Marketa Janatova

High proportion of recurrent germline mutations in the BRCA1 gene in breast and ovarian cancer patients from the Prague area.

BackgroundGermline mutations in the BRCA1 and BRCA2 genes have been shown to account for the majority of hereditary breast and ovarian cancers. The purpose of our study was to estimate the incidence and spectrum of pathogenic mutations in BRCA1/2 genes in high-risk Czech families.MethodsA total of 96 Czech families with recurrent breast and/or ovarian cancer and 55 patients considered to be at high-risk but with no reported family history of cancer were screened for mutations in the BRCA1/2 genes. The entire coding sequence of each gene was analyzed using a combination of the protein truncation test and direct DNA sequencing.ResultsA total of 35 mutations in the BRCA1/2 genes were identified in high-risk families (36.5%). Pathogenic mutations were found in 23.3% of breast cancer families and in 59.4% of families with the occurrence of both breast and ovarian cancer. In addition, four mutations were detected in 31 (12.9%) women with early onset breast cancer. One mutation was detected in seven (14.3%) patients affected with both a primary breast and ovarian cancer and another in three (33.3%) patients with a bilateral breast cancer. A total of 3 mutations in BRCA1 were identified among 14 (21.4%) women with a medullary breast carcinoma. Of 151 analyzed individuals, 35 (23.2%) carried a BRCA1 mutation and 9 (6.0%) a BRCA2 mutation. One novel truncating mutation was found in BRCA1 (c.1747A>T) and two in BRCA2 (c.3939delC and c.5763dupT). The 35 identified BRCA1 mutations comprised 13 different alterations. Three recurrent mutations accounted for 71.4% of unrelated individuals with detected gene alterations. The BRCA1 c.5266dupC (5382insC) was detected in 51.4% of mutation positive women. The mutations c.3700_3704del5 and c.181T>G (300T>G) contributed to 11.4% and 8.6% of pathogenic mutations, respectively. A total of eight different mutations were identified in BRCA2. The novel c.5763dupT mutation, which appeared in two unrelated families, was the only recurrent alteration of the BRCA2 gene identified in this study.ConclusionMutational analysis of BRCA1/2 genes in 151 high-risk patients characterized the spectrum of gene alterations and demonstrated the dominant role of the BRCA1 c.5266dupC allele in hereditary breast and ovarian cancer.

The PALB2 gene is a strong candidate for clinical testing in BRCA1 and BRCA2 negative hereditary breast cancer

Background: Several reports indicate that inherited mutations in the PALB2 gene predispose to breast cancer. However, there is little agreement about the clinical relevance and usefulness of mutation screening in this gene. We analyzed the prevalence and spectrum of germline mutations in PALB2 to estimate their contribution to hereditary breast and/or ovarian cancer in the Czech Republic. Methods: The entire PALB2 coding region was sequenced in 409 breast/ovarian cancer patients negative for BRCA1 and BRCA2 mutations. Testing for large genomic rearrangements (LGR) was performed by multiplex ligation-dependent probe amplification (MLPA) analysis. Results: We have identified 13 different pathogenic alterations including 10 truncating mutations and three LGRs in 16 of 409 patients (3.9%), whereas one truncating mutation was found in a group of 1,226 controls (0.08%; P = 2.6 × 10−9). Three novel LGRs included deletions involving exons 7–8 and 9–10, respectively, and a duplication spanning exons 9–11. Five frameshift and two nonsense mutations were novel, whereas three truncating mutations were described previously. The only recurrent mutation was the c.172_175delTTGT detected in four unrelated breast cancer individuals. Conclusions: Our analyses demonstrated the significant role of the PALB2 gene in breast cancer susceptibility. The highest frequency of PALB2 mutations (comparable with that previously reported for BRCA2) was found in a subgroup of patients with hereditary breast cancer (HBC) (13/235; 5.5%). Impact: Our results show that mutation analysis of the PALB2 gene, including the analysis of LGRs, is primarily indicated in patients with HBC in case of their BRCA1 and BRCA2 negativity. Cancer Epidemiol Biomarkers Prev; 22(12); 2323–32. ©2013 AACR.

Expression of human BRCA1Δ17-19 alternative splicing variant with a truncated BRCT domain in MCF-7 cells results in impaired assembly of DNA repair complexes and aberrant DNA damage response

Alternative pre-mRNA splicing is a fundamental post-transcriptional regulatory mechanism. Cancer-specific misregulation of the splicing process may lead to formation of irregular alternative splicing variants (ASVs) with a potentially negative impact on cellular homeostasis. Alternative splicing of BRCA1 pre-mRNA can give rise to BRCA1 protein isoforms that possess dramatically altered biological activities compared with full-length wild-type BRCA1. During the screening of high-risk breast cancer (BC) families we ascertained numerous BRCA1 ASVs, however, their clinical significance for BC development is largely unknown. In this study, we examined the influence of the BRCA1Δ17-19 ASV, which lacks a portion of the BRCT domain, on DNA repair capacity using human MCF-7 BC cell clones with stably modified BRCA1 expression. Our results show that overexpression of BRCA1Δ17-19 impairs homologous recombination repair (sensitizes cells to mitomycin C), delays repair of ionizing radiation-induced DNA damage and dynamics of the ionizing radiation-induced foci (IRIF) formation, and undermines also the non-homologous end joining repair (NHEJ) activity. Mechanistically, BRCA1Δ17-19 cannot interact with the partner proteins Abraxas and CtIP, thus preventing interactions known to be critical for processing of DNA lesions. We propose that the observed inability of BRCA1Δ17-19 to functionally replace wtBRCA1 in repair of DNA double-strand breaks (DDSB) reflects impaired capacity to form the BRCA1-A and -C repair complexes. Our findings indicate that expression of BRCA1Δ17-19 may negatively influence genome stability by reducing the DDSB repair velocity, thereby contributing to enhanced probability of cancer development in the affected families.

The BRCA1 alternative splicing variant Δ14-15 with an in-frame deletion of part of the regulatory serine-containing domain (SCD) impairs the DNA repair capacity in MCF-7 cells

The BRCA1 gene codes for a protein involved in the DNA double strand break (DDSB) repair. Alongside the dominant full-length splicing form of BRCA1, numerous endogenously expressed alternative splicing variants of unknown significance have been described in various tissues. Some of them retain the original BRCA1 reading frame but lack several critical BRCA1 structural domains, suggesting an altered function of the resulting protein in the BRCA1-regulated processes. To characterize the effect of the BRCA1Δ14-15 splicing variant (with an in-frame deletion affecting the regulatory serine-containing domain) on the DDSB repair, we constructed the MCF-7 clones stably expressing the analyzed variant with/without a shRNA-mediated downregulation of the endogenous full-length wild-type BRCA1 expression. Our results show that the expression of the BRCA1Δ14-15 variant delays the γ-radiation-induced DDSB repair, alters the kinetics of irradiation-induced foci formation/decomposition and reduces the non-homologous end-joining capacity in MCF-7 cells. Therefore, the BRCA1Δ14-15 is not able to functionally replace the full-length wt BRCA1 in the DDSB repair. Our findings indicate that the endogenously expressed BRCA1 alternative splicing variants may negatively influence genome stability and support the growing evidence of the pathological potential of the sequence variants generated by an altered or misregulated alternative splicing in the process of mammary malignant transformation.

Germline mutations 657del5 and 643C>T (R215W) in NBN are not likely to be associated with increased risk of breast cancer in Czech women

To the Editor, The NBN (formerly NBS1) gene (OMIM*602667) is located on chromosome 8q21 and encodes a 754-amino acid protein known as nibrin. Nibrin is a component of the MRE11/RAD50/NBN (MRN) complex and is involved in the DNA double-strand break repair, telomere maintenance, and cell-cycle checkpoint control. Individuals homozygous for deleterious mutations in NBN develop an autosomal recessive disorder, Nijmegen breakage syndrome (NBS; #251260), characterized by microcephaly, growth retardation, immunodeficiency, hypersensitivity to X-irradiation, and increased risk of lymphoid malignancies [1]. About 90 % of NBS patients carry the homozygous mutation 657del5 (c.657_661delACAAA) affecting exon 6 of the NBN gene that has been predominantly identified in Slavic populations [2, 3]. Another potentially deleterious NBN alteration, the missense substitution c.643C[T (p.R215W), is also located in exon 6 and has been described in two severely affected NBS siblings who were compound heterozygotes for the 657del5 and R215W mutations [4]. Previous studies suggested that heterozygous carriers of the founder mutation 657del5 or R215W have an increased risk of breast cancer (BC) in several European populations [5–7]. However, the role of these mutations was not tested in other countries and the 657del5 mutation did not contribute significantly to BC risk in the German population [7, 8]. No deleterious NBN mutations were identified in China [9]. Based on the results of our analysis, we would like to contribute to the debate on the role of the 657del5 and R215W mutations in BC susceptibility. The frequency of both NBN mutations was analyzed in 1,303 Czech patients (including 600 high-risk, hereditary and non-hereditary, BRCA1/2-negative breast/ovarian cancer patients [10], and 703 consecutive unselected BC patients) and in 915 control non-cancer individuals (Table 1) [11]. The DNA was isolated from blood samples by routine procedures. Screening for mutations affecting exon 6 and adjacent intronic regions of the NBN gene was performed by a high resolution melting (HRM) analysis using the LightCycler 480 HRM Master Kit and the LightCycler 480 (Roche) according to the manufacturer’s instructions. Sequences of the primers are available on request. Mutations in samples with an aberrant melting profile were confirmed by direct sequencing using the BigDye Terminator v3.1 in ABI PRISM 3130 genetic analyzer (Life Technologies). In addition to the truncating 657del5 mutation found in two high-risk patients, two unselected BC cases, but also in two controls, we also detected the R215W and other three different missense variants in exon 6 of the NBN gene (Table 1). In intronic regions, we observed one substitution (c.585-4A[G) only in a high-risk BC patient, while three other variants (c.585-5T[C and c.702?9G[A observed once and c.702?26C[G observed twice) were detected only in control samples. M. Mateju J. Novotny Department of Oncology, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Prague, Czech Republic

Mutation Analysis of the RAD51C and RAD51D Genes in High-Risk Ovarian Cancer Patients and Families from the Czech Republic

Recent studies have conferred that the RAD51C and RAD51D genes, which code for the essential proteins involved in homologous recombination, are ovarian cancer (OC) susceptibility genes that may explain genetic risks in high-risk patients. We performed a mutation analysis in 171 high-risk BRCA1 and BRCA2 negative OC patients, to evaluate the frequency of hereditary RAD51C and RAD51D variants in Czech population. The analysis involved direct sequencing, high resolution melting and multiple ligation-dependent probe analysis. We identified two (1.2%) and three (1.8%) inactivating germline mutations in both respective genes, two of which (c.379_380insG, p.P127Rfs*28 in RAD51C and c.879delG, p.C294Vfs*16 in RAD51D) were novel. Interestingly, an indicative family cancer history was not present in four carriers. Moreover, the ages at the OC diagnoses in identified mutation carriers were substantially lower than those reported in previous studies (four carriers were younger than 45 years). Further, we also described rare missense variants, two in RAD51C and one in RAD51D whose clinical significance needs to be verified. Truncating mutations and rare missense variants ascertained in OC patients were not detected in 1226 control samples. Although the cumulative frequency of RAD51C and RAD51D truncating mutations in our patients was lower than that of the BRCA1 and BRCA2 genes, it may explain OC susceptibility in approximately 3% of high-risk OC patients. Therefore, an RAD51C and RAD51D analysis should be implemented into the comprehensive multi-gene testing for high-risk OC patients, including early-onset OC patients without a family cancer history.

The c.657del5 variant in the NBN gene predisposes to pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is the sixth most frequent cancer type in the Czech Republic with a poor prognosis that could be improved by an early detection and subsequent surgical treatment combined with chemotherapy. Genetic factors play an important role in PDAC risk. We previously identified one PDAC patient harboring the Slavic founder deleterious mutation c.657del5 in the NBN gene, using a panel next-generation sequencing (NGS). A subsequent analysis of 241 unselected PDAC patients revealed other mutation carriers. The overall frequency of c.657del5 in unselected PDAC patients (5/241; 2.07%) significantly differed from that in non-cancer controls (2/915; 0.2%; P=0.006). The result indicates that the NBN c.657del5 variant represents a novel PDAC-susceptibility allele increasing PDAC risk (OR=9.7; 95% CI: 1.9 to 50.2). The increased risk of PDAC in follow-up recommendations for NBN mutation carriers should be considered if other studies also confirm an increased frequency of c.657del5 carriers in PDAC patients from other populations.

Mutation analysis of the PALB2 gene in unselected pancreatic cancer patients in the Czech Republic.

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis among common solid cancer diagnoses. It has been shown that up to 10% of PDAC cases have a familial component. Characterization of PDAC-susceptibility genes could reveal high-risk individuals and patients that may benefit from tailored therapy. Hereditary mutations in PALB2 (Partner and Localizer of BRCA2) gene has been shown to predispose, namely to PDAC and breast cancers; however, frequencies of mutations vary among distinct geographical populations. Using the combination of sequencing, high-resolution melting and multiplex ligation-dependent probe amplification analyses, we screened the entire PALB2 gene in 152 unselected Czech PDAC patients. Truncating mutations were identified in three (2.0%) patients. Genotyping of found PALB2 variants in 1226 control samples revealed one carrier of PALB2 truncating variant (0.08%; P = 0.005). The mean age at PDAC diagnosis was significantly lower among PALB2 mutation carriers (51 years) than in non-carriers (63 years; P = 0.016). Only one patient carrying germline PALB2 mutation had a positive family breast cancer history. Our results indicate that hereditary PALB2 mutation represents clinically considerable genetic factor increasing PDAC susceptibility in our population and that analysis of PALB2 should be considered not only in PDAC patients with familial history of breast or pancreatic cancers but also in younger PDAC patients without family cancer history.

Multiplex PCR and NGS-based identification of mRNA splicing variants: Analysis of BRCA1 splicing pattern as a model

Alternative pre-mRNA splicing increases transcriptome plasticity by forming naturally-occurring alternative splicing variants (ASVs). Alterations of splicing processes, caused by DNA mutations, result in aberrant splicing and the formation of aberrant mRNA isoforms. Analyses of hereditary cancer predisposition genes reveal many DNA variants with unknown clinical significance (VUS) that potentially affect pre-mRNA splicing. Therefore, a comprehensive description of ASVs is an essential prerequisite for the interpretation of germline VUS in high-risk individuals. To identify ASVs in a gene of interest, we have proposed an approach based on multiplex PCR (mPCR) amplification of all theoretically possible exon-exon junctions and subsequent characterization of size-selected and pooled mPCR products by next-generation sequencing (NGS). The efficiency of this method is illustrated by a comprehensive analysis of BRCA1 ASVs in human leukocytes, normal mammary, and adipose tissues and stable cell lines. We revealed 94 BRCA1 ASVs, including 29 variants present in all tested samples. While differences in the qualitative expression of BRCA1 ASVs among the analyzed human tissues were minor, larger differences were detected between tissue and cell line samples. Compared with other ASV analysis methods, this approach represents a highly sensitive and rapid alternative for the identification of ASVs in any gene of interest.

RE: frameshift variant FANCL*c.1096_1099dupATTA is not associated with high breast cancer risk.

To the Editor, In our recent publication, we noted a borderline association of the truncating variant c.1096_1099dupATTA in the FANCL gene with increased breast cancer (BC) risk in high-risk BRCA1/BRCA2/PALB2-negative BC patients (1). However, the subsequent analysis by Pfeifer et al. (2) genotyping the c.1096_1099dupATTA variant in 2370 samples from German and Macedonian BC patients and controls failed to confirm association of this variant with BC risk. We agree with Pfeifer et al. that FANCL*c.1096_ 1099dupATTA is unlikely a high-risk BC susceptibility allele with an immediate clinical utility. There are several lines of evidence that do not support strong involvement of this variant in BC susceptibility including: (i) the relative high frequency of this variant especially in European populations, (ii) functional characteristics demonstrating that cells expressing FANCL isoform coded by c.1096_1099dupATTA variant retain the residual FANCL functional capacity in vitro (3), and (iii) phenotypic characteristics that show only a mild Fanconi anemia (FA) complementation group L phenotype in the compound heterozygote carrying FANCL*c.1096_1099dupATTA (alongside an another truncating FANCL variant) (3). Moreover, we identified a male c.1096_1099dupATTA homozygote (in controls) who had no signs of FA at his age of 57 years (Table 1). We also agree that FANCL*c.1096_1099dupATTA may confer a low (or lower) risk variant. We hypothesized that this variant may represent a modifying factor because its carriers were overrepresented only in a subgroup of high-risk BC patients in our study and also four out of six c.1096_1099dupATTA carriers analyzed by a panel next-gene sequencing (NGS) carried truncating variant(s) in other known or candidate cancer-susceptibility gene(s). After publication of our study reporting 15 carriers of c.1096_1099dupATTA in 2126 analyzed samples of Czech BC patients and controls, we identified another eight carriers using the CZECANCA multicancer panel NGS (4). The individual characteristics of c.1096_1099dupATTA carriers (Table 1) indicate a relatively low mean age at diagnose [47.2 years (range 28–76 years)] in 14 carriers with BC. We also recently identified three c.1096_1099dupATTA carries with ovarian cancer diagnosed at early age. Contrary to Pfeifer et al. who reported that only one out of 10 identified c.1096_1099dupATTA carriers had a family BC history, we have noticed a known familial history of BC (in a first or second degree relative) in nine out of 23 carriers (39%) and a familial history of some cancer in 15 carriers (65%). The c.1096_1099dupATTA variant was accompanied by another truncating variant(s) in nine out of 14 cancer patients analyzed by a panel NGS (Table 1). We suppose that these characteristics indicate that c.1096_1099dupATTA may (perhaps mildly) modify the breast (or other) cancer risk or cancer onset. However, further studies are required to estimate the risk of cancer development in c.1096_1099dupATTA carriers precisely. The segregation analyses and NGS analyses in families of carriers would be also required to evaluate the involvement of this hypomorphic variant in the risk of other cancer development or in modification of cancer onset.