Human Molecular Genetics Advance Access originally published online on August 28, 2009
Human Molecular Genetics 2009 18(23):4521-4529; doi:10.1093/hmg/ddp414
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Functional analysis of 5-lipoxygenase promoter repeat variants
1 Department of Preventive Medicine, 2 Department of Biochemistry and Molecular Biology, 3 Institute for Genetic Medicine and 4 USC Epigenome Center, USC Keck School of Medicine, Los Angeles, CA 90033, USA, 5 Program in International and Community Nutrition, Department of Nutrition and 6 USDA Western Human Nutrition Research Center, University of California Davis, Davis, CA 95616, USA
* To whom correspondence should be addressed at: Institute for Genetic Medicine, USC Keck School of Medicine, 2250 Alcazar Street, CSC 206, Los Angeles, CA 90089-9075, USA. Tel: +1 3234421736; Fax: +1 3324422764; Email: hallayee{at}usc.edu
Received June 18, 2009; Revised August 12, 2009; Accepted August 26, 2009
Variants of a hexanucleotide repeat polymorphism in the promoter of the 5-lipoxygenase (5-LO) gene have been associated with cardiovascular disease traits in humans, which may be due, at least in part, to differential expression of the at-risk alleles. To more fully characterize these variants, we carried out gene expression and DNA methylation studies in primary leukocytes from healthy individuals carrying various 5-LO promoter alleles. Regardless of genotype, 5-LO and 5-LO-activating protein (FLAP) gene expression was higher in granulocytes compared with monocytes and lymphocytes, whereas leukotriene A4 hydrolase (LTA4H) expression was higher in monocytes. In all three leukocyte populations, 5-LO mRNA levels were positively correlated with those of FLAP and LTA4H, with the highest correlation observed in granulocytes. In lymphocytes, individuals homozygous for the shorter 3 and 4 repeat alleles had between 20–35% higher 5-LO, FLAP and LTA4H expression compared with homozygous carriers of the wild-type 5 repeat allele (P = 0.03–0.0001). DNA methylation analysis of four CpG islands in a 1500 bp region encompassing the 5-LO promoter and the first 
100 bp of intron 1 revealed relatively low overall DNA methylation across all genotypes and leukocyte populations. However, analysis of the promoter repeats themselves demonstrated that, regardless of cell population, the 4 allele was methylated approximately twice as much as the 3 allele (P < 0.0001). Our results demonstrate that, in lymphocytes, the shorter repeat alleles of the 5-LO promoter lead to higher gene expression, which may be regulated through differential DNA methylation of the CpGs located within these repeats.