Allele-specific transcriptional activity of the variable number of tandem repeats of the inducible nitric oxide synthase gene is associated with idiopathic achalasia

Abstract

Background: Polymorphisms of genes involved in the regulation of the immune response are risk factors for achalasia, but their contribution to disease pathogenesis is unknown. Nitric oxide is involved both in immune function and inhibitory neurotransmission.

Objective: The objective of this article is to assess the association and the functional relevance of the CCTTT-inducible nitric oxide synthase (NOS2) gene promoter polymorphism in achalasia.

Methods: Genomic DNA was isolated from 181 achalasia patients and 220 controls. Genotyping of the (CCTTT)n repeats was performed by PCR and capillary electrophoresis, and data analyzed by considering the frequency of the different alleles. HT29 cells were transfected with iNOS luciferase promoter-reporter plasmids containing different (CCTTT)n.

Results: The alleles' distribution ranged from 7 to 18, with a peak frequency at 12 repeats. Analysis of the allele frequencies revealed that individuals carrying 10 and 13 CCTTT repeats were respectively less and more frequent in achalasia (OR 0.5, 95% CI 0.3-0.5 and OR 1.6, 95% CI 1-2.4, all p < 0.05). Long repeats were also significantly associated with an earlier onset of the disease (OR 1.69, 95% CI 1.13-2.53, p = 0.01). Transfection experiments revealed a similar allele-specific iNOS transcriptional activity.

Conclusion: The functional polymorphism (CCTTT) of NOS2 promoter is associated with achalasia, likely by an allele-specific modulation of nitric oxide production.

Keywords: (CCTTT)n pentanucleotide; Idiopathic achalasia; genetic polymorphism; iNOS; nitric oxide.

Figure 1.

Allelic distribution of (CCTTT)n in achalasia and control participants. The allele distribution ranged from 7 to 18 with a peak frequency at 12 repeats in patients as well as controls. Individuals carrying 10 (CCTTT) repeats showed a reduced risk of developing achalasia, while no significant differences were observed in the allelic distribution of the other (CCTTT) repeats.

Figure 2.

Allelic distribution of long (7–11) and short (12–18) (CCTTT) repeats by gender in achalasia and healthy individuals. Males carrying the long alleles form had an increased risk of having achalasia (odds ratio (OR) 2.01, 95% confidence interval (CI) 1.16–3.46, p = 0.012).

Figure 3.

Effects of varying numbers of (CCTTT)n repeats on NOS2 gene transcription. The CCTTT repeat sequences enhance the minimal NOS2 promoter induction in response to LPS, IFNγ and TNFα stimulation, with a significant increase in luciferase activity (*p < 0.0001 vs. unstimulated). Analysis of the difference among the different (CCTTT)n within the stimulated group revealed that constructs containing 12 repeats produced a significantly greater induction of luciferase as compared to all the other constructs (°p < 0.01), whereas the 13-repeat construct produced significantly greater luciferase activity than the 9, 10, 14, 15 and 16-repeat constructs (#p < 0.01 and ## p < 0.05, respectively). Conversely, the 10-repeat construct was associated with a significantly lower transcriptional activity than 11, 12 and 13 constructs (all p < 0.01). Data are the mean of six determinations and expressed as mean ± SEM. Data analysis was performed by ANOVA with Bonferroni post-test. NOS2: nitric oxide synthase; LPS: lipopolysaccharide; IFNγ: interferon gamma; TNFα: tumor necrosis factor alpha; ANOVA: analysis of variance.