**6. SNPs in the VDR gene**

The variation in the 5'-promoter region of VDR gene can change the sequence of mRNA as well as protein levels, whereas alteration in 3' UTR sequence can disturb the stability of mRNA thereby affecting the efficacy of translated protein. Some SNPs have been existed in the VDR gene, including *Cdx*2 [106], *Fok*1 [107], *Bsm*1, *Taq*1, *EcoR*V [108], *Apa*1 [101] and *poly* (A) [109] microsatellite repeats.

#### **6.1.** *Cdx***2 SNP**

the cells to stay alive if they are the right type and present at the right place, or it helps the cells to commit suicide (apoptosis) if cells are not the right type or not present at the right place. Vitamin D also reduces the formation of blood vessels around tumours and decreases the ability of tumours to invade [98]. According to the randomized controlled trials, vitamin D

The human VDR (hVDR) gene is located at long arm of chromosome 12 bands 13-14 (12q13- 14) [101, 102]. The gene is 75 kb long and contains 11 exons [103]. This gene is divided into

The 5' promoter region of VDR lacks initiator (TATA and CAAT boxes) and is rich in GC content. It provides the putative site for binding of many transcription factors [103]. The promoter region is present at exon 1(1a, 1b, 1c, 1d, 1e, 1f). The promoter region facilitates the transcription activity of VDR target gene. The 3' UTR contains poly (A) repeats, which is

Coding region comprises of exon 2–9. Exon 2, which have translation start codons, contains DNA-binding site, whereas exon 7, 8 and 9 have ligand (vitamin D) binding site [104].

Polymorphism is defined as the presence of two or more clearly different phenotypic variants of a particular DNA sequence in the same population of a species. The most common form of polymorphism is the single nucleotide polymorphism in which variation occurs at a single base pair usually present in approximately 1% of the population. These types of changes can be present in non-coding region of genes and in introns, which would not affect the translation of proteins, but these changes can affect the degree of gene expression and levels of proteins. The changes can also be present in coding regions of DNA or exons resulting in the formation of an altered protein sequence. Sometimes variation in exons do not cause the change in the

These changes often produce or eliminate restriction sites for endonuclease to digest the DNA. As a result, fragments of DNA with a different length will be obtained which can be identified by gel electrophoresis. This process is called restriction fragment length polymorphisms (RFLPs). The produced fragments will be the undigested fragments, which is homozygous dominant, whereas the digested fragments are heterozygous and homozygous recessive.

reduced the risk of cancer, including breast cancer [99, 100].

three regions: one coding region and two non-coding regions.

reported to be associated with the mRNA stability.

**5. Single nucleotide polymorphism (SNP)**

structure of protein called synonymous polymorphisms.

**4. Vitamin D receptor gene**

144 A Critical Evaluation of Vitamin D - Clinical Overview

**4.1. Non‐coding regions**

**4.2. Coding region**

The VDR *Cdx*2 (G-1739A) is the single nucleotide polymorphism, which was recognized by the sequence analysis of promoter region. It is an adenine to guanine (A to G) SNP situated at the promoter region of VDR gene at exon 1e. It was initially reported to be located at the 3731 bp upstream exon 1a of promoter region of VDR gene among Japanese women [106], but later identified to be located at 1739 kbp upstream of 1e exon just 2 kb away from the exon 1a among many ethnic population [110]. It is the binding region of *Cdx*2 protein, a most important intestine-specific caudal-related homeodomain protein, which increases the transcription of VDR. When A allele is present in *Cdx*2 promoter, the Cdx2 protein is bound more strongly as compared to when a G allele is present. The A allele stimulates the initiation of transcription, whereas G allele inhibits [106].

#### **6.2. GATA SNP**

GATA (A-1012G) is located at exon 1a in the core sequence of DNA called AGATAT [111]. It provides the binding of GATA protein and the binding site is present in A allele and absent in G allele. The mechanism of this polymorphism is not identified yet; however, this polymorphism alters the immune responses to cancer cells. A allele is responsible to reduce cytotoxic response to cancer cells. In addition, it is also an important element that if the transcription is begun in exon 1a or 1d. In presence of G allele, exon 1d comprises an alternate start codon which results in a formation of N-ter extended protein called VDRB1. G allele is most likely associated with the VDRB1 (long) protein, whereas A allele is related with the VDRA (short) protein.

#### **6.3.** *Fok***1 SNP**

*Fok*1 polymorphism is also called start codon polymorphism (SCP). It is a thymine to cytosine (ATG to ACG) polymorphism located at the 10 bp upstream 5' end of exon 2 on the DNA- binding domain, which results in a formation of more active transcription factor that is three amino acids shorter [103, 112]. Those individuals who have ACG sequence in the start codon, the initiation of translation occurs at the second ATG site which results in a formation of three less amino acids at NH2 terminus containing 424 amino acids. If the initiation occurs at first ATG sequence, it produces full-length VDR protein containing 427 amino acids. In the presence of restriction site, alleles are designated as 'f', whereas its absence is designated as 'F' (active form) [113]. The restriction recognition site of *Fok*1 is 5'-GGATG\*-3'; 3'-CCTAC\*-5' and enzyme cleaves 9/13 nucleotide downstream of the recognition site.

### **6.4.** *Bsm***1‐***Apa***1‐***Taq***1 SNP**

Most of the functional sequence variants identified near the 3' region of VDR gene were *Bsm*1, *Apa*1 and *Taq*1 SNP. These SNPs are in linkage disequilibrium with each other and are located in the same haplotype block. Therefore, these SNPs may have the potential to influence the mRNA stability. The *Apa*1 and *Bsm*1 are located at intron 8, whereas *Taq*1 is located at exon 9 [114].

The presence of restriction enzyme site in these SNPs is designated as lower case letter such as b, a and t, whereas absence is designated as upper case letter including B, A, T. The restriction site for Bsm1 is 5'-GAATGCN\*-3', Apa1 is 5'-GGGCC\*C-3' and Taq1 is 5'-T\*CGA-3'.

#### **6.5.** *Poly* **(A) repeats**

*Poly* (A) tail is a variable number of tandem repeats (VNTR) or short tandem repeats (STR) containing variable numbers of adenine nucleotide present at the 3' UTR of VDR. *Poly* (A) is also linked with Bsm1, Apa1 and Taq1 polymorphisms and also involved in the mRNA stability of VDR. It varies in length and can be divided into two types:


Because all four polymorphisms (*Bsm*1, *Apa*1, *Taq*1 and *Poly* (A)) are present in close proximity on the VDR gene, strong linkage disequilibrium exists among them. The two most common haplotypes are:


The baTL haplotype is reported to be associated with the increase incidence of breast cancer [116].
