[CANCER RESEARCH 60, 4513– 4518, August 15, 2000]
`
`The Frequency of Germ-line Mutations in the Breast Cancer Predisposition Genes
`BRCA1 AND BRCA2 in Familial Prostate Cancer1
`
`Simon A. Gayther, Karen A. F. de Foy, Patricia Harrington, Paul Pharoah, William D. Dunsmuir,
`Stephen M. Edwards, Cheryl Gillett, Audrey Ardern-Jones, David P. Dearnaley, Douglas F. Easton, Deborah Ford,
`Robert J. Shearer, Roger S. Kirby, Anna L. Dowe, Joanne Kelly, Michael R. Stratton, The Cancer Research
`Campaign/British Prostate Group United Kingdom Familial Prostate Cancer Study Collaborators,2
`Bruce A. J. Ponder, Diana Barnes, and Rosalind A. Eeles3
`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.]
`
`ABSTRACT
`
`Predisposition to prostate cancer has a genetic component, and there
`are reports of familial clustering of breast and prostate cancer. Two highly
`penetrant genes that predispose individuals to breast cancer (BRCA1 and
`BRCA2) are known to confer an increased risk of prostate cancer of about
`3-fold and 7-fold, respectively, in breast cancer families. Blood DNA from
`affected individuals in 38 prostate cancer clusters was analyzed for germ-
`line mutations in BRCA1 and BRCA2 to assess the contribution of each of
`these genes to familial prostate cancer. Seventeen DNA samples were each
`from an affected individual in families with three or more cases of prostate
`cancer at any age; 20 samples were from one of affected sibling pairs
`where one was <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
`
`Prostate cancer is the second most common cause of cancer mor-
`tality in men in the United Kingdom. Approximately 14,000 cases/
`year and 8,742 deaths/year are reported in England and Wales (1, 2).
`Its incidence is increasing by 10% every 5 years (3), even when the
`effect of screening is taken into account, and 13% of cases occur in
`men in their preretirement years. One percent of cases occur in
`men ,55 years of age in the United Kingdom. There is increasing
`evidence that there is an inherited component to many of the common
`cancers (4), and prostate cancer is no exception. Familial clustering of
`prostate cancer has been observed, the most dramatic of which is the
`large prostate cancer kindreds described in Utah, United States (5);
`
`Received 9/20/99; accepted 6/12/00.
`The costs of publication of this article were defrayed in part by the payment of page
`charges. This article must therefore be hereby marked advertisement in accordance with
`18 U.S.C. Section 1734 solely to indicate this fact.
`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@icr.ac.uk.
`
`furthermore, case-control studies (6) show that relatives of cases have
`an increased relative risk of developing the disease, and this has been
`confirmed in two cohort studies (7, 8). This relative risk increases
`markedly when the age of the index case decreases or the number of
`affected individuals in a cluster increases, which is evidence that this
`increased risk has a genetic component. One segregation analysis has
`led to the proposed model of at least one highly penetrant gene (88%
`of gene carriers would develop prostate cancer by age 85) that
`accounts for 43% of cases diagnosed at ,55 years (9); two others
`support this model, but with a higher gene frequency of about 1% and
`a penetrance of 63% (10, 11). Other reports have suggested a reces-
`sive or X-linked model (12, 13). Recently, a highly penetrant prostate
`cancer susceptibility locus HPC1 was mapped to 1q, but this only
`accounts for up to 34% of families with four or more cases in one
`study of 91 families (14) or, at most, 20% of such large clusters in
`another study of 35 families (15). In the latter study, 1q linkage
`analysis in 101 clusters with #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). Prelim-
`inary 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 –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
`
`4 The abbreviations used are: LOH, loss of heterozygosity; PTT, protein truncation
`test; SSCA, single-stranded conformational analysis; HA, heteroduplex analysis.
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`CONTRIBUTION OF BRCA1 AND BRCA2 TO FAMILIAL PROSTATE CANCER
`
`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 (31–34) 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.
`
`MATERIALS AND METHODS
`
`Patient Material. Peripheral blood DNA from 38 affected individuals who
`were members of prostate cancer clusters was studied. The composition of the
`clusters is shown in Table 1. 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 ml of extraction buffer [13 RedHot polymerase buffer
`(Applied Biosystems), 1.5 M MgCl2, 0.45% NP40, 0.45% Tween 20, and 200
`mg/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.
`Mutation Analysis. BRCA1 and BRCA2 were both screened for germ-
`line mutations using a combination of the PTT and a nonradioactive HA to
`identify variants in the sample set. PTT is an efficient
`technique for
`screening large DNA fragments ($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 combina-
`tion of PTT and HA was considered a sensitive and efficient method of
`analysis. Direct sequence analysis was used to confirm the precise nucle-
`otide 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 frag-
`ments 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 59 and 39 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 13 mutation detection
`enhancement polyacrylamide gels as described previously (36). Syder Green
`
`Table 1 Composition of prostate cancer clusters
`
`Identifier no.
`(individual tested)
`
`Age at diagnosis of prostate
`cancer in individual
`analyzed (yrs)
`
`No. of individuals affected
`with prostate cancer in family
`
`Other cancers in cluster
`Site (age at onset; yrs)
`
`Pe(uk), Br(40, 44, uk), Bl(uk), NHL(34)
`
`Br(70), Ov(70), Bas(25)
`
`Co(78)
`Lu(57, uk), uk(uk)
`Th/B(40), Te(uk), Ey(uk)
`Co(72)
`Re(51), Co(54)
`Br(76)
`
`Lu(68), Co(85), SSC Sc(60), uk(uk)
`
`NHL(,8), Lu(64), Ov/Ut(uk)
`
`Bon(uk), Li(58), Lu(76)
`
`uk(51)
`
`Br(59), St(73)
`Th(70, uk), Br(uk), Bon(64)
`Co(uk)
`St(53), Lu(uk, uk)
`
`Age at prostate cancer diagnosis of
`other relative(s) in family (yrs)
`73, 70, uka, 73
`5
`49
`PR3380.201
`69, 63, 56, 74
`5
`65
`PRS2036.201
`87, 37, 72
`4
`43
`PR3658.201
`70, 65, 67
`4
`65
`PRS2015.205
`69, 70, 67
`4
`67
`PRS2018.201
`60, 75, 77
`4
`72
`PRS2051.201
`74, 81
`3
`67
`PR3106.201
`87, 62
`3
`71
`PR3382.201
`73, 60
`3
`64
`PRS2016.201
`41, 87
`3
`56
`PRS2024.201
`75, 65
`3
`71
`PRS2025.202
`67, 80
`3
`59
`PRS2031.202
`uk, uk
`3
`66
`PRS2039.201
`86, 67
`3
`71
`PRS2045.201
`72, 77
`3
`71
`PRS2053.201
`71, 81
`3
`76
`PRS2059.201
`58, 61
`3
`49
`PRY1061.201
`58, 77
`3
`46
`PRY1081.201
`64
`2
`63
`PR3173.201
`82
`2
`58
`PR3222.201
`71
`2
`59
`PR3378.201
`64
`2
`61
`PR3498.201
`72
`2
`54
`PR3569.201
`64
`2
`62
`PRS2001.201
`64
`2
`62
`PRS2003.201
`66
`2
`63
`PRS2005.202
`63
`2
`60
`PRS2010.201
`62
`2
`62
`PRS2012.201
`66
`2
`60
`PRS2017.202
`64
`2
`64
`PRS2047.201
`66
`2
`57
`PRS2052.201
`72
`2
`67
`PRS2058.201
`66
`2
`49
`PRY1010.201
`65
`2
`52
`PRY1026.201
`48
`2
`54
`PRY1042.201
`78
`2
`54
`PRY1052.201
`uk
`2
`53
`PRY1056.201
`Ki(51)
`69
`2
`49
`PRY1064.201
`a uk, unknown; B, brain; Bas, basaloid anal cancer; Bl, bladder; Bon, bone; Br, breast; Co, colon; Ey, eye; Ki, kidney; Li, liver; Lu, lung; NHL, Non-Hodgkin’s lymphoma; Ov,
`ovary; Pa, pancreas; Pe, penis; Re, rectum; Sc, scrotum; Sp, spine; SSC, squamous cell carcinoma; St, stomach; Te, testis; Th, throat; Ut, uterus.
`4514
`
`Sp(uk)
`Lu(66)
`
`Co(47, 63, uk), St/Bo(uk), Lu(uk), Ki(uk), uk(uk)
`St(59)
`
`Ut/Ov(55)
`Br(uk, uk, uk), Lu(uk), Co(uk)
`
`Pa(55)
`Ov(58)
`
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`CONTRIBUTION OF BRCA1 AND BRCA2 TO FAMILIAL PROSTATE CANCER
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`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 par-
`affin-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 tempera-
`ture of 18°C. Gels were visualized after silver staining as described
`previously (36).
`Immunohistochemical Staining for BRCA2 Protein. Sections (4 mm)
`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).
`
`RESULTS
`
`Mutation analysis of BRCA1 revealed no variants that appeared to
`be related to the disease phenotype in any of the 38 prostate cancer
`
`Fig. 1. The protein truncation test performed for BRCA2. a, analysis of PTT
`fragment 4 in affected individual 201 from family PRY1042 shows a truncated mutant
`protein (M) compared with only wild-type protein detected in two normal samples
`(N). b, analysis of affected individual 201 from family PRS2024 shows a truncated
`mutant protein (M) compared with only wild-type protein detected in two normal
`samples (N).
`
`Fig. 2. a, the pedigree of family PRY1042. The mutation in this family, 6710delA-
`CAA, is detected as a heteroduplex variant (het) in index case 201. This variant is not
`present in the affected brother (202) or in a normal sample (N). pa ca, pancreatic cancer;
`pr ca, prostate cancer. Analysis of tumor DNA from individual 201 shows loss of the
`wild-type homoduplex DNA band (wt-hd) and retention of the shorter, mutant homodu-
`plex band (m-hd). This is compared with wild-type homoduplex DNA seen in a normal
`sample (N). b, the mutation in family PRS2024, 5531delTT, was detected as a heterodu-
`plex variant (het) and a homoduplex conformer (hd) in blood DNA from index case 201.
`This variant is not present in blood DNA from the affected father (101) or in tumor DNA
`(T) from the affected brother (202). br ca, breast cancer.
`
`families. Several frequently observed variants were detected using
`HA, but sequencing revealed these to be either coding or noncoding
`polymorphisms that have been reported previously.5
`Analysis of BRCA2 revealed three variants that were not de-
`tected 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 pre-
`mature 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
`
`5 http://www.nchgr.nih.gov/Intramural_research/Lab_transfer/Bic/.
`
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`CONTRIBUTION OF BRCA1 AND BRCA2 TO FAMILIAL PROSTATE CANCER
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`shown). This is consistent with the observation of a germ-line
`BRCA2 mutation in one affected brother but not in the other.
`A PTT variant in exon 11 in a prostate cancer case diagnosed at 56
`years from family PRS2024 (Fig. 1) 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 pres-
`ence 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 asso-
`ciation, 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,
`
`Fig. 3. a, prostate cancer cells from the brother
`(PRY1042.202) of the individual (PRY1042.201)
`who has a germ-line deletion (6710delACAA).
`This shows weakly positive staining with antibody
`to BRCA2 protein in individual 1042.202, who
`does not carry the mutation. (3400). b, multifocal
`areas of intense staining for antibody to BRCA2
`protein in prostate cancer cells from the patient
`who has a polymorphism in BRCA2 (K2950N var-
`iant; 3400).
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`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, a and b).
`
`DISCUSSION
`
`mutations but positive in their brothers who did not have a trun-
`cating mutation and also in the individual with the K2950N vari-
`ant. 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 [name-
`ly 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 muta-
`tions 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 propor-
`tion in different prostate cancer populations.
`
`The data we have reported suggest that approximately 5% (2 of
`38) of families identified with a history of prostate cancer, based
`on either affected sibling pairs or three or more affected individ-
`uals in the family, may contain an individual with a germ-line
`mutation in the BRCA2 gene. These data also indicate that BRCA1
`does not contribute significantly to familial prostate cancer. The
`actual proportions of germ-line BRCA2 and BRCA1 mutations in
`such families may be greater; it is possible that mutations may have
`been missed using the combination of the PTT and HA, and we
`would not have detected missense mutations in the regions
`screened by PTT. It is surprising that the two disease-associated
`BRCA2 mutations that were detected were not present
`in the
`affected sibling in each of the families. In family PRY1042, the
`germ-line mutation 6710delACAA and loss of the wild-type allele
`in tumor tissue detected in the index case suggest that the mutation
`in BRCA2 is cancer-causing and acting as a tumor suppressor gene.
`This is consistent with previous reports that suggest that sporadic
`ovarian cancers with germ-line BRCA2 mutations and breast tu-
`mors from a BRCA2 linked family show nonrandom loss of the
`wild-type allele (38, 39). The germ-line mutation in family
`PRS2024 was detected in the index case diagnosed with prostate
`cancer but not in his father (who was diagnosed at 87 years),
`suggesting that the father may be a sporadic case, nor was it
`present in tumor DNA from the affected brother. The mother was
`diagnosed with breast cancer at 76 years of age, and it is probable
`that she is also a germ-line carrier of the mutation, although no
`DNA was available to test this hypothesis.
`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 sib-
`ling 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
`4517
`
`ACKNOWLEDGMENTS
`
`The contribution of all of the members of the families in this study is
`gratefully acknowledged. We are grateful to S. Osborne for data manage-
`ment.
`
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