Study On The Germline Mutation Of HPMS2 Gene In Hereditary Bonpolyposis Colorectal Cancer Families And Screening Significance Of BRAF/KRAS In HNPCC

Hereditary nonpolyposis colorectal cancer(HNPCC),also refered for Lynch syndrome,is characterized by an autosomal dominant inheritance of early-onset microsatellite instability(MSI)-positive colorectal cancer and an increased risk of other cancers,including cancers of the endometrium,stomach,ovary,urinary tract,pancreas, and small bowel,accounting for 2%～15%of all colorectal carcinomas and the penetrance reaching up 80%～90%.Presently,HNPCC is primarily due to heterozygous germline mutations in one of the mismatch repair genes,mainly hMLH1,hMSH2,hMSH6 and hPMS2.Mutations of MMR gene lead to the disfunction of MMR system,ultimately resulting in the development of neoplasm.The protein of hMSH2 can form the heterodimer with one of other mismatch repair proteins,hMSH6 or hMSH3.The protein complex can recognizes and binds any errors that may have occurred during DNA replication.Then a larger protein complex is recruited in the repair pathway.The hMLH1 protein is another component of the DNA MMR pathway,which can form a heterodimer with hMLH3,hPMS2 or hPMS1.The hMLH1 protein has no known enzymatic activity and probably acts as a "melocular matchmaker",in that it can recruit other DNA repair proteins to the mismatch repair complex.Diagnostic guidelines and criteria for molecular testing of suspected families have been proposed and are continuously updated.However, not all families fulfilling these criteria show mutations in mismatch repair genes.Because it is not enough to make a final diagnosis for HNPCC and it is the gold standard for HNPCC probands to be detected with the germline pathological mutation in one of MMR genes.Germline mutations in the coding regions of hMSH2 and hMLH1are known to be responsible for up to 45%-64%of all HNPCC families,and also hMSH6 accounting for 10%of HNPCC kindreds,whereas mutations of other genes are rare.We previously detected germline mutations and large genomic variantions of the entire coding regions of hMSH2/hMLH1/hMSH6 genes and the methylation of hMLH1 promoter in 24 AC probands,15 JC probands and 19 BG patients resulting in 29 germline mutations and 2 probands with exhaustive methylation of hMLH1 promoter(excluding 3 probands with part-methylation of hMLH1 promoter).So the final germline-variation rate of Chinese HNPCC was 53.4%(31/58).It was speculated that the remaining probands without hMSH2,hMLH1,or hMSH6 gene mutations might be associated with other disease genes such as hPMS2 gene,et al.So,this subset is the focus of current clinical and molecular research.In the past time,it is well likely that the rare prevalence of hPMS2 in HNPCC probands may have been hampered by the pseudogenes of hPMS2,like PMS2CL,which shared in the similar sequence,but having no function.Data from literature indicated that the hPMS2 exons from 1^st to 8^th and 10^th could be easily screened by sequencing of genomic DNA without interference of pseudogenes.However,for the exons 9^th and from 11^th to 15^th,sequencing of genomic DNA without interference of PMS2CL was complicated.Therefore,it seemed advisable to screen these exons by RNA-based screening methods,as recently proposed by Etzler et al.(2008) or by long-range PCR as proposed by Clendenning et al.(2006).In this study,the method of Long-range PCR was used for investigating of the germline variants in hPMS2 gene in those HNPCC probands without hMLH1/hMSH2/hMSH6 mutations.In addition,because of the complex with hMLH1 and hPMS2 protein,concomitant loss of hMLH1 and hPMS2 expression could be observed in those turnouts loss of hMLH1 expression.In our previous study of HNPCC,some probands with hMLH1 mutation showed the positive expression of hMLH1 protein.So we wanted to add hPMS2 protein to those probands in order to find the forecast for hMLH1 mutation.Some references reported that the hot mutation of BRAF V600E could only be detected in Sporadic Colorectal Cancer(SCRC),but not in diagnosed HNPCC or suspected HNPCC,followed with the advice of screening HNPCC from SCRC using the mutation of BRAF gene.And because of the mural exclusion between BRAF mutation and KRAS mutation in the development of malignant neoplasm, both of them were also investigated in SCRC and HNPCC.Comparing with SCRC),HNPCC shows its own features including the melocular mechanism,clinical features as well as the methods of treatment and fellowup.So,it is much significant benefits for us to differentiate HNPCC from SCRC,not only in the clinical aspects but also in the genetic counseling for HNPCC probands and its members. Therefore,it is very significant for this study to perfect the mutation map of MMR gene in Chinese HNPCC and to make some screening strategy suitable for Chinese HNPCC. The current research project is comprised for the following three parts.PartⅠStudy on the germline mutation of hPMS2 gene in Chinese HNPCC probands using PCR-based sequencingObjectives:To analyse the germline mutation of hPMS2 gene in unrelated HNPCC probands fulfilling defferent clinical criteria in which hMSH2 and hMLH1 as well as hMSH6 mutations were excluded.To evaluate the detection of hPMS2 gene germline mutation in the molecular genetics screening of HNPCC families.Methods:The peripheral blood was collected from the probands of 26 HNPCC families fulfilling different clinical criterial in which hMSH2/hMLH1/hMSH6 mutations were excluded,of which 5 families fulfilled AC,10 additional families fulfilled JC and the remaining 11 kindreds fit BG.Genomic DNA was extracted following the manafacturer's protocol and used as the template to amplify this four exons(Exon6,7,8 and 10) by PCR. Then four overlapping sets of primers were designed that would amplify the complete coding region of hPMS2 gene by long-range PCR and these four long-range-PCR products were used as to amplify other exons(Exon1,2,3～4,5,9,11,12,13,14,15), respectively.PCR products were purified and used as the template for direct DNA sequencing.The results of sequencing were analysed by different bioanalysis software.To further investigate the pathological effects of detected missense mutations,we analyse the same exon of hPMS2 gene using PCR-based sequencing in 130 healthy persons without family history.Results:In 26 unrelated HNPCC probands fulfilling different clinical criteria in which hMSH2/hMLH1/hMSH6 mutations were excluded,one germline putation was found in family H13.The mutation was a missense mutation at c.1532C＞T of exon11 of hPMS2 gene,which was also detected in the persons of control and the rate was approximately 2.3%(3/130),and which was a new unreported coding SNP(cSNP).At the same time, many reported SNP of hPMS2 gene were detected in the 26 HNPCC probands,which were mostly in 2^nd and 5^th exons,secondly in 7^th,11^th and 15^th exons.All of them accounted for 86.1%of detected reported SNP,including the 2^nd exon accounting for 30%(78/260),the 5^th exon for 23.1%(60/260),the 7^th exon for 10%(26/260) and the 11^th exon for 9.2%(24/260)as well as the 15^th exon for 13.8%(36/260).However,none variant was detected in these exons of 1^st,3^rd,6^th,8^th,9^th and 10^th,respectively.These two exons of 2^nd and 5^th were probably the hot SNP regions of hPMS2 gene because of them accounting for 53.1%of all reported SNP.Conclusions:The germline mutation of hPMS2 gene was probably rare in the probands of Chinese HNPCC families without hMSH2/hMLH1/hMSH6 germline mutations,and might be excluded from screening for HNPCC.The missense mutation c.1532C＞T proved to be a new cSNP unreported previously.These exons of 2^ng and 5^th were probably the hot SNP regions of hPMS2 gene,which may be significant to investigate the SNP of hPMS2 genePartⅡStudy on the expression of hPMS2 protein in the probands of HNPCC familiesObjectives:To investigate the expression rate of hPMS2 protein and to evaluate the relationship between expression of hPMS2 protein and germline mutation of hMLH1 gene.Methods:Thirty tomour tissues were collected from the probands of HNPCC families fulfilling different clinical criterial including these probands which had been investigated the germline mutations and large genomic deletions of hMLH1/hMSH2/hMSH6 genes, which of 5 probands were studied the germline promoter methylation of hMLH1 gene. Immunohistochemical staining(IHC) of hPMS2 protein was perfored using Envision two-step method.The results of microsatellite instability(MSI) status and germline methylation of hMLH1 promoter as well as the data of germline mutations and large genomic deletions in MMR genes were collected.Two well-balanced tissues were collected for the positive control of hPMS2 protein.Results:The basic information of 30 HNPCC families:In 30 unrelated families,there 17 families fulfilling Amsterdam criteria(AC) including 2 fitting ACⅡ,7 additional families fulfilling Japanese criteria(JC) and the remaining 7 fitting Bethesda Guideline(BG).Of them,23 probands of HNPCC families had been detected with germline mutations of MMR gene and/or germline methylation of hMLH1 promoter,which includes as followed: 3 germline mutation and 4 large genomic deletions of hMSH2 gene,5 probands with 6 mutaions(including family H68 with two mutations) and 4 large genomic deletions of hMLH1 gene,4 germline mutations(including family H14 with SNP) and 1 large genomic deletion of hMSH6 gene,and none family with large genomic deletion or duplication of hPMS2 gene.In addition,family H2 was possessed with two mutations of hMSH2 and hMLH1 gene,H45 holding germline mutation and large genomic deletions of hMSH6 gene,as well as families H10 and H29 holding the exhaustive germline methylation of hMLH1 promoter and families H32,H42 and H46 holding part-methylation of hMLH1 gene.Howere,family H13 found the gernmline mutation of hPMS2 gene in PartⅠwas not detected the status of MSI and expression of hPMS2 protein because the tomour tissue was lost.Except these three families H20,H48 and H54 showing microsatellite stability (MSS),the remaining probands showed high-frequency MSI.Results of IHC:four probands with abnormal expression of hPMS2 protein were detected in 30 HNPCC families including 2 weak-expression and 2 lack-expression,all of which showed MSI-H.These four abnormal-expression probands included 3 fulfilling AC AND 1 fitting JC.The abnormal-expression rate of hPMS2 protein was 15.4%(4/26). And all of 4 patients with abnormal expression of hPMS2 protein were previously detected with germline variations of hMLH1 gene(including germline mutations and methylation),which included as followed:2 patients with lack-expression of hPMS2 protein were family H6(presenting germline mutation of hMLH1 gene) and H10 (presenting exhaustive methylation of hMLH1 gene promoter),respectively,while two patients with weak-expression of hPMS2 protein were family H9(showing large genomic deletion of hMLH1 gene) and H45(showing germline mutation of hMLH1 gene and also large genomic deletion of hMSH6 gene),respectively.The above results hinted that the abnormal expression of hPMS2 protein might forecast the germline variations on hMLH1 gene and the positive-forecast rate was 28.6%(4/14) in the study.Conclusions:The abnormal rate of hPMS2 expression was lower than that of hMSH2 and hMLH1 protein(40%～60%) as well as hMSH6 protein(24%).The germline variations of hMLH1 gene counld result in the abnormal expression of hPMS2 protein and hPMS2 protein could forecast the germline variantions of hMLH1 gene(28.6%). Adding hPMS2 staining in suspected HNPCC could avoid some families missed.PartⅢStudy on the mutation of BRAF/KRAS gone in HNPCC and sporadic colorectal cancerObjectives:To analyse the mutation of BRAF and KRAS gene in both of HNPCC kindreds and sporadic colorectal cancer(CRC).Combined with the expression of MLH1 protein,to compare the rate of BRAF mutation between sporadic CRC with MSI-H and HNPCC,and in China firstly discuss the screening significance of BRAF V600E for HNPCC.Further to investigate the relationship between sporadic CRC and HNPCC during the development of neoplasm.Methods:Tissues from 94 sporadic CRC and 40 unrelated probands of HNPCC families were unselectively collected(including 16 probands with germline mutations and large genomic deletions of MMR gene and germline methylation of hMLH1 promoter,in which H14 with SNP of hMSH6 gene,H29 with exhaustive methylation of hMLH1 promoter and H32 with partly methylation of hMLH1 promoter).Genomie DNA was extracted to be used for amplify the exons 11^th and 15^th of BRAF and the 2^nd exon of KRAS gene.PCR products were purified and used for a template for direct DNA sequencing.The results of sequencing was analysed by different bioanalysis software.At the same time,the well-balanced tissue was collected for the positive control of hMLH1 protein.Results:The results of BRAF mutations:two patients were deteted with BRAF V600E mutation at the 15^th exon(c.1799T＞A) in 94 sporadic CRC while none mutation was detected in 40 probands of HNPCC at exons of 11^th and 15^th,and the rate of BRAF V600E was 2.1%(2/94) in sporadic CRC.The results of KRAS mutation:33 patients were detected with 34 mutations of KRAS gene at the 2^nd exon(including 14 being G12D,11 being g12v,6 being G13D and 2 being G12S as well as 1 being V14G,and one patient with two mutations at codon 12 and 13) and the rate of KRAS mutation was 35.1%(33/94), of which 79.4%mutation were at the 12^th) codon and 17.6%at the codon 13^th.All of above muations were previously reported.Because of the mutual exclusion between BRAF and KRAS gene during the development of neoplasm,both of two patients with BRAF V600E were not detected with the mutation of KRAS gene.Five mutations of KRAS gene were detected in 40 probands of unrelated families and the rate of mutation was 12.5%(5/40),in which the proband of family H36 had been detected with large genomic deletion of hMSH2 gene and gremline mutation of hMSH6 gene.The rate of KRAS mutation at the exon 12^th(60%,3/5)-was slightly higher than the exon 13^th(40%,2/5) in HNPCC.The results of MLH1 protein in sporadic CRC:three patients displayed abnormal expression of hMLH1 protein in 94 sporadic CRC,in which one patient(07-7992) was together detected with KRAS mutation while two patients with BRAF V600E showed positive expression of hMLH1 protein.Conclusions:The rate of BRAF mutation was only detected in sporadic CRC,which might be used for the screening marker for HNPCC.However,those that were not detected the hot-spot mutation V600E of BRAF gene also could not exclude sporadic CRC.The rate of KRAS gene mutation in sporadic CRC was higher than that in HNPCC and could be detected in diagnosed HNPCC with MMR gene germline mutations.The mutual exclusion between BRAF and KRAS gene could be seen.