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Editor—Mutations of theBRCA1 and BRCA2tumour suppressor genes have been identified in some cases of familial and early onset breast cancer.1 2 Mutations of these genes, however, account for a relatively small proportion of the total cases of female breast cancer. Male breast cancer is a very rare disease, accounting for approximately 1% of all cases of breast cancer. Less is known about the genetic influences in its development. Male breast cancer has been linked to mutations of theBRCA2 gene in some cases, with the frequency of mutations varying widely (from 4-40%) in those series studied.3 4
It has been suggested that there may be other genetic factors that confer a lower absolute risk to the person, but potentially could result in a substantial number of cases within a whole population.5 We have already shown that a polymorphism in the CYP17 gene is associated with an increased risk of male breast cancer.6
A region within exon 1 of the gene coding for the androgen receptor (located on chromosome Xq11-12) is highly polymorphic and contains a variable number of CAG repeats. The variability of the number of these repeats between different ethnic populations in the USA has been studied.7 In vitro studies have shown that a relatively short CAG repeat sequence increases the level of transactivation of the androgen receptor.8 The androgen receptor itself binds dihydrotestosterone and therefore is one factor in the regulation of the growth of prostate cells. This may account for the finding that short CAG repeat sequences have been associated with a higher risk of developing prostate cancer.9 10 Abnormally long sequences of 40 repeats or more are found in patients with X linked spinal and bulbar muscular atrophy (Kennedy's disease).11 This disease is associated with gynaecomastia and reduced fertility, suggestive of androgen insensitivity. Mutations of the androgen receptor gene may also result in reduced androgen receptor function and have been found in a few cases of male breast cancer.12 13
The aim of this study was to investigate whether increased length of the CAG repeat sequence in the androgen receptor gene is associated with the development of male breast cancer.
The selection of male breast cancer cases and controls has previously been described.6 Ethical approval for the study was obtained through the Lothian Regional Ethics Committee.
DNA extraction was from whole blood by standard phenol/chloroform extraction. DNA extraction from wax embedded tissue was from 10 μm sections incubated at 55°C with a lysis buffer and proteinase K.
Using the published sequence,14 the following primers were designed (Primer Designer v1.1 ©1990 Educational Software): ARG-F 5′-TGCGCGAAGTGATCCAGA ACC-3′, ARG-R 5′-CTCATCCAGGACCAGGTAGCC-3′. These generate PCR fragments containing the CAG repeat sequence.
PCR reactions were performed in 50 μl aliquots, each containing 1 × PCR reaction buffer, 2 mmol/l MgCl2, 5 μl dimethyl sulphoxide, 200 μmol/l deoxynucleoside triphosphates, 20 pmol of each primer, 1 unit of Taq polymerase (Life Technologies™), and approximately 100 ng DNA. The amplification was performed using an OmniGene thermal cycler (Hybaid, UK) under the following conditions: initial denaturation at 94°C for three minutes; amplification for 38 cycles, with denaturation at 94°C for 45 seconds, annealing at 56°C for 45 seconds, and extension at 72°C for 45 seconds; final extension at 72°C for 10 minutes.
The products were denatured and then run on 6% polyacrylamide gels with a 10 bp DNA ladder. The products were then ranked in order of length. Three representative products were sent for automated sequencing (DNASHEF, Department of Haematology, Royal Infirmary of Edinburgh) to confirm the number of CAG repeats, and these were used as size standards. The products were then run again, with those thought to be of equal length adjacent to each other in order to check the accuracy of the original estimation of length. A second re-run was then performed to confirm the accuracy of the results.
The lengths of the PCR products obtained varied between 224 bp and 272 bp (corresponding to 14 CAG repeats and 30 CAG repeats, respectively). PCR was unsuccessful with DNA extracted from eight of the archival wax embedded tissue sections.
The distribution of alleles among male breast cancer patients and controls is shown in fig 1. The median number of CAG repeats in both groups was 23. There were no statistically significant differences between the two groups (Mann-Whitney test, p=0.916).
Three patients showed evidence of two different alleles indicating the presence of two X chromosomes (fig 2). One of these (MBC8) was recorded on the Edinburgh Cytogenetics Register with a diagnosis of Klinefelter's syndrome. The other two patients (MBC42 and MBC 62) had died, but there was no record of clinical suspicion of Klinefelter's syndrome in their hospital case notes. Neither fathered any children. The data were reanalysed following exclusion of these three cases. The median number of CAG repeats for the remaining 53 male breast cancer patients was also 23. There was still no statistically significant difference between cases and controls (p=0.765).
We have not observed any overall difference between the median CAG repeat length of male breast cancer patients and controls. However, no males in the control group had alleles containing more than 28 CAG repeats, whereas two of the male breast cancer patients had alleles with 29 and 30 repeats respectively. Only one of the male breast cancer patients had an allele containing 18 repeats or less, compared to six of the controls. To our knowledge, the length of this CAG repeat has only been studied in one group of male breast cancer patients previously.15 There was found to be no significant difference between male breast cancer cases and controls. However, sequences of 30 repeats or more were found only among cases. Our results are consistent with these findings. In addition, it has been recently observed that women who are carriers ofBRCA1 mutations are at a significantly increased risk of breast cancer if they carry at least one androgen receptor gene allele with 28 or more CAG repeats.16 We believe that a relatively long CAG repeat sequence within the androgen receptor gene may be implicated in a few cases of male breast cancer. Conversely, a short CAG repeat sequence might offer a degree of protection against male breast cancer.
It is well recognised that Klinefelter's syndrome is associated with an increased risk of male breast cancer.17 One of the male breast cancer patients in our study was known to have had Klinefelter's syndrome. Our study of the androgen receptor gene has enabled us to identify a further two patients whom we suspect to have had Klinefelter's syndrome.
The findings presented in this study indicate that the CAG repeat sequence within the androgen receptor gene may, in some cases, be one useful molecular marker to identify males at increased risk of developing breast cancer. Larger studies are required to define the importance of this CAG repeat in male breast cancer further. An international consortium has recently been set up and we have agreed to contribute our data to this.
There is also a GGC repeat sequence within exon 1 of the androgen receptor gene. This might be an interesting area for further study.
We thank the following: Mr R Morris and Dr S Bader for technical advice; Dr T Anderson, Dr A McGregor, Dr I Nawroz, Dr K Ramesar, and Dr A M Lutfy for making available the archival wax embedded tissue sections; Miss G Kerr and the medical records staff in the Department of Clinical Oncology, Western General Hospital, Edinburgh; and staff in the Department of Blood Transfusion Medicine, Royal Infirmary of Edinburgh and in the Department of ENT Surgery, City Hospital, Edinburgh for providing some of the control samples. This work has been funded by grants from the Royal College of Surgeons of Edinburgh, the Sarah Percy Fund, the Melville Trust for the Care and Cure of Cancer, and the Robertson Trust.