Functional analysis of 5-lipoxygenase promoter repeat variants.
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 approximately 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.
Vikman
Susanna
S
Department of Preventive Medicine, USC Keck School of Medicine, Los Angeles, CA 90033, USA.
Brena
Romulo M
RM
Armstrong
Patrice
P
Hartiala
Jaana
J
Stephensen
Charles B
CB
Allayee
Hooman
H
D058927:5-Lipoxygenase-Activating Proteins; D000328:Adult; D001094:Arachidonate 5-Lipoxygenase / Q000235:genetics* / Q000378:metabolism; D044383:Black People / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D018899:CpG Islands; D019175:DNA Methylation; D004851:Epoxide Hydrolases / Q000235:genetics / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D014644:Genetic Variation; D006801:Humans; D007962:Leukocytes / Q000201:enzymology; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D018895:Microsatellite Repeats; D008875:Middle Aged; D011401:Promoter Regions, Genetic; D055815:Young Adult
D058927:5-Lipoxygenase-Activating Proteins; C548592:ALOX5AP protein, human; D002352:Carrier Proteins; D008565:Membrane Proteins; D001094:Arachidonate 5-Lipoxygenase; D004851:Epoxide Hydrolases; C043134:leukotriene A4 hydrolase