Phenotypic consequences of Phenotypic consequences of specific mutations in human specific mutations in human MLH1MLH1

Sierra Spencer

Dr. Andrew Buermeyer

Department of Environmental and Molecular Toxicology

Oregon State University

HHMI Program, Summer 2008

Image courtesy of: www.uniklinikum-saarland.de/.../forschung/hnpcc

DNA Mismatch RepairDNA Mismatch Repair

Mismatch repair (MMR) is an excision repair pathway that prevents mutations by correcting errors made in the DNA strand during replication. Errors result from:

Replication errorsRecombination pathwaysExogenous DNA damaging agents

MMR is also promotes apoptosis (cell death) in the presence of excessive DNA damage

IMPORTANCE:IMPORTANCE: COLON CANCER- Linked to deficiencies in DNA

mismatch repair (MMR)

Colorectal cancers (CRC) affect 150,000 people in the United States annually, and causes ~50,000 deaths per year.

Unfortunately, the mortality rate has shown modest reductions over the last 50 years.

This highlights the importance of preventative lifestyle choices and education.

©2005 American Cancer Society, National Cancer Institute

LYNCH SYNDROME AND LYNCH SYNDROME AND MMRMMR Lynch Syndrome, also known as

hereditary non-polyposis colorectal cancer (HNPCC), is one of the most common cancer predispositions, representing 2-5% of colorectal cancer cases in the US.

Lynch Syndrome is a disease where inactivating mutations in a single gene, that is essential for DNA mismatch repair, are inherited.

People who have Lynch Syndrome have a very high lifetime risk of developing CRC, about 90%.

Since most CRC is late onset, the elevated risk of sporadic CRC is hypothesized to be from modest defects in MMR genes.

MutLMutLαα and MMR and MMR

MutLα is a heterodimer, made up of MHL1 and PMS2 proteins.

MutLα facilitates and coordinates events in MMR.

In absence of MutLα, cells show:-increased mutation rates-decreased apoptotic response to genotoxins

Mutations in MLH1 account for 30-40% of Lynch Syndrome cases.

The Big Picture:The Big Picture:To determine the extent to which different phenotypes, caused by MMR-deficiency, contribute to the development of human cancer.To analyze the pathogenicity of many single amino acid substitution mutations identified in human patients with CRC.

Hypothesis:Hypothesis:Disease causing (or pathogenic) mutations in MLH1 cause measurable decreases in cells’ ability to perform MMR.Such changes may affect MLH1-dependant error correction and/or cytotoxic responses to DNA damage

Project Project Objectives:Objectives:Determine if the L607H variant hMLH1 gene is capable of restoring a cytotoxic response and decreasing spontaneous mutation frequencies to a level comparable to wild-type transfected cultures. Analyze phenotypic consequences for pathogenicity.

L607H MutationL607H Mutation

Images courtesy of http://www.contexo.info/DNA_Basics/Amino%20Acids_and_Mutations.htm

L607H is a mutation that was identified in two Lynch Syndrome family cases. -Limited clinical genetic analysis, therefore co-segregation of this mutation with HNCCP is unknown-Shown to have no apparent microsatellite instability-Found functionally to have no reduced MMR capacity.

It is a single amino acid substitution, located at 607, changing a leucine to a histidine.

This is a change from nonpolar, neutral amino acid to polar, weakly basic one.

Select with G418

Expression of hMLH1 in MLH1-Expression of hMLH1 in MLH1-deficient mouse fibroblast cellsdeficient mouse fibroblast cells

Positive control (WT hMHL1)

Negative control (vector)

L607H

Frequency of ouabain R mutants

MMR-dependent DNA damage response

Cytotoxic response to 6-TG and MNNG

MMR-dependant suppression of spontaneous mutation

Pass 1:10

Pass 1:10

Freeze cell lines

Pooled Assay General Outline

Experimental proceduresExperimental procedures

Maintenance of cell lines

Grown in 15% complete media (CM)

Passed 1:10 when 80% confluent into 38⁰C incubator with 5% CO2

Cytotoxic response Assay:

300 cells plated onto four 57 cm2 plates

Following day 2 plates received 7 mL dose of 1μM 6-Thioguanine. After 24 hours, four plates refed with 10 mL 15% CM. Colonies fixed and stained with 1X methylene blue stain

Plates that didn’t receive 6-TG used as negative control for plating efficiency

300 cell plated onto twelve 57 cm2 plates

Next day exposed 1 hour to 7mL dose of varying doses of MNNG (between 100μM to 0.1μM, with 0 dose control for plating efficiency)

Forward mutation assay:

Plated density of 1.0∙106 cells per 142 cm2 plate with 30mL 1mM Ouabain in 15% CM.

Fixed and stained with 1X methylene blue stain 12-14 days after plating

Colonies counted and mutation rates calculated based on survival of colonies exposed to Ouabain and the estimated cell survival determined from the efficiency plates

Immunohistochemistry

In 12 well plate, 1.0∙105 cells plated per well in 1mL media. 2 days media aspirated and cells fixed with 10% formalin

Method OverviewMethod Overview

Generally increased mutant frequencies- suggests L607H mutation may decrease MLH1 functional capacity

The MLH1 deficient vector + hMLH1 and the MLH1 deficient vector behaved within the range of our historical values.

MMR deficient

High mutant frequencies

MMR proficient

Low mutant frequencies

* - new data

MLH1-dependent suppression of spontaneous mutations

* - New data

Generally low survival within range observed with cells expressing hMLH1MMR proficient

MLH1-dependent cytotoxic response to genotoxins

MNNG Induced Cytotoxicity

Similar to 6-TG, clearly a significant cytotoxic response.

Significance of the difference in WT and vector is not clear, requires additional experimentation.

Summary/Summary/ConclusionsConclusionsOuabain Assay

Wild-type hMLH1 and the empty vector controls are consistent with historical values.

The hMLH1 cultures with the L607H mutation have elevated mutation frequencies compared to wild-type hMLH1, with some ability to reduce spontaneous mutation.

6-TG Assay

The L607H variant culture had percent survival within range of wild-type hMLH1.

The differences you see in the L607H mutations might be from how the DNA substrate was prepared, and further testing should be done.

MNNG Assay

The assay has ability to determine capacity for DNA damage response.

More trials are needed to analyze differences in functionality

Immunochemistry Assay

Did not obtain results. Cells peeled from the plates in multiple trials:

-Formalin that was used to set the cells for IHC went bad

-The cells were too confluent when setting for IHC

L607H has some capacity for MMR, most seen in cytotoxic response. More experimentation is needed to determine pathogenicity of this mutation.

Future Work and Future Work and SignificanceSignificance

To determine if L607H is likely to be pathogenic or not, more tests are needed to profile phenotypic consequences.

The next step would be to run a western blot analysis to quantify active MLH1 proteins.

Transfecting MLH1 deficient mice with L607H variant hMLH1 would be another approach to determining phenotypic consequences and their effect on pathogenicty.

Determining hMLH1 variant behavior and their pathogenicity is an important step in understanding and evaluating individual risk.

Image courtesy of: studenthealth.sa.ucsb.edu/HealthEducation/

AcknowledgementsAcknowledgementsHoward Hughes Medical Institute

Dr. Andrew Buermeyer, mentor

Dr. Kevin Ahern, program coordinator