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The Future of Whole-Genome Sequencing:
MGMT Mutations in a Family Could Be Linked to Cervical Cancer
Greenfield SMART Team: Robin Sandner, Panfua Thao, Morgan Borchardt, Hannah Flees, Joey Krasovich,
Francis DeLeon-Camacho, Alyssa Gerwig, Zoe Osberg, Mary Wojczulis
Teacher: Julie Fangmann
Mentor: Elizabeth Worthey, Ph.D., Department of Pediatrics and the Human and Molecular Genetics Center, Medical College of Wisconsin
1. Abstract
2. A Family with Cervical Cancer
In 2008, the CDC reported 4,008 cervical cancer-related deaths in the US (1). Researchers at MCW used Whole Genome
Sequencing technology to sequence the DNA of a mother and daughter diagnosed with a rapidly progressing cervical cancer.
Identifying the genetic underpinnings could explain how their cancer developed and progresses, and help develop a specific
treatment. One candidate gene, O-6-methylguanine-DNA-methyltransferase (MGMT), is a DNA repair enzyme that removes
alkylated lesions that can lead to tumor formation (2). Alkylating lesions are formed through exposure to alkylating agents,
which are produced in small quantities in numerous industrial reactions. MGMT removes a methyl group from methylated
Guanines in the DNA lesion via its Cysteine 145 residue; the normal Guanine properly pairs with Cytosine (3). Alteration of
MGMT, such as the mutations found in this family (p.Ile143Val and p.Lys178Arg), may prevent this reaction, leaving improperly
pairing methylguanines in the lesion in place. If these uncorrected lesions are in a region responsible for regulation of cell
growth, they may lead to cancer development. Understanding MGMT’s structure, specifically at these altered positions, will
assist MCW researchers in determining whether the variant MGMT is a likely cause of disease. The Greenfield SMART Team
(Students Modeling A Research Topic) modeled MGMT using 3D printing technology to analyze the likely effect of these
mutations to assist in understanding their possible role in the development of cervical cancer in this family.
A mother and her daughter have been diagnosed with cervical cancer, the third most common type of cancer in women.
Cervical cancer occurs as the squamous cells of the cervix lining (see figures to the right) mutate and overgrow, slowly
spreading deeper into the wall of the cervix (1). While there are a few symptoms of cervical cancer (abnormal vaginal
bleeding, pelvic pain, etc.), these are often missed resulting in a later diagnosis and poorer outcome. Diagnostic tests such as
a Pap smear are used to diagnose a patient in an earlier stage to improve the outcome (1). Surgical removal or radiation of the
cancer is necessary to prevent spreading to other parts of the body (1).
http://www.mdguidelines.com/cancer-cervix
Researchers at the Medical College of Wisconsin applied the technique of whole genome sequencing of the mother and
daughter’s genomes to attempt to identify genetic causes for their disease. They located two variants (mutations) in their
genomes relative to the reference genome that could contribute to their cervical cancer (see graphs in Section 4 below).
The two variants are found in a protein called MGMT, which corrects specific types of errors in the DNA that could lead to
tumor formation. These mutations could alter MGMT’s function as seen below.
http://www.encognitive.com/node/10448
3. Comparing Normal MGMT to Improperly Functioning MGMT
Normal MGMT
Properly Functioning MGMT
Cys145
MGMT is a DNA repair enzyme
that removes methyl groups from
methylated guanines that occur in
alkylated lesions due to exposure
to environmental toxins. This
MGMT repair allows the guanines
to base pair properly with
cytosine in DNA during
replication, correcting the lesions
and their associated tumor
development risk.
MGMT
Ile143
Lys178
DNA
binding
site
Methylated
guanine
DNA
Mutated MGMT
Improperly Functioning MGMT
MGMT
Cy145
Ile143Val
DNA
binding
site
Lys178Arg
Methylated
guanine
DNA
Removes methyl group
from methylated guanines
The mom and daughter have
two mutations (Ile143Val and
Lys178Arg) in their MGMT. These
mutations prevent their MGMT
from correcting mutations in the
alkylated lesions through removal
of the methyl groups on the
methylated guanine residues. This
lack of repair leaves the guanine
improperly base paired with
thymine.
MGMT
Guanine base pairs with cytosine
properly in DNA during replication
MGMT
Lys178
Ile143 Cys145
Ile143
Methyl
(CH3)
Lys178
G
T
C
T
G
C
T
A
G
T
A
A
T
T
C
A
G
A
C
G
A
T
C
A
T
T
A
www.mybiopsy.org
A
G
T
A
A
T
DNA sequence:
A
G
T
A
A
T
DNA sequence:
T
T
A
T
T
A
DNA sequence:
T
C
A
T
T
A
MGMT
A
Methyl
(CH3)
MGMT cannot remove methyl group
from methylated guanines
Lys178Arg
MGMT
Other mutations
can form in DNA
Lys178Arg
Cys145
Methyl
(CH3)
Normal functioning MGMT results in correct
DNA base pairings. Without DNA mutations ,cells
grow and function normally. This image shows
normal cervical squamous cells.
DNA lacking mutations results when MGMT
removes methyl groups from methylated
guanines. This allows guanine residues to properly
base pair with cytosine during DNA replication.
Guanine improperly base pairs
in DNA
Ile
143
Val
Cys145
How might these
mutations impact the
structure of MGMT?
Healthy cervical cells
Cys145
DNA sequence:
Ile
143
Val
Non-mutated DNA forms
A
A
T
C
T
T
T
A
G
A
A
T
C
G
T
A
A
T
A
T
Accumulation of mutations can
lead to cancer development
T
A
A
A
T
T
T
A
Methyl
(CH3)
www.mybiopsy.org
DNA sequence:
A
G
T
A
A
T
DNA sequence:
A
G
T
A
A
T
DNA sequence:
T
T
A
T
T
A
DNA sequence:
T
T
A
T
T
A
When MGMT cannot remove methyl groups
from methylated guanines, then guanine cannot
properly base pair with cytosine. Instead, an
adenine (A) – thymine (T) base pair replaces what
should be a guanine (G) – cytosine (C) pair.
Without correct DNA repair there is accumulation
of these mutation containing lesions.
As DNA mutations accumulate, cancerous
cervical cells (see image) could develop if some of
the mutations occur in important genes regulating
cell division. These cancerous cells are less
organized, overgrown, and are irregularly shaped.
This may be what is happening in the case of the
mother and daughter who have cervical cancer.
Two variants (Ile143Val and
Lys178Arg) were found. Ile143Val
is located adjacent to the alkyl
(such as a methyl group)
acceptor Cys145 in the active site
of MGMT (2). Although similar in
many regards, we hypothesis
that there is an effect due to
replacement of an Isoleucine
with a smaller Valine in this
critical region.
In addition we see that in our 3D
model, Lys178Arg lies close to
Ile143Val. Once again this change
is between two similar amino
acids, but altered hydrophobicity
might add to the altered
conformation in this critical
region.
Alteration of a protein’s shape is
known to lead to changes in
function and in the case of
MGMT, alteration in the region of
the active site is known to lead to
inability to correct DNA
mutations, as shown to the right
(3,4).
4. Genome Sequencing Data Showing the Two Variants in the Mother and Daughter with Cervical Cancer 5. What Does All of This Mean For the Future of Medicine?
Data images provided by the lab of Liz Worthey, Ph.D.
Left: This image shows the mother’s (top)
and daughter’s (bottom) DNA sequence in
this region. The reference genome is shown
at the very top. Each grey line represents a
sequence read. The MGMT amino acid
sequence is shown at the bottom. The
mother and daughter show an Adenine
(green) changed to Guanine (orange) which
causes the amino acid change p.ILE143VAL in
the MGMT protein.
Right: This image shows presence of
the second variant, where Adenine (A)
changes to Guanine (G) in DNA. This
image again shows the mother’s (top)
and daughter’s (bottom) DNA
sequence in this region, with the
reference genome at the very top. This
mutation leads to the p.LYS178ARG
amino acid change in the mom and her
daughter.
http://www.cdc.gov/cancer/cervical/
http://www.genecards.org/cgi-bin/carddisp.pl?gene=MGMT&search=mgmt#function
Pegg AE et.al. DNA Repair (Amst). 6(8):1071-8 2007
Chueh LL et.al. Carcinogenesis 13(5) (1992)
Worthey EW et. Al. Genetics in Medicine 13(3):255-62 (2011)
• Through DNA sequencing, our mentor and collaborators hope to be able to discover the cause of the
mother and daughter’s cervical cancer. Providing this link between genome sequence and disease can
be used to identify others at risk for developing cancer due to presence of specific mutations. These
individuals can be screened earlier and more frequently to detect cancers sooner. This information
can potentially be used to assist in the development of specific treatments for individuals with these
types of mutations.
• Doctors are using Whole Genome Sequencing to try to determine the genetic causes of diseases,
including cancers, that cannot be figured out otherwise. Being able to identify the genetic basis of
various medical issues can help diagnose and possibly lead to personalized treatments for many
people who have been left without an explanation or help in the past (5).
6. References
1.
2.
3.
4.
5.
• It is unclear what the role of these specific MGMT mutations are in the cervical cancer in these
individuals. There is growing evidence to support that they are not causative in other types of
cancers. However, the techniques performed to study the likely impact on function due to altered
structure are very powerful tools in making links between an individual’s diseases and genome.
The SMART Team Program (Students Modeling A Research Topic) is funded by a grant from NIH-SEPA 1R25OD010505-01 from NIH-CTSA UL1RR031973.
Background image: http://www.sciencedaily.com/releases/2008/05/080526155300.htm