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MutS/MSH2 & HNPCC

MutS/MSH2 & HNPCC. MMR & Hereditary Non-polyposis Colorectal Cancer. MMR (Mismatch Repair). Originally identified in bacteria: inactivation   ncrease in the rate of spontaneous mutations due to inability to repair replication errors.

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MutS/MSH2 & HNPCC

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  1. MutS/MSH2 & HNPCC MMR & Hereditary Non-polyposis Colorectal Cancer

  2. MMR (Mismatch Repair) • Originally identified in bacteria: • inactivationncrease in the rate of spontaneous mutations due to inability to repair replication errors. • Importance realized in early 1990s w/ discovery: inactivation of primary cause of corresponding human  Hereditary non-polyposis pathway colorectal cancers Sandrine Jacob, Francoise Praz. Biochemie 83 (2002) 27-47

  3. HNPCC Hereditary Non-Polyposis Colorectal Cancer www.endoskopischer-atlas.de/ k67e.htm

  4. Sandrine Jacob, Francoise Praz. Biochemie 83 (2002) 27-47 Discovery of HNPCC Study kindreds using linkage analysis etiology of CRC (colorectal cancer) map loci on chromosome 2p21-22 and 3p21. MutS MutL hMSH2 hMLH1 Identified regions of Loss-of-Heterozygosity (LOH) in tumors by:  MicroSatellite Instability (MSI+) cause of MSI- mutator phenotype exhibited by the MMR-deficient bacteria & yeast. Led to identification hMSH2 and hMLH1

  5. Sandrine Jacob, Francoise Praz. Biochemie 83 (2002) 27-47 The Cleveland Clinic http://www.clevelandclinic.org/registries/inherited/hnpcc.htm Key Point MSI in 90% of the HNPCCtumors  also in 15-20% of the sporadic colon cancers. MSI is not only confined to the proximal colon. Exhibited in 10-45% of • Pancreatic • Gastric • Breast • Ovarian • small-cell lung cancers

  6. MutS/MSH2’s NORMAL role in Cancer (HNPCC) is to prevent MSI from occurring: Recognize damaged bases by MMR initiate signal transduction pathway trigger apoptosis. (cell cycle checkpoint) - p53 dependent - p53 independent Sandrine Jacob, Francoise Praz. Biochemie 83 (2002) 27-47

  7. How does this (MMR) work?

  8. DNA MMR in E. Coli • MMR is mediated by 3 specialized proteins: MutS, MutL, and MutH • MutS - recognition of the base/base mismatches and small insertion or deletion loops (IDL) • MutL - stimulates the endonuclease activity of MutH when engaged in a complex with MutS • MutH- plays the final role in MMR, as we talked about previously in this class, by acting as an endonuclease to nick the nascent strand Sandrine Jacob, Francoise Praz. Biochemie 83 (2002) 27-47

  9. Molecular cell biology / Harvey Lodish ... [et al.]. 4th ed. New York : W.H. Freeman, c2000.

  10. Important Difference b/w E. Coli and Yeast/ Humans • Signal that allows discrimination b/w the template and newly synthesized strand is still doubtful, but it DOES NOT involve DNA methylation. Sandrine Jacob, Francoise Praz. Biochemie 83 (2002) 27-47 • Molecular cell biology / Harvey Lodish ... [et al.]. 4th ed. New York : W.H. Freeman, c2000.

  11. C. Richard Boland http://www.hosppract.com/genetics/9711gen.htm

  12. Role of MutS in higher organisms • Mammals • 5 homologs of MutS (MSH2-MSH6) • 4 homologs of MutL (MLH1-MLH3 and PMS1) • Each of these play specialized or partly redundant fxns in yeast and mammals. • One to remember: MSH2, the MutS homolog However, MSH2, MSH3, and MSH6 all function in stability of nuclear DNA. <How does this work?> Sandrine Jacob, Francoise Praz. Biochemie 83 (2002) 27-47

  13. MSH2 has ability to form heteroduplexes w/ both MSH6 & MSH3, forming MutS and MutS, respectively.  MSH2 + MSH6 MutS MSH2 + MSH3 MutS  • MSH2 plays different roles depending on which protein it is associated with. Sandrine Jacob, Francoise Praz. Biochemie 83 (2002) 27-47

  14. MutS: - binds most base-base mismatches ( except CC) - loops of one or a few nucleotides. MutS: - repairs heteroduplexes w/  2 extrahelical bases. - Acts w/ MHL1 & MHL3 in yeast to repair IDL’s,  freq. Of frameshift mutations HOWEVER, MutS is the main instigator of mismatch recognition, and is also expressed at higher levels than MutS. Both: • Repair insertion/deletions (1 or 2 bp, respectively) • Repair mismatches in recombination intermediates. • Inhibit recombination b/w divergent sequences. Sandrine Jacob, Francoise Praz. Biochemie 83 (2002) 27-47

  15. As a result, the DNA-synthesizing enzymes copy a region of the template strand a second time, leading to an increase in length of nine nucleotides (yellow) in this example.

  16. MSH2 Mutant Phenotypes: • “Replication ERror" (RER), or “MicroSatellite Instability” (MSI) already discussed ! 2) Tolerance to events that would normally cause a cell to commit to apoptosis. ex. alkylating agent exposure low ionizing radiation/ chronic oxidative stress temozolomide (methylating agent)

  17. MSH2 Knockout Mice: Tolerance Phenotype

  18. Tolerance: reluctance toapoptosis Mouse ES Cells Mouse ES Cells Theodore L. DeWeese. PNAS Vol. 95, No. 20 Sep. 29, 1998

  19. HNPCC Characteristics (NEXT FEW SLIDES) Inherited in autosomal dominant fashion

  20. Cont… 1)      MSH2 = tumor suppressor. 2 hit model to induce phenotypic effect 2)  Inherited predisposition to early-onsetCRC and extracolonic epithelial-derived tumors gastrointestinal and urogenital tracts. 3) 5% of all CRC assoc. w/ germline mutations of one of the MMR genes hMSH2 or hMLH1. 4) VERY HIGH gene penetrance – lifetime risk  80% 5) HNPCC patients - diagnosed before age 50 (~45) - individuals dev. tumors ~2-3 decades earlier than individuals w/ nonfamilial sporadic colon cancer. Sandrine Jacob, Francoise Praz. Biochemie 83 (2002) 27-47

  21. And……….a few more! 6) hMSH2 mutations = 40% of the mutations detected in HNPCChMLH1 account for about 55% 7) Large genomic deletions in hMSH2 cause truncating mutations  a frequent cause of HNPCC. 9) In HNPCC tumors w/ hMSH2 mutation, SOMATIC MUTATIONS LOH at the MSH2 locus, which occur in ~10% of tumors. 10) Sporadic MSI+tumors are caused by inactivation of hMLH1 in 90% of cases. Sandrine Jacob, Francoise Praz. Biochemie 83 (2002) 27-47

  22. Detection: • Blood test to test patients for the identified genes, hMSH2 or hMLH1. • However, any HNPCC family member who has previously had CRC first must be used to identify the affected gene, and some people with HNPCC will not have abnormalities of these genes. Thomas Jefferson Univ; Kimmel Cancer Center www.kcc.tju.edu/hereditarycancer/ HNPCC/hnpcc.htm The Cleveland Clinic http://www.clevelandclinic.org/registries/inherited/hnpcc.htm

  23. Men & Women Full colonoscopy every year beginning at age 25 Or 5 - 10 years before earliest cancer diagnosed in the family. Women Annual screening for endometrial cancer beginning at age 25 - 35. CRC is nearly 100% curable if diagnosed within the early stages. Thomas Jefferson Univ; Kimmel Cancer Center www.kcc.tju.edu/hereditarycancer/ HNPCC/hnpcc.htm

  24. Treatment: • Surgery: removal of portion of the intestine that contains the cancer. • Individuals with HNPCC have VERY HIGH risk of developing a 2nd colon cancer entire colon must be removed, and the healthy small intestine be connected to the rectum. Thomas Jefferson Univ; Kimmel Cancer Center www.kcc.tju.edu/hereditarycancer/ HNPCC/hnpcc.htm The Cleveland Clinic http://www.clevelandclinic.org/registries/inherited/hnpcc.htm

  25. Questions?

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