Genotyping of BRCA1, BRCA2, p53, CDKN2A, MLH1 and MSH2 genes in a male patient with secondary breast cancer
Ana Lina Vodusek1, Srdjan Novakovic2, Vida Stegel2, Berta Jereb1
1	Department of Radiation Oncology, Institute of Oncology, Ljubljana, Slovenia
2	Department of Molecular Diagnostics, Institute of Oncology, Ljubljana, Slovenia
Received 12 May 2011 Accepted 23 June 2011
Correspondence to: Ana Lina Vodušek, MD, Institute of Oncology, Zaloška 2, 1000 Ljubljana, Slovenia. Phone:+386 1 5879 629; Fax: +386 1 5879 304; E-mail: avodusek@onko-i.si
Disclosure: No potential conflicts of interest were disclosed.
Background. Some tumour suppressor genes (BRCA2) and mismatch repair genes (MSH2, MLH1) are correlated with an increased risk for male breast cancer.
Case report. Our patient developed secondary breast cancer after the treatment for Hodgkin's disease in childhood. DNA was isolated from the patients' blood and screened for mutations, polymorphisms and variants in BRCA1, BRCA2, p53, CDKN2A, MLH1 and MSH2 genes. We found no mutations but common polymorphisms, and three variants in mismatch repair genes.
Conclusions. Nucleotide variants c.2006-6T>C and p.G322D in MSH2 might be correlated with male breast cancer. Key words: gene screening; breast cancer; male; secondary neoplasm
Introduction
Secondary neoplasms (SN) are the most serious late effects of the treatment of childhood cancers. Their incidence is increasing with time of observation to 25% at 25 years from diagnosis of the primary tumor.12 Patients treated with radiotherapy for Hodgkin's disease (HD) are at highest risk for SN.3 Male breast cancer (MBC) is rare, accounting for less than 1% of all breast cancers.45 Risk factors for primary MBC include testicular disease, benign breast conditions, age, family history, the Klinefelter syndrome, gynecomasty and non-therapeutic radiation exposure.56 The highest risk for primary MBC is among the carriers of mutations in the BRCA2 gene. Besides mutations in the BRCA2 gene, additional germ line mutations in MBC have been reported also in the androgen receptor gene and PTEN7 There is likely to be a number of genes more commonly mutated correlated with a modest increase in primary MBC, such as mismatch repair (MMR) genes - MSH2, MLH1, PMS1, PMS2.78
In literature we found two case reports on secondary MBC, one after the treatment for HD and
one after the treatment for acute lymphoblastic leukaemia including total body irradiation and bone marrow transplantation.39 There are no data on genetic factors predisposing the development of secondary MBC after the treatment for HD.
The aim of this case report was to elucidate the genetic conditions in a patient with secondary MBC, screening the genes already known to be correlated with primary MBC - BRCA1, BRCA2, p53, CDKN2A, MLH1 and MSH2.
Case report
Patient
A 37 year old man developed breast cancer 24 years after the treatment for HD. In 1983, when 13 years old, he was treated with chemotherapy (6 cycles of MOPP/ABVD) and irradiation of the neck and upper thorax, retroperitoneal and inguinal nodes (25 Gy) for stage IIIBS HD. Afterwards he was followed, at the Department of Paediatrics and after 1995 at the Late Effect Clinic at the Institute of Oncology in Ljubljana.
TABLE 1. Nucleotide variations detected in male breast cancer patient
Gene BIC*	HGVS nomenclature**	Genotype	Clinical significance
MLH1	c.453+79A>G	heterozygote AG	polymorphism
	c.1668-19A>G	homozygote AA	polymorphism
	c.655A>G (p.I219V)	heterozygote AG	unclassified variant
MSH2	c.211+9G>C	homozygote GG	polymorphism
	c.1511-9A>T	heterozygote AT	polymorphism
	c.1661 + 11G>A	heterozygote GA	polymorphism
	c.2006-6T>C	heterozygote TC	unclassified variant
	c.965G>A (p.G322D]	heterozygote AG	unclassified variant
BRCA1 2201C>T	c.2082C>T (p.S694S )	heterozygote CT	polymorphism
2430T>C	c.2311T>C (p.L771L )	heterozygote TC	polymorphism
2731C>T	c.2612C>T (p.P871L )	heterozygote CT	polymorphism
3232A>G	c.3113A>G (p.E1038G )	heterozygote AG	polymorphism
3667A>G	c.3548A>G (p.K1183R )	heterozygote AG	polymorphism
4427T>C	c.4308T>C (p.F1436S )	heterozygote TC	polymorphism
4956A>G	c.4837A>G (p.S1613G )	heterozygote AG	polymorphism
BRCA2			
203G>A	c.1-25G>A	homozygote GG	polymorphism
1342C>A	c.1114C>A (p.H372N )	homozygote CA	polymorphism
3624A>G	c.3396A>G (p.L1132L )	heterozygote AG	polymorphism
4035T>C	c.3807T>C (p.V1269V]	heterozygote TC	polymorphism
7470A>G	c.7242A>G (p.S2414S )	heterozygote AG	polymorphism
IVS16-14C>T	c.7806-14C>T	heterozygote TC	polymorphism
P53	c.96 + 41_56 del CCCCAGCCCTCCAGGT c.215 C>T (p. Pro72Arg]	homozygote heterozygote CT	polymorphism polymorphism
	c.782 + 72 A>C	heterozygote AG	polymorphism
	c.782 + 92 A>G	heterozygote AC	polymorphism
CDKN2A			
	c.1-191A>G	homozygote GG	polymorphism
	c.471+69C>T	heterozygote CT	polymorphism
*Nucleotide variations described as in BIC (Breast Cancer Information Core) database. DNA variants are numerated according to NCBI reference sequence HSU14680 for mRNA of BRCA1, or U43746 for mRNA of BRCA2. First nucleotide of mRNA is numerated as 1.
**Description of nucleotide variations is in accordance with HGVS (Human Genome Variation Society) nomenclature. DNA variants are numerated according to NCBI reference sequence NM_000249 for MLH1, NM_000251 for MSH2, NM_007294.2 for BRCA1, NM_000059.3 for BRCA2, NM_000546 for p53 and NM_000077.3 for CDKN2A. First nucleotide of start codon ATG is numerated as 1.
Eleven years after the treatment we observed primary hypogonadism with low levels of testosterone, bilateral gynecomasty and azoospermia and at the age of 37 a palpable tumour 6 cm in diameter in the central part of the left breast, infiltrating the skin, with an inverted nipple. Ultrasounds of both axillae, of the abdomen, chest X-ray films and Technetium bone scan were normal.
Histology showed an invasive ductal carcinoma, grade III, with negative progesterone and estrogen receptors and positive Her2 status. His family history was negative. He was treated with neoadju-vant chemotherapy, left mastectomy with axillary node dissection, adjuvant treatment with trastuzu-mab and postoperative irradiation of the left mammary region.
Methods of genetic investigations
Patient's DNA was isolated from peripheral blood using the DNA blood isolation kit Quiagen (Hilden, Germany). It was screened for variants in tumour suppressor genes (BRCA1, BRCA2, p53, CDKN2A) and MMR genes (MLH1 and MSH2).
Four methods were used: DGGE (denaturizing gradient gel electrophoresis) or HRM (high resolution melting), direct sequencing and MLPA (multiplex ligation-dependent probe amplification). The screening of BRCA1/2 genes was performed for all exons by the DGGE while MLH1 and MSH2 genes were screened using the HRM. Positive fragments were subsequently sequenced to determine the nucleotide change. Genes BRCA1, BRCA2, MLH1 and MSH2 were also screened for large deletions or insertions using the MLPA method. All coding regions of p53 and CDKN2A were sequenced.
HRM was used for discrimination between two DNA molecules with different sequences for the detection of SNPs (single nucleotide polymorphism) and small deletions and insertions. PCR (polymer-ase chain reaction) was performed on LC480 instrument using the LC 480 High-resolution Melting Master Kit (Roche, Manheim, Germany) according to manufacturer's instructions.
DGGE was used for detection of SNPs and small deletions or insertions. The PCR amplification of DNA samples was performed using a set of GC-clamped primers (Ingeny International BV, Goes, Netherlands) according to the cycling conditions provided by the primer-manufacturer. Different denaturing and running conditions were used for specific primer sets. When electrophore-sis was complete, gels were stained with EtBr and documented using the GelDoc system.
Direct sequencing was performed using the ABI PRISM Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Warrington, UK) and products analyzed on the ABI Prism® 310 Genetic Analyzer (Applied Biosystems). Data were collected with ABI Prism 310 software (Applied Biosystems), and the results analyzed with the ABI Prism DNA sequencing analysis software (Applied Biosystems). Sequence data were analyzed utilizing the Gene Runner software tool.
The MLPA was applied for the detection of large deletions and insertions. The probe-mixes labelled as P003 for MLH1 and MSH2, P045-B1 for BRCA2, and P002 for BRCA1 from MRC Holland (MRC Holland, Amsterdam, Netherlands) were used, according to manufacturer's instructions.
Results of genetic investigations
BRCA1 in BRCA2
With MLPA, DGGE and direct sequencing we did not find any changes in the sample. We detected some polymorphisms at the frequently polymorphic loci but no mutations.
However, we found a rare polymorphism in BRCA2 gene p.V1269V which did not code a different amino acid; therefore, it did not affect protein function (Table 1). The sample was also tested for the presence of mutation 1100delC in CHEK2 gene with MLPA kit P045-B1. In the samples mutation 1100delC was not detected.
P53
Tumour suppressor gene p53 was sequenced in total. No mutations were found, but some common polymorphisms were (Table 1).
CDKN2A
By direct sequencing of exons of CDKN2A gene (p16 and p14ARF) we detected two polymorphisms: c. 1-191 A>G in 5'-UTR (untranslated region) and c.471+69C>T in 3'-UTR (Table 1).
MLH1 in MSH2
We detected no changes by means of the MLPA. The screening of the patients' DNA was therefore continued through HRM and sequencing. With these two methods no mutations were discovered, but we found some polymorphisms which were frequently detected in a general population. We also detected three variants of DNA which were described as variants with unknown influence on the protein function (Table 1).
Discussion
Secondary breast cancer is the most common SN among women who have received high dose radi-otherapy.10 However, secondary breast cancer is a rare disease in men and very little is known about its aetiology. It has been suggested that the carcinogenic effect of ionizing radiation may be similar in the male and prepubertal female breast.11
Beside the radiation exposure, some of the risk factors for primary MBC6 were also found in our
patient with secondary MBC - i.e. hormonal imbalances and gynecomasty.
Genetic factors associated with an increased risk of primary MBC include BRCA2 mutations that account for 4% to 14% of all primary MBC.612 We found no such mutations; only a rare V1269V polymorphism that does not affect protein function.
Since p53 is often mutated in female breast cancers, it could be mutated also in MBC. Even though the mutations in tumour suppressor gene p53 are correlated with numerous malignancies13,14, no mutations were found in our patient while screening this gene. Also in MMR genes (MLH1 and MSH2) and in CDKN2A no deleterious mutations have been detected (Tablel).
We found polymorphisms in MMR genes -MLH1 and MSH2, frequently detected in a general population, but also three variants (two in MSH2 and one in MLH1) which are described as variants with unknown influence on the protein function. This finding could be of interest since recent reports allude that two of these variants in MSH2 (c.2006-6T>C and p.G322D) might influence the process of cancer development. Nucleotide variant c.2006-6T>C may be associated with an increased risk for of Non-Hodgkin's Lymphomas.15 Additionally, the risk of some haematological malignancies in individuals carrying c.2006-6T>C variant is increased after the treatment with alkylating agents such as procarbazine, dacarbazine, cyclophospahmide.16
According to some recent reports the variant p.G322D is associated with changes in MSH2 protein function. Individuals with this variant have slightly reduced release efficiency of mismatched targeted DNA compared to the wild type.17
Conclusions
To our knowledge there are no reports of genetic screening in secondary MBC. We found three unclassified variants that could be correlated with an increased risk of secondary MBC but further studies should be performed.
References
1.	Jazbec J, Todorovski L, Jereb B. Classification tree analysis of second neoplasms in survivors of childhood cancer. BMC Cancer 2007; 7: 1-6.
2.	Metayer C, Lynch CF, Clarke EA, Glimelius B, Storm H, Pukkala E, et al. Second cancers among long-term survivors of Hodgkin's disease diagnosed in childhood and adolescence. J Clin Oncol 2000; 18: 2435-43.
3.	Boussen H, Kochbati L, Besbes M, Dhiab T, Makhlouf R, Jerbi G, et al. [Male secondary breast cancer after treatment for Hodgkin's disease. Case report and review of the literature]. [French]. Cancer Radiother 2000; 4: 465-8.
4.	Ozet A, Yavuz AA, Kömürcü S, Oztürk B, Safali M, Arpaci F, et al. Bilateral male breast cancer and prostate cancer: a case report. Jpn J Clin Oncol 2000; 30: 188-90.
5.	Weiss JR, Moysich KB, Swede H. Epidemiology of male breast cancer. Cancer Epidemiol Biomarkers Prev 2005; 14: 20-26.
6.	Giordano SH, Buzdar AU, Hortobagyi GN. Breast cancer in men. Ann Intern Med 2002; 137: 678-87.
7.	Leinung S, Horn LC, Backe J. [Male breast cancer: history, epidemiology, genetic and histopathology]. [German]. Zentralbl Chir 2007; 132: 379-85.
8.	Murata H, Khattar NH, Gu L, Li GM. Roles of mismatch repair proteins hMSH2 and hMLH1 in the development of sporadic breast cancer. Cancer Lett 2005; 223: 143-50.
9.	Latz D, Alfrink M, Nassar N, Beyerle C. Breast cancer in a male patient after treatment of acute lymphoblastic leukemia including total body irradiation and bone marrow transplantation. Onkologie 2004; 27: 477-9.
10.	Zebic-Sinkovec M, Kadivec M, Podobnik G, Skof E, Snoj M. Mammographycally occult high grade ductal carcinoma in situ (DCIS) as second primary breast cancer, detected with MRI: a case report. Radiol Oncol 2010; 44: 228-31.
11.	Thomas DB, Rosenblatt K, Jimenez LM, McTiernan A, Stalsberg H, Stemhagen A, et al. Ionizing radiation and breast cancer in men (United States). Cancer Causes and Control 1994; 5: 9-14.
12.	Eeles RA. Future possibilities in the prevention of breast cancer: intervention strategies in BRCA1 and BRCA2 mutation carriers. Breast Cancer Res 2000; 2: 283-90 .
13.	Malkin D. The role of p53 in human cancer. J Neurooncol 2001; 51: 231-43.
14.	Zager V, Cemazar M, Hreljac I, Lah TT, Sersa G, Filipic M. Development of human cell biosensor system for genotoxicity detection based on DNA damage-induced gene expression. Radiol Oncol 2010; 44: 42-51.
15.	Cesar Paz-y-Mino, Perez CJ, Fiallo BF, Leone PE. A polymorphism in the hMSH2 gene (gIVS12-6T>C) associated with non-Hodgkin lymphomas. Cancer Genet Cytogenet 2002; 133: 29-33.
16.	Worrillow LI, Travis LB, Smith AG, Rollinson S, Smith AJ, Wild CP, et al. An intron splice acceptor polymorphism in hMSH2 and risk of leukemia after treatment with chemotherapeutic alkylating agents. Clin Cancer Res 2003; 9: 3012-20.
17.	Ollila S, Delmadi Bebek D, Jiricny J, Nystrom M. Mechanisms of pathogenic-ity in human MSH2 missense mutants. Human Mutation 2008: 29: 1355-63.