PREFERENTIAL ALLELIC EXPRESSION CAN LEAD TO REDUCED EXPRESSION
OF BRCA1 IN SPORADIC BREAST CANCERS
Hilmi O¨ZC ¸ELIK1,2,3, Minh D.TO1,4, Jean COUTURE1,5, Shelley B. BULL1,6and Irene L. ANDRULIS1,2,3,4,7*
1Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada
2Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada
3Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada
4Department of Molecular and Medical Genetics, University of Toronto, Toronto, Canada
5Department of Surgery, Mount Sinai Hospital, Toronto, Canada
6Department of Public Health Sciences, University of Toronto, Toronto, Canada
7Cancer Care Ontario, Toronto, Canada
BRCA1 is considered to be a tumor-suppressor gene, yet
mutations in this gene are uncommon in sporadic breast
tumors. W e investigated whether mechanisms other than
DNA mutations that affect the coding region might be
involved in breast carcinogenesis. Since loss of expression of
the BRCA1 gene would lead to lack of protein, we evaluated
the level of BRCA1 mRNA in 21 normal epithelial specimens
and in 74 breast carcinomas using quantitative reverse-
transcription-polymerase-chain-reaction (RT-PCR). All nor-
mal breast epithelial samples expressed BRCA1 mRNA. On
the other hand, the tumor specimens exhibited approxi-
mately 10-fold range oflevelsofBRCA1, with some specimens
expressing barely detectable amounts of BRCA1 mRNA. The
distribution in levels was significantly higher in normal breast
epithelial cellsthanintumor specimens(p ? 0.004).Examina-
tion of the BRCA1 locus indicated that deletion of the BRCA1
gene may account for low levels of BRCA1 in a number of
specimens. In addition, analysis of samples with relatively
reduced levels of BRCA1 expression revealed preferential
allele-specific expression in a number of cases, suggesting the
presence of regulatory mutations. Our data suggest that the
BRCA1 gene may be involved in sporadic breast carcinogen-
esis through a reduction in gene expression. Int. J. Cancer
?1998 Wiley-Liss, Inc.
The identification of mutations in the breast- and ovarian-cancer-
susceptibility gene BRCA1 that co-segregated with tumors in
high-risk families was an early criterion for associating the BRCA1
gene with cancer predisposition (Castilla et al., 1994; Freidman et
al., 1994; Miki et al., 1994; Simard et al., 1994; Shattuck-Eidens et
al., 1995). Since breast and ovarian tumors from carriers with
germline BRCA1 mutations were observed to have lost the second
wild-type allele (Smith et al., 1992) it has been suggested that
BRCA1 acts as a tumor-suppressor gene. Several studies have
shown that loss of heterozygosity (LOH) is a common event at the
17q21 region in the sporadic form of these diseases, as well as in
hereditary breast and ovarian cancers (Saito et al., 1993; Cropp et
al., 1994), and it has therefore been postulated that BRCA1 may
have an important role not only in hereditary but also in sporadic
breast and ovarian cancers.
BRCA1 somatic mutations have been observed in a small
number of ovarian tumors (Hosking et al., 1995; Merajver et al.,
1995), but not in sporadic breast tumors (Futreal et al., 1994).
Nevertheless, it is possible that BRCA1 plays a role in sporadic
breast carcinomas through alterations other than coding-region
mutations. One mechanism of aberrant regulation may be through
lower levels of BRCA1-mRNA expression, as has been observed
for breast-adenocarcinoma cells in comparison with normal mam-
mary epithelial cells (Thompson et al., 1995). Inhibition of BRCA1
expression with anti-sense oligonucleotides indicated that reduced
expression of BRCA1 increased the proliferative rate of benign and
of malignant breast epithelial cells (Thompson et al., 1995). These
findings suggest that BRCA1 may play a role in the development of
sporadic breast cancer through growth stimulation induced by low
levels of BRCA1 mRNA.
The BRCA1 gene encodes a 1863-amino-acid polypeptide that
has a potential granin motif (Jensen et al., 1996) as well as a
possible RING finger motif (Lovering et al., 1993), suggestive of a
role in secretory pathway and transcriptional regulation respec-
tively. Chen et al. (1995) have reported an aberrant sub-cellular
localization of the BRCA1 protein in breast-cancer cell lines and
biopsies. Moreover, the BRCA1-protein staining was significantly
low in 4% of the breast-cancer biopsies, supporting the hypothesis
of reduced BRCA1 expression in the etiology of sporadic breast
To investigate the importance of BRCA1 expression in sporadic
breast cancers, we have evaluated the level of expression in 21
normal breast epithelial specimens and in 74 primary tumors from
axillary-node-negative (ANN) breast-cancer cases. We found that
the levels of BRCA1 mRNA expression were reduced in some
tumor specimens, and we further investigated the possible roles of
deletions, preferential allelic expression and regulatory mutations
in the low levels of BRCA1 expression.
MATERIAL AND METHODS
Axillary-node-negative breast-carcinoma samples were obtained
in collaboration with Dr. D. Sutherland (Sunnybrook Steroid-
Receptor Laboratory, Toronto) and hospitals in Toronto. Normal
tissue used in this study was extracted from mammary-reduction
samples and normal tissue adjacent to the tumor. DNA and RNA
were extracted from quick-frozen tumor and adjacent histopatho-
Quantitation of BRCA1 mRNA levels
cDNA was transcribed from cellular RNA by using random
leukemia-virus reverse transcriptase (GIBCO-BRL, Gaithersburg,
MD). Quantitative RT-PCR (Noonan et al., 1990) was performed
by multiplexing a region of BRCA1 cDNAin exons 12 and 13 with
a region of an internal control gene, porphobilinogen deaminase
(PBGD) (Finke et al., 1993). Expression of the housekeeping gene,
PBGD, was evaluated in each reaction and compared with that of
BRCA1 to control for variations in the quality and quantity of RNA
obtained from the tumor specimens. Although PBGD is located on
the long arm of chromosome 11 at q23.3, it does not map to regions
Grant sponsors: Canadian Breast-Cancer Research Initiative/National
Cancer Institute of Canada.
*Correspondence to: Samuel Lunenfeld Research Institute, Mount Sinai
Hospital, 600 University Avenue, Room 870, Toronto, Ontario, M5G 1X5,
Canada. Fax: (416) 586 8663. E-mail: firstname.lastname@example.org
Received 10 October 1997; Revised 2 March 1998
Int. J. Cancer: 77, 1–6 (1998)
?1998 Wiley-Liss, Inc.
Publication of the International UnionAgainst Cancer
Publication de l’Union Internationale Contre le Cancer
found to be amplified (11q13; Kallioniemi et al., 1994) or deleted
(11q22-q23.1 and 11q25-qterm; Koreth et al., 1997) in breast
tumors. The primers for BRCA1 (BRCA1-1F, 5?-GGCTATCCTCT-
TGGA-3?) and PBGD (PBGD-1, 5?-TGTCTGGTAACGGCAAT-
GCG-3? and PBGD-2A, 5?-TTGCCACCACACTGTCCGTCT-3?)
amplify fragments of 201 and 120 bp respectively.
PCR reactions were carried out in a 12-µl reaction volume
containing 1? PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl,
side triphosphate (dNTP), 10 pmol of each BRCA1 and PBGD
primers, 1 µ of AmpliTaq DNA polymerase (Perkin Elmer/Cetus,
Foster City, CA) and 4 µl of cDNA reaction containing 50 ng of
total RNA. The thermal cycling conditions were 94°C for 30 sec,
55°C for 30 sec and 72°C for 1 min. The PCR products were
separated on 10% polyacrylamide gels.
For each sample, PCR was carried out at 3 different cycles in the
exponential phase of amplification to ensure quantitative evalua-
tion of the PCR reaction.The band intensities ofBRCA1 and PBGD
were determined by densitometry and the ratio of BRCA1/PBGD
mRNA expression was averaged over at least 2 cycles in the
logarithmic phase of amplification.
Deletion analysis at the BRCA1 locus
The presence of deletions at the BRCA1 locus was evaluated by
multiplex analysis of the PCR products of the BRCA1 gene and an
internal control gene, asparagine synthetase (AS). The AS gene
serves as a control for DNA copy number, since it maps to
chromosome 7q21.3 (Heng et al., 1994), a region that is not
commonly amplified or deleted in breast cancers.Although there is
a region on the long arm of chromosome 7 (7q31) that is often
subject to loss of heterozygosity (LOH) in breast cancers, AS is
located beyond the region of LOH. Reactions were carried out in a
15-µl volume containing 1? PCR buffer, 1.6 mM MgCl2, 100 µM
of each dNTPs, 10 pmoles of each BRCA1 and AS primers, 1 µ of
AmpliTaq DNA polymerase (Perkin Elmer/Cetus) and 50 ng of
genomic DNA. The BRCA1 (BRCA1-3F, 5?-AGCAGAGGGA-
CACTCCGCGAC-3? and AS-5, 5?-TGCAACTTTGCCATTTG-
GCT-3?) primers yield fragments of 168 and 96 bp respectively.
The cycling conditions and evaluation of the PCR results were
carried out as described above. The quality of the genomic DNA
samples was assessed by running 50 ng of each sample on 1.2%
agarose gels; samples that appeared to have been degraded were
excluded from the study.
Allelic expression analysis
The BRCA1 primers for DNA(BRCA1-3F and BRCA1-4R, 168
bp) and RNA(BRCA1-1F and BRCA1-4R, 201 bp) amplifying the
region containing the C-T polymorphism at nucleotide 4427 in the
BRCA1 gene (Miki et al., 1994) were used to carry out a PCR
reaction as described above. PCR products (8 µl) were digested
with 5 µ EarI restriction enzyme at 37°C overnight. The DNA(128
and 40 bp) and the RNA (161 bp and 40 bp) products were run on
10% polyacrylamide gels. The intensity of the digested PCR
fragments representing each allele were measured by densitometry
for DNA and for RNA. PCR reaction and digestion analysis for
each RNA and DNA sample were repeated twice, and results were
in complete concordance.
Analysis of the promoter mutations
The potential promoter region of the BRCA1 gene was analyzed
by single-strand-conformation polymorphism (SSCP) using 4
sets of primers, BRCA1-P1 (5?-GGCTACCGCTAAGCAGCAGC-3?)
3P (5?-CGAAAGGCCTTGGCCACACT-3?) and BRCA1-4P
(5?-GTCTTCCTAGGAGTTGTAGG-3?), BRCA1-5P (5?-GGGC-
CCACTGTCCCTTTTCC-3?) and BRCA1-6P (5?-TCCCATCC-
TCTGATTGTACC-3?), BRCA1-7P (5?-GGATGACGTAAAAG-
GAAAGA-3?) and BRCA1-8P (5?-CATTAGGGCGGAAAGAG-
TGG-3?) producing 170-, 220-, 262- and 222-bp fragments respec-
tively. The PCR reaction for each primer set was carried out in a
30-µl volume containing 1? PCR buffer, 50 µM of each dNTPs, 6
pmol of each primers, 2 µCi of [a-32P]deoxycytidine triphosphate
(dCTP; 3000 Ci/mmol, Du Pont, Markham, Canada), 1 µ of
AmpliTaq DNA polymerase (Perkin Elmer/Cetus) and 50 ng of
genomic DNA. The PCR reaction was performed at 94°C for 40
sec, 57°C for 40 sec and 72°C for 70 sec for 33 cycles. The PCR
products were diluted 1:3 with formamide-loading buffer (95%
formamide, 2 mM EDTA, pH 8.3, 0.05% bromophenol blue, 0.05%
xylene cyanol), loaded on 6% non-denaturing acrylamide (40:1
acrylamide:bisacrylamide) gels in 10% glycerol and 0.5? Tris-
borate EDTA(TBE) buffer and electrophoresed for 17 hr at 15°C.
Evaluation of BRCA1 mRNA expression in ANN
Since the limitation in material is a major concern in studies of
human primary-tumor specimens, we developed a quantitative
RT-PCR assay for the analysis of BRCA1 mRNA levels, as
described in ‘‘Material and Methods’’.The level of BRCA1 mRNA
was determined in 74 ANN breast carcinomas (Fig. 1). BRCA1
FIGURE 1 – Evaluation of BRCA1 mRNA expression in ANN breast carcinomas (T800, T510, T105, T693) by quantitative RT-PCR. Each
sample was analyzed at 3 different PCR cycles (24-26-28) and the intensities of bands were measured by densitometry. The BRCA1/PBGD ratio
was calculated at each cycle, and only values within the logarithmic phase of PCR amplification were used in determining the average relative
expression level as indicated for each sample (see ‘‘Results’’).
O¨ZC ¸ELIK ET AL.
mRNAexpression varied greatly among tumor specimens (relative
units of 0.02 to 2.3), with some tumors exhibiting barely detectable
amounts of BRCA1 mRNA. In contrast, analysis of 21 histopatho-
logically normal breast epithelial samples indicated that normal
specimens expressed BRCA1 mRNA at levels ranging from 0.8 to
2.5 relative units. Comparison of the levels of BRCA1 expression
in the tumor and the normal samples (Fig. 2) revealed that the
distribution of BRCA1-expression levels exhibited by tumors was
significantly lower than the distribution of mRNA levels for the
normal tissues (2-sided Wilcoxon-Mann-Whitney
value ? 0.004).
RNA degradation, which can lead to preferential degradation of
larger RNA fragments, can be a concern when analyzing mRNA
expression from primary tumors. To control for this variable,
quantitative multiplex PCR assays were also performed in which a
BRCA1 fragment smaller than that of the PBGD internal control
was amplified (data not shown). The results of the latter were in
complete concordance with those of the first set of experiments,
and exclude the presence of RNA degradation as a cause for low
levels of BRCA1 expression observed in some tumors.
Deletion analysis of the BRCA1 gene
To address the possibility that the reduction in BRCA1 mRNA
expression resulted from allelic loss, a sub-group of 47 tumor
samples was analyzed for DNA deletion at the BRCA1 locus, by
means of a quantitative PCR assay (Fig. 3). Of the 47 tumor DNAs,
40 (85%) exhibited a ratio of copies of BRCA1 to asparagine
synthetase (AS) above 0.69 (range 0.7–1.2), and were interpreted
FIGURE 2 – Distribution of relative expression levels of BRCA1 mRNA expression in tumor (n ? 74) and normal breast epithelial (n ? 21)
samples. The reduced expression in the tumors was found to be statistically significant (p ? 0.004 by the Wilcoxon-Mann-Whitney test).
FIGURE 3 – Deletion analysis of BRCA1 gene by quantitative PCR. For each DNAsample, the BRCA1/AS ratio was analyzed at 2 different PCR
cycles within the logarithmic phase of the PCR reaction.The average ratios of BRCA1/AS are shown in representative tumor (T780,T681,T1200,
T1609) and normal (C8168) samples in the figure. Samples T681 and T1200 had markedly lower BRCA1/AS ratio than normal samples,
indicating the presence of deletions.
LOW LEVELS OF BRCA1-mRNAEXPRESSION IN HUMAN BREAST CANCERS
as not having deletion at the BRCA1 locus. The mean BRCA1/AS
ratio of 0.93 ? 0.12 for these 40 samples was comparable with the
value of 0.99 ? 0.3 found for non-cancerous DNAsamples. On the
other hand, 7 tumors (15%) had a BRCA1/AS ratio below 0.69
(range 0.35–0.65), and were interpreted as having potential small
intragenic or large chromosomal deletions at the BRCA1 locus.
Examination of the levels of BRCA1 mRNA in the 7 latter
specimens (tumors 850, 932, 1200, 800, 617, 681 and 1035 inTable
I) indicated that the distribution of levels of BRCA1 mRNA in
these specimens with deletions was significantly different from that
in the specimens without deletions (2-sided Wilcoxon-Mann-
Whitney test; p value ? 0.001), in agreement with the hypothesis
that allelic deletion may lead to low BRCA1 mRNAlevels in some
cases. On the other hand, DNA deletion did not account for all
cases of low BRCA1 mRNA, suggesting that additional mecha-
nisms contribute to a reduction in BRCA1 expression.
Allelic expression analysis of the BRCA1 gene
We explored the role of regulatory mutations, including those
affecting mRNAstability, in decreasing BRCA1 expression.Allelic
analysis was performed by taking advantage of a C-T polymor-
phism at nucleotide 4427 that changes the recognition site of the
EarI restriction enzyme. Tumor-DNAsamples (65 in all) represent-
ing cases of low and high BRCA1 mRNA levels were analyzed by
enzyme digestion of PCR products, and 21 were found to be
heterozygous at the EarI recognition site. Digestion analyses of
PCR products from these 21 informative cases were carried out for
DNA and for cDNA in order to detect differences in the allelic
expression patterns (Fig. 4). The intensities of the bands represent-
ing each allele were evaluated by densitometry both for RNA and
for DNA samples. In the 21 informative cases, 12 showed
tial allelic expression was observed in 9 samples; 7 of these showed
an approximately 2–5-fold, and 2 a more than 5-fold, difference in
expression of one allele. Comparison of BRCA1 mRNAexpression
in these 21 cases (Table I) revealed that the distribution of
expression levels in tumors with preferential allelic expression was
significantly lower than in tumors with equivalent allelic expres-
sion(2-sidedWilcoxon-Mann-Whitneytest;exactpvalue ? 0.001).
Although preferential allelic expression can be due to allelic
deletion, this does not appear to be the case for 4 of the samples.
These 4 specimens also exhibited significantly lower mRNA
expression (2-sidedWilcoxon-Mann-Whitney test; exact p value ?
0.03) without reduced amounts of BRCA1 DNA, suggesting that
other allelic silencing mechanisms are involved.
Analysis of mutations in the potential promoter region
of the BRCA1 gene
tional process and considerably reduce the amount of mRNA
produced. Although not well characterized, the DNA sequence
upstream of the 5? region of exon 1 of the BRCA1 gene has been
shown to lie in head-to-head orientation with the adjacent 1A1-3B
gene, the 5? region of which contains several potential regulatory
sequences (Brown et al., 1994). To assess the possibility of
regulatory mutations as a cause of reduced expression, this
potential 5? promoter region of the BRCA1 gene was analyzed in 25
samples exhibiting relatively reduced levels of BRCA1 mRNA.
Single-strand-conformation-polymorphism analyses were per-
formed for the entire exon 1 of the BRCA1 gene, the region lying
between exon 1 of BRCA1 and exon 1A of the 1A1-3B gene and
most of exon 1A of the 1A1-3B gene. In addition, the region
between exons 1A and 1B of 1A1-3B was analyzed, since it
contains CAT-regulatory elements. We did not observe any SSCP
shifts in the regions analyzed (data not shown).
In this study, we found that a proportion of human primary breast
expression has been associated with a proliferative phenotype in
mammary epithelial cells, and may have an important role in
that down-regulation of BRCA1 expression through anti-sense
Thus, reduction in mRNA expression may be a way in which
BRCA1 is involved in the development of sporadic breast cancer.
In investigating the possible mechanisms underlying the ob-
served reduction in BRCA1 expression, we evaluated the BRCA1
locus for deletion, since LOH has been shown to be a common
event at the 17q21 region. A reduction in BRCA1 DNA level was
detected in 15% of specimens, and was associated with relatively
low levels of BRCA1 mRNA expression. Thus, LOH or deletion
events at the BRCA1 locus may contribute to sporadic-breast-
cancer tumorigenesis through down-modulation of expression of
the BRCA1 gene.
Since low BRCA1 mRNA expression was not always accompa-
down-regulating the expression of BRCA1. Other studies have
described preferential expression of mRNAfrom one allele, despite
the presence of both copies of the gene at the DNA level (Miki et
al., 1994; Thompson et al., 1995). Our analysis showed that a
number of tumors with low BRCA1 mRNA levels exhibited
preferential allelic expression. However, not all tumors displaying
allelic-specific expression can be accounted for by DNA deletion,
suggesting the presence of mutations that affect either the transcrip-
tional regulation of the gene or the stability of the mRNA.
To further explore the possibility of transcriptional regulatory
defects, we analyzed the 5? end of the BRCA1 gene for the presence
of potential promoter mutations by SSCP. We did not observe any
SSCPshifts, suggesting that mutation is not a frequent event in this
region of the BRCA1 gene. However, the transcriptional regulatory
elements of BRCA1 have not been fully characterized, and we
TABLE I – ALLELIC EXPRESSION STATUSAND mRNALEVELS OF BRCA1*
Tumor specimen Allelic expressionExpression levels
*NI, not informative; EE, equivalent allele expression; DE, differen-
tial allele expression.
O¨ZC ¸ELIK ET AL.
cannot rule out the possibility of mutations in other regulatory
regions affecting BRCA1 transcription.
The data presented here support the hypothesis that BRCA1 may
be involved in sporadic breast cancer through a reduction in
BRCA1 mRNA levels. Several mechanisms can lead to reduced
expression of a gene in vivo. In this study, we found deletion at the
BRCA1 locus and preferential allelic expression in tumors with
low BRCA1 mRNA levels. Future studies should involve more
stringent analysis of the promoter and mRNAstability elements, as
well as investigation of the clinical significance of low levels of
We thank the pathologists and the surgeons at the co-operating
hospitals (Mount Sinai Hospital, Toronto Hospital, Women’s
College Hospital, Toronto East General Hospital, Saint Joseph’s
Hospital, North York General Hospital, and Credit Valley Hospi-
tals), also Dr. P. Watson (Manitoba Breast Tumor Bank) for the
normal epithelial cDNA samples. This work was supported by the
Canadian Breast-Cancer Research Initiative/National Cancer Insti-
tute of Canada (I.L.A. and S.B.B.). Dr. S.B. Bull is a National
Health Research Scholar (Health Canada).
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FIGURE 4 – Allelic expression analysis of the BRCA1 gene. PCR products from DNA(D) and RNA(R) from each sample were digested with the
EarI enzyme (diagram below the picture) and the band intensities of the corresponding alleles in DNA and RNA of each sample were compared.
Sample T1226 represents equivalent expression, whereas samples T1647, T1192 and T1835 show preferential expression of one allele. M, marker
is a 123-bp ladder.
LOW LEVELS OF BRCA1-mRNAEXPRESSION IN HUMAN BREAST CANCERS
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O¨ZC ¸ELIK ET AL.