MLH1 and HNPCC
March 29, 2005
Tammy
Jernigan
http://www.macgn.org/cc_hnpcc1.html
HNPCC
Hereditary Non-Polyposis Colon Cancer
Lynch Syndrome

Type I

Site Specific
Colorectal Cancer
Type 2
Colorectal Cancer
Extracolonic Cancers:
-Endometrial (80%)
-Ovarian
-Stomach, Urologic
http://my.webmd.com/hw/colorectal_cancer/nord953.asp
HNPCC
Hereditary Non-Polyposis Colon Cancer

~5% Colorectal Cancers


Autosomal, dominant
inheritance (follows TumorSuppressor 2-Hit rule)
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)

80-90% Penetrance
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
Genetic
Counseling
Age
Suspected
Diagnosis
Interval
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
US National Library of Medicine
http://ghr.nlm.nih.gov/gene=mlh1#name

Chromosome 3
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On short arm (p) at
location 21.3 (3p21.3)
Mutations in MLH1
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Over 300 have been characterized
Common 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
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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.shtml
http://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. 7 735-740
http://saturn.roswellpark.org/cmb/huberman/DNA_Repair/mmr.html
Base-Base Mismatches
1)
2)
3)
-
MSH2 and MSH6 bind to
mispaired DNA segment.
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.
Gap filled by DNA polymerase
and sealed by DNA ligase.
E. coli studied in depth for this
mechanism (methylation)
Lodish et al, Figure 23-28
http://saturn.roswellpark.org/cmb/huberman/DNA_Repair/mmr.html
Insertion-deletion loops



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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. 7 735-740
http://saturn.roswellpark.org/cmb/huberman/DNA_Repair/mmr.html
Model Organisms

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Mouse
E. coli
S. cerevisiae
Drosophila
Knockout Mouse: MLH1 and PMS2
http://www.mdc-berlin.de/~gfactor/maus.jpg

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.ohsu.edu/tech-transfer/technologies/wct_389.shtml
http://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


http://www.bio.unc.edu/courses/2005Spring/Biol169/
Connection between clinical
human data and yeast results
Supported feasibility of
constructing genes that encode
functional hybrid human-yeast
MLH1 proteins
Ellison AR, Lofing J, Bitter GA.
MLH1 and HNPCC Summary
MLH1 and HNPCC
MLH1
MMR gene
repairs base-base mispairs
eliminates insertion-deletion loops
Spontaneous
Mutations throughout
genome
- RAS
MSI or MIN
loss or gain of
microsatellites
within DNA sequence
Common Cancers
Colorectal
Endometrial
Ovarian and Stomach
Cancer Treatment Summary

Colectomy (presently used)

Hybrid human-yeast MLH1 gene introduction

Drug development that would target
MLH1 -/- cells and cause cell death
Sources
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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
=HNPCC
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
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