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A GENETIC AND EPIDEMIOLOGICAL STUDY OF HEREDITARY NON-POLYPOSIS COLORECTAL CANCER IN COLORECTAL CARCINOMA PATIENTS PRESENTING TO A TERTIARY CARE HOSPITAL Department of SGE NIMS Hyderabad 23-03-2009 THIS STUDY IS BEING DONE IN COLLABORATION WITH CENTER FOR DNA FINGERPRINTING AND DIAGNOSTICS (CDFD) Abbreviations CRC colorectal carcinoma HNPCC Hereditary Non-polyposis Colorectal Cancer APC MSI MMR PCR adenomatous polyposis coli micro satellite instability mismatch repair polymerase chain reaction Outline Background Aim Materials Methods DNA isolation Genomic Tumor Microsatellite instability Immunohistochemistry for MMR genes PCR Sanger’s sequencing What is HNPCC (Lynch Syndrome) Definition is based on demonstration of mutation in MMR genes No clinical definition Amsterdam’s criteria helps in clinically defining HNPCC Amsterdam II criteria: What is Amsterdam's criteria What are Bethesda guidelines Tests for HNPCC Screening tests Confirmatory tests Background Colorectal carcinoma in India is biologically a different disease compared to western population. Occurs in young age Rectal carcinoma constitutes half of the cases Incidence is less Majority of young CRC patients are sporadic Late presentation No studies on CRC genetics from India No reported cases of HNPCC from India Background 90% of CRC in west occur after 50years India 50-60% <50years WHY? Background In west 10% occur in young 30% of these are HNPCC This stimulated us to study about HNPCC and colorectal carcinoma in our own population Vogelstein’s pathway Background HNPCC is caused by MSI, the hallmark of HNPCC MSI is caused by MMR genes mutation MMR genes are also called caretaker genes MMR gene mutation causes HNPCC Background Bethesda guidelines are for MSI screening It is likely that Bethesda guidelines are not useful in Indian population. This hypothesis will be either proved or disproved by our study. Life time risk of cancer in HNPCC Primary objective To study the incidence of HNPCC in CRC patients presenting to NIMS. To study the mutations causing HNPCC To identify any new mutation Study the usefulness of MSI & IHC in diagnosing HNPCC To screen first degree relatives of HNPCC patients for MMR mutation Secondary objective To assess age distribution , sex distribution , site distribution of CRC To assess pathologic characteristics of CRC To assess stage of presentation INCLUSION CRITERIA 1. CRC, diagnosed in patients < 50 years 2. Synchronous or metachronous CRC or other tumors associated with HNPCC, regardless of age 3. CRC with of MSI-H or specific histology diagnosed < 60 years. 4. HNPCC related cancer <50yrs in 1 first degree relative 5. HNPCC associated tumor at any age in two first- or second-degree relatives Exclusion criteria All patients with neoadjuvant CT/RT are excluded from study. All patients who are HIV/HBsAg +ve. Patients with FAP Methods DNA isolation Genomic Tumor Microsatellite instability Immunohistochemistry for MMR genes PCR Sanger’s sequencing Case 1 24yr female with bleeding PR Sigmoid carcinomas at two synchronous locations-T2N0M0 Left hemi-colectomy T2N0M0 x2 3 yrs later - multiple carcinomas Caecum –T3N0M0 Duodenum-T2N0M0 Jejunum –T2N0M0 DNA isolation Genomic DNA Blood – easy to isolate Normal tissue Tumor DNA H&E stained 5micron slide Identify tumor cells Scrape the cells Isolate DNA DNA isolation from blood 3ml blood Potassium EDTA bottle Store at 40⁰ c Should not be frozen If frozen - should not be thawed Blood should not be stored for more than 7 days because yield will come down DNA isolation from blood Centrifuge blood after adding erythrocyte lysis buffer –30min Centrifuge @10000 rpm for 10min Discard supernatant fluid WBC pellet Add proteinkinase , DDS Incubate at 37⁰ C for 12hours Purification of DNA DNA isolation from blood Purification is a lengthy procedure WBC pellet is digested with proteinkinase Intra cellular contents are released Repeated purification and centrifugation Protein heaviest at bottom DNA next to protein Need careful extraction Once DNA is isolated it can be stored at -20⁰ c for a long time for many reactions Genomic DNA is highly concentrated Should be very careful otherwise it will contaminate other reactions PCR First described in 1985, Nobel Prize for Kary Mullis in 1993 The technique was made possible by the discovery of Taq polymerase, the DNA polymerase that is used by the bacterium Thermus aquaticus, discovered in hot springs. PCR The primary materials, or reagents, used in PCR are: DNA nucleotides, the building blocks for the new DNA Template DNA, the DNA sequence that you want to amplify Primers, single-stranded DNAs between 20 and 50 nucleotides long (oligonucleotides) that are complementary to a short region on either side of the template DNA DNA polymerase, a heat stable enzyme that drives, or catalyzes, the synthesis of new DNA PCR Sample tray and micropipettor. Each tray holds 96 samples PCR The result is a dramatic amplification of a the DNA that exists between the primers. The amount of amplification is 2 raised to the n power; n represents the number of cycles that are performed. After 20 cycles, this would give approximately 1 million fold amplification. After 40 cycles the amplification would be 1 x 1012 STEP-1 Denaturation at around 94°C : During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for example the extension from a previous cycle). STEP-2 Annealing at around 54°C : Hydrogen bonds are constantly formed and broken between the single stranded primer and the single stranded template. If the primers exactly fit the template, the hydrogen bonds are so strong that the primer stays attached STEP-3 Extension at around 72°C : The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template) THERMOCYCLER Sitamahalakshmi AGAROSE GEL ELECTROPHORESIS A method used to separate macromolecules like proteins and nucleic acids (ie DNA/RNA) based on their size and electric charge Sequencing “Sequencing” means finding the order of nucleotides on a piece of DNA . Nucleotide order determines Amino acid order, and by extension, protein structure and function (proteomics) An alteration in a DNA sequence can lead to an altered or non functional protein, and hence to a harmful effect Sequencing Historically there are two main methods of DNA sequencing: Maxam & Gilbert, using chemical sequencing Sanger, using dideoxynucleotides. Modern sequencing equipment uses the principles of the Sanger technique. The Sanger Technique Uses dideoxynucleotides (dideoxyadenine, dideoxyguanine, etc) These are molecules that resemble normal nucleotides but lack the normal -OH group. Because they lack the -OH (which allows nucleotides to join a growing DNA strand), replication stops. Normally, this would be where another phosphate Is attached, but with no -OH group, a bond can not form and replication stops The Sanger method requires Multiple copies of single stranded template DNA A suitable primer (a small piece of DNA that can pair with the template DNA to act as a starting point for replication) DNA polymerase (an enzyme that copies DNA, adding new nucleotides to the 3’ end of the template A ‘pool’ of normal nucleotides A small proportion of dideoxynucleotides labeled in some way ( radioactively or with fluorescent dyes) The template DNA pieces are replicated, incorporating normal nucleotides, but occasionally and at random dideoxy (DD) nucleotides are taken up. This stops replication on that piece of DNA The result is a mix of DNA lengths, each ending with a particular labeled DDnucleotide. Because the different lengths ‘travel’ at different rates during electrophoresis, their order can be determined. A Nanodrop readout :DNA purity & concentration is checked by spectrophotometer Preparing the wells The Sample wells are loaded with DNA to be sequenced. Great care needs to be taken to ensure that each sample goes into its assigned well. Reagents are added (water, dye, primers) in required amounts The sample wells are ‘spun’ to ensure that the DNA and reagents are mixed and at the bottom of the sample wells. The samples are run through a cycle sequencing process to get the fluorescent dyes incorporated by the DNA. The DNA and reagents are alternately heated and cooled over a 2 1/2 hour period. Mlh1 amplicons Exon 1; amplicon 1 5’ UTR: Tggggctggatggcgtaagctacagctgaaggaagaacgtgagcacgaggcactgaggtg Exon 1: ATTGGCTGAAGGCACTTCCGTTGAGCATCTAGACGTTTCCTTGGCTCTT CTGGCGCCAAA ATGTCGTTCGTGGCAGGGGTTATTCGGCGGCTGGACGAGACAGTGGTG AACCGCATCGCG GCGGGGGAAGTTATCCAGCGGCCAGCTAATGCTATCAAAGAGATGATT GAGAACTG Intron1: gtacggagggagtcgagccgggctcacttaagggctacgacttaacgggccgcgtcactc aatggcgcggacacgcctctttgcccgggcagaggcatgtacagcgcatgcccacaacgg cggaggccgccgggttccctgacgtgccagtcaggccttctccttttccgcagaccgtgtgt Microsatellites Microsatellites also known as variable nucleotide tandem repeats (VNTRs), are loci throughout the genome in which a short motif, such as a dinucleotide or mononucleotide sequence, is repeated at least several times. AAAAAAAAAAAAAAAAAAAAAAAAAAAAA CACACACACACACACACACACACACACACA Microsatellites Microsatellites refer to long segments of nontranscribed DNA that are composed of a repeating mononucleotide (eg, AAAAAAAAAAAAAAAA) or dinucleotide sequences (eg, GTGTGTGTGTGTGT). These long DNA sequences provide a simple indicator of genetic mutation rate and risk because mutations are readily apparent in these long repeating sequences. MSI MSI is the hallmark of lynch syndrome occuring in >90% of lynch syndrome. Aaltonen L.A et.al science 1993;260:812-816 Altonen et.al cancer res-1994;54;1645 MSI Only 20% to 25% of patients who have MSI-H colon cancer have an MMR gene mutation and Lynch syndrome . Barnetson RA, Tenesa A, et al. . N Engl J Med 2006;356(26): 2751–63. MSI The finding of microsatellite instability should lead to genetic testing because it is found in more than 90% of patients who have Lynch syndrome,but in only 15% to 20% of patients who have sporadic colon cancer. ICG-HNPCC Mismatch Repair Genes MMR are called caretaker genes because of their important role in policing the integrity of the genome and correcting DNA replication errors. MMR genes that undergo a loss of function contribute to carcinogenesis by accelerating tumor progression. Mismatch Repair Genes Mutations in MMR genes produce microsatellite instability. Microsatellites are repetitive sequences of DNA that appear to be randomly distributed throughout the genome. Mismatch Repair Genes Stability of microsatellite sequences is a good measure of the general integrity of the genome. MMR gene mutations result in errors in S phase when DNA is newly synthesized and copied. Microsatellite instability exists in 10% to 15% of sporadic tumors and in 95% of tumors in patients with HNPCC. Even so, only 50% of patients diagnosed with HNPCC have readily identifiable MMR mutations. Mismatch Repair Genes Stability of these sequences is a good measure of the general integrity of the genome. MMR gene mutations result in errors in S phase when DNA is newly synthesized and copied. Microsatellite instability exists in 10% to 15% of sporadic tumors and in 95% of tumors in patients with HNPCC. Even so, only 50% of patients diagnosed with HNPCC have readily identifiable MMR mutations. Age </=20 21-30 31-40 41-50 51-60 61-70 71-80 total Number 2 4 9 10 8 4 3 41 Observations Total number of cases till now Male Female <50 yrs >50 yrs Rectal ca Colonic 21 20 25 16 20 21 41 Site Caecum Ascending colon Hepatic flx. Transverse colon Splenic flex. Descending colon Sigmoid colon Rectum Total Number 4 4 2 +1 1 4+1 3 20 41 Sex distribution number male female FAMILY HYSTORY SIGNIFICANT IN 10 PATIENTS 1st degree relatives with cancer 8 patients Second primary in 2 patients Patient no 14 Patient no 14 Patient no 6,8 Patient no 2,3,4,5 for 5 exons Patient no 2,3,4,5 for 5 exons Our plan of work for next 3 months 12 patients suspected HNPCC – priority Complete investigation DNA isolation Tumor Genomic MSI IHC DNA amplification of specific gene all exons Sequencing Final result Next All patients who are included in study – MSI IHC MSI+ IHC + DNA-SEQUENCING FINAL RESULT THANK YOU