Valentina Silvestri

FANCM c.5791C>T nonsense mutation (rs144567652) induces exon skipping, affects DNA repair activity and is a familial breast cancer risk factor

Numerous genetic factors that influence breast cancer risk are known. However, approximately two-thirds of the overall familial risk remain unexplained. To determine whether some of the missing heritability is due to rare variants conferring high to moderate risk, we tested for an association between the c.5791C>T nonsense mutation (p.Arg1931*; rs144567652) in exon 22 of FANCM gene and breast cancer. An analysis of genotyping data from 8635 familial breast cancer cases and 6625 controls from different countries yielded an association between the c.5791C>T mutation and breast cancer risk [odds ratio (OR) = 3.93 (95% confidence interval (CI) = 1.28-12.11; P = 0.017)]. Moreover, we performed two meta-analyses of studies from countries with carriers in both cases and controls and of all available data. These analyses showed breast cancer associations with OR = 3.67 (95% CI = 1.04-12.87; P = 0.043) and OR = 3.33 (95% CI = 1.09-13.62; P = 0.032), respectively. Based on information theory-based prediction, we established that the mutation caused an out-of-frame deletion of exon 22, due to the creation of a binding site for the pre-mRNA processing protein hnRNP A1. Furthermore, genetic complementation analyses showed that the mutation influenced the DNA repair activity of the FANCM protein. In summary, we provide evidence for the first time showing that the common p.Arg1931* loss-of-function variant in FANCM is a risk factor for familial breast cancer.

PALB2 mutations in male breast cancer: a population-based study in Central Italy.

To the Editor, Male breast cancer (MBC) is a rare disease compared to female breast cancer (FBC). However, MBC shares many similarities with FBC, including genetic predisposition factors such as BRCA1/2 mutations. The frequency of BRCA1/2 mutations is quite different in ethnically diverse populationand clinic-based MBC series, ranging between 4 and 40% for BRCA2 and up to 10% for BRCA1 [1]. In a population-based series of 108 MBCs from Central Italy, we reported that about 9% of MBCs were BRCA1/2 carriers [2], suggesting the contribution of additional susceptibility genes. Several studies identify PALB2 as a moderate-penetrance breast cancer (BC) susceptibility gene, accounting for about 1% of BRCA1/2 negative familial/early onset BCs [3, 4]. PALB2 mutations were found in families with both FBC and MBC cases, suggesting that PALB2 may be involved in MBC risk [3]. As for BRCA1/2, there are evidences that PALB2 might play a role in prostate cancer (PC) [5]. Interestingly, PC patients have an increased risk of BC [6] and, on the other hand, MBC patients show an increased risk of PC [7], suggesting that these tumours may share risk factors. The role of PALB2 in MBC predisposition is still largely unknown. An interesting evaluation about this latter is provided in the recent article by Sauty de Chalon et al. [8]. Yet in the above mentioned study PALB2 pathogenic mutations were not found in 25 MBCs belonging to 25 BRCA1/2 negative families and, based on PALB2 mutation frequency (*1%), it cannot be excluded that these findings may indeed be due to the small series analysed. By using direct sequencing, we performed a screen for germline mutations of PALB2 coding region and intron– exon boundaries in 97 BRCA1/2 negative MBCs, previously identified in a population-based series of 108 MBCs [2]. Age at first BC diagnosis ranged between 35 and 90 years (mean 63.9 years), 25.8% of the patients reported a first-degree family history (FH) of breast/ovarian cancer and 14.4% had a personal history of cancer(s) at sites other than breast. In particular, prostate (28.5%) and bladder (28.5%) carcinomas were the most frequently observed metachronous cancers. Table 1 shows all the PALB2 variants identified in our study. No truncating mutations were found. A novel missense variant, I1180T, was identified in a young man diagnosed with BC at 42 years of age and with negative FH. In silico analysis (PolyPhen, SIFT, Align-GVGD and Pmut) showed that I1180T is likely to be deleterious, since it occurs in the WD40-4 conserved domain that regulates PALB2 interaction with BRCA2. This variant was not found in 90 male population controls analysed (Table 1). Three additional missense variants (Q559R, E672Q and G998E) and one synonymous (T1100T) variant, all previously Valentina Silvestri and Piera Rizzolo equally contributed to the work.

Mutation screening of RAD51C in male breast cancer patients

We read with great interest the paper by Akbari and colleagues [1] in a recent issue of Breast Cancer Research. The authors reported on the absence of RAD51C mutations in 454 patients with BRCA1/2-negative familial breast cancer/ovarian cancer (BC/OC). In the initial report by Meindl and colleagues [2], RAD51C mutations were identified in 6 out of 480 patients with BRCA1/2-negative familial BC/OC. Interestingly, on the basis of histopathologic features, including intermediate grade (G2), estrogen receptor-positive (ER+), progesterone receptor-positive (PR+), and HER2-negative (HER2-) expression, RAD51C-associated BCs were found to be similar to BRCA2-associated BCs [2]. BRCA2 is known to play a significant role in male BC (MBC); however, no occurrence of MBC was observed in the six RAD51C families described [2]. To investigate the role of RAD51C in MBC, we screened for RAD51C mutations in 97 MBC patients selected from our population-based series of 126 cases because they were previously found negative for BRCA1/2, CHEK2, PALB2, and BRIP1 mutations [3,4]. Notably, 25.8% of cases showed a positive first-degree family history of BC or OC or both. The majority of MBCs were invasive ductal carcinomas (74.5%), G2 (53.5%), ER+ (90.7%), PR+ (82.6%), or HER2- (85.4%). Overall, 66% of the MBCs showed an ER+/PR+/HER2- phenotype. All patients provided informed consent to the study. We carried out mutation screening of the nine exons and intron/exon boundaries of RAD51C by high resolution melting (HRM) analysis, a rapid closed-tube mutation scanning method with high sensitivity and specificity. Cases displaying abnormal profiles were evaluated by direct sequencing. Primers are available upon request. We found no truncating RAD51C mutations. We identified a novel intronic variant, IVS3 c.738-16G>T, in 1 out of 97 MBCs (1%). By in silico analysis, performed with Splice Site Prediction (BDGP, Berkeley Drosophila Genome Project, Lawrence Berkeley National Laboratory, Berkeley, CA, USA) and NetGene 2 Server software (CBSA, Center for Biological Sequence Analysis, Technical University of Denmark, Lyngby, Denmark), the IVS3 c.738-16G>T variant is predicted not to affect splicing. This variant was also identified in 2 out of 173 (1.2%) population controls examined. We also found a neutral polymorphic intronic variant, IVS6 c.904+34T>C (rs28363318), in 16 out of 97 (16.5%) MBCs. Overall, our results, which are based on a relatively large MBC series, are consistent with the findings by Akbari and colleagues [1] and with data on 92 patients with hereditary gynecological cancer in which no deleterious RAD51C mutations were identified [5] and would suggest that the impact of RAD51C mutations on BC predisposition might be more limited than initially reported. In conclusion, we found no evidence that RAD51C mutations may contribute to MBC susceptibility. Further studies on larger MBC series are needed to confirm our findings.

Gene promoter methylation and DNA repair capacity in monozygotic twins with discordant smoking habits

The influence of DNA repair capacity, plasma nutrients and tobacco smoke exposure on DNA methylation was investigated in blood cells of twenty-one couples of monozygotic twins with discordant smoking habits. All study subjects had previously been characterized for mutagen sensitivity with challenge assays with ionizing radiation in peripheral blood lymphocytes. Plasma levels of folic acid, vitamin B12 and homocysteine were also available from a previous investigation. In this work DNA methylation in the promoter region of a panel of ten genes involved in cell cycle control, differentiation, apoptosis and DNA repair (p16, FHIT, RAR, CDH1, DAPK1, hTERT, RASSF1A, MGMT, BRCA1 and PALB2) was assessed in the same batches of cells isolated for previous studies, using the methylation-sensitive high-resolution melting technique. Fairly similar profiles of gene promoter methylation were observed within co-twins compared to unrelated subjects (p= 1.23 × 10(-7)), with no significant difference related to smoking habits (p = 0.23). In a regression analysis the methylation index of study subjects, used as synthetic descriptor of overall promoter methylation, displayed a significant inverse correlation with radiation-induced micronuclei (p = 0.021) and plasma folic acid level (p = 0.007) both in smokers and in non-smokers. The observed association between repair of radiation-induced DNA damage and promoter methylation suggests the involvement of the DNA repair machinery in DNA modification. Data also highlight the possible modulating effect of folate deficiency on DNA methylation and the strong influence of familiarity on the individual epigenetic profile.

HeR2-positive male breast cancer: an update

Although rare, male breast cancer (MBC) remains a substantial cause for morbidity and mortality in men. Based on age frequency distribution, age-specific incidence rate pattern, and prognostic factor profiles, MBC is considered similar to postmenopausal breast cancer (BC). Compared with female BC (FBC), MBC cases are more often hormonal receptor (estrogen receptor/progesterone receptor [ER/PR]) positive and human epidermal growth factor receptor 2 (HER2) negative. Treatment of MBC patients follows the same indications as female postmenopausal with surgery, systemic therapy, and radiotherapy. To date, ER/PR and HER2 status provides baseline predictive information used in selecting optimal adjuvant/neoadjuvant therapy and in the selection of therapy for recurrent or metastatic disease. HER2 represents a very interesting molecular target and a number of compounds (trastuzumab [Herceptin®; F. Hoffmann-La Roche, Basel, Switzerland] and lapatinib [Tykerb®, GlaxoSmithKline, London, UK]) are currently under clinical evaluation. Particularly, trastuzumab, a monoclonal antibody which selectively binds the extracellular domain of HER2, has become an important therapeutic agent for women with HER2-positive (HER2+) BC. Currently, data regarding the use of trastuzumab in MBC patients is limited and only few case reports exist. In all cases, MBC patients received trastuzumab concomitantly with other drugs and no severe toxicity above grade 3 was observed. However, MBC patients that would be candidate for trastuzumab therapy (ie, HER2+/ER+ or HER2+/ER- MBCs) represent only a very small percentage of MBC cases. This is noteworthy, when taking into account that trastuzumab is an important and expensive component of systemic BC therapy. Since there is no data supporting the fact that response to therapy is different for men or women, we concluded that systemic therapy in MBC should be considered on the same basis as for FBC. Particularly in male patients, trastuzumab should be considered exclusively for advanced disease or high-risk HER2+ early BCs. On the other hand, lapatinib (Tykerb), a novel oral dual tyrosine kinase inhibitor that targets both HER2 and epidermal growth factor receptor, may represent an interesting and promising therapeutic agent for trastuzumab-resistant MBC patients.

Novel and known genetic variants for male breast cancer risk at 8q24.21, 9p21.3, 11q13.3 and 14q24.1: Results from a multicenter study in Italy

Increasing evidence indicates that common genetic variants may contribute to the heritable risk of breast cancer (BC). In this study, we investigated whether single nucleotide polymorphisms (SNPs), within the 8q24.21 multi-cancer susceptibility region and within BC-associated loci widespread in the genome, may influence the risk of BC in men, and whether they may be associated with specific clinical-pathologic characteristics of male BC (MBC). In the frame of the ongoing Italian Multicenter Study on MBC, we performed a case-control study on 386 MBC cases, including 50 BRCA1/2 mutation carriers, and 1105 healthy male controls, including 197 unaffected BRCA1/2 mutation carriers. All 1491 subjects were genotyped by Sequenom iPLEX technology for a total of 29 susceptibility SNPs. By logistic regression models, we found a significant association with MBC risk for five SNPs: rs1562430 (p=0.002) and rs445114 (p=0.026) both within the 8q24.21 region; rs1011970/9p21.3 (p=0.011), rs614367/11q13.3 (p=0.016) and rs1314913/14q24.1 (p<0.0001). Differences in the distribution of rs614367/11q13.3 genotypes according to oestrogen receptor (ER) status (p=0.006), and of rs1011970/9p21.3 genotypes according to human epidermal growth factor receptor 2 (HER2) status (p=0.002) emerged. Association of rs1011970/9p21.3 risk genotype with HER2+MBC was confirmed by a multivariate analysis. rs1314913/14q24.1 was associated with increased MBC risk in analyses restricted to male BRCA1/2 mutation carriers (p=0.041). In conclusion, we provided the first evidence that the 8q24.21 region is associated with MBC risk. Furthermore, we showed that the SNPs rs1562430/8q24.21 and rs1314913/14q24.1 strongly influence BC risk in men and suggested that the SNP rs1314913/14q24.1 may act as a risk modifier locus in male BRCA1/2 mutation carriers.

SULT1A1 gene deletion in BRCA2-associated male breast cancer: a link between genes and environmental exposures?

SULT1A1, a member of sulfotransferase superfamily, is a drug and hormone metabolizing enzyme involved in the metabolism of a variety of potential mammary carcinogens of endogenous and exogenous origin. Interestingly, the metabolic activity of SULT1A1 can be affected by variations in gene copy number. Male Breast Cancer (MBC) is a rare disease and less investigated disease compared to female BC (FBC). As in FBC, the concurrent effects of genetic risk factors, particularly BRCA2 mutations, increased exposure to estrogens and environmental carcinogens play a relevant role in MBC. By quantitative real‐time PCR with TaqMan probes, we investigated the presence of SULT1A1 gene copy number variations (CNVs) in a series of 72 MBCs. SULT1A1 gene deletion was observed in 10 of the 72 MBCs (13.9%). In a multivariate analysis association between BRCA2 mutation and SULT1A1 gene deletion emerged (p = 0.0005). Based on the evidence that the level of SULT1A1 enzyme activity is correlated with CNV, our data suggest that in male breast tumors SULT1A1 activity may be decreased. Thus, it can be hypothesized that in a proportion of MBCs, particularly in BRCA2‐associated MBCs, the level of estrogens and environmental carcinogens exposure might be increased suggesting a link between gene and environmental exposure in the pathogenesis of MBC.

Whole-exome sequencing and targeted gene sequencing provide insights into the role of PALB2 as a male breast cancer susceptibility gene

Male breast cancer (MBC) is a rare disease whose etiology appears to be largely associated with genetic factors. BRCA1 and BRCA2 mutations account for about 10% of all MBC cases. Thus, a fraction of MBC cases are expected to be due to genetic factors not yet identified. To further explain the genetic susceptibility for MBC, whole‐exome sequencing (WES) and targeted gene sequencing were applied to high‐risk, BRCA1/2 mutation–negative MBC cases.

Smyd3 open & closed lock mechanism for substrate recruitment: The hinge motion of C-terminal domain inferred from μ-second molecular dynamics simulations

BACKGROUND The human lysine methyltransferase Smyd3, a member of the SET and MYND domain containing protein family, harbors methylation activity on both histone and non-histone targets in a tightly regulated manner. The mechanism of how Smyd3 dynamically regulates substrate recognition is still not fully unveiled. METHODS Here, we employed molecular dynamics simulations on full length human Smyd3, performed to a total of 1.2 μ-second, in the presence (holo) and absence (apo) of the S-Adenosyl methionine (AdoMet) cofactor. The dynamical features of Smyd3 in apo and holo states have been examined and compared via examining geometrical and electrostatic properties. RESULTS The results show a distinct dynamics of the C-terminal domain (CTD) in the two states. In the apo state, the CTD undergoes a large hinge like motion and samples more opened configurations, thus acting like a loosened clamp and resulting in expanded substrate binding crevice. In the holo state, the CTD exhibits a restricted motion while the overall structure remains compact, mimicking a closed clamp. This leads to a localized increase in the negative potential at the substrate binding cleft. Further, solvent accessibility of critical residues at the target lysine access channel, important for methylation activity, is increased. CONCLUSIONS We postulate that AdoMet cofactor acts like a key and locks Smyd3 in a closed conformation. In effect, the cofactor binding restricts the elasticity of the CTD, presenting a compact substrate binding cleft with high negative potential, which may have implications on substrate recruitment via long range electrostatics. GENERAL SIGNIFICANCE The deletion of the CTD from Smyd3 has been shown to abolish the basal histone methylation activity. Our study highlights the importance of the CTD elasticity in shaping the substrate binding site for recognition and supports the previously proposed role of the CTD in stabilizing the active site for methylation activity.

Mutational profiling in melanocytic tumors: multiple somatic mutations and clinical implications.

In this study, we analyzed multiple somatic mutations in 10 genes relevant in melanoma tumorigenesis and targeted therapies. Overall, 45% of the tumors showed mutations and, in particular, 33% had multiple mutations. Based on our results, we conclude that the assessment of mutation status of multiple genes, including CDKN2A, could provide a genetic profile that can be useful as a prognostic and therapeutic marker in melanocytic tumors.