Association between ccttt pentanucleotide repeat in the inducible nitric oxide synthase promoter polymorphism and achalasia

Background and aim: It is suggested that achalasia represents an autoimmune disorder in which a triggering factor (probably a virus) is the starter of an uncontrolled destruction of inhibitory neurons of the LES (myenteric plexus), mainly those expressing nitric oxide synthase (nNOS). Nitric oxide may represent an ideal candidate to explain the spread of inflammation and inhibitory nerve degeneration occurring in achalasia patients because: a) depending upon its concentration, it is involved in both defence against infections and inhibitory neurotransmission; b) excessive concentrations of NO have been demonstrated In Vitro to be neurotoxic, particularly for NOS expressing neurons; c) its production by different isoforms of NOS is genetically regulated. The aim of the present study was to assess whether the functional polymorphism in the pentanucleotide repeat (CCTTT) of iNOS gene promoter is involved in susceptibility to achalasia. Methods: Genomic DNA was isolated from peripheral leukocytes of 181 unrelated Italian Caucasian patients with sporadic achalasia and 220 healthy subjects matched for age (+/-5 yr) and gender. Genotyping of the pentanucle- otide repeat (CCTTT)n, in the iNOS promoter was performed by PCR and by capillary electrophoresis. PCR fragments were sized by Genescan software. Data were analysed by Fisher's exact test : a) considering allele frequencies (i.e. n of repeats) ; b) dichomotizing the subjects in those having long and short repeats form (> 11 and < 12, respectively). Results: The distribution of alleles having various repeat numbers ranged from 8 to 15, with a peak frequency at 12 repeats for both controls and patients. Analysis of the allele frequencies revealed that individuals carrying 10 and 12 CCTTT repeats were respectively less and more frequent in achalasia (16 vs 28%, OR 0.5, 95% CI 0.3-0.5, p= 0.008 and 34 vs. 25% OR 1.6, 95% CI 1-2.4, p= 0.04). Nosignificant differences were observed for the other (CCTTT)n. Moreover achalasia individuals having more than 11 repeats were more frequent (47 vs. 34%, OR 1.7, 95% CI 1.1-2.6, p= 0.009). Conclusion: We showed that higher pentanucleo- tide repeats in the iNOS gene promoter are involved in the susceptibility to sporadic achalasia. Since In Vitro data showed that the iNOS promoter activity increases in parallel with the repeat number of (CCTTT)n, we conclude that individuals carrying longer forms have an increased risk of achalasia by higher nitric oxide productio