title: Genotyping of vitamin D receptor gene polymorphisms using mismatched amplification mutation assay in neonatal sepsis patients of Odisha, eastern India.
abstract: 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<0.0001) respectively, which indicated that vitamin D insufficiency was mostly prevalent in cases. We found no evidence of association between genotypes of the Apa1 polymorphism and neonatal sepsis or 25(OH)D serum levels. The distributions of the Fok1, Bsm1, and Taq1 genotypes were not consistent with Hardy-Weinberg equilibrium in the control group. Future studies in larger populations are required to establish whether the VDR polymorphisms can be potentially used as genetic markers for early screening towards predisposition to neonatal sepsis risk. In this study, we describe a simple, inexpensive and rapid screening of VDR gene polymorphisms using VDR MAMA-PCR, which can be used in both clinical and research laboratories.
authors.affiliation: Infection Biology Laboratory, KIIT School of Biotechnology, KIIT University, Bhubaneswar, India. Electronic address: dviswadeep1983@gmail.com.
authors.forename: Biswadeep
authors.initials: B
authors.lastname: Das
authors.affiliation: Infection Biology Laboratory, KIIT School of Biotechnology, KIIT University, Bhubaneswar, India.
authors.forename: Saumya
authors.initials: S
authors.lastname: Patra
authors.affiliation: Department of Pediatrics, Kalinga Institute of Medical Science, KIIT University, Bhubaneswar, India.
authors.forename: Chinmay
authors.initials: C
authors.lastname: Behera
authors.affiliation: Infection Biology Laboratory, KIIT School of Biotechnology, KIIT University, Bhubaneswar, India.
authors.forename: Mryutunjay
authors.initials: M
authors.lastname: Suar
mesh_terms: D016022:Case-Control Studies; D005787:Gene Frequency; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D007194:India / Q000453:epidemiology; D007231:Infant, Newborn; D009154:Mutation / Q000235:genetics*; D000071074:Neonatal Sepsis / Q000453:epidemiology / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D018167:Receptors, Calcitriol / Q000235:genetics*
chemical_list: D018167:Receptors, Calcitriol; C579457:VDR protein, human
keywords: Allele; Genotyping; MAMA-PCR; Neonatal sepsis; Polymorphism; Vitamin D receptor