Presentation on theme: "Keele University Medical School Staffordshire, England"— Presentation transcript:
1 Keele University Medical School Staffordshire, England What are the frequency, distribution and functional effects of vitamin D receptor polymorphisms as related to cancer risk?Dr Nicholas J RukinKeele University Medical SchoolStaffordshire, EnglandThank you. Firstly, I would like to thank Dr Cindy Davis & the organisers for the invitation to speak today.This morning sessions have all been very interesting.Today I am going to briefly discuss the role of the VDR in relation to SNP, there functional effect and how are they related to cancer risk using prostate cancer as a paradime.The work which I talk about today is a results of a good clinical scientific relationship.Prostate cancer StatisticsProf Richard Strange Prof Peter JonesMr Chris Luscombe Prof Maurice ZeegersDr Tony Fryer Dr Melisa BlagojevicDr Paul Hoban
2 UVR and Age at Onset of Prostate Cancer 1.00All other quartilesof exposure67.7 years72.1 years(p=0.006, hazard ratio 1.52)Proportion tumour free0.50Lowest 25%cumulative exposure0.005060708090Age at diagnosis (years)Luscombe et al. Lancet 2001
3 7 dehydroxycholesterol Vitamin D3 UVRSkin7 dehydroxycholesterol Vitamin D3KidneyLiver1α hydroxylase1,25(OH)2D325(OH)D3Prostate25(OH)D3OthertissuesThe formation and metabolism of Vit D is widely understood. Pre-vit D hydroxylated to 25, further hydroxylated to 1,25 via 1aplha hydroxylase.This then moves intracellular and binds within the target cell nucleus to the VDR/retinoid X receptor.This complex then initiates transcription vie binding to VDRE.This complex pathway mean that the effect of UVR can be mediated at several different stages, inc hydroxylation, binding to the VDR and RXR and the binding to the VDRE.Rest of this presentation I am going to concentrate on the VDR recpetor.Target cell nucleusRetinoid Xreceptor1,25(OH)2D3VDRVDRETranscription
4 Vitamin D Receptor Gene – Chr 12q13 9 exons, alternatively spliced promoter regionBinding domains: DNA binding domain – binds VDRELigand binding domain – 1,25(OH)2 Vit DVDR is a large gene, > base pairs. It is situated on the Long/short arm of chromosome 12 and is a well studied gene.The gene itself comprises of an alternatively spliced promoter region which accounts for approximately half the gene.The other half of the gene contains 9 exons and the 3’UTR region of the gene.We know that the VDR gene transcribes the VDR protein and our increased understanding of the gene has showed several areas to be of interest.Exons 2-4 encode the proportion of the peptide that is associated with binding to the VD response elements in the target gene.Exons 6-9 encoded the proportion of the peptide that the 1,25 (OH)2 Vit D binding domain of the VDR protein.Promoter regionDNABindingdomainLigandBindingdomain
5 Single Nucleotide Polymorphism in the VDR gene To date there are over 490 VDR SNPs known and if can be difficult to decided which to study.A lot of these SNPs have very low allele frequencies and are therefore not suited for genetic epidemiological studies.You can see the commonly studied SNPs in the VDR are shown here, this includes ………Cdx-2 GATA Fok Taq Apa1 Poly AHuman VDR >470 reported SNPsMany have low allele frequency
6 VDR Linkage Disequilibrium Blocks 5’3’1f e 1a 1d 1b c ’UTRC3C2C1BALD BlocksOur understanding of the gene has been much advanced by the work from Nejentsev and Uitterlinden who have helped define LD and haplotype blocks in the gene.I particularly enjoyed reading these papers as they have good methodology and there work had dramatically helped and improve our understanding of the VDR gene.Both these studies have helped improve our understanding of the VDR gene.Indeed, SNPs which are viable include those which are tagged as they enable us to cover a potentially larger area of the gene.We can now see that commonly studied SNPs fit within define LD blocks, except the Fok1 SNP which is interesting as it demonstrates no LD with any other SNP in the gene.Cdx-2 GATA Fok Taq Apa1 Poly ANejentsev et al Hum Mol Gen 2004Uitterlinden et al. Gene 2004
7 Here is a list of common SNPs which we and others have previously studied. There is a slight disproportionate spread of these SNPs, with more in the 5’ promoter region of the gene. Reflects our experience. These are all potentially interesting and may have functional properties.Allele frequencies reported here are taken for The International HapMap Project data.These figures compare the allele frequencies between 2 groups: CEU Utah residents with Northern & Western European ancestry and Yoruba in Ibada, Nigeria (Sub Saharian Africa).I make no excuses for showing this data since I want to highlight one important point. Marked ethnic difference is allele frequencies: e.g. Gata mutant allele & Fok mutant alleleC/A63935G/A64922
8 Functional Effects of VDR Polymorphisms in the VDR Null Mice Calcium HomeostasisAssociated with hypocalcaemia, hyperparathyroidism, rickets, osteomalacia and alopeciaCarcinogenesisDMBA-induced carcinogenesis: Increased hormone independent tumours, epidermal and lymphoid tumourFunctional effects are often difficult to fully understand and interpret what it means on a patient basis.With scientific experiments we can only study one a time, but in practice combinations of SNPs may alter function.We have already heard this morning of the varied effect which the VDR maybe responsible for, therefore it is likely that some of the SNPs in the gene may have functional significance.The best way to discuss to functionality is to study the null mice model, and the VDr null mice is a relatively poor specimen.As expected he is unable to maintain calcium homeostasis and has resulting rickets, osteomalacia and other sequelae of VDR deficiency.Give the cancer content of this meeting, what are his risk in relation to carcinogenesis. A study by Zinser et al has shown that if VDR null mice are given DMBA (def) then they are at increased risk from tumoursWell, this is all and good in VDR deficient mice, but humans are different and we need to try and establish the effects within our species.Zinser et al. J Steroid Biochem Mol Biol. 2005
9 Functional VDR Polymorphism Cdx-2GATAFok 1Poly (A) repeats≥17 A’s scored as ‘long’ (L)≤15 A’s considered as ‘short’(S)Cdx-2 A allele associated with increased binding Cdx-2 protein in vitroIncreased transcription activity of VDRIf we go back to the VDR gene structure, I am now going to review the evidence for functional SNPs within the gene.The promoter region of the gene is of interest as there appears to be transcription factor binding sites in it.The Cdx-2 SNP, first reported in 2001, is found at a Cdx2 protein binding site. Cdx2 is a transcription factor that regulates VDR gene expression in the small intestines and other tissues. The mutant A allele has been associated with increased binding of the Cdx2 transcription factors in vitro and has been associated with increased transcriptional activity of the VDR.The GATA SNP also lies in the C2 LD block of the promoter region. This SNP lies within the core sequence of a likely GATA-3 binding site. This SNP has been associated with susceptibility and outcome in MM patients. Electrophoretic mobility shift assay has demonstrated changes in protein-DNA complex formation & two fold increases in the VDR promoter activity.The Fok1 SNP, situated in exon 2, gives rise to an alteration in the start codon position resulting in a 3 amino acid longer protein been produced by the F allele. This factor also displays moderaely higher transcriptional activity and greater in vitro binding with TF 2B.The poly A repeat is situated in LD block A. It is classified as been either long or short depending on the number of A repeats found. The long version has been associated with mRNA with more stability andGATA EMSA demonstrate changes in protein-DNA complex formation & increases VDR promoter activityFok residue VDR protein is producedF allele displays moderately higher transcriptional activity, as well as greater interaction in vitro with transcription factor IIBPoly (A) repeat L allele may produce receptor mRNA that is more stable and/or is translated more efficiently into VDR protein than the S allele
10 VDR SNPs and Prostate Cancer Risk 430 prostate cancer / 320 BPH controlsValidated UVR questionnaireDNA samplesGenotypeOdds ratio (95% CI)P value1aGGGCCCReference1.13 ( )0.65 ( )0.4500.060GATAAAAG1.01 ( )0.62 ( )0.9400.030C1-1CTTT1.03 ( )0.97 ( )0.8300.890C1-20.91 ( )0.71 ( )0.6000.210Fok1TC1.13 ( )1.11 ( )0.4350.653All these functional results are interesting, but what effects do these have in populations. The best way to study this would be to compare the effects of these allele in different disease populations. As the aim of this meeting is to discuss the associations with cancer risk, then I will share with you our experience with prostate cancer and the VDR.CaP is a common cancer is men in the Western world and has been associated in epidemiological, case control and cohort studies to be associated with UVR exposure, therefore it would be logical to study VDR SNPs in relation to CaP susceptibility.We performed a C/C study in 430 CaP and 320 BPH patients. Each patient had blood taken and a DNA sample extracted for tissue banking. Each patient also completed a validated UVR questionnaire which assessed short and long term UV exposure patterns.We chose to study the 5’ end of the gene: 1. Previous studies showed this of be an area of interest2. Possible functional role of 5’ SNPsThe studied SNP and there position in the relative LD blocks are shown here.Despite an adequate sample size our initial results were slightly disappointing. We showed an association, all be it a weak one, with the GATA GG genotype in relation to CaP susceptibility. We expected this as this SNP has been associated with both susceptibility and outcome in MM.But apart from this there were no other significant results. 1a site showed some promise, but with low numbers in the mutant population we couldn’t demonstrate significance.We know prostate cancer demonstrates much hetrogeneity, therefore are certain subgroups more likely to be associated with susceptibility and if so how can we define them.1a GATA C1-1 C Fok1Data not shown: Intron 3 and Taq P>0.05Rukin et al. Cancer Letters 2007; 247:
11 VDR effect is likely to work through a GENE-ENVIRONMENT interaction Intensity of exposureDuration of exposureVDR GeneticsGenetic factors VDR polymorphismEnvironmental No foreign holidays<median exposureLow sunbathing scoreDietLow RiskHigh Risk
12 VDR-Environment Interactions Low SunbathingLow sunbathingNo foreign holidays< Median exposureProstate CancerBPHOR (95% CI)P value1a GGGCCC143 (33)238 (55.4)50 (11.6)105 (32.8)159 (49.7) 56 (17.5)Reference1.13 ( )0.65 ( )0.4500.0600.88 ( )0.40 ( )0.5910.0050.62 ( )0.26 ( )0.220GATA AAAGGG140 (32.6)226 (52.6)64 (14.9)98 (30.6)155 (48.4)67 (20.9)1.01 ( )0.63 ( )0.9400.0300.97 ( )0.42 ( )0.9000.46 ( )0.24 ( )0.077C1-1 CCCTTT191 (44.5)192 (44.6)47 (11.0)142 (44.4)36 (11.3)1.03 ( )0.97 ( )0.8300.8900.98 ( )0.56 ( )0.9220.0640.58 ( )0.22 ( )0.1800.002C1-2 CC209 (48.6)181 (42.1)40 (9.3)147 (45.9)136 (42.4)37 (11.7)0.91 ( )0.71 ( )0.6000.2100.74 ( )0.32 ( )0.2060.58 ( )0.18 ( )0.241Fok1 TT166 (38.7)203 (47.1)61 (14.2)135 (42.3)141 (43.9)44 (13.8)1.11 ( )0.4350.6531.17 ( )1.08 ( )0.4260.7920.49 ( )0.56 ( )0.1430.336I am going to spend some time talking through this table.Lets look at some of this data. Here is the same data as before, in tabular form. I have highlighted the P values so we can easily look for significance.As before, only the GATA SNP looks interesting, but its significance is marginal.As we mentioned we are trying to look at subgroups in which gene environmental interactions maybe occurring.Firstly lets see what happens to those with participate in low amounts of sunbathing. As we define these smaller subgroups of patients out group numbers decrease, BUT LOOK at the significance levels.We can see that ALL the 5’ promoter region markers have become significant. Not just significant, but comfortably significant.Fascinating results, but what does this mean!Can we push the data further! Can we define a group with even low levels of exposure. This may pushing the data but lets try it.Created a new group who have very low exposure: 1. low sunbathing2. <median cumulative exposure per year3. no regular foreign holidayThese factors have been chosen specifically since demonstrate very little evidence of correlation and they allow different type of exposure to be assed.Again, we can see that our P-values are comfortably significant and our OR’s have again improved, despite reduced numbers.Note, what ever we do with the Fok1 SNP we do not show any trends in this group. (previously Fok1 has been shown to have an effect in those with high exposure, therefore we would not really expect any result in those with low exposure).
13 VDR Haplotypes and Outcome Block C Haplotypes Low sunbathingNo foreign holidays< Median exposureSNPHaplotypeCopiesOdds ratio (95% CI)P-Value1-a + GATAC-G12Reference0.44 ( )0.19 ( )0.0360.001C1-1 + C1-2G-G0.35 ( )0.23 ( )0.0210.006GATA + C1-1G-T0.37 ( )0.20 ( )0.0110.005Do we get similar results when studying haplotypes?Here I just show 2 haplotypes in the very low exposure group. The ORs are similar suggesting that haplotype combinations show similar results.Similar associations are seen with other haplotypes, EXCEPT those containing Fok SNPSimilar effects for other haplotypes, except those with Fok
14 Conclusion VDR structure, SNP choice and haplotypes Gene-environmental interaction importantReplication of resultsFuture?VDR structure, SNP choice and haplotypes: VDR gene structure well established and is polymorphicFew well chosen tagged SNPs can cover large areas of the geneGene-environmental interaction important: Importance highlighted here of G-E interaction, we may miss important markersf we do not study theseReplication of results: Reassuring that these results have been replicated as confirmatory evidenceFuture?: Very interestingIn relation to CaP ethnicity studies may help our understanding of the mechanistics behind the VDR in CaP. Would be fascinating to look at VDR SNPs in African American as they are at greater risk, this effect maybe modified at different latitudes.Thank you very much for your time and I would like to invite any questions at this stage?
16 Functional Effects as Related to Cancer? Vitamin D receptor polymorphism and risk of prostate cancer: A meta-analysis. Ntais et al. Cancer Epidemiol Biomarkers Prev. 2003, 12,Taq1, Bsm1, poly (A) micro satellite repeats and Fok1‘The meta-analysis shows that these four polymorphisms are unlikely to be a major determinants of susceptibility to prostate cancer on a wide population basis.’A systematic review of vitamin D receptor gene polymorphisms and prostate cancer riskBerndt et al. J Urol. 2006, 175(5),‘The results of this meta-analysis suggest that the vitamin D receptor TaqI,poly (A), Bsm1, Apa1 and Fok1 polymorphisms are not related to prostatecancer risk.’Clinical example of CaP. The commonest diagnosed CaP excluding skin cancers.Associated with UVR exposure, both environmental and case/control studies.BUT, the results of VDR SNP have varied. Indeed, two recent meta-analysis have suggested that the commonly studied are not associated with prostate cancer risk.