... to the development of breast, ovarian, prostate, colo

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BRCA2 Mutant R2520X Allele Screen Ryan Kloch
Dr. Michael Black
ABSTRACT The BRCA2 breast cancer gene is a tumor
suppressor gene that acts as both a transcription
factor and as part of the cells DNA repair
system. Disruption of this gene may lead to the
development of breast, ovarian, prostate, colon
and/or pancreatic cancer. The following
experiment deals with a specific mutation within
the BRCA2 gene located at amino acid 2520 of
3418. The R2520X mutation has been classified as
a family-inherited factor, which resides on my
(R.Kloch) maternal side of the family. The
purpose of the following experiment was to run
an allelic screening test using my own DNA to
determine if I am a positive carrier of the
R2520X allele.
Introduction Breast cancer is the second
deadliest form of cancer for women (superceded
only by lung cancer). Each year around 40,000
lives are lost due to this disease. Although only
0.5 of these lives lost will be men, there are
still many aspects that surround breast cancer
which should not be ignored by men or women.
After age and gender, a known family history of
breast cancer is the highest predictable risk
factor. Due to the recent developments in
genetic mapping, most of the inherited risks have
been traced to specific mutations in single
cancer susceptibility genes. With breast cancer,
the BRCA1 and BRCA2 susceptibility genes have
been tightly linked to the inherited form of this
disease. At the cellular level, both BRCA1 and
BRCA2 act as tumor-suppressor genes in that they
encode proteins that can stop cancerous cell
growth if necessary (see Figure 1 for role in DNA
repair). Fortunately, there are several other
tumor-suppressor genes that interact with the
functions of both BRCA cancer genes, so a
mutation in one of the genes does not necessarily
mean that cancer will develop. However, if an
additional mutation arises in another
tumor-suppressor gene (e.g., p53), the risk of
developing cancer increases significantly. As
for the specific R2520X mutant allele the R
stands for the amino acid arginine at position
2520 and the X refers to any amino acid (or stop
codon) other than arginine that may be translated
due to the presence of a mutation. The nucleotide
sequence that encodes for the wild-type arginine
at this position is CGA. Any mutation leading
to a sequence that encodes for an amino other
than arginine (or a stop codon) would indicate
that the person is positive for the R2520X allele
mutant. There are several reasons why allelic
screening can be a very important process for
males and females of high-risk families. Although
the amount of males that develop breast cancer
due to mutations within the BRCA genes is very
low, there are several other implications that
should be recognized it also leads to an
increased risk in developing colon, prostate and
pancreatic cancer (see figure 2 for R.Klochs
family tree).
Results The first step taken was to see which
of four DNA isolation protocols extracted the
greatest amount of genomic DNA. Of the protocols
tested, only the QIAmp Buccal cell isolation
produced a template that allowed amplification of
the BRCA2 locus (see Figure 3). The band size is
the expected length considering our primers were
developed to amplify a region 405 base pairs
long. The DNA in the band that corresponded
to the QIAmp buccal cell isolation was purified
and ligated into the pCR2.1 vector using the TOPO
TA cloning kit (Invitrogen). Native
(unrestricted) plasmid clones 1-4 and 6 (see
Figure 4) were cycle sequenced. The sequences
from clones 1-3 and 6 show that RK contains the
wild-type allele (condon encodes arginine). As a
control for the amplification and sequencing
procedures, six sequences from LK (a known
carrier of the mutant allele) and two from MB (a
negative control) gave clear sequence results of
the expected codons at this locuse (see samples
in Figure 5). Of the six sequences from LK, 3
showed a transition mutation from CGA to TGA, a
known stop codon. The fact that LK showed a 50/50
ratio of maternal (mutant) and paternal (wild
type) alleles reassures us that there is no
allelic bias in the amplification/cloning
procedures.
Figure 1 BRCA2 functions as a component of the
DNA repair machinery. This cartoon shows the
different complexes that the BRCA alleles form
under wild type conditions and the consequences
upon loss of one or both of the gene products.
Ld 1 2 3 4 5
1 2 3 4 5 6 7 Ld
Figure 5 Sample Electropherograms of BRCA2
sequences. Comparing the R2520 codon sequence
between experimental sequences (RK2 6) with the
negative (MB1) and positive (LK2 6) controls.
The positive control sequence illustrates the TGA
mutated sequence of R2520X allele while the
negative control and experimental sequence show
the wild type sequence of CGA.
Figure 3 PCR results from various methods of
isolating genomic DNA for BRCA2 amplification.
Lanes are as follows (1) QIAmp Blood Isolation
Protocol (2) QIAmp Buccal Cell Isolation
Protocol/ (3) NaOH (FBI) Blood Isolation
Protocol (4) NaOH (FBI) Buccal Cell Isolation
Protocol (5) 5?l H20 Control. Using the 1kb
Promega DNA standard ladder (Ld), the amplicon
product in lane 2 is approximately 500 bp in
length.
Figure 4 Recovered plasmid clones from ligation
of BRCA2 amplicon. Lanes 1-6 correspond to all
six clones. Only lane 5 (clone 5) shows an
insert that is not the correct size. All other
clones have the 4Kb vector along with the 500bp
insert. Lane 7 corresponds to the undigested
control (5Kb). Lanes 1-6 were compared to the
Promega 1Kb DNA ladder standard in lane Ld.
Discussion Although the sequences from only five
clones does not definitively prove that I am not
a carrier of this allele, the results from our
positive control suggest that there is at least
no more than a 3 chance. Out of the five
different sequences that were produced from my
own DNA, none of them contained the TGA mutation.
When performing an allelic screen for a
mutation, it is very important that there is 100
assurance and confidence in the results. With
only five of my clones producing clear results,
it is hard to say exactly how sure I am about not
being a carrier. Even if I were 100 sure that I
am not a carrier of the mutation, this would not
eliminate my concerns about cancer. Until cancer
and its effects are fully understood, it is
important to be aware not only of the
family-inherited implications, but also of the
other factors and risks that may be associated
with this deadly disease.