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
Results
Breast Cancer- have developed breast cancer
Colon Cancer- have developed colon cancer
Prostate Cancer- have developed prostate
cancer
BRCA2- are positive for the BRCA2 mutation
but have not developed cancer
?- most likely developed cancer but are unsure
Male
?
Male
Colon
Cancer
Male
?
Female
Breast
Cancer
Female
Breast
Cancer
Male
Prostate
Cancer
Female
Breast
Cancer
Female
Breast
Cancer
Grandma
Colon
Cancer
Female
Breast
Cancer
Female
BRCA2
Male
Colon
Cancer
Female
BRCA2
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.
Ld 1
Male
Colon
Cancer
Female
Breast
Cancer
Female
Breast
Cancer
Female
Breast
Cancer
Female
Female
Breast
Cancer
Male
Colon
Cancer
Female
Breast
Cancer
Jaime
BRCA2
Female
Breast
Cancer
Female
BRCA2
Female
2
3
4
5
1
Female
Female
Breast
Cancer
LeeAnn
BRCA2
Ryan
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 tumorsuppressor gene (e.g., p53), the risk of developing cancer increases significantly.
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.Kloch’s family
tree).
Legend
Great
Grandma
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.
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.
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.
Female
Breast
Cancer
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.
Female
Figure 2: Family tree. Starting with my great grandmother on my mothers side of the family. Not all family members are included.
Those family members with nothing in their box either indicates that they have been tested negative for the mutation or have not yet
developed any cancer.
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.
2 3
4 5 6 7 Ld
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.
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.
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.