[show abstract][hide abstract] ABSTRACT: on behalf of the ENIGMA consortium BACKGROUND: Accurate evaluation of unclassified se-quence variants in cancer predisposition genes is essen-tial for clinical management and depends on a multi-factorial analysis of clinical, genetic, pathologic, and bioinformatic variables and assays of transcript length and abundance. The integrity of assay data in turn re-lies on appropriate assay design, interpretation, and reporting. METHODS: We conducted a multicenter investigation to compare mRNA splicing assay protocols used by mem-bers of the ENIGMA (Evidence-Based Network for the Interpretation of Germline Mutant Alleles) consor-tium. We compared similarities and differences in re-sults derived from analysis of a panel of breast cancer 1,
[show abstract][hide abstract] ABSTRACT: The PALB2 gene, also known as FANCN, forms a bond and co-localizes with BRCA2 in DNA repair. Germline mutations in PALB2 have been identified in approximately 1% of familial breast cancer and 3-4% of familial pancreatic cancer. The goal of this study was to determine the prevalence of PALB2 mutations in a population of BRCA1/BRCA2 negative breast cancer patients selected from either a personal or family history of pancreatic cancer.
132 non-BRCA1/BRCA2 breast/ovarian cancer families with at least one pancreatic cancer case were included in the study. PALB2 mutational analysis was performed by direct sequencing of all coding exons and intron/exon boundaries, as well as multiplex ligation-dependent probe amplification.
Two PALB2 truncating mutations, the c.1653T>A (p.Tyr551Stop) previously reported, and c.3362del (p.Gly1121ValfsX3) which is a novel frameshift mutation, were identified. Moreover, several PALB2 variants were detected; some of them were predicted as pathological by bioinformatic analysis. Considering truncating mutations, the prevalence rate of our population of BRCA1/2-negative breast cancer patients with pancreatic cancer is 1.5%.
The prevalence rate of PALB2 mutations in non-BRCA1/BRCA2 breast/ovarian cancer families, selected from either a personal or family pancreatic cancer history, is similar to that previously described for unselected breast/ovarian cancer families. Future research directed towards identifying other gene(s) involved in the development of breast/pancreatic cancer families is required.
PLoS ONE 01/2013; 8(7):e67538. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: BACKGROUND: Accurate evaluation of unclassified sequence variants in cancer predisposition genes is essential for clinical management and depends on a multifactorial analysis of clinical, genetic, pathologic, and bioinformatic variables and assays of transcript length and abundance. The integrity of assay data in turn relies on appropriate assay design, interpretation, and reporting.METHODS: We conducted a multicenter investigation to compare mRNA splicing assay protocols used by members of the ENIGMA (Evidence-Based Network for the Interpretation of Germline Mutant Alleles) consortium. We compared similarities and differences in results derived from analysis of a panel of breast cancer 1, early onset (BRCA1) and breast cancer 2, early onset (BRCA2) gene variants known to alter splicing (BRCA1: c.135-1G>T, c.591C>T, c.594-2A>C, c.671-2A>G, and c.5467+5G>C and BRCA2: c.426-12_8delGTTTT, c.7988A>T, c.8632+1G>A, and c.9501+3A>T). Differences in protocols were then assessed to determine which elements were critical in reliable assay design.RESULTS: PCR primer design strategies, PCR conditions, and product detection methods, combined with a prior knowledge of expected alternative transcripts, were the key factors for accurate splicing assay results. For example, because of the position of primers and PCR extension times, several isoforms associated with BRCA1, c.594-2A>C and c.671-2A>G, were not detected by many sites. Variation was most evident for the detection of low-abundance transcripts (e.g., BRCA2 c.8632+1G>A Delta19,20 and BRCA1 c.135-1G>T Delta5q and Delta3). Detection of low-abundance transcripts was sometimes addressed by using more analytically sensitive detection methods (e.g., BRCA2 c.426-12_8delGTTTT ins18bp).CONCLUSIONS: We provide recommendations for best practice and raise key issues to consider when designing mRNA assays for evaluation of unclassified sequence variants.
[show abstract][hide abstract] ABSTRACT: Fanconi anemia is a genetically heterogeneous autosomal recessive disorder characterized by development abnormalities, bone marrow failure, and childhood cancers. Compelling evidence indicates a common genetic basis for FA and breast/ovarian cancer susceptibility. Recently, biallelic germ-line mutations in SLX4 have been demonstrated to cause a previously unknown FA subtype (FA-P). We address the role of SLX4/FANCP in breast/ovarian cancer susceptibility by conducting a comprehensive mutation scanning in 486 index cases from non-BRCA1/BRCA2 multiple-case breast and/or ovarian cancer families (non-BRCA1/2 families) from Spain. We detected one unequivocal loss-of-function mutation (p.Glu1517X). In addition, one missense change (p.Arg372Trp) predicted to be pathogenic by in silico analysis co-segregates with disease in one family. Overall, the study indicates that SLX4 mutation screening will have a very low impact (if any) in the genetic counseling of non-BRCA1/2 families.European Journal of Human Genetics advance online publication, 5 December 2012; doi:10.1038/ejhg.2012.268.
European journal of human genetics: EJHG 12/2012; · 3.56 Impact Factor
[show abstract][hide abstract] ABSTRACT: Alterations in telomere maintenance mechanisms leading to short telomeres underlie different genetic disorders of ageing and cancer predisposition syndromes. It is known that short telomeres and subsequent genomic instability contribute to malignant transformation, and it is therefore likely that people with shorter telomeres are at higher risk for different types of cancer. Recently, the authors demonstrated that the genes BRCA1 and BRCA2 are modifiers of telomere length (TL) in familial breast cancer. The present study analysed TL in peripheral blood leucocytes of hereditary and sporadic ovarian cancer cases, as well as in female controls, to evaluate whether TL contributes to ovarian cancer risk.
TL was measured by quantitative PCR in 178 sporadic and 168 hereditary ovarian cases (46 BRCA1, 12 BRCA2, and 110 BRCAX) and compared to TL in 267 controls.
Both sporadic and hereditary cases showed significantly shorter age adjusted TLs than controls. Unconditional logistic regression analysis revealed an association between TL and ovarian cancer risk with a significant interaction with age (p<0.001). Risk was higher in younger women and progressively decreased with age, with the highest OR observed in women under 30 years of age (OR 1.56, 95% CI 1.34 to 1.81; p=1.0×10(-18)).
These findings indicate that TL could be a risk factor for early onset ovarian cancer.
Journal of Medical Genetics 04/2012; 49(5):341-4. · 5.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: It has been demonstrated that monoallelic PALB2 (Partner and Localizer of BRCA2) gene mutations predispose to familial breast cancer. Some of the families reported with germline PALB2 mutations presented male breast cancer as a characteristic clinical feature. Therefore, we wanted to investigate the contribution of germline PALB2 mutations in a set of 131 Spanish BRCA1/BRCA2-negative breast/ovarian cancer families with at least one male breast cancer case. The analysis included direct sequencing of all coding exons and intron/exon boundaries as well as a Multiplex Ligation-dependent Probe Amplification-based analysis of genomic rearrangements. For the first time we have identified a genomic rearrangement of PALB2 gene involving a large deletion from exon 7 to 11 in a breast cancer family. We have also identified several PALB2 variants, but no other obvious deleterious PALB2 mutation has been found. Thus, our study does not support an enrichment of PALB2 germline mutations in the subset of breast cancer families with male breast cancer cases. The identification of intronic and exonic variants indicates the necessity of assessing the implications of variants that do not lead to PALB2 truncation in the pathoghenicity of the PALB2 gene.
Breast Cancer Research and Treatment 11/2011; 132(1):307-15. · 4.47 Impact Factor
[show abstract][hide abstract] ABSTRACT: Classification of intronic and predicted missense changes in the breast cancer susceptibility genes BRCA1 and BRCA2 remains a significant challenge for manage-ment of patients carrying these variants. Defective mRNA splicing is established as a pathway to disease, and mRNA analysis of unclassified variants has been shown to assist in classification and genetic counselling. However the interpretation of splicing assay results can be difficult, particularly for those variants that give rise to aberrations in a background of naturally occurring isoforms. The ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) consortium was set up to facilitate research and improve research methods used to classify rare variants in the BRCA1 and BRCA2 (and potentially other) breast cancer predisposition genes.
Familial Aspects of Cancer 2011 Research and Practice; 08/2011 · 1.71 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mutations in BRCA1 and BRCA2 predispose carriers to early onset breast and ovarian cancer. A common problem in clinical genetic testing is interpretation of variants with unknown clinical significance. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium was initiated to evaluate and implement strategies to characterize the clinical significance of BRCA1 and BRCA2 variants. As an initial project of the ENIGMA Splicing Working Group, we report splicing and multifactorial likelihood analysis of 25 BRCA1 and BRCA2 variants from seven different laboratories. Splicing analysis was performed by reverse transcriptase PCR or mini gene assay, and sequencing to identify aberrant transcripts. The findings were compared to bioinformatic predictions using four programs. The posterior probability of pathogenicity was estimated using multifactorial likelihood analysis, including co-occurrence with a deleterious mutation, segregation and/or report of family history. Abnormal splicing patterns expected to lead to a non-functional protein were observed for 7 variants (BRCA1 c.441+2T>A, c.4184_4185+2del, c.4357+1G>A, c.4987-2A>G, c.5074G>C, BRCA2 c.316+5G>A, and c.8754+3G>C). Combined interpretation of splicing and multifactorial analysis classified an initiation codon variant (BRCA2 c.3G>A) as likely pathogenic, uncertain clinical significance for 7 variants, and indicated low clinical significance or unlikely pathogenicity for another 10 variants. Bioinformatic tools predicted disruption of consensus donor or acceptor sites with high sensitivity, but cryptic site usage was predicted with low specificity, supporting the value of RNA-based assays. The findings also provide further evidence that clinical RNA-based assays should be extended from analysis of invariant dinucleotides to routinely include all variants located within the donor and acceptor consensus splicing sites. Importantly, this study demonstrates the added value of collaboration between laboratories, and across disciplines, to collate and interpret information from clinical testing laboratories to consolidate patient management.
Breast Cancer Research and Treatment 07/2011; 132(3):1009-23. · 4.47 Impact Factor
[show abstract][hide abstract] ABSTRACT: The c.156_157insAlu BRCA2 mutation has so far only been reported in hereditary breast/ovarian cancer (HBOC) families of Portuguese origin. Since this mutation is not detectable using the commonly used screening methodologies and must be specifically sought, we screened for this rearrangement in a total of 5,443 suspected HBOC families from several countries. Whereas the c.156_157insAlu BRCA2 mutation was detected in 11 of 149 suspected HBOC families from Portugal, representing 37.9% of all deleterious mutations, in other countries it was detected only in one proband living in France and in four individuals requesting predictive testing living in France and in the USA, all being Portuguese immigrants. After performing an extensive haplotype study in carrier families, we estimate that this founder mutation occurred 558 ± 215 years ago. We further demonstrate significant quantitative differences regarding the production of the BRCA2 full length RNA and the transcript lacking exon 3 in c.156_157insAlu BRCA2 mutation carriers and in controls. The cumulative incidence of breast cancer in carriers did not differ from that of other BRCA2 and BRCA1 pathogenic mutations. We recommend that all suspected HBOC families from Portugal or with Portuguese ancestry are specifically tested for this rearrangement.
Breast Cancer Research and Treatment 06/2011; 127(3):671-9. · 4.47 Impact Factor
[show abstract][hide abstract] ABSTRACT: Although in recent years deleterious BRCA1 mutations have been extensively studied as a prostate cancer risk factor, results are inconclusive. To assess the contribution of the BRCA1 Galician founder mutation c.211A>G in prostate cancer morbidity we conducted a case-control study. Moreover, to better elucidate whether deleterious BRCA1 mutations are involved in the development of prostate cancer, we performed a systematic review and a meta-analysis of BRCA1 studies on prostate cancer.
A total of 905 unselected men diagnosed with adenocarcinoma of the prostate and a control group of 936 unrelated men without history of prostate cancer were evaluated for c.211A>G. Adjusted by age Odds ratios (OR) and 95% confidence intervals (CIs) were estimated using logistic regression. To construct the meta-analysis, genotype-based epidemiological studies reporting BRCA1 founder mutations on prostate cancer were identified by comprehensive and systematic bibliographic search. After extraction of relevant data, main and subgroup analysis by mutation were performed to assess the effect of BRCA1 on prostate cancer risk.
Four c.211A>G heterozygous individuals, one patient and three controls, were detected (OR = 0.27; 95% CI: 0.01-2.36; P = 0.28). Meta-analysis results from the integration of our data and other seven studies with BRCA1 genotyping data (5,705 prostate cancer cases and 13,218 controls) did not detect an association with prostate cancer risk (OR = 1.36; 95% CI: 0.87-2.14; P = 0.18).
Our conclusive trial demonstrates the lack of association between Galician splicing mutation c.211A>G in the BRCA1 gene and prostate cancer risk. Moreover, the result of the meta-analysis also discards the involvement of BRCA1 mutations in the development of prostate cancer.
The Prostate 04/2011; 71(16):1768-79. · 3.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: Biallelic inactivation of ATM gene causes the rare autosomal recessive disorder Ataxia-telangiectasia (A-T). Female relatives of A-T patients have a two-fold higher risk of developing breast cancer (BC) compared with the general population. ATM mutation carrier identification is laborious and expensive, therefore, a more rapid and directed strategy for ATM mutation profiling is needed. We designed a case-control study to determine the prevalence of 32 known ATM mutations causing A-T in Spanish population in 323 BRCA1/BRCA2 negative hereditary breast cancer (HBC) cases and 625 matched Spanish controls. For the detection of the 32 ATM mutations we used the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technique. We identified one patient carrier of the c.8264_8268delATAAG ATM mutation. This mutation was not found in the 625 controls. These results suggest a low frequency of these 32 A-T causing mutations in the HBC cases in our population. Further case-control studies analyzing the entire coding and flanking sequences of the ATM gene are warranted in Spanish BC patients to know its implication in BC predisposition.
Breast Cancer Research and Treatment 03/2011; 128(2):573-9. · 4.47 Impact Factor
[show abstract][hide abstract] ABSTRACT: Environmental or lifestyle factors are likely to explain part of the heterogeneity in breast and ovarian cancer risk among BRCA1 and BRCA2 mutation carriers. We assessed parity as a risk modifier in 515 and 503 Spanish female carriers of mutations in BRCA1 and BRCA2, respectively. Hazard ratios (HR) and their corresponding 95% confidence intervals (CI) were estimated using weighted Cox proportional hazards regression, adjusted for year of birth and study centre. The results for ever being parous and number of live-births were very similar for carriers of mutations in both genes. For all mutation carriers combined, the estimated HR associated with ever having had a live-birth was 0.74 (95% confidence interval [CI] = 0.55-1.01, P = 0.06), and that associated with each live-birth was 0.87 (95%CI = 0.77-0.98, P = 0.02). The latter association was observed only in women aged 40 and above (HR = 0.81, 95%CI = 0.70-0.94, P = 0.004 vs. HR = 0.99, 95%CI = 0.83-1.18, P = 0.9 for women under age 40), and this trend was highly consistently observed for carriers of mutations in each gene. There was no evidence of an association between breast cancer risk and age at first birth for parous BRCA1 or BRCA2 mutation carriers (P-trend >or= 0.3). The power to detect associations with ovarian cancer risk was much lower, especially for BRCA2 mutation carriers. Nevertheless, having a live-birth was associated with protection for BRCA1 mutation carriers (HR = 0.41, 95%CI = 0.18-0.94, P = 0.03), and a strong and consistent protective effect of age at first birth was observed for parous carriers of mutations in both genes (HR = 0.65, 95%CI = 0.52-0.83, P < 0.001). This is the third independent study to find that, as in the general population, parity appears to be associated with protection from breast cancer in women with mutations in BRCA1 and BRCA2. Parity appears to be protective for ovarian cancer in BRCA1 mutation carriers, but its role in BRCA2 mutation carriers remains unclear. Whether later age at first birth is also protective for ovarian cancer in mutation carriers requires further confirmation.
Breast Cancer Research and Treatment 04/2009; 119(1):221-32. · 4.47 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
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.
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.
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.
Clinical Cancer Research 05/2008; 14(9):2861-9. · 7.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: The signaling pathways involving class I phosphatidylinositol 3-kinases (PI3K) and the phosphatidylinositol-(3,4,5)-trisphosphate phosphatase PTEN regulate cell proliferation and survival. Thus, mutations in the corresponding genes are associated to a wide variety of human tumors. Heterologous expression of hyperactive forms of mammalian p110alpha and p110beta in Saccharomyces cerevisiae leads to growth arrest, which is counterbalanced by coexpression of mammalian PTEN. Using this in vivo yeast-based system, we have done an extensive functional analysis of germ-line and somatic human PTEN mutations, as well as a directed mutational analysis of discrete PTEN functional domains. A distinctive penetrance of the PTEN rescue phenotype was observed depending on the levels of PTEN expression in yeast and on the combinations of the inactivating PTEN mutations and the activating p110alpha or p110beta mutations analyzed, which may reflect pathologic differences found in tumors with distinct alterations at the p110 and PTEN genes or proteins. We also define the minimum length of the PTEN protein required for stability and function in vivo. In addition, a random mutagenesis screen on PTEN based on this system allowed both the reisolation of known clinically relevant PTEN mutants and the identification of novel PTEN loss-of-function mutations, which were validated in mammalian cells. Our results show that the PI3K/PTEN yeast-based system is a sensitive tool to test in vivo the pathologic properties and the functionality of mutations in the human p110 proto-oncogenes and the PTEN tumor suppressor and provide a framework for comprehensive functional studies of these tumor-related enzymes.
Cancer Research 11/2007; 67(20):9731-9. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Ligneous conjunctivitis (LC) is a rare disease characterized by wood-like pseudomembranes developing on the ocular and extraocular mucosae secondary to plasminogen (PLG) deficiency. In this paper, we report two cases of LC in two sisters of 57 and 62 years of age that presented with recurrent, bilateral pseudomembranes on conjunctiva and a history of consanguinity and deafness. Pseudomembranes showed superficial and/or subepithelial deposits of eosinophilic amorphous hyaline, amyloid-like material with a variable proportion of granulation tissue, and inflammatory cells. The eosinophilic deposits were negative for Congo red stain, immunoreactive for fibrinogen, and consistently negative for amyloid A component, transthyretin, beta(2)-microglobulin, albumin, fibronectin, collagen type IV, vimentin, and cytokeratins. Among inflammatory cells, a percentage of positivity of roughly 60% for lymphocytes T (CD3+) and 40% for lymphocytes B (CD8+), with a relation of cytotoxic/helper (CD8/4) T cells of 3:2, was found. In one case, nasal polyps and recurrent gastric peptic ulcer were also characterized by the same subepithelial hyaline deposits. A novel homozygous point mutation c.1856 C>T was found in exon 15 of the PLG gene in both patients. Amniotic membrane transplantation was done in one case with promising results.
Archiv für Pathologische Anatomie und Physiologie und für Klinische Medicin 10/2007; 451(4):815-21. · 2.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: It is estimated that 5-10% of all breast cancer are hereditary and attributable to mutations in the highly penetrance susceptibility genes BRCA1 and BRCA2. The genetic analysis of these genes is complex and expensive essentially because their length. Nevertheless, the presence of recurrent and founder mutations allows a pre-screening for the identification of the most frequent mutations found in each geographical region. In Spain, five mutations in BRCA1 and other five in BRCA2 account for approximately 50% of the mutations detected in Spanish families.
We have developed a novel PCR multiplex SNaPshot reaction that targets all ten recurrent and founder mutations identified in BRCA1 and BRCA2 in Spain to date.
The SNaPshot reaction was performed on samples previously analyzed by direct sequencing and all mutations were concordant. This strategy permits the analysis of approximately 50% of all mutations observed to be responsible for breast/ovarian cancer in Spanish families using a single reaction per patient sample.
The SNaPshot assay developed is sensitive, rapid, with minimum cost per sample and additionally can be automated for high-throughput genotyping. The SNaPshot assay outlined here is not only useful for analysis of Spanish breast/ovarian cancer families, but also e.g. for populations with Spanish ancestry, such as those in Latin America.
BMC Medical Genetics 02/2007; 8:40. · 2.54 Impact Factor