusmn SD) 5026 plusmn 1268 4475 plusmn 1563 4722 plusmn 1063

Gender 119899 ()Male 18 (409) 32 (571) 51 (418)Female 26 (591) 24 (429) 71 (582)

Histological characteristic of tumorsAstrocytoma 119899 () 11 (196)Glioblastoma multiforme 119899 () 22 (393)Oligodendroglioma 119899 () 8 (143)Oligoastrocytomas 119899 () 6 (107)lowastOthers 119899 () 9 (161)

Values as average plusmn standard deviation lowastEpendymoma hemangioblastoma paraganglioma and so forth

Table 2 Genotype and allele frequencies of meningioma cases glioma cases and controls

SNP Controls 119899 ()(119899 = 122)

Meningioma 119899 ()(119899 = 44) 120594

2119875 value Glioma 119899 ()

(119899 = 56) 1205942

119875 valuelowastFok-I genotype

FF 56 (459) 19 (432) 28 (500)Ff 63 (516) 18 (409) 10527 0005 23 (411) 4598 01ff 3 (25) 7 (159) 5 (89)

lowastFok-1 allelesF 175 (717) 56 (636) 79 (705)f 69 (283) 32 (364) 1997 0157 33 (295) 0052 0818

Taq-I genotypeTT 65 (533) 23 (523) 32 (571)Tt 44 (360) 18 (409) 0703 0704 18 (322) 0275 0872tt 13 (107) 3 (68) 6 (107)

Taq-I allelesT 174 (713) 64 (727) 82 (732)t 70 (287) 24 (273) 0064 0800 30 (268) 0137 0710

Chi-square test was used to compare alleles and clinic pathological characteristics in the study group 119899 number of individualslowastFor Fok 1 polymorphism (rs2228570) F is referred to as T allele and f is referred to as C alleleFor Taq 1 polymorphism (rs731236) T is referred to as T allele and t is referred to as C alleleDue to the same base changes T-C or C-T for both polymorphisms it should be shown as the initial letter of the polymorphism

Table 3 The risk of meningioma and glioma associated with VDR genotypes

SNP Controls Meningioma OR (95 CI) 119875 value Glioma OR (95 CI) 119875 valueFok-1

FF 56 (459) 19 (432) 1050 (0774ndash1425) 0756 28 (50) 1089 (0787ndash1508) 0611Ff + ff 66 (541) 25 (568) 28 (50)FF + Ff 119 (975) 37 (841) 6470 (1749ndash23926) 0001 51 (911) 3631 (0899ndash14665) 0053ff 3 (25) 7 (159) 5 (89)

Taq-ITT 65 (533) 23 (523) 1022 (0711ndash1468) 0909 32 (571) 1073 (0810ndash1421) 0631Tt + tt 57 (467) 2 (477) 24 (429)TT + Tt 109 (893) 41 (932) 1043 (0943ndash1153) 0460 50 (893) 1005 (0403ndash2508) 0991tt 13 (107) 3 (68) 6 (107)


haplotypes were evaluated for association with brain cancersHaplotype analysis revealed that there was no relationshipbetween VDR polymorphisms and brain cancers (for gliomacases 1198631015840 017 LOD 108 and 119903-squared 0029 and formeningioma cases1198631015840 0199 LOD 106 and 119903-squared 0034)(Tables 4 and 5)

4 Discussion

For the first time we demonstrated the positive associationof VDR Fok-I gene variants with meningioma cases We alsodetermined that VDR Fok-I ff genotype might affect devel-opment of meningioma but we found that there was no sta-tistically significant difference between VDR polymorphismswith glioma

Vitamin D is an important factor in the regulation ofcell division and differentiation The VDR gene is a memberof nuclear receptor superfamily The VDR gene located onchromosome 12 For a long time several polymorphisms inVDR gene have been investigated for functional significanceand potential effects on disease susceptibility [17]Many stud-ies reported that vitamin D has an antiproliferative effect onmany cancer types which is promoting apoptosis in a varietyof malignant cells such as glioma neuroblastoma leukemialymphoma cells breast cancer and colon cancer [28 30 31]and numerous vitamin D analogs have been produced for thetreatment of several cancer types Alternations of vitamin Dlevels may be related to the changing in the expression of sev-eral transcription factors cell cycle arrested proteins growthfactor and other genes [32] Several studies implicated thatthe ff and Ff genotypes of the VDR gene are associated with adecreased transcriptional activity VDR Fok-I polymorphismchanges the size of the VDR protein [33ndash36] The shorterVDR variant could be less active therefore this variationmay lead to more aggressive disease prognosis [28 37]Thereare some studies showing that vitamin D deficiency affectedthe brain morphology and cellular proliferation and growthfactor signaling in other tissue [19 38ndash40] Brain tumorsare relatively rare than other cancer types but some of thebrain cancers are fatal cancer types [41]Meningiomas are oneof the most frequent neoplasms of the brain tumors whichaccount for nearly 13ndash26 and originate from the arachnoidcells or meningothelial cells [5 42 43] Most of the menin-gioma cases are sporadic tumors Today we have a very littleknowledge about genetic risk factors of meningioma Thepathophysiology ofmeningiomamay be associatedwith a fewgenes such as NF2 ATM GST CYP450 TP53 KRAS andMNI Especially these genes are related to DNA repair cellcycle regulation tumor suppressor and hormone metabolicpathways [44] Malmer et al reported that there may be arelationship between the ATM gene variants which regulatefor cellular response against DNA damage and meningiomarisk [45] Ting et al reported that the activation of theATM gene is mediated with VDR phosphorylation whengenotoxic stress furthermore VDR genemutation associatedwith inhibition of ATM gene expression [46]

Sadetzki et al reported that the genotype of KRAS wasrelated to the increased the risk of (nearly 2 fold)meningioma

Table 4 Haplotype frequencies of glioma cases and controls

Block Haplotype Case control ratiocounts 120594

2119875

Block 1TT 0547 0534 0553 0114 07359TC 0170 0198 0157 0892 03448CT 0168 0171 0166 0017 08968CC 0115 0097 0124 0546 046

lowastFor Fok-1 polymorphism (rs2228570) F is referred to as T allele and f isreferred to as C alleleFor Taq-1 polymorphism (rs731236) T is referred to as T allele and t isreferred to as C alleleDue to the same base changes T-C or C-T for both polymorphisms it shouldbe shown as the initial letter of the polymorphism

Table 5 Haplotype frequencies of meningioma cases and controls


2119875

Block 1CT 0543 0512 0554 0476 04904CC 0183 0250 0159 3581 00584TT 0153 0125 0163 0726 0394TC 0121 0114 0124 0062 08032


[47] VDR expression rates were associated with KRASmuta-tion in several cancer types Several studies suggest that cel-lular effects of VDR may be associated with MAPK signalingpathways especially KRAS mutation in several cancer typessuch as breast and colorectal cancers [48 49] Some menin-giomas might originate from arachnoids cells within thecranium and other meningioma types may associated withincreased bone density [50] Furthermore vitamin D alsoacts directly on osteoblasts which ismodulate differentiationand regulates mineralization of the extracellular matrix inosteoblastic cells [33] Previous studies reported that MN1gene was a candidate genetic risk factor for sporadic menin-gioma cases [51] Sutton et al demonstrated that protein levelsof MN1 was significantly in a relationship with vitamin Dmediated transcription mechanism in osteoblastic cells [32]All of these findings gave us an idea that vitamin D genevariations may be a genetic risk factor for the developmentof meningioma cases Finally we found that there wasstatistically significant difference between the control andmeningioma patients for Fok-I ff genotypes The individualswho had VDR Fok-I ff genotype had an increased risk formeningioma

Despite an extensive literature study there was no studythat reported the relationship between VDR polymorphismsand brain cancer susceptibility In conclusion the presentresults suggest that the Fok-I polymorphism in the VDR gene


may be related to the risk of meningioma However furtherstudies with large number of subjects are needed to explainthis kind of relationship between VDR-Fok-I genetic variantsand meningioma risk

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgment

This study was supported by a grant from the University ofIstanbul Research Foundation Turkey

References

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[2] S R Chandana S Movva M Arora and T Singh ldquoPrimarybrain tumors in adultsrdquo American Family Physician vol 77 no10 pp 1423ndash1430 2008

[3] J L Fisher J A SchwartzbaumMWrensch and J LWiemelsldquoEpidemiology of brain tumorsrdquo Neurologic Clinics vol 25 no4 pp 867ndash890 2007

[4] M L Goodenberger and R B Jenkins ldquoGenetics of adultgliomardquo Cancer Genetics vol 205 no 12 pp 613ndash621 2012

[5] C Marosi M Hassler K Roessler et al ldquoMeningiomardquo CriticalReviews in OncologyHematology vol 67 no 2 pp 153ndash1712008

[6] P Rajaraman B S Melin ZWang et al ldquoGenome-wide associ-ation study of glioma and meta-analysisrdquo Human Genetics vol131 no 12 pp 1877ndash1888 2012

[7] H Pinarbasi Y Silig andM Gurelik ldquoGenetic polymorphismsof GSTs and their association with primary brain tumorincidencerdquo Cancer Genetics and Cytogenetics vol 156 no 2 pp144ndash149 2005

[8] K T Amor R M Rashid and P Mirmirani ldquoDoes D matterThe role of vitamin D in hair disorders and hair follicle cyclingrdquoDermatology Online Journal vol 16 no 2 article 3 2010

[9] L Vuolo C Di Somma A Faggiano and A Colao ldquoVitamin Dand cancerrdquo Frontiers in Endocrinology vol 3 p 58 2012

[10] Y Amano K Komiyama and M Makishima ldquoVitamin D andperiodontal diseaserdquo Journal of Oral Science vol 51 no 1 pp11ndash20 2009

[11] A W Norman ldquoFrom vitamin D to hormone D fundamentalsof the vitamin D endocrine system essential for good healthrdquoAmerican Journal of Clinical Nutrition vol 88 no 2 2008

[12] J C Mathers G Strathdee and C L Relton ldquoInduction of epi-genetic alterations by dietary and other environmental factorsrdquoAdvances in Genetics vol 71 pp 4ndash39 2010

[13] M L Slattery ldquoVitamin D receptor gene (VDR) associationswith cancerrdquo Nutrition Reviews vol 65 no 8 pp S102ndash1042007

[14] K Kostner N Denzer C S L Muller R Klein W Tilgen andJ Reichrath ldquoThe relevance of Vitamin D Receptor (VDR) genepolymorphisms for cancer a review of the literaturerdquo Anti-cancer Research vol 29 no 9 pp 3511ndash3536 2009

[15] K W Colston and C M Hansen ldquoMechanisms implicated inthe growth regulatory effects of vitamin D in breast cancerrdquoEndocrine-Related Cancer vol 9 no 1 pp 45ndash59 2002

[16] M L Slattery C Sweeney M Murtaugh et al ldquoAssociationsbetween vitamin D vitamin D receptor gene and the androgenreceptor gene with colon and rectal cancerrdquo International Jour-nal of Cancer vol 118 no 12 pp 3140ndash3146 2006

[17] R D Mittal P K Manchanda S Bhat and H K Bid ldquoAsso-ciation of vitamin-D receptor (Fok-I) gene polymorphism withbladder cancer in an Indian populationrdquo BJU International vol99 no 4 pp 933ndash937 2007

[18] A A Bhanushali N Lajpal S S Kulkarni S S Chavan S SBagadi and B R Das ldquoFrequency of fokI and taqI polymor-phism of vitamin D receptor gene in Indian population and itsassociation with 25-hydroxyvitamin D levelsrdquo Indian Journal ofHuman Genetics vol 15 no 3 pp 108ndash113 2009

[19] E Garcion N Wion-Barbot C N Montero-Menei F Bergerand D Wion ldquoNew clues about vitamin D functions in thenervous systemrdquo Trends in Endocrinology and Metabolism vol13 no 3 pp 100ndash105 2002

[20] E Abe CMiyaura andH Sakagami ldquoDifferentiation ofmousemyeloid leukemia cells induced by 112057225-dihydroxyvitaminD

3rdquo

Proceedings of the National Academy of Sciences of the UnitedStates of America vol 78 no 8 pp 4990ndash4994 1981

[21] P Naveilhan F Berger K Haddad et al ldquoInduction of gliomacell death by 125(OH)

2vitamin D

3 towards an endocrine ther-

apy of brain tumorsrdquo Journal of Neuroscience Research vol 37no 2 pp 271ndash277 1994

[22] C Baudet G Chevalier P Naveilhan L Binderup P Brachetand D Wion ldquoCytotoxic effects of 112057225-dihydroxyvitamin D

3

and synthetic vitamin D3analogues on a glioma cell linerdquo Can-

cer Letters vol 100 no 1-2 pp 3ndash10 1996[23] C Baudet G Chevalier A Chassevent et al ldquo125-Dihydroxy-

vitamin D3induces programmed cell death in a rat glioma cell

linerdquo Journal of Neuroscience Research vol 46 no 5 pp 540ndash550 1996

[24] N Davoust D Wion G Chevalier M Garabedian P Brachetand D Couez ldquoVitamin D receptor stable transfection restoresthe susceptibility to 125-dihydroxyvitamin D

3cytotoxicity in a

rat glioma resistant clonerdquo Journal of Neuroscience Research vol52 no 2 pp 210ndash219 1998

[25] P Trouillas J Honnorat P Bret A Jouvet and J P GerardldquoRedifferentiation therapy in brain tumors long-lasting com-plete regression of glioblastomas and an anaplastic astrocytomaunder long term 1-alpha-hydroxycholecalciferolrdquo Journal ofNeuro-Oncology vol 51 no 1 pp 57ndash66 2001

[26] I Neveu P Naveilhan C Menaa D Wion P Brachet andM Garabedian ldquoSynthesis of 125-dihydroxyvitamin D

3by rat

brain macrophages in vitrordquo Journal of Neuroscience Researchvol 38 no 2 pp 214ndash220 1994

[27] S A Miller D D Dykes and H F Polesky ldquoA simple saltingout procedure for extracting DNA from human nucleated cellsrdquoNucleic Acids Research vol 16 no 3 p 1215 1988

[28] S Raimondi H Johansson P Maisonneuve and S GandinildquoReview and meta-analysis on vitamin D receptor polymor-phisms and cancer riskrdquo Carcinogenesis vol 30 no 7 pp 1170ndash1180 2009

[29] I Yaylim-Eraltan H A Ergen S Arikan et al ldquoInvestigation ofthe VDR gene polymorphisms association with susceptibility tocolorectal cancerrdquoCell Biochemistry and Function vol 25 no 6pp 731ndash737 2007

[30] D Eyles J Brown A Mackay-Sim J McGrath and F FeronldquoVitamin D

3and brain developmentrdquoNeuroscience vol 118 no

3 pp 641ndash653 2003


[31] E Giovannucci ldquoThe epidemiology of vitamin D and cancerincidence and mortality a review (United States)rdquo CancerCauses and Control vol 16 no 2 pp 83ndash95 2005

[32] A L M Sutton X Zhang T I Ellison and P N MacDonaldldquoThe 125(OH)

2D3-regulated transcription factor MN1 stimu-

lates vitamin D receptor-mediated transcription and inhibitsosteoblastic cell proliferationrdquoMolecular Endocrinology vol 19no 9 pp 2234ndash2244 2005

[33] H Arai K IMiyamoto Y Taketani et al ldquoA vitaminD receptorgene polymorphism in the translation initiation codon effecton protein activity and relation to bone mineral density inJapanese womenrdquo Journal of Bone andMineral Research vol 12no 6 pp 915ndash921 1997

[34] N Swapna U M Vamsi G Usha and T Padma ldquoRisk con-ferred by FokI polymorphism of vitamin D receptor (VDR)gene for essential hypertensionrdquo Indian Journal of HumanGenetics vol 17 no 3 pp 201ndash206 2011

[35] P W Jurutka L S Remus G K Whitfield et al ldquoThe poly-morphic N terminus in human vitamin D receptor isoformsinfluences transcriptional activity by modulating interactionwith transcription factor IIBrdquoMolecular Endocrinology vol 14no 3 pp 401ndash420 2000

[36] E M Colin A E A M Weel A G Uitterlinden et al ldquoConse-quences of vitamin D receptor gene polymorphisms for growthinhibition of cultured human peripheral blood mononuclearcells by 125-dihydroxyvitamin D

3rdquo Clinical Endocrinology vol

52 no 2 pp 211ndash216 2000[37] C Sweeney K Curtin M A Murtaugh B J Caan J D Potter

and M L Slattery ldquoHaplotype analysis of common vitaminD receptor variants and colon and rectal cancersrdquo CancerEpidemiology Biomarkers and Prevention vol 15 no 4 pp 744ndash749 2006

[38] J J McGrath F P Feron T H Burne A Mackay-Sim and DW Eyles ldquoVitaminD

3-implications for brain developmentrdquoThe

Journal of Steroid Biochemistry and Molecular Biology vol 89-90 no 1ndash5 pp 557ndash560 2004

[39] M Tseng R A Breslow B I Graubard and R G ZieglerldquoDairy calcium and vitamin D intakes and prostate cancer riskin the National Health and Nutrition Examination Epidemi-ologic Follow-up Study cohortrdquo American Journal of ClinicalNutrition vol 81 no 5 pp 1147ndash1154 2005

[40] D Feskanich J Ma C S Fuchs et al ldquoPlasma vitamin Dmetabolites and risk of colorectal cancer in womenrdquo CancerEpidemiology Biomarkers and Prevention vol 13 no 9 pp 1502ndash1508 2004

[41] K M Reilly ldquoBrain tumor susceptibility the role of genetic fac-tors and uses of mousemodels to unravel riskrdquo Brain Pathologyvol 19 no 1 pp 121ndash131 2009

[42] X-B Jiang C Ke Z-A Han et al ldquoIntraparenchymal papillarymeningioma of brainstem case report and literature reviewrdquoWorld Journal of Surgical Oncology vol 10 p 10 2012

[43] A Mogi J Hirato T Kosaka E Yamaki and H Kuwano ldquoPri-mary mediastinal atypical meningioma report of a case andliterature reviewrdquoWorld Journal of Surgical Oncology vol 21 no10 p 17 2012

[44] J S Barnholtz-Sloan and C Kruchko ldquoMeningiomas causesand risk factorsrdquo Neurosurgical Focus vol 23 no 4 p E2 2007

[45] B S Malmer M Feychting S Lonn et al ldquoGenetic variation inp53 and ATM haplotypes and risk of glioma and meningiomardquoJournal of Neuro-Oncology vol 82 no 3 pp 229ndash237 2007

[46] H J Ting S Yasmin-Karim S J Yan et al ldquoA positive feedbacksignaling loop between ATM and the vitamin D receptor is

critical for cancer chemoprevention by vitamin Drdquo CancerResearch vol 72 no 4 pp 958ndash968 2012

[47] S Sadetzki P Flint-Richter S Starinsky et al ldquoGenotyping ofpatients with sporadic and radiation-associated meningiomasrdquoCancer Epidemiology Biomarkers and Prevention vol 14 no 4pp 969ndash976 2005

[48] X Qi R Pramanik J Wang et al ldquoThe p38 and JNK pathwayscooperate to trans-activate vitamin D receptor via c-JunAP-1and sensitize human breast cancer cells to vitamin D

3-induced

growth inhibitionrdquo Journal of Biological Chemistry vol 277 no29 pp 25884ndash25892 2002

[49] S Kure K Nosho Y Baba et al ldquoVitaminD receptor expressionis associated with PIK3CA and KRAS mutations in colorectalcancerrdquo Cancer Epidemiology Biomarkers and Prevention vol18 no 10 pp 2765ndash2772 2009

[50] S Budhdeo R A Ibrahim M Hofer and M Gillies ldquoPrimaryintraosseous osteoblastic meningiomardquo JRSM Short Reportsvol 2 no 652 2011

[51] M A Meester-Smoor M Vermeij M J L van Helmond et alldquoTargeted disruption of the Mn1 oncogene results in severedefects in development of membranous bones of the cranialskeletonrdquo Molecular and Cellular Biology vol 25 no 10 pp4229ndash4236 2005

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Enzyme Research



Microbiology


12q12-14 [14] Various studies suggested the effect of VDRgene polymorphisms in the development of several typesof carcinoma such as breast prostate and colon carcinoma[9 15ndash17] One of the VDR gene polymorphisms is the Fok-I (rs2228570) polymorphism which is located in exon 2 andresults in an alternative transcription initiation site leadingto alter the activity of VDR protein The polymorphic Fok-Isite in exon 2 results in different translation initiation regiondue to thymine (T) to cytosine (C) substitution The one ofthe most well known of these polymorphisms such as Taq-I(rs731236) which are located in exons 9 and a TC nucleotidesubstitution (ATT to ATC) leading to a synonymous changeat codon 352 (isoleucine) [18] It has been reported that VDRslocalized in neuronal and glial cells affect the metabolismof brain cells and change the expression of VDR [18 19]It has been reported that the potential effect of vitamin Don the treatment of cancer was first identified in myeloidleukemic cells [20] Synthetic vitaminD analogues are amongthe preferred options in the treatment of central nervoussystem tumors In addition phase II clinical studies havecorrelated with positive effects of vitamin D therapy onglioblastoma cells [21ndash26]The increased vitamin D synthesisin glioma cells after treatment can regulate cell proliferation[19] However the consist of this effect is required in VDRgene expression [20 25] Several studies have determinedthe contribution of VDR polymorphisms in various types ofcancer The aim of this study was to evaluate the associationbetween polymorphisms in the VDR Fok-I and Taq-I and therisk of brain cancer in Turkish patients

2 Materials and Methods

Subject Selection We investigated the VDR Taq-I and VDRFok-I gene polymorphisms in 100 brain cancer patients(including 44 meningioma cases and 56 glioma cases) and122 age-matched healthy control subjects who were in thefollow-up Cerrahpasa Faculty of Medicine Department ofNeurosurgery in IstanbulUniversityThemean ages of gliomaand meningioma patients and control group were 4475 plusmn1563 5026 plusmn 1268 and 4722 plusmn 1063 years respectively

The specimens were taken after obtaining informed con-sent and the study was conducted prospectivelyTheMedicalEthics Committee of Istanbul Medical Faculty approval wasobtained for the study The protocol followed was consistentwith the World Medical Association Declaration of Helsinki(Ethical Principles for Medical Research Involving HumanSubjects)

21 Polymorphism Analysis Blood specimens were collectedin tubes containing EDTA and DNA samples were extractedfrom the whole blood by a salting out procedure (Miller etal 1988) [27] Genotyping was performed by the polymerasechain reaction (PCR) and restriction fragment length poly-morphism (RF LP) For Taq-I polymorphism the followingprimers were used to amplify the VDR gene 51015840-CAG AGCATG GAC AGG GAG CAA G-31015840 51015840-GCA ACT CCT CATGGGCTGAGGTCTCA-31015840 For detection of the Taq-I RFLP50ndash100 ng genomic DNA was amplified with 1x polymerase

chain reaction (PCR) buffer 3mM MgCl2 02mM of each

dNTP 02mM of each primer and Taq polymerase in a 50120583Lreaction volume The PCR conditions were as follows Initialdenaturation step of 94∘C for 4min followed by 5 cycles of94∘C for 45 sec 64∘C for 60 sec and 72∘C for 2min and afurther 25 cycles of 94∘C for 30 sec 64∘C for 30 sec and 72∘Cfor 45 sec PCR products were digested with TaqI restrictionenzyme at 65∘C electrophoresed on 2 agarose gels andstained with ethidium bromide Genotypes were determinedas TT (490 245 bp) Tt (490 290 245 and 205 bp) or tt (290245 and 205 bp) for Taq-I polymorphism [28] The primers(MBI Fermentas Lithuania) for Fok-I polymorphism were51015840-GAT GCC AGC TGG CCC TGG CAC TG-31015840 51015840-ATGGAA ACA CCT TGC TTC TTC TCC CTC-31015840 The DNAtemplate was amplified by PCR using 3mM MgCl

2 02mM

of each dNTP 025mM of each primer and Taq polymerase(MBI Fermentas Lithuania) in a 50 120583L final volumeThePCRconditions involved an initial denaturation of 4min at 94∘Cand followed by 30 cycles of 94∘C for 1min annealing at 60∘Cfor 1min and extension at 72∘C for 1min A final extensionstep at 72∘C for 4min was also studied PCR productswere digested with FokI restriction enzyme (MBI FermentasLithuania) at 37∘C for 3 h followed by electrophoresis in a 2agarose gelThe FF genotype (homozygote of common allele)shows only one band of 272 bp in agarose gelThe ff genotype(homozygote of infrequent allele) generates two fragmentsof 198 and 74 bp The heterozygote displays three fragments(272 198 and 74 bp) [29]

22 Statistical Analysis Statistical analyses were performedusing the SPSS software package (revision 16 SPSS IncChicago IL USA) Data are expressed as means + SD Dif-ferences in the distribution of genotypes or alleles betweencases and controls were tested using the chi-square statisticOdds ratios (ORs) and 95 confidence intervals (95 CI)were calculated to estimate the risk of glioma and menin-gioma Values of119875 lt 005were considered statistically signif-icant Haplotype frequencies1198631015840 and 1199032 were calculated usingHaploview 40 programme

3 Results

The analysis included 100 brain cancer (including 44 menin-gioma cases 56 glioma cases and 122 healthy controlsTable 1 shows the clinical characteristics of the study groupsGenotype and allele frequencies ofmeningioma cases gliomacases and controls are shown in Table 2 The risks ofmeningioma and glioma associated with VDR genotypes areshown in Table 3 There was a significant difference in thedistribution of VDR Fok-I genotypes inmeningioma patientsbut no significant difference was detected in glioma patients(119875 gt 005) (Table 2) VDR Fok-I ff genotype was significantlyincreased in meningioma patients (159) compared withcontrols (25) and carriers of Fok-I ff genotype had anincreased risk for meningioma cases (119875 = 0004) (1205942 1033OR 6470 95 CI 1749ndash23926) The VDR Taq-I genotypefrequencies for meningioma glioma and control cases werenot significantly different (119875 gt 005) In addition vitamin D








SNP Controls 119899 ()(119899 = 122)

Meningioma 119899 ()(119899 = 44) 120594

2119875 value Glioma 119899 ()

(119899 = 56) 1205942


FF 56 (459) 19 (432) 28 (500)Ff 63 (516) 18 (409) 10527 0005 23 (411) 4598 01ff 3 (25) 7 (159) 5 (89)



Taq-I allelesT 174 (713) 64 (727) 82 (732)t 70 (287) 24 (273) 0064 0800 30 (268) 0137 0710








4 Discussion






2119875

Block 1TT 0547 0534 0553 0114 07359TC 0170 0198 0157 0892 03448CT 0168 0171 0166 0017 08968CC 0115 0097 0124 0546 046




2119875

Block 1CT 0543 0512 0554 0476 04904CC 0183 0250 0159 3581 00584TT 0153 0125 0163 0726 0394TC 0121 0114 0124 0062 08032








Acknowledgment


References





















3rdquo



2vitamin D




3






3cytotoxicity in a




3by rat







3 pp 641ndash653 2003



























3-induced












Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








SNP Controls 119899 ()(119899 = 122)

Meningioma 119899 ()(119899 = 44) 120594

2119875 value Glioma 119899 ()

(119899 = 56) 1205942


FF 56 (459) 19 (432) 28 (500)Ff 63 (516) 18 (409) 10527 0005 23 (411) 4598 01ff 3 (25) 7 (159) 5 (89)



Taq-I allelesT 174 (713) 64 (727) 82 (732)t 70 (287) 24 (273) 0064 0800 30 (268) 0137 0710








4 Discussion






2119875

Block 1TT 0547 0534 0553 0114 07359TC 0170 0198 0157 0892 03448CT 0168 0171 0166 0017 08968CC 0115 0097 0124 0546 046




2119875

Block 1CT 0543 0512 0554 0476 04904CC 0183 0250 0159 3581 00584TT 0153 0125 0163 0726 0394TC 0121 0114 0124 0062 08032








Acknowledgment


References





















3rdquo



2vitamin D




3






3cytotoxicity in a




3by rat







3 pp 641ndash653 2003



























3-induced












Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



4 Discussion






2119875

Block 1TT 0547 0534 0553 0114 07359TC 0170 0198 0157 0892 03448CT 0168 0171 0166 0017 08968CC 0115 0097 0124 0546 046




2119875

Block 1CT 0543 0512 0554 0476 04904CC 0183 0250 0159 3581 00584TT 0153 0125 0163 0726 0394TC 0121 0114 0124 0062 08032








Acknowledgment


References





















3rdquo



2vitamin D




3






3cytotoxicity in a




3by rat







3 pp 641ndash653 2003



























3-induced












Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Acknowledgment


References





















3rdquo



2vitamin D




3






3cytotoxicity in a




3by rat







3 pp 641ndash653 2003



























3-induced












Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



























3-induced












Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article the vitamin d receptor (vdr) gene...

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