Abstract
We studied functional effect of rs12722489 single nucleotide polymorphism located in the first intron of human IL2RA gene on transcriptional regulation. This polymorphism is associated with multiple autoimmune conditions (rheumatoid arthritis, multiple sclerosis, Crohn's disease, and ulcerative colitis). Analysis in silico suggested significant difference in the affinity of estrogen receptor (ER) binding site between alternative allelic variants, with stronger predicted affinity for the risk (G) allele. Electrophoretic mobility shift assay showed that purified human ERα bound only G variant of a 32-bp genomic sequence containing rs12722489. Chromatin immunoprecipitation demonstrated that endogenous human ERα interacted with rs12722489 genomic region in vivo and DNA pull-down assay confirmed differential allelic binding of amplified 189-bp genomic fragments containing rs12722489 with endogenous human ERα. In a luciferase reporter assay, a kilobase-long genomic segment containing G but not A allele of rs12722489 demonstrated enhancer properties in MT-2 cell line, an HTLV-1 transformed human cell line with a regulatory T cell phenotype.
Highlights
New technologies, modern computational capacities and collection of large study populations through international collaboration brought new hope for understanding the mechanisms of complex diseases with strong genetic component [1]
Genome-wide association studies (GWAS) include over a million single-nucleotide polymorphisms (SNP) and thousands of participants, allowing identification of SNPs with small effect sizes and putative risk loci in or near genes not previously suspected of being involved in the etiology of a particular disease [2]
Materials and methods Computational analysis of transcription factor binding sites We analyzed preselected SNPs with flanking sequences (25 nucleotides at each side) using PERFECTOS-APE, a specialized software to predict effects of SNPs on transcription factor binding using predefined collection of position weight matrices [15] with the following parameters: HOCOMOCO v10 [16] collection of mono- and dinucleotide position weight matrices (PWMs), 0.0005 as a threshold for motif P-value for any of two alternative alleles, and 4 as a threshold for the ratio between the motif P-values for two alternative alleles.Triallelic SNPs were virtually split into three twoallelic variants and each pair was analyzed separately
Summary
Modern computational capacities and collection of large study populations through international collaboration brought new hope for understanding the mechanisms of complex diseases with strong genetic component [1]. Genome-wide association studies (GWAS) include over a million single-nucleotide polymorphisms (SNP) and thousands of participants, allowing identification of SNPs with small effect sizes and putative risk loci in or near genes not previously suspected of being involved in the etiology of a particular disease [2]. A strongly associated SNP may not be the causative polymorphism by itself; instead, it could be in a strong linkage disequilibrium with the actual functional SNP or may be linked to the causative copy number variant [2]. Even if the causative variant has been identified, the molecular mechanisms that connect the genetic variant and the disease remain undetermined
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