| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Molecular Biology and Genetics |
Department of Laboratory Medicine and Pathology, Mayo Clinic and Foundation, Rochester, Minnesota, 55905
| ABSTRACT |
|---|
|
|
|---|
| INTRODUCTION |
|---|
|
|
|---|
In an early study of seven large Icelandic breast cancer families, two with linkage to BRCA1, prostate cancer was second to breast cancer in occurrence (15) . In the two BRCA1-linked families, 44% of the presumed paternal carriers of BRCA1 mutations had been diagnosed with prostate cancer. Additional evidence for the involvement of BRCA1 in prostate cancer was identified in a cooperative study of 33 BRCA1-linked families (16) . An estimated relative risk of prostate cancer of 3.33 was calculated for men carrying a BRCA1 mutation when compared with the general population. A relative risk of 2.89 was later identified by Easton et al. (17) for men carrying a mutant BRCA2 gene. Recently, a relative risk of prostate cancer of 7.33 was reported for men <65 years of age carrying a mutant BRCA2 gene (13) .
In light of the apparent elevated relative risk of prostate cancer associated with BRCA1, a number of prostate cancer populations have been screened for BRCA1 and BRCA2 mutations to assess the possible role of these genes in the development of prostate cancer. Forty-nine men <65 years of age from a population-based case-control study of prostate cancer were screened for germ-line BRCA1 mutations (18) . One protein truncating mutation, the Ashkenazi Jewish founder mutation 185delAG, and six rare sequence variants in coding and noncoding regions were identified from the population. Recently, Uchida et al. (19) studied 24 prostate cancer samples and found only 1 to contain a BRCA1 mutation. This particular sample came from a man whose sister died of ovarian cancer at age 57. In a separate study, Edwards et al. (20) screened for allelic loss of markers, both flanking and within the BRCA2 gene. Of the 73 sporadic and familial prostate cancer samples screened, 11 had loss of one marker and 6 had loss of more than one marker. The 17 samples with loss of a marker were associated with a poorer prognostic phenotype. There was, however, no statistically significant difference in the frequency of allelic loss between sporadic cases, 24% (10 of 41), and familial cases, 22% (7 of 32).
The BRCA1 185delAG mutation was first identified in Ashkenazi Jewish breast and ovarian cancer families and is estimated to be carried by 0.9% of the Ashkenazi population (21) . 185delAG and the BRCA2 6174delT mutation, which is found in 1% of the Ashkenazi population (22) , are considered founder mutations in this ethnic population. Lehrer et al. (23) screened 60 Ashkenazi Jewish men with prostate cancer for both the 185delAG and the 6174delT mutations to determine whether these mutations had a role in prostate cancer in this population. None of the men screened carried either of the mutations. Wilkens et al. (24) screened 47 individuals from 18 Ashkenazi Jewish families with three or more first-degree relatives with prostate cancer for the BRCA1 185delAG and 5382insC and BRCA2 6174delT mutations. Only one unaffected individual carried a mutation, 6174delT. In a more recent study, Nastiuk et al. (25) screened Ashkenazi Jewish prostate cancer patients, those diagnosed with the disease at a young age, for the 185delAG and 6174delT mutations. One of the 83 patients carried the 185delAG mutation. Similarly, 1 of the 82 patients carried the 6174delT mutation. Both Wilkens et al. (24) and Nastiuk et al. (25) suggest that mutations in BRCA1 and BRCA2 do not have a significant role in prostate cancer in the Ashkenazi Jewish population.
Several studies have also examined the role of the BRCA2 Icelandic founder mutation, 999del5, in prostate cancer. This mutation has an estimated frequency of 0.4% in the general Icelandic population (26) . Johannesdottir et al. (26) screened 65 Icelandic men diagnosed with prostate cancer <65 years of age and 499 controls. Only 2 of the 65 (2.7%) prostate cancer patients and 2 of the 499 (0.4%) controls carried the 999del5 mutation. The differences between the prostate cancer patients and the population controls were statistically insignificant. In contrast, a study by Thorlacius et al. (27) found that prostate cancer was the second most prevalent cancer in male first-degree relatives of 999del5 mutation carriers affected with breast cancer. Relative risk of prostate cancer in first-degree relatives was estimated at 3.46. Sigurdsson et al. (28) analyzed men affected with prostate cancer from 16 BRCA2 999del5 families and a random group of men diagnosed with prostate cancer over a 1-year period. Relative risk of prostate cancer was calculated to be 4.6 in first-degree relatives and 2.5 in second-degree relatives. The study also noted that 8 of 12 familial prostate cancer cases and 2 of 65 random prostate cancer cases carried the 999del5 founder mutation, and each of the 10 carriers developed advanced prostate cancer and died. The authors proposed that the 999del5 mutation may be useful as a prostate cancer marker for poor prognosis in the Icelandic population.
Although these studies have found some evidence of BRCA1 and BRCA2 involvement in prostate cancer etiology, a clear understanding of the importance of these genes in familial prostate cancer remains to be elucidated. To assess the involvement of BRCA1 and BRCA2 mutations in familial prostate cancer, we selected families with at least two cases of breast and/or ovarian cancer from a large collection of high-risk prostate cancer families. We selected these families to maximize the odds of detecting mutations in BRCA1 and BRCA2. Mutation screening was performed on the prostate cancer proband and an additional family member affected with breast or prostate cancer from each of the 22 selected families.
| MATERIALS AND METHODS |
|---|
|
|
|---|
A total of 21 families with at least two cases of breast and/or ovarian
cancer in each pedigree were identified (Table 1)
. One additional family with a single case of breast cancer was also
included in the analysis. This subset was selected for BRCA1
and BRCA2 mutation screening of two individuals/pedigree.
All 22 probands plus an additional affected (either prostate or breast
cancer) relative from 21 of the pedigrees were screened.
|
CSGE3
Mutations in BRCA1 and BRCA2 have been found
throughout the entire coding regions of the genes. To screen the entire
coding region of each gene, we used intron-based primers for PCR
amplification of the 22 and 26 coding exons of BRCA1 and
BRCA2, respectively. To ensure complete screening of larger
exons, BRCA1 exon 11 and BRCA2 exons 10, 11, and
27 were divided into multiple overlapping fragments, resulting in a
total of 31 BRCA1 primer sets and 42 BRCA2 primer
sets.
PCR was performed using standard conditions containing 25 ng of genomic DNA, 1x buffer (Promega), 1.5 mM MgCl2 (Promega), 0.2 mM deoxynucleotide triphosphates, 0.5µM each primer, and 1 unit of Taq DNA polymerase (Promega). Amplification conditions were as follows: denaturing at 93°C for 3 min, 35 cycles of denaturing at 93°C for 30 s, annealing for 30 s, and extension at 72°C for 1 min, followed by a final extension at 72°C for 5 min. For heteroduplex formation, the products were then denatured at 98°C for 5 min and allowed to reanneal at 68°C for 30 min. A 10-µl volume of the PCR product was combined with 2 µl of nondenaturing loading dye, and the entire 12-µl sample was loaded onto a CSGE gel. Gels were prepared with 10% polyacrylamide [99:1 acrylamide:1,4-bis(acroyl)piperazine (Fluka)], 10% ethylene glycol, 15% formamide, 0.5x TTE buffer (44.4 mM Tris, 14.25 mM taurine, and 0.1 mM EDTA, pH 9.0). Gels were run at 400V overnight in 0.5x TTE buffer, stained with ethidium bromide (0.5 mg/µl), and photographed with UV illumination.
Variant Sequencing
PCR products displaying variant gel migration patterns were purified as
follows. One µl exonuclease I (Amersham Pharmacia Biotech) was added
to 5 µl of PCR product and incubated at 37°C for 15 min and 80°C
for 15 min. The incubation was then repeated after the addition of 1
µl of shrimp alkaline phosphatase (SAP) (Amersham Pharmacia
Biotech). Upon completion of the incubation cycles, 4 µl of distilled
H2O and 3.2 pM of the appropriate
primer were added. The sample was then sequenced by the Molecular Core
Facility at Mayo on an ABI 377 sequencer and analyzed for sequence
alterations.
| RESULTS |
|---|
|
|
|---|
We identified one previously reported missense mutation in
BRCA2, C1206A, which results in the substitution of an
arginine for a serine at codon 326. This BRCA2 missense
mutation has been identified previously on three occasions as reported
on the BIC
website.4However, it is not known if the mutation is disease associated. To
address this uncertainty, DNA samples from five additional members of
this family were examined (Fig. 1)
for segregation of the mutation with cancer. The additional five
members consisted of the probands brother, two nieces, and two
nephews. The brother and one nephew were both diagnosed with prostate
cancer at age 72 and 63, respectively, and one niece was diagnosed with
skin cancer at age 54. We identified the missense mutation in the
probands brother only. Unfortunately, we were unable to obtain DNA
from the probands father or remaining sibling, both of whom had been
diagnosed with prostate cancer. The availability of these samples would
have facilitated a more complete analysis of segregation of the
mutation with cancer in this family.
|
|
| DISCUSSION |
|---|
|
|
|---|
The missense mutation, C1206A, results in the substitution of a charged arginine residue for a polar serine residue at codon 326. The introduction of a charged arginine residue may alter the protein conformation and as a result affect the function. Nucleotide 1206 is located in BRCA2 exon 10 at the site reported to be required for interaction with the transcriptional coactivator protein, P/CAF (33) . The C1206A missense mutation is located 36 nucleotides into the 163 nucleotides essential for P/CAF interaction. It is possible that the introduction of a positively charged amino acid through the substitution of arginine for serine may disrupt P/CAF binding to BRCA2. Loss of P/CAF binding may inhibit histone acetylation and chromatin remodeling and lead to disruption of BRCA2-dependent transcription and/or DNA repair.
We were unable to perform Southern blot analysis of each sample to check for BRCA1 gene rearrangements that cannot be detected by PCR screening methods because of a lack of sufficient DNA. Because genomic rearrangements may account for up to 15% of all BRCA1 mutations (34) , it is possible that we may have missed one or more mutations. We did identify an interesting CT repeat region in BRCA2 intron 7 when we screened exon 7 for mutations. Previously reported intron-based primers for the exon 7 region are located upstream of the CT repeats. Our reverse primer for exon 7 is located downstream of the repeats. Although any sequence alterations in this repeat region are unlikely to affect the actual coding sequence or splicing, it is interesting to note that every one of the 43 samples we screened had one of three sequence variants in this region.
Three of the families (nos. 2, 3, and 14) studied had one individual
with male breast cancer in addition to two, six, and two female
relatives affected with breast and/or ovarian cancer, respectively
(Table 1)
. Mutations in BRCA2 have been associated with
14% of male breast cancer cases with a family history
(31)
and 4% of male breast cancer cases unselected for
family history (30)
. Recent studies in Sweden and Hungary
reported BRCA2 mutations in 21% (35)
and 33%
(36)
of male breast cancer patients without a family
history of breast cancer. None of the three families with cases of male
breast cancer in our population carried BRCA2 sequence
variants that might be disease associated; therefore, it is unlikely
that BRCA2 plays any role in the etiology of the male breast
cancers in this population.
It is possible that the breast cancer in these prostate cancer families may be sporadic cases. No evidence has been found to date to show that BRCA1 or BRCA2 have a significant role in sporadic forms of female breast cancer. Therefore, if the breast cancers are sporadic, selection of families with cases of breast and/or ovarian cancer along with prostate cancer would not be expected to enrich for carriers of BRCA1 and BRCA2 mutations. From the data presented here, it would appear that BRCA1 and BRCA2 have little or no role in hereditary prostate cancer. However, it remains possible that other tumor suppressor genes account for the cancers in these families. Sixteen of the 22 families have other cancers in addition to breast, ovarian, and prostate. Although there is no obvious pattern to the cancer occurrences, it is premature to dismiss these as unrelated in breast and prostate cancer families. The search is ongoing for prostate and breast cancer predisposition genes. Once identified, it will quickly be determined whether these genes cause both prostate and breast cancer in families.
| ACKNOWLEDGMENTS |
|---|
| FOOTNOTES |
|---|
1 This study was supported in part by a grant from
the Breast Cancer Research Foundation and Grant CA72818. ![]()
2 To whom requests for reprints should be
addressed, at the Mayo Clinic and Foundation, Hilton Building 970,
Rochester, MN 55905. Phone: (507) 284-4696; Fax: (507) 284-0043;
E-mail: Stephen.Thibodeau{at}mayo.edu ![]()
3 The abbreviations used are: CSGE,
conformation-sensitive gel electrophoresis; BIC, Breast Cancer
Information Core. ![]()
4 Internet address: http://www.nhgri.nih.gov/Intramural_research/Lab_transfer/Bic.
Received 8/24/99. Accepted 1/ 5/00.
| REFERENCES |
|---|
|
|
|---|
This article has been cited by other articles:
![]() |
H. Schayek, K. Haugk, S. Sun, L. D. True, S. R. Plymate, and H. Werner Tumor Suppressor BRCA1 Is Expressed in Prostate Cancer and Controls Insulin-like Growth Factor I Receptor (IGF-IR) Gene Transcription in an Androgen Receptor-Dependent Manner Clin. Cancer Res., March 1, 2009; 15(5): 1558 - 1565. [Abstract] [Full Text] [PDF] |
||||
![]() |
I. Agalliu, R. Gern, S. Leanza, and R. D. Burk Associations of High-Grade Prostate Cancer with BRCA1 and BRCA2 Founder Mutations Clin. Cancer Res., February 1, 2009; 15(3): 1112 - 1120. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. A. Douglas, A. M. Levin, K. A. Zuhlke, A. M. Ray, G. R. Johnson, E. M. Lange, D. P. Wood, and K. A. Cooney Common Variation in the BRCA1 Gene and Prostate Cancer Risk Cancer Epidemiol. Biomarkers Prev., July 1, 2007; 16(7): 1510 - 1516. [Abstract] [Full Text] [PDF] |
||||
![]() |
F. J. Couch, M. R. Johnson, K. G. Rabe, K. Brune, M. de Andrade, M. Goggins, H. Rothenmund, S. Gallinger, A. Klein, G. M. Petersen, et al. The Prevalence of BRCA2 Mutations in Familial Pancreatic Cancer Cancer Epidemiol. Biomarkers Prev., February 1, 2007; 16(2): 342 - 346. [Abstract] [Full Text] [PDF] |
||||
![]() |
I. Agalliu, E. M. Kwon, D. Zadory, L. McIntosh, J. Thompson, J. L. Stanford, and E. A. Ostrander Germline Mutations in the BRCA2 Gene and Susceptibility to Hereditary Prostate Cancer Clin. Cancer Res., February 1, 2007; 13(3): 839 - 843. [Abstract] [Full Text] [PDF] |
||||
![]() |
H. A. Risch, J. R. McLaughlin, D. E. C. Cole, B. Rosen, L. Bradley, I. Fan, J. Tang, S. Li, S. Zhang, P. A. Shaw, et al. Population BRCA1 and BRCA2 Mutation Frequencies and Cancer Penetrances: A Kin-Cohort Study in Ontario, Canada J Natl Cancer Inst, December 6, 2006; 98(23): 1694 - 1706. [Abstract] [Full Text] [PDF] |
||||
![]() |
K. A. Zuhlke, J. J. Madeoy, J. Beebe-Dimmer, K. A. White, A. Griffin, E. M. Lange, S. B. Gruber, E. A. Ostrander, and K. A. Cooney Truncating BRCA1 Mutations Are Uncommon in a Cohort of Hereditary Prostate Cancer Families with Evidence of Linkage to 17q Markers Clin. Cancer Res., September 15, 2004; 10(18): 5975 - 5980. [Abstract] [Full Text] [PDF] |
||||
![]() |
C. A. Heinlein and C. Chang Androgen Receptor in Prostate Cancer Endocr. Rev., April 1, 2004; 25(2): 276 - 308. [Abstract] [Full Text] [PDF] |
||||
![]() |
D. J. Schaid The complex genetic epidemiology of prostate cancer Hum. Mol. Genet., April 1, 2004; 13(90001): R103 - 121. [Abstract] [Full Text] [PDF] |
||||
![]() |
A. Liede, B. Y. Karlan, and S. A. Narod Cancer Risks for Male Carriers of Germline Mutations in BRCA1 or BRCA2: A Review of the Literature J. Clin. Oncol., February 15, 2004; 22(4): 735 - 742. [Abstract] [Full Text] [PDF] |
||||
![]() |
S. A. Krum, G. A. Miranda, C. Lin, and T. F. Lane BRCA1 Associates with Processive RNA Polymerase II J. Biol. Chem., December 26, 2003; 278(52): 52012 - 52020. [Abstract] [Full Text] [PDF] |
||||
![]() |
T Ikonen, M P Matikainen, K Syrjakoski, N Mononen, P A Koivisto, A Rokman, E H Seppala, O-P Kallioniemi, T L J Tammela, and J Schleutker BRCA1 and BRCA2 mutations have no major role in predisposition to prostate cancer in Finland J. Med. Genet., August 1, 2003; 40(8): e98 - 98. [Full Text] [PDF] |
||||
![]() |
J. Simard, M. Dumont, P. Soucy, and F. Labrie Perspective: Prostate Cancer Susceptibility Genes Endocrinology, June 1, 2002; 143(6): 2029 - 2040. [Full Text] [PDF] |
||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |