Mercedes Durán

The Average Cumulative Risks of Breast and Ovarian Cancer for Carriers of Mutations in BRCA1 and BRCA2 Attending Genetic Counseling Units in Spain

Purpose: It is not clear that the published estimates of the breast and ovarian cancer penetrances of mutations in BRCA1 and BRCA2 can be used in genetic counseling in countries such as Spain, where the incidence of breast cancer in the general population is considerably lower, the prevalence of BRCA2 mutations seems to be higher, and a distinct spectrum of recurrent mutations exists for both genes. We aimed to estimate these penetrances for women attending genetic counseling units in Spain. Experimental Design: We collected phenotype and genotype data on 155 BRCA1 and 164 BRCA2 mutation carrier families from 12 centers across the country. Average age-specific cumulative risks of breast cancer and ovarian cancer were estimated using a modified segregation analysis method. Results: The estimated average cumulative risk of breast cancer to age 70 years was estimated to be 52% [95% confidence interval (95% CI), 26-69%] for BRCA1 mutation carriers and 47% (95% CI, 29-60%) for BRCA2 mutation carriers. The corresponding estimates for ovarian cancer were 22% (95% CI, 0-40%) and 18% (95% CI, 0-35%), respectively. There was some evidence (two-sided P = 0.09) that 330A>G (R71G) in BRCA1 may have lower breast cancer penetrance. Conclusions: These results are consistent with those from a recent meta-analysis of practically all previous penetrance studies, suggesting that women with BRCA1 and BRCA2 mutations attending genetic counseling services in Spain have similar risks of breast and ovarian cancer to those published for other Caucasian populations. Carriers should be fully informed of their mutation- and age-specific risks to make appropriate decisions regarding prophylactic interventions such as oophorectomy.

Whole Exome Sequencing Suggests Much of Non-BRCA1/BRCA2 Familial Breast Cancer Is Due to Moderate and Low Penetrance Susceptibility Alleles

The identification of the two most prevalent susceptibility genes in breast cancer, BRCA1 and BRCA2, was the beginning of a sustained effort to uncover new genes explaining the missing heritability in this disease. Today, additional high, moderate and low penetrance genes have been identified in breast cancer, such as P53, PTEN, STK11, PALB2 or ATM, globally accounting for around 35 percent of the familial cases. In the present study we used massively parallel sequencing to analyze 7 BRCA1/BRCA2 negative families, each having at least 6 affected women with breast cancer (between 6 and 10) diagnosed under the age of 60 across generations. After extensive filtering, Sanger sequencing validation and co-segregation studies, variants were prioritized through either control-population studies, including up to 750 healthy individuals, or case-control assays comprising approximately 5300 samples. As a result, a known moderate susceptibility indel variant (CHEK2 1100delC) and a catalogue of 11 rare variants presenting signs of association with breast cancer were identified. All the affected genes are involved in important cellular mechanisms like DNA repair, cell proliferation and survival or cell cycle regulation. This study highlights the need to investigate the role of rare variants in familial cancer development by means of novel high throughput analysis strategies optimized for genetically heterogeneous scenarios. Even considering the intrinsic limitations of exome resequencing studies, our findings support the hypothesis that the majority of non-BRCA1/BRCA2 breast cancer families might be explained by the action of moderate and/or low penetrance susceptibility alleles.

A High Proportion of DNA Variants of BRCA1 and BRCA2 Is Associated with Aberrant Splicing in Breast/Ovarian Cancer Patients

Purpose: Most BRCA1/2 mutations are of unknown clinical relevance. An increasing amount of evidence indicates that there can be deleterious effects through the disruption of the splicing process. We have investigated the effect of aberrant splicing of BRCA1/2 on hereditary breast/ovarian cancer (HBOC). Experimental Design: DNA variants were analyzed with splicing prediction programs to select putative splicing mutations. Splicing assays of 57 genetic variants were done by lymphocyte reverse transcription-PCR and/or hybrid minigenes in HeLa and nontumor breast epithelial cells. Results: Twenty-four BRCA1/2 variants of Spanish HBOC patients were bioinformatically preselected. Functional assays showed that 12 variants induced anomalous splicing patterns, 6 of which accounted for 58.5% of BRCA1 families. To further evaluate the defective splicing of BRCA1/2, we analyzed 31 Breast Cancer Information Core Database (BIC) and two artificial variants that were generated by mutagenesis. Sixteen variants induced different degrees of aberrant splicing. Altogether, anomalous splicing was caused by 28 BRCA1/2 variants of all types, indicating that any DNA change can disrupt pre-mRNA processing. We show that a wide range of regulatory elements can be involved, including the canonical and cryptic splice sites, the polypyrimidine tract, and splicing enhancers/silencers. Twenty mutations were predicted to truncate the BRCA proteins and/or to delete essential domains, thus supporting a role in HBOC. Conclusions: An important fraction of DNA variants of BRCA1/2 presents splicing aberrations that may represent a relevant disease-causing mechanism in HBOC. The identification of splicing disruptions by functional assays is a valuable tool to discriminate between benign polymorphisms and pathogenic mutations. Clin Cancer Res; 16(6); 1957–67

Germline Mutations in FAN1 Cause Hereditary Colorectal Cancer by Impairing DNA Repair

Identification of genes associated with hereditary cancers facilitates management of patients with family histories of cancer. We performed exome sequencing of DNA from 3 individuals from a family with colorectal cancer who met the Amsterdam criteria for risk of hereditary nonpolyposis colorectal cancer. These individuals had mismatch repair-proficient tumors and each carried nonsense variant in the FANCD2/FANCI-associated nuclease 1 gene (FAN1), which encodes a nuclease involved in DNA inter-strand cross-link repair. We sequenced FAN1 in 176 additional families with histories of colorectal cancer and performed in vitro functional analyses of the mutant forms of FAN1 identified. We detected FAN1 mutations in approximately 3% of families who met the Amsterdam criteria and had mismatch repair-proficient cancers with no previously associated mutations. These findings link colorectal cancer predisposition to the Fanconi anemia DNA repair pathway, supporting the connection between genome integrity and cancer risk.

Heteroduplex analysis by capillary array electrophoresis for rapid mutation detection in large multiexon genes

Heteroduplex analysis (HA) has proven to be a robust tool for mutation detection. HA by capillary array electrophoresis (HA-CAE) was developed to increase throughput and allow the scanning of large multiexon genes in multicapillary DNA sequencers. HA-CAE is a straightforward and high-throughput technique to detect both known and novel DNA variants with a high level of sensitivity and specificity. It consists of only three steps: multiplex-PCR using fluorescently labeled primers, heteroduplex formation and electrophoresis in a multicapillary DNA sequencer. It allows, e.g., the complete coding and flanking intronic sequences of BRCA1 and BRCA2 genes from two patients (approximately 25 kb each) to be scanned in a single run of a 16-capillary sequencer, and has enabled us to detect 150 different mutations to date (both single nucleotide substitutions, or SNSs, and small insertions/deletions). Here, we describe the protocol developed in our laboratory to scan BRCA1, BRCA2, MLH1, MSH2 and MSH6 genes using an ABI3130XL sequencer. This protocol could be adapted to other instruments or to the study of other large multiexon genes and can be completed in 7–8 h.

High proportion of novel mutations of BRCA1 and BRCA2 in breast/ovarian cancer patients from Castilla-León (central Spain)

AbstractA total of 264 unrelated breast/ovarian cancer patients and 45 healthy individuals with familial antecedents referred for genetic testing were scanned for germ-line mutations in BRCA1 and BRCA2 by conformation-sensitive gel electrophoresis (CSGE) and heteroduplex analysis by capillary array electrophoresis (HA-CAE). We detected 101 distinct mutations (41 in BRCA1 and 60 in BRCA2); ten of them have not been previously reported. These mutations were c.2411_2429dup19, c.2802_2805delCAAA and c.5294A>G (p.E1725E) of BRCA1; and c.667C>T (p.Q147X), c.2683C>T (p.Q819X), c.5344_5347delAATA, c.5578_5579delAA;insT, c.8260_8261insGA, c.744+14C>T and c.8099A>G (p.Y2624C) of BRCA2. Twenty-four different mutations, including seven of the new mutations (five frameshift and two nonsense), were classified as pathogenic. These 24 alterations were found in 39 families (12.6% of all families). A remarkable proportion of deleterious mutations were found in BRCA2: 25 families carried a mutation in BRCA2 (BRCA2+; 64.1%) compared with 14 families BRCA1+ (35.9%). The highest incidences of deleterious mutations were found in families with three or more cases of site-specific breast cancer (BC) (27.4%) and families with BC and ovarian cancer (22.2%). Finally, four recurrent mutations, 3036_3039delACAA, c.5374_5377delTATG of BRCA2, as well as c.5272-1G>A and c.5242C>A (p.A1708E) of BRCA1, accounted for 44% of all of the deleterious mutations.

Comprehensive splicing functional analysis of DNA variants of the BRCA2 gene by hybrid minigenes.

IntroductionThe underlying pathogenic mechanism of a large fraction of DNA variants of disease-causing genes is the disruption of the splicing process. We aimed to investigate the effect on splicing of the BRCA2 variants c.8488-1G > A (exon 20) and c.9026_9030del (exon 23), as well as 41 BRCA2 variants reported in the Breast Cancer Information Core (BIC) mutation database.MethodsDNA variants were analyzed with the splicing prediction programs NNSPLICE and Human Splicing Finder. Functional analyses of candidate variants were performed by lymphocyte RT-PCR and/or hybrid minigene assays. Forty-one BIC variants of exons 19, 20, 23 and 24 were bioinformatically selected and generated by PCR-mutagenesis of the wild type minigenes.ResultsLymphocyte RT-PCR of c.8488-1G > A showed intron 19 retention and a 12-nucleotide deletion in exon 20, whereas c.9026_9030del did not show any splicing anomaly. Minigene analysis of c.8488-1G > A displayed the aforementioned aberrant isoforms but also exon 20 skipping. We further evaluated the splicing outcomes of 41 variants of four BRCA2 exons by minigene analysis. Eighteen variants presented splicing aberrations. Most variants (78.9%) disrupted the natural splice sites, whereas four altered putative enhancers/silencers and had a weak effect. Fluorescent RT-PCR of minigenes accurately detected 14 RNA isoforms generated by cryptic site usage, exon skipping and intron retention events. Fourteen variants showed total splicing disruptions and were predicted to truncate or eliminate essential domains of BRCA2.ConclusionsA relevant proportion of BRCA2 variants are correlated with splicing disruptions, indicating that RNA analysis is a valuable tool to assess the pathogenicity of a particular DNA change. The minigene system is a straightforward and robust approach to detect variants with an impact on splicing and contributes to a better knowledge of this gene expression step.

Frequency of rearrangements in lynch syndrome cases associated with MSH2: Characterization of a new deletion involving both EPCAM and the 5′ part of MSH2

Lynch syndrome is caused by germline mutations in MSH2, MLH1, MSH6, and PMS2 mismatch repair genes and leads to a high risk of colorectal and endometrial cancer. It was recently shown that constitutional 3′ end deletions of EPCAM could cause Lynch syndrome in tissues with MSH2 deficiency. We aim to establish the spectrum of mutations in MSH2-associated Lynch syndrome cases and their clinical implications. Probands from 159 families suspected of having Lynch syndrome were enrolled in the study. Immunohistochemistry and microsatellite instability (MSI) analyses were used on the probands of all families. Eighteen cases with MSH2 loss were identified: eight had point mutations in MSH2. In 10 Lynch syndrome families without MSH2 mutations, EPCAM-MSH2genomic rearrangement screening was carried out with the use of multiplex ligation–dependent probe amplification and reverse transcriptase PCR. We report that large germline deletions, encompassing one or more exons of the MSH2 gene, cosegregate with the Lynch syndrome phenotype in 23% (8 of 35) of MSI families tested. A new combined deletion EPCAM-MSH2 was identified and characterized by break point analysis, encompassing from the 3′ end region of EPCAM to the 5′ initial sequences of the MSH2 (c.859-1860_MSH2:646-254del). EPCAM-MSH2 fusion transcript was isolated. The tumors of the carriers show high-level MSI and MSH2 protein loss. The clinical correlation provided evidence that the type of mutation and the extension of the deletions involving the MSH2 gene could have different implications in cancer predisposition. Thus, the identification of EPCAM-MSH2 rearrangements and their comprehensive characterization should be included in the routine mutation screening protocols for Lynch syndrome. Cancer Prev Res; 4(10); 1556–62. ©2011 AACR.

The TP53 Arg72Pro and MDM2 309G > T polymorphisms are not associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers

Background:The TP53 pathway, in which TP53 and its negative regulator MDM2 are the central elements, has an important role in carcinogenesis, particularly in BRCA1- and BRCA2-mediated carcinogenesis. A single nucleotide polymorphism (SNP) in the promoter region of MDM2 (309T>G, rs2279744) and a coding SNP of TP53 (Arg72Pro, rs1042522) have been shown to be of functional significance.Methods:To investigate whether these SNPs modify breast cancer risk for BRCA1 and BRCA2 mutation carriers, we pooled genotype data on the TP53 Arg72Pro SNP in 7011 mutation carriers and on the MDM2 309T>G SNP in 2222 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). Data were analysed using a Cox proportional hazards model within a retrospective likelihood framework.Results:No association was found between these SNPs and breast cancer risk for BRCA1 (TP53: per-allele hazard ratio (HR)=1.01, 95% confidence interval (CI): 0.93–1.10, Ptrend=0.77; MDM2: HR=0.96, 95%CI: 0.84–1.09, Ptrend=0.54) or for BRCA2 mutation carriers (TP53: HR=0.99, 95%CI: 0.87–1.12, Ptrend=0.83; MDM2: HR=0.98, 95%CI: 0.80–1.21, Ptrend=0.88). We also evaluated the potential combined effects of both SNPs on breast cancer risk, however, none of their combined genotypes showed any evidence of association.Conclusion:There was no evidence that TP53 Arg72Pro or MDM2 309T>G, either singly or in combination, influence breast cancer risk in BRCA1 or BRCA2 mutation carriers.

Evaluation of Rare Variants in the New Fanconi Anemia Gene ERCC4 (FANCQ) as Familial Breast/Ovarian Cancer Susceptibility Alleles

Recently, it has been reported that biallelic mutations in the ERCC4 (FANCQ) gene cause Fanconi anemia (FA) subtype FA‐Q. To investigate the possible role of ERCC4 in breast and ovarian cancer susceptibility, as occurs with other FA genes, we screened the 11 coding exons and exon–intron boundaries of ERCC4 in 1573 index cases from high‐risk Spanish familial breast and ovarian cancer pedigrees that had been tested negative for BRCA1 and BRCA2 mutations and 854 controls. The frequency of ERCC4 mutation carriers does not differ between cases and controls, suggesting that ERCC4 is not a cancer susceptibility gene. Interestingly, the prevalence of ERCC4 mutation carriers (one in 288) is similar to that reported for FANCA, whereas there are approximately 100‐fold more FA‐A than FA‐Q patients, indicating that most biallelic combinations of ERCC4 mutations are embryo lethal. Finally, we identified additional bone‐fide FA ERCC4 mutations specifically disrupting interstrand cross‐link repair.