Rama D. Mittal, Sanjay Gandhi PGIMS, Urology and Renal Transplantation, Lucknow, Uttar Pradesh, India. e-mail: email@example.com
To explore the association of vitamin-D receptor (VDR) genotypes and haplotypes (variants at the Fok-I, and Taq-I sites) with the risk of bladder cancer, as vitamin D is antiproliferative and reported to induce apoptosis in human bladder tumour cells in vitro.
PATIENTS, SUBJECTS AND METHODS
A case-control study using polymerase chain reaction-restriction fragment length polymorphism was conducted in 130 patients with bladder cancer and 346 normal healthy individuals in a north Indian population. Patients were also categorized according to grade and stage of tumour.
There was a significant difference in genotype and allelic distribution of VDR (Fok-I) polymorphism in the patients (P = 0.033 and = 0.017, respectively). The FF genotype was associated with twice the risk for bladder cancer (odds ratio 2.042, 95% confidence interval, CI, 0.803–5.193). There was no significant difference in genotypic distribution or allelic frequencies of the VDR (Taq-I) polymorphism (P = 0.477 and 0.230) when compared with the controls. The stage and grade of the bladder tumours had no association with VDR (Fok-I and Taq-I) genotypes. There was a significant difference in the frequency distribution of the haplotypes FT and fT (P < 0.001); these haplotypes had a protective effect in the control group (odds ratio 0.167, 95% CI 0.096–0.291, and 0.079, 0.038–0.164).
These data suggest that VDR (Fok-I) polymorphism is associated with the risk of bladder cancer. Further, the results for the haplotype FT and fT indicate that patients with this haplotype have a lower risk of developing bladder cancer than those with other haplotypes.
Bladder cancer is a complex and multifactorial disease and the second most common genitourinary malignancy . Like many other types of solid tumours, TCC of the bladder develops through a series of genetic changes that lead to tumour progression . The molecular and genetic changes in TCC of the bladder involves chromosomal alterations, tumour proliferation caused by loss of cell-cycle regulation, and metastasis, bringing into play processes such as angiogenesis and loss of cellular adhesion . 1,25-dihydroxyvitamin D3 (vitamin D) is important in the regulation of cell division and differentiation . The vitamin D receptor (VDR) is a member of the steroid hormone family of nuclear receptors, which are responsible for the transcriptional regulation of several hormone-responsive genes. Vitamin D has antiproliferative and differentiating effects on various human tissues and tumours ; the antiproliferative effects are evident from several studies showing inhibition of growth in myeloid leukaemia, melanoma, osteosarcoma, breast, prostate, colon and head and neck carcinoma cell models [6,7]. The VDR gene is ≈ 75 kb long and is made up of 11 exons together with intervening introns. The gene encoding the VDR is on chromosome 12q, and has several known allelic variants including a Fok-I restriction fragment length polymorphism (RFLP) in intron 2, Bsm-I and Apa-I polymorphisms in the intron 8, Taq-I variant in intron 9, and a mononucleotide [(A)n] repeat polymorphism in the 3′ untranslated region [8,9]. The idea that the VDR gene might influence the occurrence of bladder cancer is mainly based on the notion that vitamin D is implicated in the regulation of cell proliferation and cell differentiation, and superficial TCC of the bladder expresses VDR . Indeed, the most frequent haplotypes at the 3′ end of the VDR gene have been shown to produce slightly different gene activities in a reporter gene construct . The present study was conducted to explore the association of the Fok-I and Taq-I polymorphisms of the VDR gene with bladder cancer risk in an Indian population. Further, the relationship was evaluated by using haplotypes and linkage disequilibrium (LD) within Fok-I and Taq-I sites.
PATIENTS, SUBJECTS AND METHODS
The VDR gene polymorphism was analysed in 130 patients with bladder cancer (all TCC, of which 38% were grade I tumours, 28% grade II and 34% grade III; 112 men and 18 women; mean age 62.5 years, sd 10.2); and 346 unrelated normal individuals (305 men and 41 women) of similar ethnicity and from the same geographical area (mean age 60 years, sd 11.5) having no family history of cancer were selected as controls. Criteria for selecting the patients were based on a questionnaire covering medical, pathological, and histopathological records from the outpatient department of the state’s tertiary-care hospital between 2002 and 2005. When the patients were categorized for staging, 62% were T1, 27% were T2 and 11% were T3. All patients and controls were interviewed using a standardized questionnaire pertaining to smoking, dietary habits, alcohol consumption and demographic characteristics. The Institutional Review Board approved the protocol, and informed consent was obtained from the patients and the controls participating in the study. The hospital ethical committee approved the study.
Genomic DNA was extracted from peripheral blood by ‘salting out’. Reaction mixtures of 25 µL were used in PCR for the VDR gene (Fok-I and Taq-I) polymorphism and DNA samples were amplified in a Peltier thermal cycler (MJ Research, now BioRad, Hercules, CA, USA). Gels were visualized under ultraviolet light and photographed with an appropriate camera and software. The primers of the VDR gene used were as reported earlier [13,14].
For the Fok-I polymorphism, the PCR cycle conditions were: initial denaturation at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, 61 °C for 30 s and 72 °C for 1 min, and one final cycle of extension at 72 °C for 7 min. The PCR product was digested with 1.0 unit of Fok-I restriction enzyme and the reaction buffer, incubated at 37 °C for 4 h and verified by 9% Page stained with ethidium bromide. The FF genotype (homozygote of common allele) lacked a Fok-I site and showed only one band of 265 bp. The ff genotype (homozygote of infrequent allele) generated two fragments of 196 and 69 bp. The heterozygote had three fragments of 265, 196 and 69 bp, designated as Ff.
For the Taq-I polymorphism, the PCR cycle conditions were: denaturation at 94 °C for 3 min, followed by 35 cycles at 94 °C for 60 s, 63 °C for 60 s and 72 °C for 2 min, and one final cycle of extension at 72 °C for 5 min. The PCR products (740 bp) were digested with Taq-I (Fermentas, Lithuania) restriction enzyme at 65 °C for 3 h and verified on 9% PAGE. Taq-I digestion gave one obligatory restriction site, the homozygous TT (absence of the specific Taq-I restriction site) and yielded bands of 245 bp and 495 bp. The homozygous tt had 205, 245, 290 bp and the heterozygous Tt 495, 205, 245, 290 bp fragments.
The chi-square test was used to assess the association between VDR polymorphisms and risk of bladder cancer. Odds ratios (ORs) and 95% CI were calculated to determine the risk of bladder cancer associated with a given VDR genotype by binary logistic regression model. Allele frequencies were assessed for deviation from the expected Hardy–Weinberg equilibrium using the chi-square test, with P < 0.05 considered to indicate statistical significance. Allele frequency was calculated as the number of occurrences of the test allele in the population divided by the total number of alleles. The carriage rate was calculated as the number of individuals carrying at least one copy of the test allele divided by the total number of individuals. Haplotype frequencies and LD were estimated using the expectation-maximization algorithm.
The genotype frequency of VDR (Fok-I and Taq-I) polymorphisms are presented in Table 1 and Figs 1 and 2; there was a significant difference in genotype frequencies of Fok-I between patients and controls (P = 0.033, chi-square). The genotype FF (low producer of vitamin D) had twice the risk of bladder cancer (OR 2.042, 95% CI = 0.803–5.193). The frequency distribution of Taq-I polymorphism of the VDR gene was similar in patients and controls and hence no association was detected (Table 1), but the TT genotype, which is known to be a low producer, had a slightly higher risk of bladder cancer (OR 1.565, 95% CI 0.739–3.313). The association of allele frequencies and carriage rates of VDR (Fok-I and Taq-I) polymorphism is also shown in Table 1; the frequency distribution of the F allele in Fok-I was significantly higher in patients (P = 0.017). The carriage rate of this allele showed a slightly increasing trend but the association was not significant (P = 0.109). The frequency of the T allele in Taq-I polymorphism, although tending to be higher in patients, was not statistically significant (P = 0.230). The carriage rate of this allele also tended to be higher but again the association was not significant (P = 0.468). We also investigated whether any of the VDR polymorphisms were associated with particular clinical/pathological characteristics of the patients (Table 2), but there was no significant association with bladder tumour grading and staging. We also evaluated the association of VDR Fok-I and Taq-I genotypes in smokers with the risk of bladder cancer, but again there was no significant association (data not shown).
Table 1. The frequency distribution of VDR (Fok-I and Taq-I) gene polymorphism, and the allelic frequencies and carriage rates, in healthy controls and patients with bladder cancer
Haplotype frequencies between the sites of VDR gene (Fok-I and Taq-I) in patients and controls are shown in Table 3. All the four possible haplotypes whose frequencies were >1% were present in controls and patients, and there was a significant difference in haplotype frequency distribution, for F-T and f-T (P < 0.001, respectively). These haplotypes had a protective effect in the control group (Table 3). The LD between Fok-I and Taq-I was not significant in controls or patients (D′ = 0.040; P = 0.120, and D′ = 0.002; P = 0.876).
Table 3. The distribution of haplotypes of the VDR gene (Fok-I and Taq-I) in patients and healthy controls
OR (95% CI)
There are several known genetic polymorphisms in the VDR gene that cluster at the 3′ end and are in strong LD. However, it remains ambiguous whether they represent functional differences in the gene and are therefore marked as risk alleles [15,16]. We selected two of these polymorphisms, the Taq-I and Fok-I RFLP, for our study. The Fok-I polymorphism is the least studied and is not in LD with those at the 3′ end of the gene . This polymorphism results in a new start codon 9 bp upstream of the usual one, thus creating a longer protein, which is considered of potential functional relevance .
The Taq-I polymorphism has been examined most extensively for prostate cancer [9,18] and to the best of our knowledge there is no study in bladder cancer until the present case-control study, which showed a significant association between Fok-I and a non-significant association for Taq-I with bladder cancer (P = 0.033 and 0.477 respectively). The FF genotype of Fok-I, which is a low producer, was associated with a greater risk of bladder cancer. Correa-Cerro et al. reported a significant association of the TT genotype of Taq-I (a low producer of vitamin D) with prostate cancer but no association with the Fok-I genotype. The human bladder is a potential target for VDR ligands and has VDR expression similar to that in the prostate, which is a well-characterized VDR ligand-sensitive tissue. We also assessed the clinical stage and/or pathological grade for Fok-I and Taq-I genotypes for the risk of bladder cancer; the polymorphisms did not influence the biological properties of bladder cancer, as there was no association with clinicopathological variables (staging and grading of bladder tumours). In a previous study, although the FF genotype of Fok-I was significantly associated with prostate cancer there was no association with Gleason grade . Samanic et al. reported that tobacco smoking was associated with bladder cancer, but there was no association between VDR Fok-I and Taq-I genotypes and smoking in the present study. Our previous findings for the GSTM1, T1 and NAT2 genotypes also showed a relationship of smoking with the risk of bladder cancer [22,23].
Vitamin D is involved in the regulation of cell proliferation and differentiation in vitro and in vivo. Genetically, it is important to understand which alleles are linked to each other. In the present study the haplotype F-T and f-T had a protective effect for bladder cancer. The non-significant value for LD showed that there was no association of Fok-I and Taq-1 among the variants. Our results are in accord with those of Zeminik et al., who reported no LD between Fok-I and Taq-I in a Dalmatian population. However, a strong LD at the 3′ end of the gene was reported for the Bsm-I, Apa-I, EcoRV and Taq-I RFLPs . It follows that the LD and haplotype structure of a certain candidate gene are important for association analysis, to understand how polymorphic variation in such a gene can contribute to the risk of disease and the population variance of phenotypes of interest. Knowing which haplotype carries this risk allele, it is possible to determine by cell biological and molecular biological functional analysis which of the variants on that haplotype allele truly cause the effect. The present study shows for the first time an association of Fok-I restriction polymorphism in bladder cancer. The VDR gene was also reported to be involved in different cancers [25–28].
As bladder cancer is a polygenic disease, the search for candidate genes must include panels of various polymorphisms to elucidate genes that might be useful in screening and risk evaluation of bladder cancer. Vitamin D and its analogues are potential antiproliferative agents and their effects are mediated by VDRs ; it reduces the high mitotic rate of cancer cells to that of normal cells . Thus, the polymorphisms in the VDR gene could alter receptor function and affect susceptibility for cancer. If these genes could be identified and associated mutations characterized, then genetic screening for bladder cancer susceptibility could become a reality. The association between bladder cancer and various genetic markers has helped to increase knowledge of the genetics and pathogenesis of bladder cancer; this problem provides a target for further studies.
In conclusion, the present results indicate that the Fok-I polymorphism in the VDR gene are associated with bladder cancer. Additional studies on larger cohorts are warranted to verify the correlation among Indians and other racial-ethnic groups. Further investigations into the mechanisms of interactions of the VDR with other environmental and/or genetic influences affecting the risk of bladder cancer could lead to a new understanding of the role of vitamin D in the control of cellular and developmental pathways. Early identification of individuals at increased risk of bladder cancer would allow targeted, aggressive screening and treatment in the population.
Parmeet K. Manchanda is thankful to the Council of Scientific and Industrial Research, New Delhi, India for providing junior research fellowship (J.R.F.).