Original 65J Contemp Med Sci | Vol. 8, No. 1, January-February 2022: 65–68 Association of TaqI Polymorphism of Vitamin D Receptor (VDR) Gene with Anemia in Saudi Women Maryam A. AL-Ghamdi1,2* 1Department of Biochemistry, Faculty of Science, King Abdulaziz University Jeddah 21589, Saudi Arabia. 2Vitamin D Pharmacogenomics Research Group, King Abdulaziz University, Jeddah, Saudi Arabia. *Correspondence to: Maryam A. AL-Ghamdi (E-mail: maaalghamdi3@kau.edu.sa) (Submitted: 13 December 2021 – Revised version received: 16 January 2022 – Accepted: 23 January 2022 – Published online: 26 February 2022) Introduction Fat soluble vitamin D is required by the body for normal bone development and maintenance. It acts by increasing absorp- tion of phosphate, calcium and magnesium. Vitamin D defi- ciency is implicated in a wide range of diseases, most notably in relation to bones.1 Vitamin D deficiency is a health concern worldwide. Recent studies have shown vitamin D deficiency to play a major role in many metabolic as well as physiological disorders such as diabetes, cardiovascular disease, cancer and thyroid disorders.2 Sunlight accounts for about 50 to 90% of supply of vitamin D.3 Vitamin D regulates bone turnover by the stimulation of osteoclastic and osteoblastic cells.4 Variations of the VDR gene, that is present in chromo- some 12, have been associated with various disorders and the polymorphisms vary to a great extent among different popula- tions of the world.5 Four frequently occurring single nucleo- tide polymorphisms (SNPs) namely: ApaI, BsmI, FokI, and TaqI, found at the 3´ end of the VDR gene have been studied in detail.6,7 The VDR suppresses activation of T cells, therefore, T-cell mediated autoimmune diseases are associated with pol- ymorphisms in this receptor gene.8 Polymorphisms in the VDR gene have been extensively studied.9 Deficiency of iron leads to anemia. There are various var- iants of anemia, such as pernicious anemia, nutritional anemia, sickle cell anemia and hemolytic anemia. While some anemias are genetic, the most common form observed in a large section of the population worldwide is due to iron defi- ciency, in turn manifested as low levels of hemoglobin. According to a WHO report in 2008, 1;62 billion of the world population is anemic. In females, the main cause of anemia is blood loss during menstruation and pregnancy. Since nutri- tional deficiency is the most common cause of iron deficiency anemia, this study is aimed at studying the possible associa- tion between anemic, vitamin D deficient women with VDR gene polymorphism at three restriction sites ApaI, TaqI and BsmI. We found a significant association between the TaqI polymorphisms and occurrence of anemia in the study population. Materials and Methods Study Subjects The study involved volunteers: 50 women in the age group 25–35 who were anemic and had low serum vitamin D levels. The control group consisted of 50 women in age group 25–35 who had sufficient blood hemoglobin and serum vitamin D levels. Care was taken to only include women in both groups who did not have any other health conditions such as dia- betes, hypertension, cardiac disorders, thyroid disorders etc. Informed consent of the volunteers was obtained. Hemo- globin levels of all the volunteers were measured using CBC. Women who had hemoglobin levels lesser than 10 grams/dL were classified as anemic, while those with hemoglobin levels greater than 10 grams/dL were classified as healthy. Serum vitamin D levels were measured by blood test for 25-hydroxyvitamin D: 25(OH)D. Levels of less than 20 ng/mL are considered deficient, 20–30 ng/mL are consid- ered as insufficient and levels greater than 30 ng/mL are con- sidered sufficient. Extraction of DNA and Amplification of Vitamin D Receptor Gene The protocol followed is the same as the one in our previous study.9 Genomic DNA was extracted from whole blood using QIAamp DNA blood mini kit (QIAGEN, USA, Cat. no.51104). The extracted DNA was stored at −20˚C for fur- ther PCR reactions (Table 1). Nanodrop2000c instrument from Thermo Scientific (USA) was used to calculate Abstract Objectives: This study aims to correlate occurrence of anemia in a population of Saudi women with polymorphisms in the VDR gene. Methods: The study sample consisted of 50 anemic women with vitamin D deficiency and 50 healthy women with normal hemoglobin and sufficient serum vitamin D levels. Restriction fragment length polymorphism (RFLP) analysis of the VDR genes was done for Single Nucleotide Polymorphism (SNP) sites namely ApaI, BsmI and TaqI. Statistical correlation was done between gene polymorphisms at these three sites and hemoglobin levels. Results: We found a significant association between TaqI site polymorphisms of the VDR gene and presence of anemia in the study population. Conclusion: This is the first report of a significant association between vitamin D receptor gene polymorphisms and anemia in saudi population. Further studies on a larger population size will pave way to elucidation of the mechanism in which VDR gene polymorphisms exert an influence on anemia. Keywords: VDR polymorphism, anemia, TaqI polymorphism, anemia, vitamin D deficiency ISSN 2413-0516 http://orcid.org/0000-000-0861953x Association of Taqi Polymorphism of Vitamin D Receptor (VDR) Gene with Anemia in Saudi Women Original Maryam A. AL-Ghamdi 66 J Contemp Med Sci | Vol. 8, No. 1, January-February 2022: 65–68 Table 1. Represents the PCR reaction mixture 10–30 ng DNA template 2X reaction buffer 4 mMMg+2 4 µM dNTPs 0.2 µM each of forward and reverse primers 0.45 U taq DNA polymerase Final volume made up with nuclease free water to 50 µl Table 2. Represents the PCR conditions for 30 cycles6 Denaturation Annealing Extension Final extension Hold 95˚C for 30 sec 60˚C for 1 min 68˚C for 2 min 72˚C for 5 min 4˚C∞ Table 3. PCR – RFLP products for the restriction sites ApaI BsmI TaqI AA – 2229 bp Aa – 2229 bp, 1700 bp and 529 bp aa – 1700 bp, 259 bp BB - 2229 bp Bb – 2229 bp, 1579 bp, and 650 bp bb – 1579 bp and 650 bp TT - 1982 bp and 247 bp Tt – 1982 bp, 1780 bp, 202 bp, and 247 bp tt – 1780 bp, 202 bp, and 247 bp Table 4. Distribution of various alleles in healthy women (sufficient hemoglobin and vitamin D levels) Restriction site Genotype Number of samples % Frequency ApaI AA Aa aa 18 21 11 (total 50) 36 42 22 (total 100%) BsmI BB Bb bb 20 17 13 (total 50) 40 34 26 (total 100%) TaqI TT Tt tt 22 18 10 (total 50) 44 36 20 (total 100%) Table 5. Distribution of various alleles in women who were anemic as well as deficient in vitamin D Restriction site Genotype Number of samples % Frequency ApaI AA Aa aa 19 21 10 (total 50) 38 42 20 (total 100%) BsmI BB Bb bb 22 16 12 (total 50) 44 32 24 (total 100%) TaqI TT Tt tt 9 10 31 (total 50) 18 20 62 (total 100%) Fig. 1 Agarose gel (1%) showing the results of PCR-RFLP compar- ison between the three restriction enzymes in anemia (P) and the control (C). Lane M: DNA marker. Lane 2: negative control. Lane 3: PCR product of size 2229 bp. Lane 4, 5: RFLP of ApaI digestion in control and patient. Lane 7, 8: RFLP of TaqI digestion in control and patient. Lane 10, 11: RFLP of BsmI digestion in control and patient. Results PCR-RFLP Analyses and Distribution of the Genotypes The restriction profiles obtained after PCR-RFLP are summa- rized in Table 3. The distribution of various alleles in control group and anemic group are presented in Tables 4 and 5. From Table 4, we observe that in the control group which comprised of healthy women with sufficient hemoglobin and serum vitamin D levels, there is no significant difference between the genotype frequencies in the Single nucleotide polymorphisms (SNPs) at any of the three sites. In contrast, as seen in Table 5, the study group of women with anemia and vitamin D deficiency clearly show a significant difference in concentration and purity of the extracted DNA (Table 2). The polymerase chain reaction (PCR) was carried out as described in our previous study.9 PCR Amplification The amplification products were resolved on 1% agarose gels and 1X of Tris-borate-EDTA (TBE) buffer followed by staining with ethidium bromide and visualized under uv light. Genotyping of the VDR Gene After its amplification, PCR product of ~2229 bp was exposed to enzymatic restriction digestion by three enzymes which were purchased from Thermo Scientific: ApaI, BsmI, and TaqI (Figure 1). The conditions for restriction digestion were fol- lowed according to Iyer et al., 2017. The digested DNA frag- ments and the DNA size marker were segregated by electrophoresis on a 1% agarose gel stained with ethidium bro- mide and 1X TBE electrophoresis buffer. Maryam A. AL-Ghamdi Original Association of Taqi Polymorphism of Vitamin D Receptor (VDR) Gene with Anemia in Saudi Women 67J Contemp Med Sci | Vol. 8, No. 1, January-February 2022: 65–68 genotype frequency at the TaqI site. The genotype frequency of homozygous recessive alleles aa is 62% compared to TT and Tt genotypes, while in control subjects the homozygous recessive genotype tt is observed in only 10% of the study subjects. Discussion In recent times, anemia has been recognized as a world wide health issue. Nutritional as well as metabolic disorders have been attributed to low hemoglobin levels associated with anemia. Although we are well aware of the role of vitamin D in bone formation, metabolic regulation and overall growth and development, studies over the past few years have cate- gorically suggested the role of vitamin D in erythropoiesis.10 Studies by Sim et al., 2010.,11 have shown significant association between vitamin D deficiency and anemia. Cusato et al.,12 2015 proved the role of VDR gene polymorphisms in the ribavirin-induced anemia in HCV-patients at 2 and 4 weeks of medication. The VDR gene polymorphisms could attribute to significant receptor dysfunction thereby causing various disorders such as low bone mineral density, autoim- munity, infections, cardiovascular disease and cancers.13 Moreover, studies on VDR gene polymorphism in various populations around the world have pointed out the significant role of the VDR gene in vitamin D deficiency.14 Our own research group has established association of BsmI polymor- phisms with type I diabetes, ApaI polymorphisms with type II diabetes and TaqI polymorphisms with gestational diabetes in Saudi population.9 These findings encouraged us to probe the possible relation between anemia and VDR gene polymor- phisms. With this as the premise, our current study was designed to look for a possible correlation between anemia and vitamin D deficiency with polymorphisms of the VDR gene at three SNP sites namely ApaI, TaqI and BsmI. We have taken extreme care to include only those subjects who were anemic as well as had low serum vitamin D levels as the study group while the control group were women with sufficient levels of hemoglobin as well as serum vitamin D. We have clearly observed that at the TaqI site, there is a significant dif- ference in genotype frequency between study group and con- trol group. The homozygous recessive genotype tt was observed in 62% of the study group as against mere 20% in the control group. A study by Yassin et al.,15 in an Egyptian popu- lation showed no correlation between anemia and VDR poly- morphism at ApaI and TaqI sites. Another study by Yu et al.,16 showed a significant association between VDR polymor- phisms and Aplastic anemia in a Chinese population. According to Erturk et al.,17 BsmI polymorphism may have a role in anemia in hemodialysis patients with BB genotype being strongly associated with lower hemoglobin levels. To the best of our knowledge, our study is a pioneering finding that strongly establishes the association of homozygous reces- sive alleles tt of TaqI polymorphism (rs731236) SNP with anemia. The study is a very strong support for this finding because all the patients recruited for the study were anemic as well as vitamin D deficient, while the control group was suffi- cient in both hemoglobin as well as serum vitamin D level, clearly establishing the connection between the two deficien- cies at the genotypic level. Medrano et al.,18 previously have proved a role of calcitrol, the active form of vitamin D in hematopoiesis. This strongly supports our observation that links anemia and vitamin D anemia. Conclusion Our study is the first of its kind reporting a genotypic associa- tion between anemia and vitamin D deficiency with the homozygous tt genotype of the TaqI SNP showing significant association in the study group as compared to healthy control group. Our study was conducted in women in the age group of 25–35, who are in active menstruation and childbirth age. It would be interesting to extend the study to a larger population, such as post menopausal women as well as children and observe any genotypic variation. Acknowledgments The authors acknowledge King Abdulaziz University, Jeddah, for scientific research. Disclosure Statement The author declares no conflict of interest.  References 1. Nair R, Maseeh A. Vitamin D: The ‘sunshine’ vitamin. J Pharmacol Pharmacother 2013;3:118–26. 2. Sizar O, Khare S, Goyal A, et al. Vitamin D Deficiency. In: StatPearls [Internet]. [Updated 2021 Jul 21] 3. Naeem Z. Vitamin D deficiency- an ignored epidemic. Int J Health Sci 2010 Jan;4(1):V–VI. 4. Mukhtar M, Sheikh N, Suqaina SK, Batool A, Fatima N, Mehmood R, Nazir S. Vitamin D Receptor Gene Polymorphism: An Important Predictor of Arthritis Development. Biomed Res Int. 2019 Mar 18;2019:8326246. 5. Nurminen V, Seuter S, Carlberg C. 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