AIM： TO detect the germline mutations Of hMLH1 and hMSH2 based on mRNA sequencing to identify hereditary non polyposis oolorectal cancer （HNPCC） families. METHODS： Total RNA was extracted from peripberal blood of ...AIM： TO detect the germline mutations Of hMLH1 and hMSH2 based on mRNA sequencing to identify hereditary non polyposis oolorectal cancer （HNPCC） families. METHODS： Total RNA was extracted from peripberal blood of 14 members from 12 different families fulfilling Amsterdam criteria II. mRNA of hMLH1 and hMSH2 was reversed with special primers and heat-resistant reverse tmnscriptase, cDNA was amplified with expand long template PCR and cDNA sequendng analysis was followed. RESULT： Seven germline mutations were found in 6 families （6/12, 50%）, in 4 hMLH1 and 3 hMSH2 mutations （4/12, 33.3%）; （3/12, 25%）. The mutation types involved 4 missense, 1 silent and 1 frame shift mutations as well as 1 mutation in the non-coding area. Four out of the seven mutations have not been reported previously. The 4 hMLH1 mutations were distributed in exons 8, 12, 16, and 19. The 3 hMSH2 mutations were distributed in exons 1 and 2. Six out of the 7 mutations were pathological, which were dislTibuted in 5 HNPCC families. CONCLUSION： Germline mutations of hMLH1 and hMSH2 can be found based on cDNA sequencing so as to identify HNPCC family, which is highly sensitive and has the advantages of cost and time saving.展开更多
AIM： To establish and validate the mutation testing for identification and characterization of hereditary non-polyposis colorectal cancer （HNPCC） in suspected Chinese patients. METHODS： Five independent Chinese ki...AIM： To establish and validate the mutation testing for identification and characterization of hereditary non-polyposis colorectal cancer （HNPCC） in suspected Chinese patients. METHODS： Five independent Chinese kindreds with HNPCC fulfilling the classical Amsterdam criteria were collected. Genomic DNA was extracted after informed consent was obtained. The coding region of hMSH2 and hMLH1 genes was detected by polymerase chain reaction （PCR） and denaturing high-performance liquid chromatography （DHPLC）. Mutations identified in the proband by DHPLC were directly sequenced using a 377 DNA sequencer, analyzed with a basic local alignment tool （BLAST）, and tested in the corresponding family members by direct DNA sequencing. RESULTS： Mutations were identified in two Chinese HNPCC kindreds. One was the missense mutation of hMSH2 c.1808A→G resulting in Asp 603 Gly identified in the proband of the fifth HNPCC （HNPCCS） kindred. In the HNP5 kindred, three family members were found to have this mutation and two of them had colorectal cancer. The other mutation of hMLH1 c.1882A→G was identified in the HNP2 kindred＇s proband, which might be the nonsense mutation analyzed by BLAST. CONCLUSION： Pedigree investigation and mutation testing of hMSH2 and hMLH1 are the practical methods to identify high-risk HNPCC patients in China.展开更多
AIM: To study the status of hMLH1 gene point mutations of gastric cancer kindreds and gastric cancer patients from northern China, and to find out gene mutation status in the population susceptible to gastric cancer. ...AIM: To study the status of hMLH1 gene point mutations of gastric cancer kindreds and gastric cancer patients from northern China, and to find out gene mutation status in the population susceptible to gastric cancer. METHODS: Blood samples of 120 members from five gastric cancer families, 56 sporadic gastric cancer patients and control individuals were collected. After DNA extraction,the mutations of exon 8 and exon 12 of hMLH1 gene were investigated by PCR-SSCP-CE, followed by DNA sequencing.RESULTS: In the five kindreds, the mutation frequency was 25% (5/16) for the probands and 18% (19/104) for the non-cancerous members, which were significantly higher than the controls (P＜0.01 x2 = 7.71, P＜0.01 x2 = 8.65, respectively). In the sporadic gastric cancer, the mutation frequency was 7% (4/56), which was similar to that (5/100) in the healthy controls. The mutation point of exon 8 was at 219 codon of hMLH1 gene (A-G), resulting in a substitution of Ile-Val (ATC-GTC), whereas the mutation of exon 12 was at 384 codon of hMLH1 gene (T-A) resulting in a substitution of Asp-Val (GTT-GAT), which were the same as previously found in hereditary nonpolyposis colorectal carcinoma.CONCLUSION: The members of gastric cancer families from northern China may have similar genetic background of hMLH1 gene mutation as those of hereditary nonpolyposis colorectal carcinoma.展开更多
基金Supported by the Key Programs of Shanghai Medical Subjects, No. 05 Ⅲ 004
文摘AIM： TO detect the germline mutations Of hMLH1 and hMSH2 based on mRNA sequencing to identify hereditary non polyposis oolorectal cancer （HNPCC） families. METHODS： Total RNA was extracted from peripberal blood of 14 members from 12 different families fulfilling Amsterdam criteria II. mRNA of hMLH1 and hMSH2 was reversed with special primers and heat-resistant reverse tmnscriptase, cDNA was amplified with expand long template PCR and cDNA sequendng analysis was followed. RESULT： Seven germline mutations were found in 6 families （6/12, 50%）, in 4 hMLH1 and 3 hMSH2 mutations （4/12, 33.3%）; （3/12, 25%）. The mutation types involved 4 missense, 1 silent and 1 frame shift mutations as well as 1 mutation in the non-coding area. Four out of the seven mutations have not been reported previously. The 4 hMLH1 mutations were distributed in exons 8, 12, 16, and 19. The 3 hMSH2 mutations were distributed in exons 1 and 2. Six out of the 7 mutations were pathological, which were dislTibuted in 5 HNPCC families. CONCLUSION： Germline mutations of hMLH1 and hMSH2 can be found based on cDNA sequencing so as to identify HNPCC family, which is highly sensitive and has the advantages of cost and time saving.
基金The Special Funds of China Education Ministry for Returnees, No. 2003-14
文摘AIM： To establish and validate the mutation testing for identification and characterization of hereditary non-polyposis colorectal cancer （HNPCC） in suspected Chinese patients. METHODS： Five independent Chinese kindreds with HNPCC fulfilling the classical Amsterdam criteria were collected. Genomic DNA was extracted after informed consent was obtained. The coding region of hMSH2 and hMLH1 genes was detected by polymerase chain reaction （PCR） and denaturing high-performance liquid chromatography （DHPLC）. Mutations identified in the proband by DHPLC were directly sequenced using a 377 DNA sequencer, analyzed with a basic local alignment tool （BLAST）, and tested in the corresponding family members by direct DNA sequencing. RESULTS： Mutations were identified in two Chinese HNPCC kindreds. One was the missense mutation of hMSH2 c.1808A→G resulting in Asp 603 Gly identified in the proband of the fifth HNPCC （HNPCCS） kindred. In the HNP5 kindred, three family members were found to have this mutation and two of them had colorectal cancer. The other mutation of hMLH1 c.1882A→G was identified in the HNP2 kindred＇s proband, which might be the nonsense mutation analyzed by BLAST. CONCLUSION： Pedigree investigation and mutation testing of hMSH2 and hMLH1 are the practical methods to identify high-risk HNPCC patients in China.
基金Supported by the Knowledge Innovation Program of the Chinese Academy of Sciences, No. DICP K2001A4
文摘AIM: To study the status of hMLH1 gene point mutations of gastric cancer kindreds and gastric cancer patients from northern China, and to find out gene mutation status in the population susceptible to gastric cancer. METHODS: Blood samples of 120 members from five gastric cancer families, 56 sporadic gastric cancer patients and control individuals were collected. After DNA extraction,the mutations of exon 8 and exon 12 of hMLH1 gene were investigated by PCR-SSCP-CE, followed by DNA sequencing.RESULTS: In the five kindreds, the mutation frequency was 25% (5/16) for the probands and 18% (19/104) for the non-cancerous members, which were significantly higher than the controls (P＜0.01 x2 = 7.71, P＜0.01 x2 = 8.65, respectively). In the sporadic gastric cancer, the mutation frequency was 7% (4/56), which was similar to that (5/100) in the healthy controls. The mutation point of exon 8 was at 219 codon of hMLH1 gene (A-G), resulting in a substitution of Ile-Val (ATC-GTC), whereas the mutation of exon 12 was at 384 codon of hMLH1 gene (T-A) resulting in a substitution of Asp-Val (GTT-GAT), which were the same as previously found in hereditary nonpolyposis colorectal carcinoma.CONCLUSION: The members of gastric cancer families from northern China may have similar genetic background of hMLH1 gene mutation as those of hereditary nonpolyposis colorectal carcinoma.
