Investigation Of Prevalence Of Mismatch Repair Gene Biology Essay
Colorectal cancer is one of the leading causes of cancer related mortality and morbidity, ranking fourth worldwide. In Sri Lanka colorectal cancer accounts for 6.8% of cancer cases in males and 4.8% in females.() The overall incidence is increasing worldwide, therefore it is important to identify, validate and implement appropriate diagnostic and prognostic indicators. Thus, this study hopes to identify the relative risk of hereditary factors with regard to onset of colorectal cancer with special emphasis on Hereditary Nonpolyposis Colorectal Cancer (HNPCC) and the predictive value of selected molecular markers.
2.2 Approximately 20% of all colorectal cancer has a hereditary component () Hereditary Nonpolyposis Colorectal Cancer is the most common form of hereditary colorectal cancer with an incidence of 5-7 %(). HNPCC is an autosomal dominant syndrome, characterized by neoplastic lesions and microsatellite instability (MSI) (). The lacks of characteristic diagnostic features have led to a certain criteria to identify HNPCC: Amsterdam criteria and Bethesda criteria. According to the Amsterdam criteria, there should be at least three relatives with an HNPCC-associated cancer (colorectal cancer, cancer of the endometrium, smallbowel, ureter, or renal pelvis, which is the most specific tumors for HNPCC). One affected person should be a first-degree relative of the other two. At least two successive generations should be affected with these cancers. At least one should be diagnosed before age50. Tumors should be verified by pathological examination and familial adenomatous polyposis (FAP) should be excluded (). As HNPCC has a close correlation with MSI the Bethesda criteria was introduces in 1999 with revision in 2002.
2.3 HNPCC is caused by germline mutations in the DNA mismatch repair genes (MMR genes). In the absence of normally functioning MMR genes there is accumulation of microsatellite replication errors () causing microsatellite instability (MSI). In HNPCC the main MMR genes found are MLH1, MSH2, MSH6 and PMS2. Of these four genes the frequency of mutations in HNPCC families of MSH2 is 60%, () while mutations in MLH1 is found in 30% (). Frequency of mutations in MSH6 and PMS2 are 5-7% and <5% repectively.() Most of these microsatellites are situated in the intron regions, but selected genes have microsatellite regions in the exon or coding regions. These target genes include receptors for growth factors (transforming growth factor – β receptor, insulin – like growth factor II receptor, activin receptor type 2), cell cycle regulators (E2F4), regulators of apoptosis (BAX), and some of the MMR genes themselves (MSH3 and MSH6). Hence, mutations of MMR genes do not directly transform the cell, but rather creates defective growth regulatory mechanisms. This is called the MSI pathway of tumourigenesis()
2.4 Oncogenic activation of signaling pathways downstream of the EGFR, such as mutation of KRAS , BRAF , or PIK3CA oncogenes, or inactivation of the PTEN tumor suppressor gene is central to the progression of colorectal cancer. () BRAF V600E hot spot mutation, which is a kinase encoding gene from the RES/RAF/MAPK has been recently been found in association with colorectal cancers with MSI (). Interestingly BRAF mutations are not associated with germline mutation of MLH1, MSH2 and MSH6 mismatch repair genes.() This indicates that BRAF mutations occur in sporadic onset colorectal cancers, and thereby can be used to differentiate MSI colorectal tumors of HNPCC origin or sporadic origin.
2.5 Alternatively, BRAF V600E is also being explored for possible resistance to EGFR targeted monoclonal antibody treatment (12) The anti–epidermal growth factor receptor (EGFR) monoclonal antibodies cetuximab (Erbitux®) and panitumumab (Vectibix®) have been recently introduced to treat CRC. Clinical evidence suggests that benefit from the EGFR monoclonal antibody inhibitors cetuximab and panitumumab is limited to a subgroup of only 10% to 30% of CRC patients[ ].As they have serious side effects, prior predictive mechanism to assess the response rate to these treatments would be of clinical significance. The frequency of the BRAF p.Val600Glu variant was 5% to 12% in CRC populations, and the presence of this variant has been found to be associated with poor survival in patients with CRC in several studies in which patients were not treated with anti-EGFR monoclonal antibodies . Therefore a analysis of response rate to EGFR monoclonal antibody in BRAF V600E mutational positive patients will be of clinical significance.
To genotype 50 patients with a history of HNPCC for the prevalence of mutations in the Mismatch Repair Genes (MLH1, MSH2,MSH6 and PMS2)
To genotype for BRAF V600E mutation prevalence in the 50 patients with HNPCC (cases) and 50 patients with colorectal cancer without a hereditary component (control) groups to determine whether the mutation of this region is a significant marker for the determination of sporadic or germline mutations in the mismatch repair genes.
To perform a subgroup analysis on those with a positive BRAF V600E mutation and response to EGFR targeted monoclonal antibody treatment
Subjects would comprise of 50 patients with hereditary nonpolyposis colorectal cancer according to the Amsterdam criteria II. Subjects for the control groups would be matched for sex, ethnicity and geographical area (district level). Subjects (cases and controls) would be recruited from University surgical wards of National Hospital of Sri Lanka, Colombo North Teaching Hospital and Human Genetics Unit, Faculty of Medicine, Colombo, Sri Lanka. The inclusion and exclusion criteria given below would apply when recruiting cases and controls.
Subjects who fulfill the following criteria would be recruited as cases,
Sex – Male and Female
HNPCC according to the Amsterdam Criteria II
Country of origin and ethnicity – Sri Lankan, Sinhalese
Being able to provide written informed consent
Sex – Male and Female
Colorectal cancer with no familial/ hereditary origin
Country of origin and ethnicity – Sri Lankan, Sinhalese
Being able to provide written informed consent
Sex – Worldwide colorectal cancer incidence does not show a sex preference. In Sri Lanka population, according to the National Cancer Control Programme prevalence of colorectal cancer in males and females is 6.8 and 4.8% respectively. Therefore, we are including both females and males in the study.
Country of origin and ethnicity - Previous studies have shown that prevalence of alleles of individual polymorphisms between different ethnic populations within the Sri Lankan population vary . Thus, to prevent any population admixture, only Sri Lankan, Sinhalese will be chosen for this study. Those of mixed ethnicity would not be recruited. The ethnicity would be established by inquiring into the paternal and maternal grandparental ethnicity of the subject.
Amsterdam criteria II - The Amsterdam criteria arose because of a meeting of the International Collaborative Group on Hereditary Non-Polyposis Colon Cancer in Amsterdam, in 1990.[HYPERLINK "#cite_note-bellizzi-4"] Following this, some of the genetic mechanisms underlying Lynch syndrome were elucidated during the 1990s and the significance of tumours outside the colon, such as those of the endometrium, small intestine and ureter, became more clear. These changes in the knowledge of the syndrome lead to a revision of the Amsterdam criteria in 1998. Amsterdam criteria were a series of clinical criteria formulated to serve as a common starting point for future research into the genetics underlying the disease.
Cases and controls with the following criteria would be excluded from the study
Subjects unable to give written informed consent
Obtaining Written Informed Consent
Patients with a positive clinical history compatible with HNPCC will be recruited based on their clinical records from each of the units. Controls will be recruited from the surgical units. Each subject will be given an information sheet which includes details about the study and a consent form to be read and signed before participating in the study. If the subjects have any questions regarding the study they will be answered before signing the consent form. Any subject may withdraw his/her consent to participate in the study at any time, with no penalty or effect on medical care or loss of benefits. Moreover, all participants will also be given contact details of the investigators and the contact details of the Ethics Review Committee (ERC) in case they need to clarify any doubts about the study.
4.3 Blood Samples
At recruitment, a sample of 10ml of venous blood will be obtained from subjects. These blood samples would be stored at -80°C. These samples would be used to carry out genotyping analysis for the genes selected. The genotyping analysis is described in section 4.6. The left over samples after genotyping would be stored in a -80°C freezer under the supervision of the supervisors of this study indefinitely for future research into genetics of colorectal cancer. Appropriate consent would be obtained for this purpose.
4.4 Data Collection
At recruitment, subjects would be personally interviewed to gather details on demographic data and clinical data. In addition, clinical data will be gathered by examining the medical records as well. Data that is collected will be entered into an electronic database in order to organize the data. Personal data will be collected to keep a track of the participants for follow up. The personal data will be kept separate from the database and not entered into the electronic database. All measures would be taken to maintain confidentiality by the investigators. No information by which subjects can be identified will be released or published. The data would not be used in a way that the subjects would be identified in any public presentation or publication. The electronic database would be kept under the care of the supervisors at the end of the study.
4.5 Molecular Genetic Analysis
DNA will be extracted from peripheral blood leucocytes from all subjects and selected mismatch repair genes will be amplified by Polymerase Chain Reaction and sequenced for mutation analysis. Polymerase Chain Reaction amplification of each exon region of MLH1, MSH2, MSH6 and PMS2 including the splice donor and acceptor sites (exon/intron boundaries) will be carried out and then sequences. The methods are described below.
4.5.1 DNA Extraction
3 -5 ml venous blood will be obtained from cases and controls as described above. Blood will be collected into EDTA containing tubes and stored at -4°C prior to DNA extraction. DNA extraction will be done using Promega Wizard® Genomic DNA purification kit according to the manufacturers protocol.
The Promega Wizard® Genomic DNA purification procedure will be carried out as follows: 300 µl of whole blood will be added to 900 µl of cell lysis solution. The mixture will be incubated for 10 minutes at room temperature to lyse the red blood cells. Following incubation, the mixture will be centrifuged briefly at 14,000 rpm for 20 seconds. Then the supernatant will be discarded without disturbing the white pellet formed. This will be followed by vigorous vortexing for about 10-15 seconds until the white blood cells are resuspended. Next 300 µl of nucleus lysis solution will be added to the microcentrifuge tubes containing the resuspended cells. This solution will be pipetted 5-6 times to lyse the white blood cells. The extraction process will be continued by adding 1.5 µl of RNase solution to the nuclear lysate and incubating at 37°C for 15 minutes and by cooling back to room temperature. This will then be followed by adding 100 µl of protein precipitation solution and vortexting vigorously for 10-20 seconds. Next the DNA extraction process will be followed by centrifugation at 14,000 rpm for 3 minutes and the supernatant will be transferred to 1.5µl microcentrifuge tubes containing 300 µl of room temperature isopropanol. This mixture will be then gently mixed until thread-like strands of DNA form a visible mass. This will be again centrifuged at 14,000 rpm for 1 minute and the supernatant will be discarded. Next 300 µl of room temperature 70% ethanol will be added to the DNA and the tubes will be gently inverted to wash the DNA pellet and the sides of the microcentrifuge tubes. Then the ethanol will be carefully aspirated and the tubes will be inverted on clean absorbent paper and the pellets will be air-dried for 10-15 minutes. Lastly, 100 µl of DNA rehydration solution will be added to the microcentrifuge tubes containing DNA and the DNA will be rehydrated by incubating the solution overnight at 4°C. The eluted DNA samples will be labeled and stored at -20°C. These samples will be used for PCR-sequencing experiments, which is described next. The left over blood samples would be stored in a -80°C freezer under the supervision of the supervisors of this study indefinitely for future research into genetics of colorectal cancer.
4.5.2 Polymerase Chain Reaction
Isolated Genomic DNA of all subjects will be genotyped by the PCR technique for each gene selected. PCR can selectively and repeatedly replicate selected segments from a complex DNA mixture. PCR will be conducted in a reaction mix containing 100-200 ng of template genomic DNA, Forward and Reverse primers, 10x PCR reaction buffer (10mM Tris-HCL, 50 mM KCl, 1.5mM MgCl2), 200 µM deoxynucleotides (dNTPs: dATP, dCTP, dGTP and dTTP) and 1U Taq DNA polymerase. PCR amplification will involve repeated cycles of heat, which will be done using a thermal cycler ABI2910??. At the end of the process the presence of amplified PCR products will be confirmed by the ABI3130 sequencer.
4.5.3 Genetic Sequencing
5µl of PCR product will be purified to remove excess primers and DNTPs by using 1U SAP and 5U of Exonuclease with 10x SAP buffer, Exonuclease Buffer and distilled water in a 10µl reaction mixture and incubated at 370C for 15minutes. The reaction will be stopped by inactivating the enzyme by further incubation for 15minutes at 800C. Sample tubes will then be cooled on ice and spun down before being subject to cycle sequencing.
Cycle sequencing reactions will be set up in a final volume of 10 µl, containing 3 µl of the enzyme treated (purified) PCR products, 4µl bigdye ready reaction mix, and approximately 5pmol (12.5µM) of the sequencing primer in a final volume of 10µl. All reaction will be set up in the dark (and samples were covered with an aluminium foil) to prevent degeneration of light sensitive dyes. The conditions for cycle sequencing will be initial denaturation for 1 minute at 960C and 25 cycles of 960C for 30seconds, annealing at 500C for 15seconds and 600C for 4minutes. The temperature cycles will be concluded with 280C for 1 minute followed by cooling. Following cycle sequencing, products will be cleaned by loading into a Centriflex® gel filteration cartridge (Edge Biosystems, USA) and subjecting it to centrifugation at 3600rpm for 2 minutes. This will be evapourated to dryness at 900C and then stored at -200C. Before sequencing 10µl of formamide will be added to the tube and sample will be denatured at 950C for 5 minutes and stored on ice till loading in to the 96 well sequencing plate.
Results will be analysed using the Statistical Package for the Social Sciences (SPSS) program.
4.6 Publication Plan
It is planned to publish the results of this study at local as well as international conferences and in peer-reviewed journals.
4.7 Ethical Issues Relevant to the Study
The cases will be recruited from those who have undergone surgical procedures due to colorectal cancer who have clinical histories compatible with HNPCC. Controls will be those treated surgically for colorectal cancer without a hereditary component in the clinical history. There will be no direct benefit to the study subjects by participating in this study. However, they will be contributing to the advancement of generalisable knowledge regarding HNPCC that may be useful in future clinical practice. There is a minimal risk of bruising due to venapuncture, which will be minimized by venapuncture being conducted by an experienced by an experienced nurse. Appropriate measures will be taken to ensure privacy of patients and to ensure confidentiality regarding the data gathered. The samples obtained will be used for future research in colorectal cancer. Appropriate consent will be obtained for this purpose such studies would be subjected to ethics review prior to commencement. Once results of the study is published, a written summary of the results, in lay language would be posted to all subjects.
4.8 Declaration of Conflict of Interests
The research team declares there is no conflict of interests in relation to this study.
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