Investigating Polymorphisms in the NADSYN1/DHCR7 Locus (rs1790349 and rs12785878) as Novel Genetic Markers for Cardiovascular Disease Sally I. Hassanein, Mohamed A. Abu el Maaty, Hameis M. Sleem, Mohamed Z. Gad Clinical Biochemistry Unit, Faculty of Pharmacy and Biotechnology , The German University in Cairo (GUC), Cairo, Egypt Background: Vitamin D has been widely known to maintain calcium homeostasis. It has been shown to play a role in almost every major disease which has led to a substantial interest in the determination of the vitamin D status. DHCR7/NADSYN1 is a novel locus for association with vitamin D status. DHCR7 encodes the enzyme 7-dehydrocholesterol (7-DHC) reductase, which converts 7-DHC to vitamin D3 by exposure to UVB irradiation in the skin (1). While NADSYN1 gene encodes NAD synthetase I involved in cholesterol synthesis (2). The NADSYN1 gene is located on chromosome 11 close to the DHCR7 gene. A recent GWA study revealed that variants near genes involved in cholesterol synthesis influence the vitamin D status (3). Objectives: Examining the influence of DHCR7/NADSYN1 gene variants on the serum levels of 25(OH)D and the incidence of coronary artery disease (CAD). Methodology: Subjects: 63 male patients, 35–50 years of age, with single- or multi-vessel CAD, were recruited as well as age- and sex-matched controls (n = 31) with no diagnostic signs of CAD. Exclusion criteria for this cohort study was the presence of chronic diseases such as chronic kidney, heart and liver disease as well as diabetes mellitus. None of the subjects were receiving vitamin D. Genotyping: PCR was performed for both SNPs rs1790349 and rs12785878. The purified PCR products were sequenced to identify the gene variants. 25-hydroxyvitamin D measurement: An in house developed and validated HPLC with UV detection was adopted for 25(OH)D analysis [4]. Normal = 25(OH)D concentrations ≥ 30 ng/ml, deficient ≤ 20 ng/ml and insufficient with concentrations between 20 - 30 ng/ml . Results: Table 1. Genotypic and allelic distribution of both SNPs in controls and patients Table 3.Association of the observed genotypes and alleles for both SNPs with circulating 25(OH)D2, 25(OH)D3 and total 25(OH)D levels SNPs Genotype/ Alleles 25(OH)D3 ng/ml p value 25(OH)D2 Total 25(OH)D rs12785878 Controls GG (n = 17) GT (n = 8) TT (n = 6) 26.34 ± 8.55 10.45 ± 2.84 13.4 ± 2.82 0.003 13.78 ± 7 12.68 ± 4.2 4.35 ± 0.21 0.19 40.13 ± 5.44 23.12 ± 2 17.75 ± 2.62 0.002 Patients GG (n = 27) GT (n = 30) 8.49 ± 6.82 8.94 ± 5.52 7.2 ± 0.17 0.57 18.96 ± 8.42 13.64 ± 9.76 10.4 ± 0.34 0.08 26.71 ± 10.56 22.3 ± 11.32 21.27 ± 0.23 0.4 rs1790349 GG (n = 2) AG (n = 9) AA (n = 20) 21.48 ± 11.99 8.79 ± 6.22 9.46 ± 5.42 14.38 ± 6.27 18 ± 8 12.93 ± 8.98 35.83 ± 9.54 26.16 ± 9.56 22.58 ± 9.55 0.0001 GG (n = 4) AG (n = 20) AA (n = 39) 5.2 ± 1.7 8.49 ± 6.8 9 ± 5.5 0.17 8.5 ± 7.5 13.75 ± 8.98 0.15 13.7 ± 5.8 26.72 ± 10.56 23 ± 9.87 0.41 G= 42 T= 20 G = 22.8 ± 2.4 T = 11.5 ± 1 0.0064 G = 13.5 ± 5.5 T = 9.9 ± 2.2 0.2 G = 36.35 ± 2 T = 21.3 ± 1.4 0.0024 G= 84 T= 42 G = 8.7 ± 1.2 T = 8.6 ± 1.2 0.9 G = 15.89 ± 1.8 T = 13 ± 2 G = 24 ± 2.2 T = 22 ± 2.3 0.5 A= 49 G= 13 A = 8.7 ± 1.2 G = 14 ± 2.3 A = 15.89 ± 1.8 G = 18.5 ± 1.5 A = 25.2 ± 2 G = 33.2 ± 2.3 0.01 A= 100 G= 26 A = 9 ± 1.2 G = 7.6 ± 1.5 0.3 A = 15.5 ± 1.6 G = 16.1 ± 2.3 0.8 A = 24.3 ± 1.8 G = 23.2 ± 2.8 0.6 SNPs Controls (n = 31) Patients (n = 63) OR (95% CI) p value rs1790349 AA AG GG A G 20 9 2 49 13 39 4 100 26 1.23 (0.58 – 2.6) 0.9 0.7 rs12785878 GT TT T 17 8 6 42 27 30 84 1.58 (0.88-2.85) 0.097 0.14 Conclusion: 1- A lack of association between the rs12785878 and rs1790349 SNP polymorphisms and CAD. 2- Association between total 25(OH)D levels and the SNPs in the control subjects. 3- The allelic distribution of the rs12785878 was found to be closer to that of the other African populations as well as the Japanese population. References: 1- Holick, M. F., J. A. MacLaughlin and S. H. Doppelt (1981). ‘’Regulation of cutaneous previtamin D3 photosynthesis in man: skin pigment is not an essential regulator. ‘Science 211(4482): 590-593. 2- Bu FX, Armas L, Lappe J, Zhou Y, Gao G, Wang HW, Recker R, Zhao LJ: Comprehensive association analysis of nine candidate genes with serum 25-hydroxy vitamin D levels among healthy caucasian subjects. Hum Genet 2010, 128(5):549-556. 3- Wang TJ, Zhang F, Richards JB, Kestenbaum B, van Meurs JB, Berry D, Kiel DP, Streeten EA, Ohlsson C, Koller DL, et al: Common genetic determinants of vitamin D insufficiency: a genome-wide association study. Lancet 2010,376(9736):180-188. 4- Abu el Maaty MA, Hanafi RS, El Badawy S, Gad MZ: Association of suboptimal 25-hydroxyvitamin D levels with knee osteoarthritis incidence in post-menopausal Egyptian women . Rheumatol Int 2013; 33: 2903–2907. Table 2. Allelic frequencies of both SNPs in different HapMap populations as well as the controls included in this study SNP Allele Allele frequencies in population European Chinese Japanese African Sub-Saharan African Egyptian rs1790349 A G 0.832 0.168 0.709 0.291 0.640 0.360 0.668 0.332 0.765 0.235 0.790 0.209 rs12785878 T 0.274 0.726 0.512 0.488 0.923 0.077 0.836 0.164 0.677 0.323 Contact: Sally.ibrahim@guc.edu.eg