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Molecular Biology and Genetics |
Cancer Research Campaign Human Cancer Genetics Research Group, University of Cambridge, Addenbrooke’s Hospital, Cambridge CB2 2QQ, United Kingdom [S. A. G., K. A. F. d. F., P. H., P. P., B. A. J. P.]; Institute of Cancer Research, Surrey SM2 5NG, United Kingdom [W. D. D., S. M. E., D. P. D., J. K., M. R. S., R. A. E.]; Department of Urology, St George’s Hospital, London SW17 0QT, United Kingdom [W. D. D., R. S. K.]; Hedley Atkins/Imperial Cancer Research Fund Breast Pathology Laboratory, Guy’s Hospital, London SE1 9RT, United Kingdom [C. G., D. B.]; Royal Marsden National Health Service Trust, Surrey SM2 5PT, United Kingdom [A. A-J., D. P. D., R. J. S., A. L. D., R. A. E.]; Cancer Research Campaign Genetic Epidemiology Unit, Strangeways Research Laboratory, Cambridge CB1 8RN, United Kingdom [D. F. E.]; and Cancer Research Campaign Section of Epidemiology, Institute of Cancer Research, Surrey SM2 5NG, United Kingdom [D. F.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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67 years at
diagnosis. No germ-line mutations were found in BRCA1.
Two germ-line mutations in BRCA2 were found, and both
were seen in individuals whose age at diagnosis was very young (56
years) and who were members of an affected sibling pair. One is
a 4-bp deletion at base 6710 (exon 11) in a man who had prostate cancer
at 54 years, and the other is a 2-bp deletion at base 5531 (exon 11) in
a man who had prostate cancer at 56 years. In both cases, the wild-type
allele was lost in the patient’s prostate tumor at the
BRCA2 locus. However, intriguingly, in neither case did
the affected brother also carry the mutation. Germ-line mutations in
BRCA2 may therefore account for about 5% of prostate
cancer in familial clusters. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3 cases showed
no evidence of linkage to 1q. Recent studies have suggested that other
loci exist: (a) one at 1q42 (16)
that has not
yet been confirmed; (b) one at Xq27–28 (17)
that accounts for 16% of families; and (c) one at 1p36
(18)
. Preliminary evidence from analysis of 187 prostate
cancer clusters in our laboratory indicates that these loci do not
account for all of familial prostate cancer. Other genes therefore
remain to be located. There is an association between breast and prostate cancer in families; a higher incidence of prostate cancer among male relatives of breast cancer patients has been reported previously (19, 20, 21) . Anderson and Badzioch (22) report a doubling of familial breast cancer risk when prostate cancer is present in the family history. BRCA1 and BRCA2 are located on chromosomes 17q12–21 and 13q12–13, respectively. LOH4 studies in prostate cancer have shown that 52% of tumors have LOH at 17q; in one study, 44% of tumors had LOH with a marker intragenic in the breast cancer predisposition gene BRCA1 (23) . BRCA1 carriers also have a 3-fold increased risk of mortality from prostate cancer (24) . We have demonstrated a 25% incidence of LOH at the BRCA2 locus in familial and sporadic prostate cancer (25) . Tonin et al. (26) calculated that there was a relative risk of 7.2 of prostate cancer in BRCA2 carriers but did not mention an age-at-onset effect.
One family with four prostate cancer cases but no breast cancer has been reported to have a germ-line BRCA1 mutation (27) that is 185delAG, a common BRCA1 mutation in Ashkenazi Jewish families with breast cancer (28) . This family was indeed of this ethnic origin. Workers from Iceland (29) have reported a common BRCA2 mutation (999del5) in nine Icelandic cancer families with multiple cases of breast cancer. Some of these families also had multiple cases of prostate cancer. Icelandic studies have shown 2.7% of prostate cancer cases in Iceland carry this mutation (30) . Four studies (31, 32, 33, 34) have reported that there is no increased frequency of the founder Ashkenazi BRCA1 and BRCA2 mutations over that expected in this population when germ-line DNA from prostate cancer cases with and without a family history are analyzed.
The Cancer Research Campaign/British Prostate Group United Kingdom
Familial Prostate Cancer Study aims to investigate the role of genetic
susceptibility to prostate cancer. As part of the study of high
penetrance genes, prostate cancer cases with an increased chance of
harboring a prostate cancer susceptibility gene are being collected.
Those clusters with a relative risk of developing prostate cancer of
4 are targeted for collection (35)
; these are clusters
of 3 prostate cancers at any age or in sibling pairs, preferably
where one is <65 years at diagnosis. The first 38 of these
clusters were analyzed in this study; BRCA1 and
BRCA2 were analyzed from germ-line DNA to assess the
contribution of BRCA1 and BRCA2 germ-line
mutations to familial prostate cancer. This is the first study to
analyze the entire coding region of BRCA1 and
BRCA2 in a non-Ashkenazi series of prostate cancer clusters.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Wherever possible, DNA from the youngest available member of each
cluster was studied. Tumor DNA was prepared after microdissection of
tumor tissue from paraffin sections. Microdissected tissue was removed
into 200 µl of extraction buffer [1x RedHot polymerase buffer
(Applied Biosystems), 1.5 M
MgCl2, 0.45% NP40, 0.45% Tween 20, and 200
µg/ml proteinase K] and incubated at 55°C for 12 h.
After incubation, proteinase K was deactivated by heating to 99°C for
10 min.
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1 kb) for truncating
mutations and was used to analyze exon 11 of BRCA1 (which
represents approximately 60% of the coding sequence) and exons 10 and
11 of BRCA2 (60% of the coding region). The remaining exons
and splice boundaries of both genes were screened using HA. The
majority of germ-line mutations reported in BRCA1 and
BRCA2 result in truncation of the predicted protein as a
result of frameshift, nonsense, or splice site alterations; therefore,
the combination of PTT and HA was considered a sensitive and efficient
method of analysis. Direct sequence analysis was used to confirm the
precise nucleotide alteration associated with PTT and/or HA variants.
The primer sequences for BRCA2 and their respective product sizes and
amplification conditions have been described previously
(36)
. PTT was performed for the largest two exons of BRCA2 and for the largest exon only for BRCA1. Primers were designed to PCR amplify exons 10 and 11 of BRCA2 and exon 11 of BRCA1 from genomic DNA in overlapping fragments ranging in size from 1.0–1.3 kb. PTT was performed as described previously (36) .
Coding exons 2, 3, 5–10, and 12–24 of BRCA1 and 2–9 and 12–27 of BRCA2 were amplified from genomic DNA. The 5' and 3' splice boundaries for exon 11 of BRCA1 and exons 10 and 11 of BRCA2 were also amplified from genomic DNA. SSCA/HA was performed in 1x mutation detection enhancement polyacrylamide gels as described previously (36) . Syder Green staining was used for DNA detection. Sequence analysis of variant PTT and SSCA/HA samples was performed using the ABI 373A DNA sequencer by dye terminator cycle sequencing with AmpliTaq DNA polymerase FS (Perkin-Elmer).
Haplotype Analysis.
Peripheral blood DNA and tumor DNA from paraffin-embedded tumor
tissue was PCR amplified with three polymorphic microsatellite markers,
D13S260, D13S263, and D13S267, which flank the BRCA2 gene on
chromosome 13q12. PCR products were electrophoresed at 250 V on 8–12%
polyacrylamide gels for 14–16 h at a constant temperature of 18°C.
Gels were visualized after silver staining as described previously
(36)
.
Immunohistochemical Staining for BRCA2 Protein.
Sections (4 µm) were cut from blocks of prostate cancer tissue,
picked up on adhesive-coated slides (Vector Laboratories, Burlingame,
CA), and baked overnight at 56°C before staining. The BRCA2 antigen
was unmasked by placing the sections in a pressure cooker containing
boiling 0.01 M citrate buffer (pH 6.0) and boiling
under pressure for 2 min. Sections were cooled in running tap water and
rinsed in Tris-buffered saline before the application of the rabbit
polyclonal BRCA2 antibody (courtesy of N. Spurr and D. M. Barnes,
Imperial Cancer Research Fund) for 1 h. Antibody binding
was detected using a conventional peroxidase-conjugated streptavidin
biotin complex method (Dako Ltd., High Wycombe, United Kingdom). Sites
of peroxidase activity were detected using diaminobenzidine as the
chromogen. A breast carcinoma known to express BRCA2 was used as a
positive control. A negative control in which the primary antibody was
replaced with Tris-buffered saline was included for each case. The
presence of any nuclear staining was recorded as positive.
The BRCA2 antibody used was raised against a COOH terminus peptide synthesized using the sequence published by Wooster et al. (37) . The peptide, which contained residues 2301–2320 (DGKGKEEFYRALCDVKAT) with a peak corresponding to a calculated Mr of 2101, was prepared using the fastmoc HBTU method to a standard purity (25) .
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Analysis of BRCA2 revealed three variants that were
not detected in any other individuals from the sample set. Two of these
variants were detected as truncated proteins by PTT; the third was
detected as a heteroduplex variant in exon 22. In family PRY1042, from
individual 201, a PTT variant in exon 11 (Fig. 1
) was characterized as a 4-bp deletion beginning at nucleotide 6710
(6710delACAA) that is predicted to cause a frameshift and premature
truncation of the predicted protein at codon 2166. This mutation has
not been reported previously. HA using primers designed to amplify the
region flanking this mutation confirmed the presence of this alteration
in the index case and also showed loss of the wild-type allele in DNA
from tumor tissue from the same individual. However, HA of DNA prepared
from tumor tissue from the affected sibling, who was diagnosed with
prostate cancer at 48 years, indicated that this individual did not
carry the BRCA2 mutation (Fig. 2a
). Haplotype analysis using three polymorphic microsatellite
markers flanking the BRCA2 gene at chromosome 13q12–13 was
performed on DNA from the two affected brothers from family PRY1042.
Although it was not possible to phase the haplotypes, the allele sizes
of each marker indicate that both copies of chromosome 13 differ
between the two brothers (data not shown). This is consistent with the
observation of a germ-line BRCA2 mutation in one affected
brother but not in the other.
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) was characterized as a 2-bp deletion
beginning at nucleotide 5531 (5531delTT). This is a novel mutation and
is predicted to result in frameshift and truncation at codon 1772. The
family history of PRS2024 with respect to the index case consists of
the father diagnosed with prostate cancer at 87 years, the mother
diagnosed with breast cancer at 76 years, and a brother diagnosed with
prostate cancer at 41 years. HA confirmed the presence of the mutation
in the index case but indicates that the same alteration is not present
in the father’s constitutional DNA or in tumor DNA from the affected
brother (Fig. 2b
). No DNA was available from the proband’s
mother. Neither deletion was found in over 100 normal
individuals tested. A heteroduplex variant was detected in exon 22 in a prostate cancer case diagnosed at 46 years, from family PRS1081. This variant was characterized as a single-base substitution (G to T at nucleotide 9078) that is predicted to convert a lysine amino acid residue to an asparagine residue (K2950N) and has not been reported previously. DNA was not available from a second affected individual from the family to confirm segregation of this alteration with the disease. To examine whether this alteration is a putative missense mutation or merely a rare variant without disease association, DNA was analyzed from a series of normal individuals for the presence of the sequence change. The identification of the K2950N alteration in 2 of 340 (0.59%) normal chromosomes suggests that this change is a rare polymorphism that is not associated with the disease. Several other heteroduplex variants were observed throughout BRCA2 in the sample set. However, these all occurred relatively frequently and were characterized as either previously reported coding or intronic polymorphisms that are not considered to be disease related.
The BRCA2 antibody stains 25% (25)
of sporadic
prostate cancer samples. We found that the two individuals with
deletions in BRCA2 did not exhibit any staining in their
prostate tumors, but their siblings without mutation and the individual
with the K2950N variant did so. In the latter case, multifocal areas of
intense nuclear staining were observed within tumor areas (Fig. 3 and b
).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The fact that several reports have now shown that germ-line
mutations in the BRCA2 gene are associated with an increased
risk of prostate cancer (29
, 40
, 41)
makes
BRCA2 a putative candidate gene for familial prostate cancer
in general. Our data using linkage analysis at the BRCA2
locus in 100 affected sibling pairs with prostate cancer has estimated
that up to 30% of such pairs (95% confidence interval, 0–70%) may
be due to the BRCA2 gene (42)
. Although the
Cancer Research Campaign/British Prostate Group United Kingdom Familial
Prostate Cancer Study ascertained sibling pairs with at least one of
the affected siblings at age <67 years at diagnosis, the two mutations
described here have occurred in prostate cancer cases occurring at 56
years, and BRCA2 germ-line mutations may therefore
contribute to a significant proportion of young cases within these
pairs. We were surprised to find that in both of the sibling pairs, the
BRCA2 mutation was not present in the affected brother. This
was unexpected because both brothers affected who did not carry the
mutation were younger than those who did. There are two possible
explanations for this. The first is that the BRCA2 mutation
is not cancer causing. This is unlikely because both mutations are
deletions and would be expected to have a major effect on the function
of the protein. Furthermore, the wild-type allele was lost in the
subsequent prostate tumor in PRY1042, individual 201. It is interesting
that BRCA2 antibody staining was negative in both patients
with BRCA2 germ-line mutations but positive in their
brothers who did not have a truncating mutation and also in the
individual with the K2950N variant. The overall frequency of positive
BRCA2 staining in sporadic prostate cancer is 25%
(25)
. The second possible explanation is that there is
another gene segregating in the prostate cancer clusters PRY1042 and
PRS2024 and that the BRCA2 mutation is acting as a modifier.
There is some evidence for this in Icelandic families with
BRCA2 mutations, in which prostate cancer incidence is
inversely proportional to male breast cancer incidence in branches of
the same family with the same germ-line mutation (29)
.
Both families with BRCA2 mutations contained an individual
with a cancer at another site. PRY1042 contained a case of pancreatic
cancer, and PRS2024 contained a case of breast cancer. In PRS2024, the
father of the prostate cancer case with the germ-line BRCA2
mutation did not have the mutation, despite being affected with the
disease. If it had been inherited and was not a novel mutation, then
this is presumed to have been inherited from the case’s mother. This
raises the possibility that BRCA2 germ-line mutations are
only seen in the context of prostate cancer families with associated
cancers known to occur in BRCA2 families [namely breast,
pancreatic, ovarian, and gall bladder cancer (41)
]. Of
the 38 families, 25 (66%) had prostate cancer and other cancers. Table 1
lists the other cancers. Of 25 prostate cancer families with prostate
and other cancers, two had germ-line mutations in BRCA2
(8%). None of the 13 families with prostate cancer alone had
BRCA2 mutations. Our data suggest that a proportion of
prostate cancer families may harbor germ-line mutations in the
BRCA2 gene. Because the clusters we analyzed were small, it
is possible that we have underestimated the contribution of germ-line
mutations in BRCA2 to prostate cancer overall. Additional
studies are warranted in larger series of both prostate cancer clusters
and isolated cases at varying ages to determine the size of this
proportion in different prostate cancer populations.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Cancer Research Campaign; The
Institute of Cancer Research, Prostate Cancer Research, United Kingdom;
and Imperial Cancer Research Fund. S. M. E. was supported by Royal
Marsden National Health Service Trust Charitable Funds and is
now supported by the Cancer Research Campaign. D. P. D. is supported
by the Bob Champion Cancer Trust, and W. D. D. is supported by
Zeneca, Yamanouchi, and Merck Sharpe & Dohme Pharmaceuticals.
B. A. J. P. is a Gibb Fellow of the Cancer Research Campaign. 
2 The Cancer Research Campaign/British Prostate
Group United Kingdom Familial Prostate Cancer Study
collaborators. List available on request.
3 To whom requests for reprints should be
addressed, at Institute of Cancer Research, 15 Cotswold Road, Sutton,
Surrey SM2 5NG, United Kingdom. Phone: 44-0-2081-643-8901, ext. 3642;
Fax: 44-0-2081-770-1489; E-mail: ros{at}icr.ac.uk
4 The abbreviations used are: LOH, loss of
heterozygosity; PTT, protein truncation test; SSCA, single-stranded
conformational analysis; HA, heteroduplex analysis.
5 http://www.nchgr.nih.gov/Intramural_research/Lab_transfer/Bic/.
Received 9/20/99. Accepted 6/12/00.
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