vRare VCAM1 Promoter Haplotypes Prevalent in African Americans Are Hyperinducible.

Cell adhesion molecules direct the inflammatory response through cell-cell interactions between leukocytes and endothelial cells, leukocyte trafficking, and cell signaling. Vascular cell adhesion molecule 1 (VCAM1) is a member of the CAM immunoglobulin superfamily whose expression at the cell surface is highly tissue and mitogen specific. Because genetic variation in VCAM1 has been implicated in the pathogenesis of a variety of human diseases, we sought a method for rapidly identifying functional simple nucleotide polymorphsisms (SNPs) present within a discrete non-coding regulatory regions of this gene. Using a novel transfection-based transcriptional pathway profiling method, we show that several uncommon variant haplotypes are functionally hyperactive. Initially, DNA sequencing across the 2.5 kb VCAM1 promoter in a screening population of 40 healthy African Americans identified 21 SNPs that define 18 different promoter haplotypes. Eight of these promoter haplotypes, defined by 13 SNPs and representing 80% of the haplotypes present in the screening population, were then evaluated for response to 5 hour stimulation with known mitogens in transient transfections of Jurkat T cells. Transcriptional reporter activity in response to combinations of phorbol ester, lectins and ionophore were clustered by inducibility using principal component analysis (PCA). Three uncommon haplotypes, each with frequencies of less than 5% in the controls, were clearly identified by PCA as hyperactive in response to mitogens. Next, in vitro haplotype activity was correlated with a bioinformatic analysis of transcription factor binding site gains or losses created by 11 of the 13 variant nucleotide sites and unique to each haplotype. Using this approach, a low frequency regulatory allele (A-540G), present on one of the hyperactive VCAM1 promoter haplotypes (haplotype 5), was identified as a putative binding site for the transcription factor ETS2. The selective gain of the ETS binding site was confirmed in vivo by chromatin immunoprecipitation experiments comparing two lymphblastoid cell lines (LCLs) of known genotype. An LCL heterozygous for the hyperactive VCAM1 haplotype 5 demonstrated nearly an 8 fold enrichment in ETS2 complexes at the VCAM1 promoter relative to a cell line homozygous for the wild type alleles. Together, these results suggest that some variants in the VCAM1 promoter alter function by changing the affinity of specific transcription factors for their DNA binding sites. This study provides the first functional evaluation of VCAM1 promoter polymorphisms and establishes a hypothetical foundation for investigating specific VCAM1 functional variants in the pathogenesis of complex genetic diseases that disproportionately afflict African Americans, including hemoglobinopathies, asthma, and hematopoietic malignancies. Overall, this study demonstrates feasibility of combining a series of genetic, bioinformatic, and wet lab methodologies for rapid identification of functional genetic variants within a larger pool of SNPs present across non-coding and regulatory regions of human genes.