Colin Sunde

4/23/16

Specific Aims

Lynch Syndrome, also known as hereditary non-polyposis colorectal cancer, is

an autosomal dominant genetic disorder that is responsible for 3% of worldwide
colorectal cancer cases1. Those affected have an increased risk of many types of
cancer. One of the genes associated with Lynch Syndrome is MutL homolog 1
(MLH1). While the genetic deletion of MLH1 is well studied, as of yet there is very
little research on missense mutations and their effects on tumor suppression.
MLH1 is involved in DNA replication, specifically in mismatch repair 1. MLH1
binds to other mismatch repair proteins to form a complex that cuts the new DNA
strand around the mismatch site2. The mismatch repair process fixes random
mistakes in DNA replication, and plays an important role in the induction
of apoptosis1,4. Without proper mismatch repair, not only do mutations during cell
replication build up, but cell apoptosis is also impaired; leading to an increased
chance of tumor formation1,4. I will be focusing on the E23D missense mutation,
which causes Lynch Syndrome in humans with a single base pair substitution of the
MLH1 gene5. A mouse model will be used because of its similar mismatch repair
function to humans.
A Recent study suggested that missense mutations in the MLH1 gene may
affect the mismatch repair function in a tissue specific manner 4. My primary goal
is to determine if E23D MLH1 causes tumorigenesis in a tissue specific manner that
is different than wild type MLH1 in mice. The overall objective is to find the cancer
susceptibility of different tissues related to the E23D mutation, using a mouse. I am
testing the central hypothesis that MLH1 expression levels will change in tissues
because of the missense mutation, causing changes in the tissues tumor
susceptibility.
Aim 1: Identify the MLH1s E23D mutation homolog in mice.
Hypothesis: The mouse MLH1 protein will have a homologous mutation site that will
replicate the human MLH1 E23D mutation.
Approach:
1. Using sequences from NCBI and protein domains from Pfam, compare the MLH1
homologs
2. Find the E23D base pair substitution in humans, and where it would be in mice
Rationale: To determine if it is possible to replicate the E23D missense mutation in
mice.
Aim 2: Determine if MLH1 with the E23D mutation is differentially expressed than wildtype
MLH1 in mice.
Hypothesis: The E23D mutation will cause the expression of MLH1 to change in
some tissues.
Approach:
1. Using CRISPR/cas9, create a mouse model with the base pair substitution replicating the
E23D mutation
2. Preform RNA sequencing to determine the expression of MLH1 in different
tissues
3. Compare the expression of the mutated MLH1 to the expression of wild type
MLH1
Rationale: To see if the missense mutation effects the MLH1s expression in
different tissues.

Colin Sunde
4/23/16
Aim 3: Determine if E23D MLH1 causes a different pattern of tumorigenesis in
mouse tissues.
Hypothesis: The E23D mutation will cause a different pattern of tumorigenesis, with higher
levels of cancerous cells where MLH1 was under expressed in aim 2.
Approach:
1. Using the mice model created in aim 2, determine which tissues develop tumors most
often.
2. Compare these results to mice with wild type and knock out MLH1
Rationale: To determine if tumors are more likely to develop in different tissues when MLH1 is
mutated.
Outcomes: Understanding the tissue sensitivity to the E23D MLH1 mutation in mice will give
insight into what tissues are most susceptible to tumor formation in humans. This will further
push the information available to researchers in the hopes of better treatment and possibly a
cure in the future.

Colin Sunde
4/23/16

[1]: Kohlmann W, Gruber SB. Lynch Syndrome. 2004 Feb 5 [Updated 2014
May 22]. In: Pagon RA, Adam MP, Ardinger HH, et al., editors. GeneReviews
[Internet]. Seattle (WA): University of Washington, Seattle; 1993-2016.
Available from: http://www.ncbi.nlm.nih.gov/books/NBK1211/
[2]: A.B. Buermeyer, S.M. Deschenes, S.M. Baker, R.M. Liskay, Mammalian
DNA mismatch repair, Annu Rev Genet, 33 (1999), pp. 533564
[3]: T.E. Raevaara, M.K. Korhonen, Functional significance and clinical
phenotype of nontruncating mismatch repair variants of MLH1,
Gastroenterology, 129 (2005), pp. 537549
[4]: E. Avdievich, C. Reiss, S.J. Scherer, Y. Zhang, S.M. Maier, B. Jin, H. Hou Jr.,
A. Rosenwald, H. Riedmiller, R. Kucherlapati, P.E. Cohen, W. Edelmann, B.
Kneitz Distinct effects of the recurrent Mlh1G67R mutation on MMR
functions, cancer, and meiosis. Proc. Natl. Acad. Sci. U. S. A., 105 (2008),
pp. 42474252
[5]: Takahashi M., et al. (2007) Functional analysis of human MLH1
variants using yeast and in vitro mismatch repair assays. Cancer Res.,
67, 45954604.