1. A CANCER SUSCEPTIBILITY SYNDROME:
SEQUENCING FOUR MISMATCH REPAIR GENES IN BOXERS
 To date, no additional mutations have been identified in the intronic or
exonic regions that are predicted to have an affect on splicing or the
function of the MMR genes; however, sequencing is not complete.
 We are currently in the process of sequencing MSH6 and PMS2 and
finishing the exons in MSH2 and MLH1.
• Once mutations are identified in our boxer samples, we will need to
screen a larger number of boxers in order to determine the prevalence of
specific mutations.
 If consistent mutations can be correlated with the occurrence of cancer,
boxers can be screened in the future for this mutations to help predict
whether or not they are predisposed to developing cancer.
This work was supported by the NIH T-35 Interdisciplinary Biomedical Research Training Program Grant as well as
supports of the Hauck Laboratory.
Introduction
ReferencesAcknowledgements
Results
 Blood samples where collected from 9 dogs that visited the NCSU CVM Hospital.
• 3 samples from non-boxer dog breeds that are not predisposed to mast cell tumors.
• 3 samples from boxers with few mutations in c-kit.
• 3 samples from boxers with a higher incidence of mutations in c-kit.
 Primers were designed for each exon of the 4 MMR genes using the NCBI database for
the published canine gene sequence.
 PCR was run using optimized conditions for each exon and product size was verified
using acrylamide gel electrophoresis.
 Samples were commercially sequenced (Genewiz©) using Sanger sequencing.
 Chromatograms were viewed on Chromas Lite (Softpedia©) to verify quality of
sequencing while the sequences were aligned on Clone Manager© in reference to the
published sequence for each exon to verify that PCR obtained the exon of interest.
 When mutations were identified, PCR products were cloned to ensure the mutations
were not introduced by PCR.
 In silico analysis of mutations was performed using two programs: Panther2
to predict
the likelihood that a mutation within an exon would negatively impact the gene’s
function and Human Splicing Finder3
to determine if an intronic mutation would affect
the splicing of the mRNA.
Figure 3
 Cancer susceptibility syndromes (CSS) occur when a genetic mutation is inherited
within related individuals leading to an increased risk and frequency of cancer types .
• CSS are better defined in people, with over 50 syndromes identified, and often affect
genes involved in cell cycle regulation, signaling and DNA repair.1
• Lynch syndrome in people occurs when individuals have a mutation in a mismatch
repair (MMR) gene. Mostly commonly this mutation occurs in MSH2 or MLH1 but
can also occur in MSH6 and PMS2.1
 Boxers demonstrate a high incidence of multiple cancer types (lymphoma, mast cell
tumors, brain tumors etc.).
• It has be suggested that there is a genetic component to this frequent development of
cancer, which would be similar to cancer susceptibility syndromes occurring in
people.
 Previously, our lab identified an increased number of single nucleotide variations in c-
kit in boxers compared to other dogs; c-kit is an oncogene.
• When an oncogene is mutated or over-expressed, cancer is more likely to develop.
• This directed our interest to MMR genes as one of their main roles is to identify and
repair single nucleotide mistakes in DNA replication.
• If a mutation where to occur in MMR genes, mistakes can accumulate in DNA and
cancer may follow.
 Last summer, the majority of MSH2 was sequenced in our lab as well as a few exons
within MLH1. One mutation was identified in the boxer samples that was predicted to
have an affect on the function of the gene.
 We hypothesized that mutations in MMR genes MSH2, MLH1, MSH6, and PMS2 may
be more prevalent in boxers compared to dog breeds with lower mast cell tumor
incidence.
Figure 1
Figure 2
Figure 3. Sample alignment for MLH1 exon 9. This figure demonstrates an alignment
of 4 samples, each with their forward and reverse complement sequence, with the
reference sequence, the intronic regions surrounding the exon of interest in row 1, and the
exonic region in row 2. Rows 3-4 contains the sequence from a non-boxer dog, rows 5-6 a
low mutator dog, rows 7-8 and 9-10 high mutator dogs. The orange highlighted area
represents a deviation from the reference sequence and the dashes signify that a sequence
has terminated.
Conclusions
 A total of 13 exons have been sequenced thus far.
 For MLH1, exons 2, 3, 4, 5, 6, 7, 8, 9, 11 have been completed.
• Leaving exons 1, 10, and 12-19 to be sequenced.
• A single nucleotide variation (c>g) was identified in intron 10 at position
15,760 within MLH1. This was determined to be a single nucleotide
polymorphism as 2 of our non-boxer samples contained this variation, as
well as 2 low mutator and 1 high mutator boxers.
 For MSH2, exon 6, 13, 17, and 19 have been completed.
• Leaving exon 1 to be sequenced as the others were completed previously.
Methods
Figure 2. Sample chromatogram for MLH1 exon 4. Peaks are color coated according to the
nucleotide in the sequence, which is indicated above each peak. The number scale across the top
represents the position within the sequence. This sample contains few background peaks, indicating the
sequence is reliable for analysis.
Figure 1. Sample 2% agarose gel of PCR products for MLH1 exon 3. Lane designations are shown
at the top of the figure. Lane 1 is a 100 base pair molecular weight ladder and lane 11 is the negative
control (contains all components of the master mix except for the template DNA). A band
corresponding to the predicted molecular weight of exon 3 (838 base pairs) is visible in all sample lanes
2-10, respectively representing one of each of our 9 samples.
1
Lindor NM et. al J Natl Cancer Inst Monogr (38) 1-93, 2008.
2
http://www.pantherdb.org/
3
http://www.umd.be/HSF/
1
2
3
4
5
6
7
8
9
10
Alicia Braxton1
, Marlene Hauck1,2
1
Comparative Oncology Laboratory, 2
Department of Clinical Sciences, College of Veterinary Medicine,
North Carolina State University, Raleigh, NC