Effect of heme oxygenase-1 polymorphisms on lung function and gene expression

Abstract

Background: Oxidative stress induced by smoking is considered to be important in the pathogenesis of Chronic Obstructive Pulmonary Disease (COPD). Heme oxygenase-1 (HMOX1) is an essential enzyme in heme catabolism that is induced by oxidative stress and may play a protective role as an antioxidant in the lung. We determined whether HMOX1 polymorphisms were associated with lung function in COPD patients and whether the variants had functional effects.

Methods: We genotyped five single nucleotide polymorphisms (SNPs) in the HMOX1 gene in Caucasians who had the fastest (n = 278) and the slowest (n = 304) decline of FEV1 % predicted, selected from smokers in the NHLBI Lung Health Study. These SNPs were also studied in Caucasians with the lowest (n = 535) or the highest (n = 533) baseline lung function. Reporter genes were constructed containing three HMOX1 promoter polymorphisms and the effect of these polymorphisms on H2O2 and hemin-stimulated gene expression was determined. The effect of the HMOX1 rs2071749 SNP on gene expression in alveolar macrophages was investigated.

Results: We found a nominal association (p = 0.015) between one intronic HMOX1 SNP (rs2071749) and lung function decline but this did not survive correction for multiple comparisons. This SNP was in perfect linkage disequilibrium with rs3761439, located in the promoter of HMOX1. We tested rs3761439 and two other putatively functional polymorphisms (rs2071746 and the (GT)n polymorphism) in reporter gene assays but no significant effects on gene expression were found. There was also no effect of rs2071749 on HMOX1 gene expression in alveolar macrophages.

Conclusions: We found no association of the five HMOX1 tag SNPs with lung function decline and no evidence that the three promoter polymorphisms affected the regulation of the HMOX1 gene.

Figure 1

Single-nucleotide polymorphisms (SNPs) in the region of the heme oxygenase 1 (HMOX1) gene whose allele frequency was > 10% in European-American Coriell samples. Shading represents the strength of pair-wise linkage disequilibrium (LD) with a black-to-white gradient reflecting higher to lower r² values. Bin: a group of SNPs where the alleles are highly associated. *: tag SNPs; †: SNPs which were excluded from the study.

Figure 2

HMOX1 gene promoter analysis of 9 different constructs. Each test construct was co-transfected into A549 cells with Renilla luciferase vector as an internal control. The basal promoter activity of each plasmid is indicated as luciferase activity relative to that of construct 1. The relative inducibility by H₂O₂ (300 μM) or Hemin (100 μM) is shown for each construct. Data are presented as means + SD from three to four independent experiments. There was no significant difference in induction of reporter gene expression among these constructs.