muts/msh2 & hnpcc mmr & hereditary non-polyposis colorectal cancer

MutS/MSH2 & HNPCCMutS/MSH2 & HNPCC

MMR & Hereditary Non-polyposis MMR & Hereditary Non-polyposis Colorectal CancerColorectal Cancer

MMR (Mismatch Repair)MMR (Mismatch Repair)

• Originally identified in bacteria:Originally identified in bacteria:• inactivationinactivation ncrease in the rate of spontaneous ncrease in the rate of spontaneous

mutations due to inability to repair replication errors.mutations due to inability to repair replication errors.• Importance realized in early 1990s w/ discovery:Importance realized in early 1990s w/ discovery:

inactivation of inactivation of primary cause of primary cause of corresponding human corresponding human Hereditary non- Hereditary non-

polyposispolyposis pathway pathway colorectal cancers colorectal cancers

Sandrine Jacob, Francoise Praz. Biochemie 83 (2002) 27-47

HNPCCHNPCC

HereditaryHereditary

Non-PolyposisNon-Polyposis

ColorectalColorectal

CancerCancer

www.endoskopischer-atlas.de/ k67e.htm

Discovery of HNPCCDiscovery of HNPCCStudy kindreds using Study kindreds using linkage analysislinkage analysis

etiology of etiology of CRCCRC ( (colorectal cancercolorectal cancer))

map loci on chromosome map loci on chromosome 2p21-222p21-22 and and 3p213p21..

MutS MutLMutS MutL

hMSH2 hMLH1hMSH2 hMLH1Identified regions of Identified regions of Loss-of-Heterozygosity Loss-of-Heterozygosity ((LOHLOH) in tumors by:) in tumors by: MicroSatellite InstabilityMicroSatellite Instability ( (MSIMSI++)) cause of cause of MSIMSI- - mutator phenotypemutator phenotype exhibited by the exhibited by the MMR-deficientMMR-deficient bacteria & bacteria & yeast. yeast. Led to identification Led to identification hMSH2hMSH2 and and hMLH1hMLH1


Key PointKey PointMSIMSI in in 90%90% of the of the HNPCCHNPCC tumorstumors also in also in 15-20%15-20% of the sporadic colon cancers. of the sporadic colon cancers.

MSIMSI is not only confined to the is not only confined to the proximalproximal colon. colon.Exhibited in Exhibited in 10-45%10-45% of of • PancreaticPancreatic• GastricGastric• Breast Breast • OvarianOvarian• small-cell lung cancerssmall-cell lung cancers


The Cleveland Clinic http://www.clevelandclinic.org/registries/inherited/hnpcc.htm

MutS/MSH2’s NORMAL role in Cancer (HNPCC) is to MutS/MSH2’s NORMAL role in Cancer (HNPCC) is to prevent MSI from occurring:prevent MSI from occurring:

Recognize damaged bases by Recognize damaged bases by MMRMMR

initiate signal transduction pathwayinitiate signal transduction pathway

trigger apoptosistrigger apoptosis..

(cell cycle checkpoint)(cell cycle checkpoint) - p53 dependent- p53 dependent

- p53 independent- p53 independent


How does this (MMR) How does this (MMR) work?work?

DNA MMR in E. ColiDNA MMR in E. Coli

MMRMMR is mediated by 3 specialized proteins: is mediated by 3 specialized proteins: MutSMutS, , MutLMutL, and , and MutHMutH

1)1) MutS - MutS - recognition of the base/base mismatches and recognition of the base/base mismatches and small small insertion or deletion loopsinsertion or deletion loops ( (IDLIDL))

2)2) MutL - MutL - stimulates the endonuclease activity of stimulates the endonuclease activity of MutHMutH when engaged in a complex with when engaged in a complex with MutSMutS

3)3) MutHMutH -- plays the final role in plays the final role in MMRMMR, as we talked , as we talked about previously in this class, by acting as an about previously in this class, by acting as an endonuclease to nick the nascent strand endonuclease to nick the nascent strand


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

Important Difference b/w E. Coli and Important Difference b/w E. Coli and Yeast/ HumansYeast/ Humans

Signal that allows Signal that allows discrimination b/w the discrimination b/w the template and newly template and newly synthesized strand is still synthesized strand is still doubtful, but it DOES doubtful, but it DOES NOT involve DNA NOT involve DNA methylation.methylation.

Sandrine Jacob, Francoise Praz. Biochemie 83 (2002) 27-471. Molecular cell biology / Harvey Lodish ... [et al.]. 4th ed. New York : W.H. Freeman, c2000.

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

Role of MutS in higher organismsRole of MutS in higher organisms

MammalsMammals 55 homologs of homologs of MutSMutS ( (MSH2-MSH6MSH2-MSH6) ) 44 homologs of homologs of MutL MutL ((MLH1-MLH3MLH1-MLH3 and and PMS1PMS1))

Each of these play specialized or partly redundant fxns in Each of these play specialized or partly redundant fxns in yeast and mammals.yeast and mammals.One to remember: One to remember: MSH2MSH2, the , the MutS homologMutS homolog

However, However, MSH2, MSH3, and MSH6MSH2, MSH3, and MSH6 all function in all function in stability of nuclear DNA.stability of nuclear DNA.

<<How does this work?How does this work?>>


MSH2MSH2 has ability to form heteroduplexes has ability to form heteroduplexes w/ both w/ both MSH6MSH6 & & MSH3MSH3, forming , forming MutSMutS and and MutSMutS, respectively., respectively.

MSH2 MSH2 plays different roles depending plays different roles depending on which protein it is associated with.on which protein it is associated with.


MutSMutS: - : - binds most base-base mismatches ( except CC) binds most base-base mismatches ( except CC)

- loops of one or a few nucleotides.- loops of one or a few nucleotides.

MutSMutS:: - repairs heteroduplexes w/ - repairs heteroduplexes w/ 2 extrahelical bases. 2 extrahelical bases.

- Acts w/ - Acts w/ MHL1MHL1 & & MHL3MHL3 in yeast to repair in yeast to repair IDL’s,IDL’s,

freq. Of frameshift mutationsfreq. Of frameshift mutations

HOWEVER, MutSHOWEVER, MutS is the main instigator of mismatch is the main instigator of mismatch recognition, and is also expressed at higher levels than recognition, and is also expressed at higher levels than MutSMutS..

Both:Both:

1)1) Repair insertion/deletions (Repair insertion/deletions (1 or 2 bp, respectively)1 or 2 bp, respectively)

2)2) Repair mismatches in recombination intermediates.Repair mismatches in recombination intermediates.

3)3) Inhibit recombination b/w divergent sequences.Inhibit recombination b/w divergent sequences.


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.

MSH2 Mutant Phenotypes:MSH2 Mutant Phenotypes:

1)1) ““Replication ERror" (RER)Replication ERror" (RER), or , or “MicroSatellite “MicroSatellite Instability” (MSI)Instability” (MSI)

already discussed ! already discussed !

2) 2) Tolerance to events that would normally cause a Tolerance to events that would normally cause a cell to commit to apoptosis.cell to commit to apoptosis.

ex. alkylating agent exposureex. alkylating agent exposure low ionizing radiation/ chronic oxidative stresslow ionizing radiation/ chronic oxidative stress temozolomide (methylating agent)temozolomide (methylating agent)

MSH2Knockout Mice:Tolerance Phenotype

Tolerance: reluctance toTolerance: reluctance to apoptosisapoptosis

Mouse ES Cells Mouse ES CellsTheodore L. DeWeese. PNAS Vol. 95, No. 20 Sep. 29, 1998

HNPCC Characteristics HNPCC Characteristics (NEXT FEW SLIDES)(NEXT FEW SLIDES)

Inherited in autosomal dominant fashionInherited in autosomal dominant fashion

Cont…Cont…11) MSH2) MSH2 = tumor suppressor. = tumor suppressor. 2 hit model to induce 2 hit model to induce

phenotypic effect phenotypic effect

2) 2) Inherited predisposition to Inherited predisposition to early-onsetearly-onset CRCCRC and and extracolonic extracolonic epithelial-derived tumorsepithelial-derived tumors

gastrointestinalgastrointestinal and and urogenitalurogenital tracts. tracts.

3) 3) 5%5% of all of all CRC CRC assoc. w/ germline mutations of one of the assoc. w/ germline mutations of one of the MMRMMR genes genes hMSH2hMSH2 or or hMLH1hMLH1..

4) VERY HIGH gene penetrance4) VERY HIGH gene penetrance – lifetime risk – lifetime risk 80% 80%5) HNPCC5) HNPCC patients patients

- diagnosed before age - diagnosed before age 50 (~45)50 (~45) - individuals dev. tumors - individuals dev. tumors ~2-3 decades~2-3 decades earlier than earlier than individuals w/ nonfamilial sporadic colon cancer.individuals w/ nonfamilial sporadic colon cancer.


And……….a few more!And……….a few more!6) hMSH26) hMSH2 mutations = mutations = 40%40% of the mutations detected in of the mutations detected in HNPCCHNPCC

hMLH1hMLH1 account for about account for about 55%55%

7) Large genomic deletions7) Large genomic deletions in in hMSH2hMSH2 cause truncating mutations cause truncating mutations a frequent cause of a frequent cause of HNPCCHNPCC..

9) 9) In In HNPCCHNPCC tumors w/ tumors w/ hMSH2hMSH2 mutation, mutation, SOMATIC MUTATIONSSOMATIC MUTATIONS LOHLOH at the at the MSH2 locusMSH2 locus, which , which

occur in occur in ~10%~10% of tumors. of tumors.

10) Sporadic MSI10) Sporadic MSI++ tumors are caused by inactivation of tumors are caused by inactivation of hMLH1hMLH1 in in 90%90% of cases. of cases.


Detection:Detection:

Blood test to test patients for Blood test to test patients for the identified genes, the identified genes, hMSH2hMSH2 or or hMLH1hMLH1. .

However, any However, any HNPCCHNPCC family family member who has previously member who has previously had had CRCCRC first must be used first must be used to identify the affected gene, to identify the affected gene, and some people with and some people with HNPCCHNPCC will not have will not have abnormalities of these genes.abnormalities of these genes.

The Cleveland Clinic

http://www.clevelandclinic.org/registries/inherited/hnpcc.htm

Thomas Jefferson Univ; Kimmel Cancer Center www.kcc.tju.edu/hereditarycancer/ HNPCC/hnpcc.htm

CRCCRC is nearly 100% curable if diagnosed is nearly 100% curable if diagnosed within the early stages.within the early stages.

Men & WomenMen & Women Full Full colonoscopy colonoscopy every every

year beginningyear beginning at age 25 at age 25 Or 5 - 10 years before Or 5 - 10 years before

earliest cancer diagnosed earliest cancer diagnosed in the family.in the family.

WomenWomen Annual screening for Annual screening for

endometrial cancerendometrial cancer beginning at age 25 - 35. beginning at age 25 - 35.


Treatment:Treatment: Surgery:Surgery: removal of removal of

portion of the intestine portion of the intestine that contains the cancer.that contains the cancer.

Individuals with Individuals with HNPCC HNPCC have have VERY HIGHVERY HIGH risk of risk of developing a 2developing a 2ndnd colon colon cancer cancer entire colon must be entire colon must be

removed, and the healthy removed, and the healthy small intestine be small intestine be connected to the rectum.connected to the rectum.

The Cleveland Clinic

http://www.clevelandclinic.org/registries/inherited/hnpcc.htm


Questions?Questions?

muts/msh2 & hnpcc mmr & hereditary non-polyposis colorectal cancer

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