Single-nucleotide promoter polymorphism alters transcription of neuronal nitric oxide synthase exon 1c in infantile hypertrophic pyloric stenosis

Abstract

Infantile hypertrophic pyloric stenosis (IHPS), characterized by enlarged pyloric musculature and gastric-outlet obstruction, is associated with altered expression of neuronal nitric oxide synthase (nNOS). Here we have studied molecular mechanisms by which nNOS gene expression is altered in pyloric tissues of 16 infants with IHPS and 9 controls. A significant decreased expression of total nNOS mRNA was found by quantitative RT-PCR in IHPS after normalization against GAPDH, which predominantly affected exon 1c with a reduction of 88% compared with controls (P < 0.001). After normalization against the neuronal-specific gene PGP9.5, expression of exon 1c was still decreased (P < 0.001), whereas expression of exon 1f was increased significantly (P ‫؍‬ I nfantile hypertrophic pyloric stenosis (IHPS), with an incidence of 1-5 per 1,000 live births in whites and a marked preponderance of males to females (4:1), is the most frequent disorder requiring surgery in the first year of life (1). A genetic contribution toward the etiology of IHPS is well established with familiar aggregation, a concordance rate of 25-40% in monogenetic twins, a recurrence risk of 10% for males and 2% for females born after an affected child, and a ratio of risk of 18 for first-degree relatives compared with the general population (2).IHPS is characterized by hypertrophy and hyperplasia of the circular muscle layer of the pylorus, leading to persistent vomiting 2-12 weeks after birth (3, 4). Defective pyloric relaxation and increased pyloric smooth muscle mass have been suggested to be responsible for gastric-outlet obstruction. In most cases, surgical pyloromyotomy is the treatment of choice, but conservative management has sometimes been used successfully (5, 6). Within a few months, hypertrophy resolves and no permanent functional or morphological abnormalities have been detected after healing (5,7,8).Recent evidence suggested a reduced expression of neuronal nitric oxide synthase (nNOS) in IHPS, because the pyloric circular muscle layer shows decreased staining of nNOS-positive neurons (9) and decreased expression of nNOS mRNA normalized against GAPDH (10). Additionally, genetic-linkage analysis suggests the nNOS gene as a susceptibility locus for hereditary IHPS (11). In addition to decreased nNOS expression, other abnormalities have been described, such as a reduced amount of interstitial cells of Cajal (12) NO, generated by nNOS, is a major inhibitory transmitter in the gut. It is involved in the intrinsic and extrinsic inhibitory innervation of the pyloric muscle (14), and genetic deletion of nNOS␣ in mice causes a phenotype closely resembling IHPS with delayed gastric emptying, enlargement of the stomach, and hypertrophy of the muscle layer of the antrum (15, 16). Furthermore, deletion of cGMP-dependent kinase I, the downstream target of the NO͞cGMP signaling pathway, leads to a similar phenotype with pyloric stenosis, delayed gastric emptying, and thickened pyloric muscle (17). Additional evidence for an important role of the NO͞cGMP...

Genome-wide linkage analysis in families with infantile hypertrophic pyloric stenosis indicates novel susceptibility loci

“…14 Furthermore, a functional polymorphism in the NOS1 gene promoter that affects the transcription was found to be associated with IHPS in a small material of 16 patients. 13 This finding could neither be confirmed in our material consisting of 83 IHPS patients, 15 nor by another recent study of 56 cases. 16 The diverging results of the NOS1 gene studies could be explained by genetic heterogeneity of IHPS.…”

“…11 Neuronal nitric oxide synthase (NOS1) is to date the only gene reported with evidence as an IHPS susceptibility locus. However, the gene has not been mutation analyzed in IHPS patients, but linkage to an intragenic marker 12 and association to a promoter polymorphism 13 are reported. Neither of these findings could be confirmed when studied in different materials [14][15][16] supporting the presence of locus heterogeneity.…”

The genetic contribution of the NO system at the glutamatergic post-synapse to schizophrenia: Further evidence and meta-analysis

“…The first casecontrol study was published in 2002 and tested a potentially functional SNP in the 3'UTR in a sample of 215 Japanese schizophrenic patients (Shinkai et al, 2002), also with positive outcome. Subsequently, our group conducted a mutation analysis, qRT PCR and haplotype analysis in Caucasian patients suffering from schizophrenia arguing that a functional promoter SNP (rs41279104), resulting in decreased expression of a reporter gene in cell culture experiments (Saur et al, 2004), is associated with disease (Reif et al, 2006a). Since then, six more case-control association studies on schizophrenia and NOS1 were published in total (Cui et al, 2010;Nicodemus et al, 2010;Okumura et al, 2009;Riley et al, 2010;Tang et al, 2008;Wang et al, 2012).…”

No association between a promoter NOS1 polymorphism (rs41279104) and Infantile Hypertrophic Pyloric Stenosis

“…Furthermore, this allele has been shown to be a risk factor for IHPS. 14 This prompted us to examine the association between the c.-84G4A SNP in the NOS1 gene and IHPS, in a large cohort of familial and sporadic cases of patients with IHPS.…”