title: In-vitro function of upstream visfatin polymorphisms that are associated with adverse cardiometabolic parameters in obese children.
abstract: Visfatin 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.
  
  
  
  A 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.
  
  
  
  Variant 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.
  
  
  
  We 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.
authors.affiliation: Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.|Division of Paediatric Endocrinology and Diabetes, Khoo Teck Puat-National University Children's Medical Institute, National University Health System, Singapore, Singapore.
authors.forename: Delicia Shu Qin
authors.initials: DS
authors.lastname: Ooi
authors.affiliation: Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.|Division of Paediatric Endocrinology and Diabetes, Khoo Teck Puat-National University Children's Medical Institute, National University Health System, Singapore, Singapore.
authors.forename: Siong Gim
authors.initials: SG
authors.lastname: Ong
authors.affiliation: Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.|Division of Paediatric Endocrinology and Diabetes, Khoo Teck Puat-National University Children's Medical Institute, National University Health System, Singapore, Singapore.
authors.forename: Chew Kiat
authors.initials: CK
authors.lastname: Heng
authors.affiliation: Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.|Division of Paediatric Endocrinology and Diabetes, Khoo Teck Puat-National University Children's Medical Institute, National University Health System, Singapore, Singapore.
authors.forename: Kah Yin
authors.initials: KY
authors.lastname: Loke
authors.affiliation: Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore. paeleeys@nus.edu.sg.|Division of Paediatric Endocrinology and Diabetes, Khoo Teck Puat-National University Children's Medical Institute, National University Health System, Singapore, Singapore. paeleeys@nus.edu.sg.|Singapore Institute for Clinical Sciences, Agency of Science, Technology and Research, Singapore, Singapore. paeleeys@nus.edu.sg.|Department of Paediatrics, Yong Loo Lin School of Medicine, NUHS tower block, Level 12, 1E Kent Ridge Road, Singapore, 119228, Singapore. paeleeys@nus.edu.sg.
authors.forename: Yung Seng
authors.initials: YS
authors.lastname: Lee
mesh_terms: D001665:Binding Sites; D015415:Biomarkers; D002460:Cell Line; D002648:Child; D020022:Genetic Predisposition to Disease; D006801:Humans; D054409:Nicotinamide Phosphoribosyltransferase / Q000235:genetics* / Q000378:metabolism; D009765:Obesity / Q000235:genetics* / Q000378:metabolism*; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014157:Transcription Factors; D015533:Transcriptional Activation
chemical_list: D015415:Biomarkers; D014157:Transcription Factors; D054409:Nicotinamide Phosphoribosyltransferase
keywords: Childhood obesity; Luciferase assay; Polymorphisms; Variants; Visfatin promoter