Abstract
BackgroundVisfatin is an adipokine associated with glucose and lipid metabolism. We previously reported two visfatin upstream single nucleotide polymorphisms (SNPs), c.-3187G > A (rs11977021) and c.-1537C > T (rs61330082), which were in perfect linkage disequilibrium, in a Singaporean cohort of severely obese children and are associated with visfatin level and adverse cardiometabolic parameters. We aim to functionally characterize the effect of c.-3187G > A and c.-1537C > T SNPs on basal transcriptional activity.MethodsA 1.6 kb and 3.7 kb upstream promoter region of the visfatin gene was amplified by polymerase chain reaction and separately cloned into luciferase reporter vectors. Successful clones were transfected into human embryonic kidney (HEK293T) and human breast carcinoma (MCF7) cells and in-vitro dual-luciferase assay was performed. Electrophoretic mobility shift assay (EMSA) was also conducted to examine the binding affinity between transcription factors and visfatin promoter sequences.ResultsVariant promoter with only c.-1537C > T SNP did not show a change in transcriptional activity as compared to the wild type. However, variant promoter with both c.-3187G > A and c.-1537C > T SNPs showed a statistically significant increase of 1.41 fold (p < 0.01) in transcriptional activity. The longer 3.7kbp visfatin promoter sequence was also shown to have significantly higher transcriptional activity (p < 0.05) as compared to the shorter 1.6kbp visfatin promoter. Both c.-3187G > A and c.-1537C > T variants showed an increased binding with nuclear protein.Discussion and conclusionsWe have demonstrated for the first time that visfatin variant promoter with both c.-3187G > A and c.-1537C > T SNPs result in an increase in transcriptional activity. This supports our previous finding and postulation that these SNPs contribute to elevated visfatin levels which may mediate higher triglyceride levels, severe systolic blood pressure and severe hypertension in obese children. These SNPs may co-operatively affect enhancer or silencer function to regulate transcriptional activity. In conclusion, this study shows that upstream visfatin SNPs could potentially affect phenotypic outcome in obese children through alteration of circulating visfatin level.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3315-9) contains supplementary material, which is available to authorized users.
Highlights
Visfatin is an adipokine associated with glucose and lipid metabolism
The binding between the visfatin variant promoter sequence and nuclear protein is assessed by densitometry and the large standard deviation values were due to comparison between 2 separate Electrophoretic mobility shift assay (EMSA) experiments (Fig. 3c)
Wang et al reported that the c.-1537C > T single nucleotide polymorphisms (SNPs) altered promoter binding to AP-1 [9] but the study did not examine the promoter binding affinity to AP-2alpha2 which can be a likely transcription factor that is affected by the c.-1537C > T SNP due to the homology between the promoter sequence and AP-2alphaA as predicted by PROMO
Summary
We previously reported two visfatin upstream single nucleotide polymorphisms (SNPs), c.-3187G > A (rs11977021) and c.-1537C > T (rs61330082), which were in perfect linkage disequilibrium, in a Singaporean cohort of severely obese children and are associated with visfatin level and adverse cardiometabolic parameters. Our group has previously reported perfect linkage disequilibrium between rs61330082 and another SNP, c.-3187C > T (rs11977021) which was found to be significantly associated with total plasma cholesterol and LDLcholesterol levels in French-Canadian subjects [5]. These two SNPs were shown to be associated with obesity, severe systolic blood pressure, severe hypertension, plasma visfatin and triglyceride levels in our cohort of local severely obese children [12]. We hypothesized that the two upstream promoter SNPs (rs61330082 and rs11977021) may have a functional effect on the transcriptional activity of the visfatin gene
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
