MLH1 and HNPCC

March 29, 2005

Tammy Jernigan

http://www.macgn.org/cc_hnpcc1.html

MLH1 and HNPCCHereditary Non-Polyposis Colon Cancer

Lynch I SyndromeSite SpecificColorectal Cancer

MLH1 and HNPCCHereditary Non-Polyposis Colon Cancer

Lynch I SyndromeSite SpecificColorectal Cancer

HNPCCHereditary Non-Polyposis Colon Cancer

Type I Site Specific

Colorectal Cancer

Type 2Colorectal Cancer

Extracolonic Cancers:

-Endometrial (80%)

-Ovarian

-Stomach, Urologic

Lynch Syndrome

http://my.webmd.com/hw/colorectal_cancer/nord953.asp

HNPCCHereditary Non-Polyposis Colon Cancer

~5% Colorectal Cancers

Autosomal, dominant inheritance (follows Tumor-Suppressor 2-Hit rule)

80-90% Penetrance

40% of these are related to the gene MLH1, a DNA Mismatch Repair gene

90% of the tumors related to MLH1 deficiency show microsatellite instability (MSI or MIN)

http://www.healthsystem.virginia.edu/uvahealth/hub_cancer/hnpcc.cfm

http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=120436

Treatment for HNPCC

Colectomy Removal of entire colon

and reconnection of small intestine to the remaining rectum

Followed by the Health Maintenance Guideline

http://www.kimmelcancercenter.org/kcc/HereditaryCancer/HNPCC/hnpcc.htm

Health Maintenance Guideline

Method Age Interval

Genetic Counseling

Suspected Diagnosis

Colonoscopy 21 years old or

10 years before age of first family diagnosis

Every 2 years to age 40, then annually

Pelvic exam, Ultrasound, blood test

At 25-35 years old

Annually

Urine Cytology

At 30 years old

Every 1-2 years

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

MLH1 Location

Chromosome 3

On short arm (p) at location 21.3 (3p21.3)

US National Library of Medicine http://ghr.nlm.nih.gov/gene=mlh1#name

Mutations in MLH1

Over 300 have been characterizedCommon Mutations include:

Entire deletion of the exon (Codons 578-672 on short arm of chromosome 3)

Frameshift mutation (new stop codon); truncation

Insertion of nucleotides

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=omim

MLH1 Function

One of the two most common genes related to HNPCC One of five genes involved in DNA Mismatch Repair

during cell division (MSH2, MLH1, MSH6, PMS1, PMS2)

MLH1 protein is part of a DNA repair complex that includes PMS2, which is part of the MMR system.

MLH1 deficiency results in increased spontaneous mutations throughout the genome due to dysfunction of the MMR system

http://www.ohsu.edu/tech-transfer/technologies/wct_389.shtmlhttp://www.exactsciences.com/pregen26/professionals/about_hnpcc/_index.htm

MLH1: A Mismatch Repair Gene

Role of Mismatch Repair System:

- Eliminate base-base mismatches

- due to replication errors or

- deamination (cytosine to thymidine)

- Eliminate insertion-deletion loops

- due to DNA polymerase pausing and template slippage

Peltomaki, Human Molecular Genetics, 2001. Vol. 10, No. 7 735-740http://saturn.roswellpark.org/cmb/huberman/DNA_Repair/mmr.html

Base-Base Mismatches

1) MSH2 and MSH6 bind to mispaired DNA segment.

2) This triggers the binding of the MLH1 endonuclease complex (includes PMS2). DNA helicase unwinds helix, daughter stand is cut, exonuclease removes many nucleotides including mismatched base.

3) Gap filled by DNA polymerase and sealed by DNA ligase.

- E. coli studied in depth for this mechanism (methylation)

Lodish et al, Figure 23-28http://saturn.roswellpark.org/cmb/huberman/DNA_Repair/mmr.html

Insertion-deletion loops

Due to DNA polymerase pausing and template slippage during DNA replication

Slippage can occur in regions of tandemly repeated units (such as (CA)12). These regions of repeated units are known as microsatellites. The gain or loss of these units due to slippage is known as microsatellite instability (MSI or MIN).

DNA melts and reanneals incorrectly and creates a loop extending off of one strand.

Repair follows similar mechanism of base-base mismatches

http://www.virtuallaboratory.net/Biofundamentals/lectureNotes/AllGraphics/slippage.jpg Peltomaki, Human Molecular Genetics, 2001. Vol. 10, No. 7 735-740http://saturn.roswellpark.org/cmb/huberman/DNA_Repair/mmr.html

Model Organisms

Mouse E. coli S. cerevisiae Drosophila

Knockout Mouse: MLH1 and PMS2

Phenotype:- normal gestation- >80% developed tumors after one year- 7 of 8 tumors did not express APC (Adenomatus polyposis coli)- Frameshift and base substitution mutations increased dramatically in all examined tissues

Double knockout of MLH1 and PMS2 revealed similar results, supporting the idea that MLH1 nullizygosity is sufficient to inactivate MMR completely

http://www.mdc-berlin.de/~gfactor/maus.jpg http://www.ohsu.edu/tech-transfer/technologies/wct_389.shtmlhttp://www.pdg.cnb.uam.es/UniPub/iHOP/gs/122799.html

S. cerevisiae

Missense Codons from human HNPCC correlated with MLH1 and MSH2 introduced into yeast to note effects:- complete loss of function- silent polymorphisms- efficiency polymorphisms

Connection between clinical human data and yeast results

Supported feasibility of constructing genes that encode functional hybrid human-yeast MLH1 proteins

http://www.bio.unc.edu/courses/2005Spring/Biol169/ Ellison AR, Lofing J, Bitter GA.

MLH1 and HNPCC Summary

M L H 1 a n d H N P C C

S p on tan eo usM uta tio n s th ro ug ho ut

g en om e- R A S

M S I o r M INlo ss o r g a in o fm icrosa te lli tes

w ith in D N A seq u en ce

C o m m o n C a nce rsC o lo rec ta l

E nd om e tria lO v ar ian an d S to m a ch

M L H 1M M R ge ne

re pa irs ba se-ba se m ispa irse lim ina te s in se rt ion -de le tio n loo ps

Cancer Treatment Summary

Colectomy (presently used)

Hybrid human-yeast MLH1 gene introduction

Drug development that would target

MLH1 -/- cells and cause cell death

Sources

http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=120436 http://www.md.ucl.ac.be/entites/mint/intr/hainaut/dossierprojet/dossierdocsem/canccolrectgen/canccolrectgen.html http://search.cancer.org/search?client=amcancer&site=amcancer&output=xml_no_dtd&proxystylesheet=amcancer&restrict=cancer&q=H

NPCC http://www.clevelandclinic.org/registries/inherited/hnpcc.htm http://www.virtuallaboratory.net/Biofundamentals/lectureNotes/AllGraphics/slippage.jpg http://www.ohsu.edu/tech-transfer/technologies/wct_389.shtml http://www.pdg.cnb.uam.es/UniPub/iHOP/gs/122799.html http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=omim http://saturn.roswellpark.org/cmb/huberman/DNA_Repair/mmr.html http://www.exactsciences.com/pregen26/professionals/about_hnpcc/_index.htm http://www.healthsystem.virginia.edu/uvahealth/hub_cancer/hnpcc.cfm http://www.kimmelcancercenter.org/kcc/HereditaryCancer/HNPCC/hnpcc.htm http://ghr.nlm.nih.gov/gene=mlh1#name http://www.mdc-berlin.de/~gfactor/maus.jpg http://www.bio.unc.edu/courses/2005Spring/Biol169/ http://my.webmd.com/hw/colorectal_cancer/nord953.asp

Ellison AR, Lofing J, Bitter GA., Functional analysis of human MLH1 and MSH2 missense variants and hybrid human-yeast MLH1 proteins in Saccharomyces cerevisiae. Hum Mol Genet. 2001 Sep 1;10(18):1889-900.

Lodish et al. Molecular Cell Biology. Ed. 5, Figure 23-28. W.H. Freeman Co, NY, 2004 Peltomaki, Paivi. Deficient DNA mismatch repair: a common etiologic factor for colon cancer. Hum Mol Genet. 2001, Vol. 10 No. 7 735-

740 Satya Narayan and Deodutta. Roy, Role of APC and DNA mismatch repair genes in the development of colorectal cancers. Molecular

Cancer 2003, 2:41