Study On The Germline Mutation Of HMSH6 Gene, Large Genomic Deletions Of Mismatch Repair Genes And The Methylation Of The HMLH1 Promoter In Hereditary Nonpolyposis Colorectal Cancer Families

Hereditary nonpolyposis colorectal cancer(HNPCC) is one of the most common autosomal dominantly inherited cancers syndrome,accounts for 2%-15%of all colorectal cancers and the penetrance reach up to 80%-90%.Compare with sporadic colorectal cancer(SCRC),HNPCC shows its own characteristics associated with the molecular mechanism,clinical features,the methods for treatment,and management of HNPCC kindreds.Therefore,there are many benefits to differentiate HNPCC from SCRC,which interests not only in the clinic but also in genetic counseling of HNPCC kindreds.Now,many countries and territories have established the clinical diagnostic criteria for HNPCC,such as,Amsterdam criteria,Amsterdam revised criteria, Japanese criteria and Bethesda guidelines and so on.However,there are not enough to make a final diagnosis families and the patients of HNPCC only rely on the above clinical diagnostic criteria.It is a gold criteria to make a final diagnosis for HNPCC dependents on the detection of pathological mutations of mismatch repair genes.There are parts of studies in germline mutations of hMSH2 and hMLH1 genes and less clinical reports in our domestic now.Up to now,it has not been reported in the molecular genetics screening of large HNPCC families and it can not make a final diagnosis to tremendous portion of HNPCC kindreds.Previously study showed that the rate of germline mutations was account for 31%and significantly less than other studies(80%-90%) in hMSH2 and hMLH1 genes of our HNPCC families fulfilling different clinical criteria.There are less reports about the other mismatch repair genes germline muations and another type abnormalities of MMR genes in the genesis of HNPCC.Therefore,it is urgent to conduct a comprehensive study in the fields,such as,the detection of germline mutation in hMSH6 gene,the detections of large genomic deletions in hMSH2/hMLH1/hMSH6/hPMS2 genes and the study of methylation of hMLH1 promoter in HNPCC families fitting with different clinical criteria.It is very important for full-scal molecular genetics screening HNPCC kindreds and avoiding omission the early diagnosis of parts of HNPCC families.The current research project is comprised of the following four parts.Part 1 Study on the germline mutation of hMSH6 gene in Chinese hereditary nonpolyposis colorectal cancer pedigrees using PCR based sequencingObjectives:To analysis the germline mutation of hMSH6 gene in unrelated HNPCC probands fufilling different clinical criteria in which hMSH2 and hMLH1 mutations were excluded.To evaluate the detection of hMSH6 gene germline mutation in the molecular genetics screening of HNPCC families.Methods:The peripheral blood was collected from the probands of 39 HNPCC families fulfilling different clinical criteria in which hMSH2 and hMLH1 mutations were excluded.11 families fulfilled ACⅠand ACⅡ,11 additional families fulfilled JC and the remaining 17 kindreds fit BG.Genomic DNA was extracted following the manufacturer's protocol.18 sets of primers were designed to amplify the entire coding region,including 10 exons and each splicing site of hMSH6.PCR products were purified and used as a template for direct DNA sequencing.The results of sequencing were bioinformatic analyzed by different bioanalysis software.To further investigate the pathological effects of detected missense mutations,we analyzed the same exons of hMSH6 gene using PCR-based sequencing in 137 healthy persons with no family history.To analysis the single nucleotide polymorphism(SNP) by detected in HNPCC families.Results:In 39 unrelated HNPCC probands fulfilling different clinical criteria in which hMSH2 and hMLH1 mutations were excluded,six germline mutation were found after SNP reported were excluded.The missense mutation in the case H14 at c.3488A＞T of exon6 of hMSH6 gene was also found in the persons of control,the rate was approximately 3.6%(5/137),it was a new unreported SNP.The remaining five mutations were new unreported mutations.Of them,one showed 1477G＞T at exon 4,which resulted in glutamine at codon 493 to a stop codon,this nonsense mutation would lead to a truncated hMSH6 protein whose normal function could be lost.One showed TTTT insertion at c4001-9 in intron 9,which was most likely result in splicing defect.The other three missense mutations are at codon 468(CGT＞ CAT,Arg＞His),codon 666(TCT＞CCT,Ser＞Pro) and codon 1284(ACG＞ATG,Thr＞Met),respectively.Of them,the missense mutation at the codon 468 of exon 4.3 of hMSH6 gene occurred in the H3 proband fufilling the typical AC HNPCC family.The SNP of germline hMSH6 gene was mainly distributed the 1^st,3^rd and 5^th exons in the probands of Chinese HNPCC families,they are at c.116G＞A,c.458-52T＞G and c.3306T＞A of hMSH6 gene,respectively.The three site and type of SNP might be the hot spot in the genesis of SNP in hMSH6 gene of Chinese HNPCC families.Conclusions:The rate of hMSH6 gene germline mutation is less account for 12.8%in the probands of Chinese HNPCC familes without hMSH2 and hMLH1 germline mutations,Perhaps,it is necessary to detect the germline mutation of hMSH6 gene for the above HNPCC families and its benefit to avoiding omission the early diagnosis parts of HNPCC pedigrees.The germline mutation of hMSH6 gene can occur in the patient with the typical clinical HNPCC features fulfilling Amsterdam criteria.The SNP of germline hMSH6 gene is mainly distributed the 1^st,3^rd and 5^th exons in the probands of Chinese HNPCC families,they are at c.116G＞A,e.458-52T＞G and c.3306T＞A,respectively,it is very important to study the SNP of hMSH6 gene.The SNP of c.3488A＞T at the codon 1163 of 6^th exon is a new SNP unreported previously.Part 2 Study on the expression of hMSH6 protein in the probands of hereditary nonpolyposis colorectal cancer familiesObjectives:To evaluate whether the six novel germline mutations in hMSH6 gene are pathological.To evaluate the relationship between the abnormal expression of hMSH6 protein and the another defect-phenotype of MMR gene(microsatellite instability,MSI).Methods:Fifty tumor tissues were collected from the probands of HNPCC families fulfilling different clinical criteria containing six HNPCC families with hMSH6 gene germline mutations.Immunohistochemical staining of hMSH6 protein was performed using Envision two-step method.The data of germline mutations in hMSH2,hMLH1 and hMSH6 genes and the results of microsatellite instability status were collected in forty HNPCC families,and the clinical data were also collected about six HNPCC kindreds with hMSH6 gene germline mutations.The function of the six novel germline mutations of hMSH6 gene were evaluated by means of combining the results of IHC and MSI with the clinical information from the different HNPCC families. The relationship was evaluated between the expreesion of hMSH6 protein and the status of microsatellite instability.Results:The basic information of forty HNPCC families:In fifty unrelated families, there are twenty four families fufilling Amsterdam criteria(AC) including one (ACⅡ),13 additional families fulfilling Japanese criteria(JC) and the remaining 13 patients fitting Bethesda guidelines(BG).Of them,seven germline mutation of hMSH2 gene,11 germline mutation of hMLH1 gene and six germline mutation of hMSH6 gene were detected.All the probands of HNPCC families showed High frequency microsatellite instability in tumor tissues except seven probands with MSS phenotype.The clinical data about six HNPCC families with hMSH6 gene germline mutations:In six families,there were one proband fulfilling Amsterdam criteria,2 additional families fulfilling Japanese criteria and the remaining 3 patients fitting Bethesda guidelines.There were 20 persons to suffering HNPCC and concerning tumors,the average age of first onset CRC was 42.8 years old.Metachronous multiple CRCs occurred in 3 patients.The ease of H61 suffered from a left hemicolon cancer at age 33,had endometrial and ovarian cancer at age 45,and had a right hemicolon cancer at age 51.Four persons suffered from right hemicolon cancer account for 36.4%(4/11),and 7 ones suffered from left hemicolon cancer account for 63.6%(7/11). Results of IHC:Twelve abnormal expressions of hMSH6 protein were detected in 50 probands of HNPCC families including 5 weak expression and 7 lack expression.In 6 probands' tumor tissures with hMSH6 gene germline mutation,5 patiens displayed the lack of hMSH6 protein expression,one displayed positive expression of hMSH6 protein(the proband with the mutation of hMSH6 gene was confirmed as a new unreported SNP).Five patients with weak expression of hMSH6 protein displayed MSI-H phenotype and germline mutations in hMSH2 gene,and the remaining 2 patients with lack expression of hMSH6 protein were the cases of H45 and H47.The H45 case displayed MSI-H phenotype,germline mutation of hMLH1 gene and the deletion of Exonl in hMSH6 gene(See part3),the H47 case displayed MSS phenotype and germline mutation of hMLH1 gene.The remaining 38 patients also displayed the positive expression of hMSH6 protein(Containing 32 patients with MSI-H phenotype and 6 persons with MSS phenotype).Conclusions:Five out of six germline mutations in hMSH6 gene are preliminarily considered to be pathological ones owing to display abnoraml expression of hMSH6 protein.The phenotype with weak expression of hMSH6 protein can concern with the germline mutation of hMSH2 gene.The defect expression of hMSH6 protein can occur in the patients of HNPCC families with MSS phenotype.The weak expression of hMSH6 protein has not prognosticate the germline mutation of hMSH6 gene,but lack expression of hMSH6 protein may be prognosticate the germline mutation of hMSH6 gene.Part 3 Detection of large genomic deletions and duplications of mismatch repair genes in Chinese HNPCC patients using MLPA-ArrayObjectives:To set up a new convenient molecular method to screening the copy number changes and its clinical application for the rapid screening the deletions and duplications of MMR genes in HNPCC families.Detection of large genomic deletions and duplications in hMSH2,hMLH1,hMSH6 and hPMS2 genes of HNPCC patients fulfilling different clinical criteria using MLPA-Array method,and comprehensive its role in the molecular genetics screening HNPCC families.Methods:The peripheral blood was collected from the probands of 80 HNPCC families fulfilling different clinical criteria,28 families fulfilled ACⅠand ACⅡ,19 additional families fulfilled JC and the remaining 33 kindreds fit BG.Genomic DNA was extracted following the manufacturer's protocol.The large genomic changes in hMSH2,hMLH1,hMSH6 and hPMS2 genes were detected by using MLPA-Array, every samples were tested again by the same method.The results of MLPA-Array detection were confirmed by the classic multiplex ligation-dependent probe amplification(MLPA).Analysis the whole large genomic changes of MMR genes and abnormal frequency of large genomic changes in mismatch repair genes.in Chinese HNPCC familiesResults:Nineteen patients with large genomic deletions of MMR genes were found in the probands of HNPCC families fulfilling different clinical criteria by using MLPA-Array platform.The abnormal rate accouted for 23.75%(19/80).Of them,8 patients fulfilling Amsterdam criteria,3 additional patients fulfilling Japanese criteria and the remaining 8 persons fitting Bethesda guidelines.Nineteen large genomic changes of mismatch repair genes were detected in 80 probands of HNPCC kindreds, they were all large genomic deletions not duplications.Among them,9 large genomic deletions were found in hMSH2 gene and they account for up to 47.4%(9/19),7 large genomic deletions were found in hMLH1 gene and they account for 36.8%(7/19) and the remaining 3 ones were hMSH6 gene which only account for 15.8%(3/19). However,large genomic deletions in hPMS2 gene were not found in all the probands of Chinese HNPCC families.Sixteen large genomic deletions of hMSH2 and hMLH1 genes were found(84.2%,16/19) in 80 HNPCC probands.The above results detected by MLPA-Array were confirmed by the classic MLPA analysis with 100% concordanceConclusions:MLPA-Array is a new convenient high-flux molecular method to rapid screening the deletions and duplications of MMR genes and it can apply for the molecular genetics screening of Chinese HNPCC families.The major type of large genomic changes is the deletion of MMR genes in the patients of HNPCC kindreds. The rate of large genomic deletions in MMR genes is approximately account for 23.75%(19/80),they are main distribute in the patients fulfilling Amsterdam criteria and Bethesda guidelines.The large genomic deletions of hMSH2 and hMLH1 genes are more common changes and they accout for 84.2%(16/19),the large deletions of hMSH6 gene is only account for 15.8%(3/19) and the large deletion of hPMS2 gene is not found.Therefore,it is necessary to detect the large genomic deletions in hMSH2, hMLH1 and hMSH6 genes for Chinese HNPCC families and it is very important to screening HNPCC kindreds.Part 4 Study on the methylation of hMLH1 gene promoter in the probands of Chinese hereditary nonpolyposis colorectal cancerObjectives:To detect the methylation of hMLH1 gene promoter in the patients of Chinese hereditary nonpolyposis colorectal cancer.To evaluate the role between the detection of methylation in hMLH1 gene promoter and the molecular genetics screening for HNPCC.Methods:Eighteen HNPCC families with the defect-phenotype of MMR gene(MSI-H) and without the germline muations in hMSH2,hMLH1 and hMSH6 genes were collected by us.At the same time,we collected 6 HNPCC kindreds with MSS phenotype and without germline mutations in hMSH2,hMLH1 and hMSH6 genes as the comparative group.The peripheral blood was collected from the probands of 24 HNPCC families fulfilling different clinical criteria.Genomic DNA was extracted following the manufacturer's protocol.Design the specific primers for methylation specific PCR of hMLH1 gene.The specific straps of methylation and unmethylation of hMLH1 gene were amplified by using MSP in every samples.The status of methylation in hMLH1 gene was evaluated according to specific straps of methylation and unmethylation of hMLH1 gene.The results of MSP were confirmed by using clone sequencing of PCR products.To ensure the reliability of the result,the DNA of H65 case with the nonsense mutation at c.2228C＞A in germline hMSH2 gene was used as negative control of methylation in hMLH1 gene,the DNA of cell lines sw48 was used as known positive control in the methylation of hMLH1 gene and the water as DNA was used blank control.Immunohistochemical staining of hMLH1 protein was performed by using Envision two-step method in the tumor tissues of patients with hMLH1 gene methylation.To judge whether the status of methylation of hMLH1 gene effect the expression of hMLH1 protein.Results:Five hMLH1 gene methylation was found in the patients with MSI-H phenotype and without germline mutations in hMSH2,hMLH1 and hMSH6 genes in Chinese HNPCC families.Of them,2 patients displayed exhaustive-methylation and the remaining 3 patients displayed part-methylation.Eight out of 13 patients with unmethylation phenotype fulfilling Japanese criteria and Bethesda guidelines,only five patient fitting Amsterdam criteria.All the patients with part-methylation phenotype fulfilling Amsterdam criteria.The patients of cases H10 and H29 displayed exhaustive-methylation of hMLH1 gene,they fulfilling Japanese criteria and Amsterdam criteria,respectively.The rate of abnor-methylation in hMLH1 gene in AC group(22.2%,4/18) was higher than its in JC groups(5.6%,1/18) in the HNPCC families with MSI-H phenotype and without germline mutations of hMSH2,hMLH1 and hMSH6 genes.However,no patients with methylation of hMLH1 gene was found in the HNPCC families with MSS phenotype and without germline mutations of hMSH2,hMLH1 and hMSH6 genes.Therefore,we believed that the methylation of hMLH1 gene might be relationship with microsatellite phenotype.Results of IHC by using Envision two-step method:The tumor tissues of two patients with exhaustive-methylation phenotype displayed lack expression of hMLH1 protein, however,the tumor tissues of the patients with part-methylation phenotype displayed positive expression of hMLH1 protein.So,we could conclude that exhaustive-methylation of hMLH1 gene can cause the defect-expression of hMLH1 protein.Conclusions:The rate of abnor-methylation of hMLH1 gene is approximately 27.8%(5/18) in the patients with MSI-H phenotype and without germline mutations of hMSH2,hMLH1 and hMSH6 genes of Chinese HNPCC families,however, exhaustive-methylation rate is only accout for 11.1%(2/18).Perhaps,the changes may be much lower in all the patients of HNPCC families.The methylation phenotype of hMLH1 gone concerns with microsatellite phenotype of MMR gone,the patient with MSI-H phenotype is easy to genesis the methylation of hMLH1 gene.The exhaustivemethylation of hMLH1 gene can effect the expression of hMLH1 protein,however, the part-methylation of hMLH1 gene has no effect with the expression of hMLH1 protein.hMLH1 promoter methylation analysis is a promising tool to molecular genetics screening for HNPCC but is not yet used in daily screening and diagnostics.