Indian Journal of Human Genetics Medknow Publications on behalf of Indian Society of Human Genetics ISSN: 0971-6866 EISSN: 1998-362x Vol. 9, Num. 2, 2003, pp. 51-54

Indian Journal of Human Genetics, Vol. 9, No. 2, July-Dec, 2003, pp. 51-54

Study of vitamin-D receptor (VDR) gene start codon polymorphism (Fok I) in healthy individuals from North India

H. K. Bid, R. D. Mittal

Department of Urology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow - 226014, India.
Address for correspondence: R. D. Mittal, Additional Professor, Department of Urology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow - 226014, India. E-mail: rmittal@sgpgi.ac.in

Code Number: hg03011

The vitamin D receptor (VDR) gene Fok I polymorphism represents a strong positional candidate susceptibility gene for different diseases like Prostate cancer, Urolithiasis, Inflammatory bowl disease and Osteoporosis. This genetic variation has also been of great interest due to its possible association with polygenic diseases. Allelic frequencies of the F/f start codon polymorphism of VDR gene vary among populations but there is no data regarding its distribution from India. The present study was carried out to determine the normal distribution of VDR gene Fok I polymorphism by using a PCR-based restriction analysis in unrelated normal healthy individuals from North India. We obtained an allelic frequency of 71.5% and 28.5% for (F) and (f) allele and the percentage of genotypes FF, Ff and ff as 46%, 51% and 3% respectively. Our results suggest that the frequency and distribution of this polymorphism in Indian population is substantially different from other populations and ethenic groups.

Key Words: VDR gene, SNP, Genetic polymorphism.

Introduction

DNA sequences of the human genome reveal that many genes are polymorphic. In coding or noncoding regions of a specific gene, there may be either a single base pair substitution of one nucleotide (SNPs) for another or a variable number of repeats of a short (VNTR), repetitive DNA sequence. These variations may influence the rate of gene transcription, the stability of the messenger RNA, or the quantity and activity of the resulting protein. Thus, the susceptibility or severity of a number of disorders will be influenced by possession of specific alleles of polymorphic genes. A single nucleotide polymorphism (SNP) is a site on the DNA in which a single base pair varies from person to person. If a SNP is found within a small, unique segment of DNA, it serves both as a physical landmark and as a genetic marker whose transmission can be followed from parent to child. SNPs have gained popularity in recent years and are touted as the genetic markers of choice for the study of complex genetic traits.^1,2

Vit-D is a member of the steroid receptor family and mediates the effects of the active metabolite 1, 25(OH)₂ Vit D₃ by regulating transcription of a number of different cellular genes.³ The action of vitamin D is mediated through binding to its nuclear receptor (VDR). In response to hormone binding, the VDR regulates the transcriptional activity of 1, 25(OH)₂D₃-responsive genes by complexing with a vitamin D response element located in the promoter region of target genes. The vitamin-D receptor (VDR) is a candidate locus for different disease such as Prostate cancer, Urolithiasis, Inflammatory bowl disease and Osteoporosis etc. due to allelic variation, which affects the activity of the receptor and subsequent downstream vitamin-D mediated effects, such as calcium absorption, excretion and modulation of cellular proliferation and differentiation. Several polymorphisms have been identified in the VDR gene (Figure 1), and their functional significance and potential effects on disease susceptibility have been investigated.⁴ These studies have also pointed out substantial allelic variations of VDR gene in different populations. One of the known DNA sequence variants is a thymine/cytosine (T/C) polymorphism in the first of two potential start (ATG) codons separated by 3 codons. This polymorphism results in two alleles that can be distinguished by RFLP using the endonuclease Fok I.⁵ Other polymorphisms found in the 3' region of the VDR gene, in the intron between exons 8 and 9, using endonucleases Bsm I and ApaI and at the 3' noncoding sequences in exon 9 is identified by the enzyme TaqI. The SNP sites most frequently reported have been the start codon (Fok I) and intron 8 (Bsm I) polymorphism in western and Asian populations. However, there are no reports of allelic variations in VDR (Fok I) gene from Indian subcontinent where populations/ethinicity are quite different. Therefore, the present study is an attempt to investigate normal distribution of VDR gene Fok I polymorphism by using a PCR-based restriction analysis in unrelated normal healthy individuals from North India.

Material and Methods

Subjects

Blood samples were collected from 200 unrelated normal individuals (Male-159 & Female-41) [Age range- 15-68] from the North Indian population with informed consent. The genomic DNA was isolated from peripheral blood by using standard phenol chloroform method.⁶

Polymerase Chain Reaction

Reaction mixtures of 50μl were used in PCR for the VDR gene start codon polymorphism. The reaction mixture consisted of 100-200ng of genomic DNA, 2-6 pmol of forward and reverse primers, 1x Taq polymerase buffer (1.5mM Mgcl₂)(Genetix Biotech Asia Pvt. Ltd.), dNTPs (200μM each) and 1.5 units of Taq DNA polymerase (Bangalore genei India). The primers of the VDR gene used were similar to as reported by Harris et al.⁷

Forward

(5'-AGCTGGCCCTGGCACTGACTCTGCTCT-3')

Reverse

(5'-ATGGAAACACCTTGCTTCTTCTCCCTC-3').

DNA samples were amplified in MJ Research PTC-100^TM (Peltier Thermal Cycler) with cycling parameters as follows: Denaturation at 94⁰C for 5 min, 35 cycles at 94⁰C for 30 s, 61⁰C for 30 s and 72⁰C for 1 min and one final cycle of extension at 72⁰C for 7 min. The T/C polymorphism in the first of two-start codon (ATG) at the translation initiation site of the VDR gene was detected by RFLP using the restriction endonuclease Fok-I. The PCR product of the 265bp band was digested with 1.0 unit of Fok I restriction enzyme (New England Biolabs) and the reaction buffer and incubated at 37⁰C for 4 hours; 10μl of the digested reaction mixture was then loaded into 9% PAGE (Polyacrylamide gel electrophoresis) containing ethidium bromide and visualized under UV and photographed with a Alpha Imager 1220 v5.5 Camera software. The sizes were determined using 100-bp ladder (Roche, Germany). Digestion of the amplified 265 bp PCR product gave two fragments, of 169 bp and 96 bp respectively if the product was excisable.⁸ Depending on the digestion pattern, individuals were scored as (ff) when homozygous for the presence of the Fok-I site, (FF) when homozygous for the absence of the Fok-I site, or (Ff) in case of heterozygosity.

Statistical Analysis

Genotype frequencies of the VDR gene polymorphism in normal healthy controls in North Indian population were determined according to Hardy-Weinberg equilibrium. Fisher's Exact Test was done to compare the allelic frequencies of different populations using NCSS 6.0 software program and data were analyzed by using the computer software SPSS for windows (version 10.0).

Results

Genotype analysis in 200 normal controls was done by PCR based restriction analysis. The bands on the gel revealed digested (ff) and undigested (FF), as well as heterozygotes (F/f) (Figure 2). The distribution of VDR genotypes and allele frequencies in north India is shown in (Table 1). The allelic frequency of `F'; `f ' was 71.5; 21.5% in our population. Genotype distribution was in agreement with Hardy-Weinberg equilibrium. Table 2 shows genotype distribution of the VDR alleles in our population with those reported in other populations. We compared the different alleles of VDR gene of different populations with reference to Indian population (Table 2) by using χ² exact test.

Discussion

SNPs are scattered throughout the genome and high degree of variability makes these informative genetic markers useful for disease susceptibility. VDR is known to regulate cell proliferation, calcium absorption from the gut, and cell differentiation and may also influence androgen and estrogen activation. The action of VDR is not only up regulated by vitamin D but also by protein kinase A, parathyroid hormone and growth factors. Any defect in the VDR gene could modulate the metabolism of calcium thereby increasing the risk of developing different diseases e.g. osteoporosis and calcium stones. The VDR gene Fok-1 polymorphism has been widely used as a genetic marker for diseases related to calcium metabolism. In this study we have reported the distribution of VDR Fok-1 genotypes in different populations worldwide and compared with our population. The frequency of the individual alleles varies among different ethnic or geographic populations shown in (Table 2) but allele (F) is more common than allele (f) in different populations including ours. Genotype (FF) was significant for populations in Finland, Australia and Black Pennsylvania (P<0.05). However for the rest of the populations P-values were nonsignificant. Genotype (Ff) was not significant for all the comparable populations (P>0.05) except Black Pennsylvania (P<0.05). Genotype (ff) was significantly different in all the populations except Black Pennsylvania and north Indian population when allelic frequencies (F & f) of VDR gene in different populations were compared; Finland, Australia and White Massachusetts were significantly different.

In the present analysis, variation at VDR gene was measured solely on the basis of PCR-RFLP of the basic core sequence. Due to their highly polymorphic content, SNPs constitute useful tools in population genetic studies in understanding population and ethnic variations. As there are large differences in allele frequency and distribution of genotypes of VDR (Fok I) SNP, it would be quite helpful to find out any linkage disequilibrium in individuals from other ethnic groups. Allelic association studies are in progress with several chronic inflammatory and degenerative diseases in which VDR may be involved. In the long run, these studies may help in determining disease susceptibility and clinical management of patients.

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