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Open Access Highly Accessed Research article

Identification of functional SNPs in the 5-prime flanking sequences of human genes

Salim Mottagui-Tabar1*, Mohammad A Faghihi1, Yosuke Mizuno1, Pär G Engström1, Boris Lenhard1, Wyeth W Wasserman2 and Claes Wahlestedt1

Author Affiliations

1 Center for Genomics and Bioinformatics, Karolinska Institutet, SE-17177 Stockholm, Sweden

2 Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, BC V5Z 4H4, Canada

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BMC Genomics 2005, 6:18  doi:10.1186/1471-2164-6-18

Published: 17 February 2005

Abstract

Background

Over 4 million single nucleotide polymorphisms (SNPs) are currently reported to exist within the human genome. Only a small fraction of these SNPs alter gene function or expression, and therefore might be associated with a cell phenotype. These functional SNPs are consequently important in understanding human health. Information related to functional SNPs in candidate disease genes is critical for cost effective genetic association studies, which attempt to understand the genetics of complex diseases like diabetes, Alzheimer's, etc. Robust methods for the identification of functional SNPs are therefore crucial. We report one such experimental approach.

Results

Sequence conserved between mouse and human genomes, within 5 kilobases of the 5-prime end of 176 GPCR genes, were screened for SNPs. Sequences flanking these SNPs were scored for transcription factor binding sites. Allelic pairs resulting in a significant score difference were predicted to influence the binding of transcription factors (TFs). Ten such SNPs were selected for mobility shift assays (EMSA), resulting in 7 of them exhibiting a reproducible shift. The full-length promoter regions with 4 of the 7 SNPs were cloned in a Luciferase based plasmid reporter system. Two out of the 4 SNPs exhibited differential promoter activity in several human cell lines.

Conclusions

We propose a method for effective selection of functional, regulatory SNPs that are located in evolutionary conserved 5-prime flanking regions (5'-FR) regions of human genes and influence the activity of the transcriptional regulatory region. Some SNPs behave differently in different cell types.