The Association Of The TMX Gene With Congenital Hypertrophic Pyloric Stenosis

Part one: Clinical Features of CHPS in Chinese Han populationObjective: To explore clinical features of Congenital Hypertrophic Pyloric Stenosis(CHPS) in Chinese Han population,and to provide the evidence for diagnosis, treatment and epidemiological investigations.Methods: Three hundred and seven hospitalized patients with CHPS were retrospectively reviewed, and data including patient's sex, onset age, body weight, other coexisting congenital defects, pyloric muscle thickness evaluated by ultrasonograph, serum electrolytes concentration and results of arterial blood gas analysis on admission were collected. The patients were divided into two groups according to the duration between first onset and admission: less than or equal to 10 days (early group)and more than 10 days(later group).Results of the arterial blood gas analysis, serum electrolyte concentration and average daily weight gain were compared between the two groups.Results: There were 262 males and 45 females in 307 patients, and the onset age ranged between 1 and 351 days. After 6 extreme cases were excluded, the mean onset age of the remaining 301 cases was 23.8±13.0 days. The birth weight ranged between 1.6 and 4.5kg, and the mean weight was 3.24±0.44kg. Coexisting congenital defects were found in 62 cases(20.2%).Pyloric muscle thickness ranged between 3 and 8 mm,and the mean was 5.4±1.1mm. For the early group, the rates of hypokalemia, hypochloraemia and hypercapnia were significantly lower than those of late group, while the daily weight increase was significantly greater.Conclusions: In Chinese Han population, the onset age of CHPS is 3~5 weeks. The mean pyloric circular muscle thickness is 5.4±1.1mm, and 20% of the patients are accompanied with other congenital difects. Infants with persistent vomiting at the age of 3~5 weeks should be suspected with CHPS, and be diagnosed as soon as possible. Part two: A whole-genome scan for CHPS in nuclear familiesObjective: To Explore the genetic risk factors of CHPS by whole genome scan.Methods: The study cohort comprised 19 nuclear families. Peripheral blood samples were collected and genomic DNA was extracted. A genome-wide scan was performed using Affymetrix Genome-Wide Human SNP6.0 array. Statistical analysis was performed by using family-based association test (FBAT) with golden helix 6.4 package.Results: One hundred and five of CHPS-related SNP loci with p values from magnitude of 10-5 were found and SNP loci whose genotypes in parents were not in agreement with Hardy-Weinberg equilibrium were excluded.and according to the possible functions of genes and P values, we found polymorphism locus rs7161242 in TMX gene was associated with CHPS.Conclusions: Polymorphism locus rs7161242 might be associated with CHPS in Chinese Han population, and TMX gene is a candidate gene for CHPS.Part three: The association of TMX gene single nucleotide polymorphism with Congenital Hypertrophic Pyloric StenosisObjective: To investigate the association of TMX gene single nucleotide polymorphism loci rs7161242 and rs7160810 with CHPS,and to explore the role of TMX gene in the pathogenesis of CHPS.Methods: In 31 CHPS patients and 60 normal controls as well as 22 CHPS nuclear families, the polymorphic loci rs7161242 and rs7160810 were genotyped with PCR and sequencing method. TDT and Chi-square test were performed for family based and case-control study, respectively. Online softwares were used to predict RNA secondary structure and to study relationships between structures and functions.Immunohistochemical assay was employed to study TMX expression in pyloric circle muscle.Results: Genotypic distributions of the two polymorphic loci in all three groups(patients, normal control and proband's parents) were in conformity with Hardy-Weinberg equilibrium(P>0.05).There were significant preferential transmission of G allele of rs7161242 from the parents to affected offspring (transmission disequilibrium test, TDT: x 2= 13.76, P = 2.0×10-4) and A allele of rs7160810 from the parents to affected offspring (TDT: x 2=16.2, P=5.699×10-5). Strong linkage disequilibrium was found between the two loci with r2 being 0.757 and D' value being 0.893.Case-control study indicated the frequencies of GG genotype and G allele of rs7161242 were significantly higher in CHPS group than those in controls(70.97% vs 36.67% and 83.87% vs 61.67%, respectively), and GG genotype increased risk for CHPS (OR=4.222,95%CI:1.655~10.772,P=0.002). The frequencies of AA genotype and A allele of rs7160810 were significantly higher in CHPS group than those in controls(70.96% vs 38.33% and 83.87% vs 36.67%, respectively), and AA genotype increased risk for CHPS (OR=3.932,95%CI:1.545~10.006,P=0.012).Online analysis indicated that polymorphic locus rs7161242 was located in exon 6 splice acceptor site, which will change entropy and free energy. The maximum entropy of genotype TT was lower than that of genotype GG in rs7161242 (-19.55 VS -22.22), and free energy of RNA secondary structure of TT was also lower than that of genotype GG. The maximum entropy of genotype GG was lower than that of genotype AA in rs7160810, and the free energy of RNA secondary structure was also lower than that of genotype AA. These data indicated genotype GG in rs7161242 and genotype AA in rs7160810 are relatively unstable. Through immunohistochemical assay, we found TMX gene was expressed at the cell membrane and cytoplasm in smooth muscle, and no expression was found in nucleus. Mean optical density of positive cells calculated by semi-quantitative analysis suggested that TMX gene expression was significantly weaker in pylorus smooth muscle in CHPS patients than that in normal controls (0.132±0.062 VS 0.213±0.029, P<0.05).Conclusions: The polymorphism loci rs7161242 and rs7160810 in TMX gene are associated with CHPS in Chinese Han population, with genotype GG in rs7161242 and AA in rs7160810 being risk factors. rs7161242 G allele and rs7160810 A allele might interfere with the stability of RNA secondary structure. TMX is expressed at the smooth muscle cell membrane and cytoplasm, and no expression is found in nucleus. TMX gene expression is significantly weaker in pylorus smooth muscle of CHPS than that in normal controls. TMX gene is a candidate gene for CHPS.