Genotyping of vitamin D receptor gene polymorphisms using mismatched amplification mutation assay in neonatal sepsis patients of Odisha, eastern India

Vitamin D has potent antimicrobial and anti-inflammatory properties. Vitamin D deficiency has been shown to be associated with the risk of vulnerability to different infectious diseases, such as neonatal sepsis. Polymorphisms in vitamin D receptor (VDR) gene can influence the expression of vitamin D in individuals. Hence, it is essential to study the vitamin D status and VDR gene polymorphisms for assessing neonatal sepsis risk. In this study, we assessed the serum 25(OH)D, the main circulating form of vitamin D and VDR polymorphism on 120 subjects in a case-control approach, recruiting 60 subjects in each category. We genotyped Fok1, Bsm1, Apa1 and Taq1 gene polymorphisms in VDR by developing a unique mismatch amplification mutation assay (MAMA) and studied their association in both populations. VDR-MAMA primers were designed by addition of dual mismatches (DM) near the 3′ end and were selected based on high ΔCt values in comparison to single mismatch (SM) primers using SYBR-Green RT-PCR, which were eventually used for VDR genotyping. Genotyping was also performed using PCR-RFLP for further confirmation. Serum 25(OH)D ELISA revealed that cases were vitamin D insufficient (Median=12.16ng/ml, 95% CI: 3.84–22.22) and controls were vitamin D sufficient (Median=30.22ng/ml, 95% CI: 20.08–46.78; p