【作者】 冯志强；

【导师】 聂玉强；

【作者基本信息】 广州医学院 ， 内科学， 2010， 博士

【摘要】 前言先天性肥厚性幽门狭窄(congenital hypertrophic pyloric stenosis, CHPS)是新生儿常见的消化道先天性畸形,其发病率具有种族、性别和地区差异,男性较多,占90%以上。多合并有其他先天性疾病。主要临床症状为出生后15～25天,于进食后10～30 min出现频繁呕吐胃内容物,无胆汁;体检于中上腹部可扪及橄榄样包块。主要原因在于幽门部肌肉肥厚所致的幽门管腔狭窄及胃出口的梗阻。目前病因及发病机理不清,研究表明其发病与遗传及环境因素有关。先后有学者认为染色体16p12-p13位点、一氧化氮合酶(NOS1)基因、胃动素基因与其发病相关,但未获更多证据支持。最近国外学者通过全基因组扫描分析,认为瞬时受体电位离子通道蛋白6(transient receptor potential channel 6, TRPC6)可能为CHPS的易感基因。以上研究基本是针对国外人群进行的,尚无我国的相关深入研究。故本研究先通过对307例住院CHPS患者进行临床数据的回顾性分析,研究其临床发病特点;然后选择19个核心家系(患者和父母双亲),进行全基因组芯片扫描,初步探索其发病相关基因位点;再扩大样本,采用测序的方法进行基因分型,针对初步确定的候选基因位点(TMX基因的多态性位点rs7161242及rs7160810)进行无关个体的病例-对照分析及核心家系的传递不平衡检验(Transmission/Disequilibrium Test,TDT),进一步验证其与CHPS的关系,并利用生物信息学方法,研究多态性位点的不同基因型对基因的结构及功能的影响;最后采用免疫组化方法,研究多态性位点所在的TMX基因在CHPS患者幽门平滑肌组织中的表达。第一部分中国汉族人群先天性肥厚性幽门狭窄临床特征目的:探讨中国汉族人群先天性肥厚性幽门狭窄(CHPS)的临床发病特点,为诊断、治疗,以及遗传易感基因研究和流行病学调查提供依据。方法:对307例CHPS住院患儿的病历资料进行回顾性总结和分析,观察项目包括患儿性别、发病年龄、体重变化、入院时电解质及动脉血气情况、B超检查结果及合并其他先天性疾病情况。将发病10天内治疗者列为早期组,而超过10天者列为晚期组,比较这两组动脉血气、电解质及日平均体重增加的差别。结果:307例患儿中男性262例,女性45例,发病年龄1~351天,去掉离散程度较大者6例,其余301例平均发病年龄23.8±13.0天。患儿出生体重1.6~4.5kg,平均3.24±0.44kg;只有1对同卵双胞胎同时患病;合并其它先天性疾病共62例(20.2%),前二位的是心血管和消化系统的先天性异常。幽门环肌B超厚度3~8mm,平均5.4±1.1mm。早期组日平均体重增加明显大于晚期组,而低钾血症、低氯血症及高碳酸血症发生率明显低于晚期组(P<0.05);血钠与血pH值无显著性差异。结论:中国汉族人群CHPS以男性为主,发病年龄为3~5周,幽门环肌厚度5.4±1.1mm,合并其它先天性疾病比例较高,但家庭聚集发病少见。对于在出生后3~5周内出现持续性呕吐患儿,应高度警惕CHPS,并应尽快诊治,以减少低氯低钾性碱中毒的发生,避免病情进一步恶化。第二部分CHPS核心家系的全基因组芯片扫描分析目的:从全基因组范围内探索CHPS发病相关基因。方法:对由19个患儿及父母组成的核心家系,采用Affymetrix公司的SNP 6.0基因芯片进行全基因组芯片扫描,并用golden helix 6.4软件包的FBAT模块进行分析,搜索与CHPS发病相关的基因及位点,对所发现的有显著性意义的位点,进一步根据基因的功能及P值,选择候选基因。结果:以p值达10-5为判定标准,总共发现105个SNP位点,通过初步筛选,发现位于硫氧还蛋白相关跨膜蛋白1基因(thioredoxin-related transmembrane protein 1,TMX)上的多态性位点rs7161242与中国汉族人群CHPS发病相关联。结论:TMX基因的rs7161242位点与中国汉族人群CHPS发病相关联。第三部分TMX基因与先天性肥厚性幽门狭窄遗传易感性的关联研究目的:探索TMX多态性位点rs7161242[c.492T>G]及rs7160810[c.648G>A]与CHPS发病的相关性。方法:应用PCR及测序的方法,对多态性位点rs7161242及rs7160810进行基因分型,对22个核心家系进行TDT分析,进一步对31例CHPS患儿与60名正常对照采用病例-对照研究,来探索这两个SNP位点与CHPS发病的相关性,并利用在线软件对这两个SNP位点进行生物信息分析,研究其多态性与TMX基因结构与功能之间的关系,进一步利用免疫组化方法来研究TMX基因表达与CHPS发病的关系。结果:测序结果验证了芯片扫描的准确性,未发现新的突变位点;患儿、父母及正常对照者组内这两个多态性位点的Hardy-Weinberg平衡检验均P>0.05,TDT检验提示多态性位点rs7161242的G等位基因及rs7160810的A等位基因的均与CHPS发病相关,其P值分别为2.0×10-4和5.699×10-5;连锁不平衡分析结果提示:这两个位点的r2为0.757,D’值为0.893,成紧密连锁。病例-对照研究提示rs7160810位点A等位基因纯合子患病风险明显增加(OR=3.932,95%CI:1.545~10.006, x 2 =8.924,P=0.012);rs7161242位点G等位基因纯合子患病风险明显增加(OR=4.222,95%CI:1.655~10.772, x 2=9.630,P=0.002)。在线分析发现多态位点rs7161242位于第6外显子剪接受点(acceptor site)区域内,会导致熵值和自由能的改变。rs7161242位点的TT基因型的最大熵低于GG基因型(-19.55 VS -22.22),所转录的RNA的二级结构的自由能也低于GG基因型;rs7160810位点GG基因型的最大熵也低于AA基因型,所转录的RNA的二级结构的自由能也低于AA基因型,提示rs7161242位点的GG基因型及rs7160810位点的AA基因型相对不稳定,且其转录的mRNA也相对不稳定。免疫组化结果提示TMX基因在幽门平滑肌细胞膜和细胞浆表达,未发现在细胞核中表达。采用Image-Pro Plus 6.0(IPP)图像分析系统测量阳性细胞平均光密度值(IOD mean,MOD),半定量分析发现TMX基因在CHPS患儿幽门部平滑肌的表达量明显弱于正常对照者(0.117±0.033 VS 0.229±0.044,P<0.001)。结论:1、TMX基因的多态性位点rs7161242[c.492T>G]及rs7160810[c.648G>A]与中国汉族人群CHPS发病密切相关。rs7161242位点GG基因型及rs7160810位点AA基因型是CHPS患病的危险因素。2、TMX基因的两个多态性位点rs7161242[c.492T>G]及rs7160810[c.648G>A]会影响RNA的二级结构。3、TMX基因在平滑肌细胞膜及细胞浆中表达,未发现在细胞核中表达。4、TMX基因在CHPS患儿的幽门环形肌中表达显著低于正常对照。更多还原

【Abstract】 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.更多还原