Study Of Screening Hereditary Nonpolyposis Colorectal Cancer Families Based On MRNA Sequencing

Hereditary nonpolyposis colorectal cancer (HNPCC) is one of the most common autosomal dominantly inherited cancer syndromes and accounts for 2%～15% of all colorectal cancers. Compared with sporadic colorectal cancer (SCRC), HNPCC shows its own characteristics associated with the molecular mechanism, clinical features, as well as the methods for treatment, and management of HNPCC families. There are practical benefits to differentiate HNPCC from SCRC, which interests not only in the clinic but in genetic counseling of HNPCC families as well. HNPCC has drawn worldwide recognition, and several developed countries have established institutions such as HNPCC genetic counseling centers. International Collaborative Group on Hereditary Nonpolyposis Colorectal Cancer (ICG-HNPCC) was founded. Based on the clinical features and family histories of HNPCC, since 1990, several clinical criteria for the diagnosis of the concerned families have been established, such as Amsterdam criteria, Japanese criteria, Bethesda Guidelines and so on. A lot of reseaching information showed wide mutation sites in MMR genes, and no hot sites of the mutations associated with HNPCC have been found. There is little information to show what the mutations are in Chinese. CRC is one of the commonest malignant tumors in China, it has been the second commonest in female, and the third commonest in male, what is more, its incidence is gradually climbing. The number of small families is increasing due to family planning in China, which makes HNPCC family histories not very clear. Therefore, it is urgent to conduct a comprehensive study in the field.The current research project is comprised of the following three parts.Objective: To set up a novel method to identify HNPCC families in China based on peripheral blood mRNA sequencing with hot-resistance reverse transcriptase and special primers . To detect the types and spectrums of mutations in MLH1 and MSH2 in Chinese.Methods: The peripheral blood was collected from the members 19 fulfilling Amsterdam Criteria (AC), 11 additional fulfilling Japanese Criteria (JC), and remaining 29 fitting Bethesda Guideline (BG) families. RNA was extracted from the blood, and reserve transcription was performed with hot-resistance reserve transcriptase and special primers of MLH1 and MSH2. cDNA was amplified, and sequencing analysis was followed. Sequencing analysis based on genomic DNA was employed to verify the several novel mutations found by means of the technique based on mRNA sequencing.Results: In 17 unrelated families selected by the different clinical criteria, 15 germ-line mutations were found with 9 (60%, 9/15) in MLH1 and 6 (40%, 6/15) in MSH2. Two mutations were found in two same families respectively. Among the 15 germ-line mutations, 6 have been reported as pathological ones, and additional 12 haven' t been reported. There was a wide spectrum of mutation type including missense, frame shift, stop-codon, silent, and noncoding area mutations. The mutations in MLH1 distributed in exons 12 (2), 2 (2), 16 (2), 8 (1), 4 (1) and 19 (1) respectively, while the mutations in MSH2 distributed in exons 3 (1), 1 (1), 2 (1), 7 (1), 12 (1), and 13 (1) respectively.Conclusions: The technique of sequencing analysis based on mRNA can find the germ-line mutations in MMR genes with a lower cost advantage compared with that of sequencing analysis based on genomic DNA. MLH1 seems to be involved more frequently than MSH2 in Chinese HNPCC families. Missense mutation might be the main type in Chinese HNPCC families. Different clinical criteria predict mutations with different sensitivities, Amsterdam Criteria are the most sensitive ones, Bethesda criteria are the lowest ones. The germ-line mutations in MLH1 seem to bemore frequently involve exons 2, 12, and 16, while the germ-line mutations in MSH2 seem to be more frequently involve exon 3.Part 2 Preliminary Functional Evaluation of the Novel Germ-line Missense Mutations in MLH1 and MSH2Objectives: To evaluate whether the two novel germ-line mutations in MLH1 and three novel ones in MSH2 are pathological. To judge whether the families with the novel mutations are HNPCC ones.Methods: The suffering spectrum of the families with the novel germ-line mutations were arranged, and the tumor tissues of the patients with novel germ-line mutations were collected. Microsatellite instability (MSI) status was studied with a panel of microsatellite markers (5 loci) proposed by ICG-HNPCC including mononucleotide repeat BAT26, BAT25 and dinucleotide repeat D2S123, D5S346, D17S250. The amplified PCR product for each locus was accomplished by capillary electrophoresis using the ABI 310 Genetic Analyzer. MSI status was analyzed with GeneScan software. Immunohistochemical staining of MSH2 and MLH1 proteins was performed using Envision two-step method. To take the stroma and lymphocytes in the tumor as the control. The function of the novel germ-line mutations was evaluated by means of combining the results of IHC and MSI with the clinical information from the families.Results: Proband information: There were 20 persons to suffer from HNPCC and concerning tumors from the families with the novel germ-line mutations, the first CRC average youngest age was 36. 7 years old at diagnosis, and 8 persons suffered from proximal colon cancer, and 10 ones suffered from distal that. Synchronous and metachronous multiple CRCs occurred in 3 patients. Results of MSI: All the six patients' tumor tissues displayed MSI, one patient had MSI in 5 loci, three patients MSI in 4 loci, and two patients in 3 loci respectively. Results of IHC: In the two patients' tumor tissues with MLH1 mutations, one displayed the lack of MLH1 protein expression, another displayed the weak expression of MLH1 protein, while MSH2 protein displayed the normal expression. All the fourpatients' tumor tissues with MSH2 mutations displayed the lack of MSH2 protein expression, while displaying the normal expression of MLH1 protein.Conclusions: The mutations in MLH1, Vall235Gly at codon 412 exon, Prol742Leu at codon 581 exon 16, as well as the mutations in MSH2, Thrl937Arg at codon 646 exon 13, Val1100Glu at codon 367 exon 7 and Asn379His at codon 127 exon 3, are preliminarily considered to be pathological ones.The six families are HNPCC ones.Part 3 Quantity analysis of MLH1 mRNA in HNPCC Clinical FamiliesObjectives: To detect mRNA expression of MLH1 gene in the HNPCC clinical families fulfilling different criteria. To set up a new molecular method to screen HNPCC families based on peripheral blood.Methods: To collect the same peripheral blood samples as these in the first part of the study, and total RNA extracting was followed. Reverse transcription was performed with hot-resistance reverse transcriptase and random primers. Real-time reverse transcription polymerase chain reaction (Real-time RT-PCR) was used to investigate the expression of MLH1 mRNA in the samples, and to take conventional gene HBA2 as the control. The results of real-time RT-PCR was analyzed with opticon monitor 1.07 software. The RNA quantity and difference were corrected with the copy number of MLH1 gene of every sample being divided by the corresponding copy number of HBA2 gene. To compare the mRNA expression differences of MLH1 gene between the different HNPCC clinical families, and the differences between the individuals with mutations and the individuals with no mutations in the families fulfilling Amsterdam Criteria.Conclusions: The technique, Real-time RT-PCR, can be used to perform quantity analysis of MLH1 mRNA. There is a significant difference between the individuals with mutations and the individuals with no mutations among the members fulfilling Amsterdam criteria. There are no significant differences between the different HNPCC clinical criteria.