Background & Aims: The role of the mismatch repair gene PMS2 in hereditary nonpolyposis colorectal carcinoma (HNPCC) is not fully clarified. To date, only 7 different heterozygous truncating PMS2 mutations have be...Background & Aims: The role of the mismatch repair gene PMS2 in hereditary nonpolyposis colorectal carcinoma (HNPCC) is not fully clarified. To date, only 7 different heterozygous truncating PMS2 mutations have been reported in HNPCC-suspected families. Our aim was to further assess the role of PMS2 in HNPCC. Methods: We performed Southern blot analysis in 112 patients from MLH1- , MSH2- , and MSH6- negative HNPCC-like families. A subgroup (n = 38) of these patients was analyzed by denaturing gradient gel electrophoresis (DGGE). In a second study group consisting of 775 index patients with familial colorectal cancer,we performed immunohistochemistry using antibodies against MLH1,MSH2, MSH6, and PMS2 proteins. In 8 of 775 tumors, only loss of PMS2 expression was found. In these cases, we performed Southern blot analysis and DGGE. Segregation analysis was performed in the families with a (possibly) deleterious mutation. Results: Seven novel mutations were identified: 4 genomic rearrangements and 3 truncating point mutations. Three of these 7 families fulfill the Amsterdam II criteria. The pattern of inheritance is autosomal dominant with a milder phenotype compared with families with pathogenic MLH1 or MSH2 mutations. Microsatellite instability and immunohistochemical analysis performed in HNPCC-related tumors from proven carriers showed a microsatellite instability high phenotype and loss of PMS2 protein expression in all tumors. Conclusions: We show that heterozygous truncatingmutations in PMS2 do play a role in a small subset of HNPCC-like families. PMS2 mutation analysis is indicated in patients diagnosed with a colorectal tumor with Absent staining for the PMS2 protein.展开更多
目的探讨新生儿重症肺炎死亡病例中大肠埃希菌(E.coli)感染的地位。方法收集北京儿童医院新生儿重症肺炎组200例和非感染性疾病(对照组)34例的尸检病理标本,应用PCR和Southern blot技术检测E.coli特异性uidA基因片断,并复习患儿临床资...目的探讨新生儿重症肺炎死亡病例中大肠埃希菌(E.coli)感染的地位。方法收集北京儿童医院新生儿重症肺炎组200例和非感染性疾病(对照组)34例的尸检病理标本,应用PCR和Southern blot技术检测E.coli特异性uidA基因片断,并复习患儿临床资料。结果肺炎组中,PCR和Southern blot对E.coli检出率明显高于对照组(PCR:5.0% vs. 0,掊2=11.37,P<0.01;Southern blot:21.5% vs. 5.9%,掊2=4.56,P<0.05);晚发型肺炎E.coli检出率明显高于早发型(PCR:8.2%vs.1.9%,掊2=4.18,P<0.05;Southern blot:26.8% vs. 16.5%,掊2=4.48,P<0.05)。1980年前E.coli检出率高于1981年后(Southern blot法:28.0%vs.15.0%,掊2=5.00,P<0.05)。E.coli检测阳性组和阴性组比较,临床表现的差异无统计学意义(P﹥0.05)。结论 E.coli是新生儿重症肺炎死亡病例中的重要病原。分子生物学方法检测E.coli对早期诊断有重要价值。展开更多
基于SYBR Green I荧光染料与双链DNA结合产生荧光的原理,建立一种痕量DNA荧光检测方法。采用不同稀释倍数的SYBR Green I荧光染料做DNA的标准曲线,确定SYBR Green I荧光染料的最佳稀释倍数。比较荧光法和Southern blotting杂交法定量DN...基于SYBR Green I荧光染料与双链DNA结合产生荧光的原理,建立一种痕量DNA荧光检测方法。采用不同稀释倍数的SYBR Green I荧光染料做DNA的标准曲线,确定SYBR Green I荧光染料的最佳稀释倍数。比较荧光法和Southern blotting杂交法定量DNA。结果表明,SYBR Green I荧光染料稀释倍数为1∶10 000时线性范围广,线性关系好(R2=0.9999);荧光法和Southern blotting杂交法可以相互印证。由此可知,SYBR Green I荧光法定量DNA是一种快速、稳定的痕量DNA检测方法。展开更多
文摘Background & Aims: The role of the mismatch repair gene PMS2 in hereditary nonpolyposis colorectal carcinoma (HNPCC) is not fully clarified. To date, only 7 different heterozygous truncating PMS2 mutations have been reported in HNPCC-suspected families. Our aim was to further assess the role of PMS2 in HNPCC. Methods: We performed Southern blot analysis in 112 patients from MLH1- , MSH2- , and MSH6- negative HNPCC-like families. A subgroup (n = 38) of these patients was analyzed by denaturing gradient gel electrophoresis (DGGE). In a second study group consisting of 775 index patients with familial colorectal cancer,we performed immunohistochemistry using antibodies against MLH1,MSH2, MSH6, and PMS2 proteins. In 8 of 775 tumors, only loss of PMS2 expression was found. In these cases, we performed Southern blot analysis and DGGE. Segregation analysis was performed in the families with a (possibly) deleterious mutation. Results: Seven novel mutations were identified: 4 genomic rearrangements and 3 truncating point mutations. Three of these 7 families fulfill the Amsterdam II criteria. The pattern of inheritance is autosomal dominant with a milder phenotype compared with families with pathogenic MLH1 or MSH2 mutations. Microsatellite instability and immunohistochemical analysis performed in HNPCC-related tumors from proven carriers showed a microsatellite instability high phenotype and loss of PMS2 protein expression in all tumors. Conclusions: We show that heterozygous truncatingmutations in PMS2 do play a role in a small subset of HNPCC-like families. PMS2 mutation analysis is indicated in patients diagnosed with a colorectal tumor with Absent staining for the PMS2 protein.
文摘目的探讨新生儿重症肺炎死亡病例中大肠埃希菌(E.coli)感染的地位。方法收集北京儿童医院新生儿重症肺炎组200例和非感染性疾病(对照组)34例的尸检病理标本,应用PCR和Southern blot技术检测E.coli特异性uidA基因片断,并复习患儿临床资料。结果肺炎组中,PCR和Southern blot对E.coli检出率明显高于对照组(PCR:5.0% vs. 0,掊2=11.37,P<0.01;Southern blot:21.5% vs. 5.9%,掊2=4.56,P<0.05);晚发型肺炎E.coli检出率明显高于早发型(PCR:8.2%vs.1.9%,掊2=4.18,P<0.05;Southern blot:26.8% vs. 16.5%,掊2=4.48,P<0.05)。1980年前E.coli检出率高于1981年后(Southern blot法:28.0%vs.15.0%,掊2=5.00,P<0.05)。E.coli检测阳性组和阴性组比较,临床表现的差异无统计学意义(P﹥0.05)。结论 E.coli是新生儿重症肺炎死亡病例中的重要病原。分子生物学方法检测E.coli对早期诊断有重要价值。
文摘基于SYBR Green I荧光染料与双链DNA结合产生荧光的原理,建立一种痕量DNA荧光检测方法。采用不同稀释倍数的SYBR Green I荧光染料做DNA的标准曲线,确定SYBR Green I荧光染料的最佳稀释倍数。比较荧光法和Southern blotting杂交法定量DNA。结果表明,SYBR Green I荧光染料稀释倍数为1∶10 000时线性范围广,线性关系好(R2=0.9999);荧光法和Southern blotting杂交法可以相互印证。由此可知,SYBR Green I荧光法定量DNA是一种快速、稳定的痕量DNA检测方法。
