AIM： To detect the MLH1 gene promoter germline- methylation in probands of Chinese hereditary non- polyposis colorectal cancer （HNPCC）, and to evaluate the role of methylation in MLH1 gene promoter and molecular ge...AIM： To detect the MLH1 gene promoter germline- methylation in probands of Chinese hereditary non- polyposis colorectal cancer （HNPCC）, and to evaluate the role of methylation in MLH1 gene promoter and molecular genetics in screening for HNPCC.METHODS： The promoter germline methylation of MLH1 gene was detected by methylation-specific PCR （MSP） in 18 probands from unrelated HNPCC families with high microsatellite-instability （MSI-H） phenotype but without germline mutations in MSH2, MLH1 and MSH6 genes. At the same time, 6 kindreds were col- lected with microsatellite-stability （MSS） phenotype but without germline mutations in MSH2, MIH1 and MSH6 genes as controls. The results of MSP were confirmed by clone sequencing. To ensure the reliability of the results, family H65 with nonsense germline mutation at c.2228C 〉 A in MSH2 gene was used as the negative control and the cell line sw48 was used as the known positive control along with water as the blank control. Immunochemical staining of MIH1 protein was performed with Envision two-step method in those patients with aberrant methylation to judge whether the status of MLH1 gene methylation affects the expression of MLH1 protein.RESULTS： Five probands with MIH1 gene promoter methylation were detected in 18 Chinese HNPCC families with MSI-H phenotype but without germline mutations in MSH2, MLH1 and MSH6 genes. Two of the five probands from families H10 and H29 displayed exhaustive-methylation, fulfilling the Japanese criteria （JC） and the Amsterdam criteria （AC）, respectively. The other 3 probands presented part-methylation fulfilling the AC. Of the 13 probands with unmethylation phenotype, 8 fulfilled the JC and the Bethesda guidelines （BG）, 5 fulfilled the AC. The rate of aberrant methylation in MLH1 gene in the AC group （22.2%, 4/18） was higher than that in the JC/BG groups （5.6%, 1/18） in all HNPCC families with MSI-H phenotype but without germline mutations in PISH2, PIIH1 and MSH6 genes. However, no proband with methylation in MLH1 gene was found in the families with MSS phenotype and without germline mutations in MSH2, MLH1 and MSH6 genes. No expression of MLH1 protein was found in tumor tissues from two patients with exhaustive-methylation phenotype, whereas positive expression of MLH1 protein was observed in tumor tissues from patients with partial methylation phenotype （excluding family H42 without tumor tissue）, indicating that exhaustive-methylation of MLH1 gene can cause defective expression of MLH1 protein.CONCLUSION： Methylation phenotype of MLH1 gene is correlated with microsatellite phenotype of MMR genes, especially with MSI-H. Exhaustive-methylation of MLH1 gene can silence the expression of MLH1 protein. MLH1 promoter methylation analysis is a promising tool for molecular genetics screening for HNPCC.展开更多
Hereditary non-polyposis colorectal carcinoma （HNPCC） is an autosomal dominant disorder associated with colorectal and endometrial cancer and a range of other tumor types. Germline mutations in the DNA mismatch repa...Hereditary non-polyposis colorectal carcinoma （HNPCC） is an autosomal dominant disorder associated with colorectal and endometrial cancer and a range of other tumor types. Germline mutations in the DNA mismatch repair （MMR） genes, particularly MLH1, MEH2, and MEH5, underlie this disorder. The vast majority of these HNPCC-associated mutations have been proven, or assumed, given the family history of cancer, to be transmitted through several generations. To the best of our knowledge, only a single case of a de novo germline MMR gene mutation （in MEH2） has been reported till now. Here, we report a patient with a de novo mutation in MLH1. We identified a MLH1 Q701X truncating mutation in the blood lymphocytes of a male who had been diagnosed with rectal cancer at the age of 35. His family history of cancer was negative for the first- and second-degree relatives. The mutation could not be detected in the patient＇s parents and sibling and paternity was confirmed with a set of highly polymorphic markers. Non-penetrance and small family size is the common explanation of verified negative family histories of cancer in patients with a germline MMR gene mutation. However, in addition to some cases explained by non-paternity, de novo germline mutations should be considered as a possible explanation as well. As guidelines that stress not to restrict MMR gene mutation testing to patients with a positive family history are more widely introduced, more cases of de novo MMR gene germline mutations may be revealed.展开更多
基金Supported by Shanghai Medical Development Fund for Major Projects, No. 05III004Shanghai Pujiang Projects for Talents, No. 06PJ14019
文摘AIM： To detect the MLH1 gene promoter germline- methylation in probands of Chinese hereditary non- polyposis colorectal cancer （HNPCC）, and to evaluate the role of methylation in MLH1 gene promoter and molecular genetics in screening for HNPCC.METHODS： The promoter germline methylation of MLH1 gene was detected by methylation-specific PCR （MSP） in 18 probands from unrelated HNPCC families with high microsatellite-instability （MSI-H） phenotype but without germline mutations in MSH2, MLH1 and MSH6 genes. At the same time, 6 kindreds were col- lected with microsatellite-stability （MSS） phenotype but without germline mutations in MSH2, MIH1 and MSH6 genes as controls. The results of MSP were confirmed by clone sequencing. To ensure the reliability of the results, family H65 with nonsense germline mutation at c.2228C 〉 A in MSH2 gene was used as the negative control and the cell line sw48 was used as the known positive control along with water as the blank control. Immunochemical staining of MIH1 protein was performed with Envision two-step method in those patients with aberrant methylation to judge whether the status of MLH1 gene methylation affects the expression of MLH1 protein.RESULTS： Five probands with MIH1 gene promoter methylation were detected in 18 Chinese HNPCC families with MSI-H phenotype but without germline mutations in MSH2, MLH1 and MSH6 genes. Two of the five probands from families H10 and H29 displayed exhaustive-methylation, fulfilling the Japanese criteria （JC） and the Amsterdam criteria （AC）, respectively. The other 3 probands presented part-methylation fulfilling the AC. Of the 13 probands with unmethylation phenotype, 8 fulfilled the JC and the Bethesda guidelines （BG）, 5 fulfilled the AC. The rate of aberrant methylation in MLH1 gene in the AC group （22.2%, 4/18） was higher than that in the JC/BG groups （5.6%, 1/18） in all HNPCC families with MSI-H phenotype but without germline mutations in PISH2, PIIH1 and MSH6 genes. However, no proband with methylation in MLH1 gene was found in the families with MSS phenotype and without germline mutations in MSH2, MLH1 and MSH6 genes. No expression of MLH1 protein was found in tumor tissues from two patients with exhaustive-methylation phenotype, whereas positive expression of MLH1 protein was observed in tumor tissues from patients with partial methylation phenotype （excluding family H42 without tumor tissue）, indicating that exhaustive-methylation of MLH1 gene can cause defective expression of MLH1 protein.CONCLUSION： Methylation phenotype of MLH1 gene is correlated with microsatellite phenotype of MMR genes, especially with MSI-H. Exhaustive-methylation of MLH1 gene can silence the expression of MLH1 protein. MLH1 promoter methylation analysis is a promising tool for molecular genetics screening for HNPCC.
文摘Hereditary non-polyposis colorectal carcinoma （HNPCC） is an autosomal dominant disorder associated with colorectal and endometrial cancer and a range of other tumor types. Germline mutations in the DNA mismatch repair （MMR） genes, particularly MLH1, MEH2, and MEH5, underlie this disorder. The vast majority of these HNPCC-associated mutations have been proven, or assumed, given the family history of cancer, to be transmitted through several generations. To the best of our knowledge, only a single case of a de novo germline MMR gene mutation （in MEH2） has been reported till now. Here, we report a patient with a de novo mutation in MLH1. We identified a MLH1 Q701X truncating mutation in the blood lymphocytes of a male who had been diagnosed with rectal cancer at the age of 35. His family history of cancer was negative for the first- and second-degree relatives. The mutation could not be detected in the patient＇s parents and sibling and paternity was confirmed with a set of highly polymorphic markers. Non-penetrance and small family size is the common explanation of verified negative family histories of cancer in patients with a germline MMR gene mutation. However, in addition to some cases explained by non-paternity, de novo germline mutations should be considered as a possible explanation as well. As guidelines that stress not to restrict MMR gene mutation testing to patients with a positive family history are more widely introduced, more cases of de novo MMR gene germline mutations may be revealed.
