Comprehensive functional assessment of MLH1 variants of unknown significance.

Lynch syndrome is associated with germline mutations in DNA mismatch repair (MMR) genes. Up to 30% of DNA changes found are variants of unknown significance (VUS). Our aim was to assess the pathogenicity of eight MLH1 VUS identified in patients suspected of Lynch syndrome. All of them are novel or not previously characterized. For their classification, we followed a strategy that integrates family history, tumor pathology, and control frequency data with a variety of in silico and in vitro analyses at RNA and protein level, such as MMR assay, MLH1 and PMS2 expression, and subcellular localization. Five MLH1 VUS were classified as pathogenic: c.[248G>T(;)306G>C], c.[780C>G;788A>C], and c.791-7T>A affected mRNA processing, whereas c.218T>C (p.L73P) and c.244A>G [corrected] (p.T82A) impaired MMR activity. Two other VUS were considered likely neutral: the silent c.702G>A variant did not affect mRNA processing or stability, and c.974G>A (p.R325Q) did not influence MMR function. In contrast, variant c.25C>T (p.R9W) could not be classified, as it associated with intermediate levels of MMR activity. Comprehensive functional assessment of MLH1 variants was useful in their classification and became relevant in the diagnosis and genetic counseling of carrier families.

Detection of a large rearrangement in PALB2 in Spanish breast cancer families with male breast cancer.

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.

Functional characterization of the novel APC N1026S variant associated with attenuated familial adenomatous polyposis.

BACKGROUND & AIMS: We identified the APC N1026S variant of unknown malignant potential in the adenomatous polyposis coli (APC) gene in a Spanish attenuated familial adenomatous polyposis (AFAP) family. The variant was located in the first of the 4 highly conserved 15-amino acid (AA) repeats within the beta-catenin union domain. Our aim was to determine its functional relevance to establish its pathogenicity. METHODS: N1026S variant was analyzed in 22 members of the AFAP family studied, in 236 sporadic colorectal cancer cases, 203 matched controls, and 205 unrelated familial colorectal cancer cases. To assess its effects on beta-catenin binding, beta-catenin/Tcf-4-mediated transcription and beta-catenin subcellular distribution we performed affinity chromatography experiments, BIAcore 1000 (BIAcore AB, Uppsala, Sweden) assays, luciferase reporter assays, assessment of c-myc messenger RNA levels, and cell fractionation. RESULTS: N1026S variant cosegregated with the disease in the AFAP family studied. None of the sporadic or familial cases as well as the controls analyzed was positive for the variant. N1026S variant completely precluded beta-catenin binding to the first 15-AA repeat and diminished it when all four 15-AA repeats were present. Expression of APC N1026S in SW480 and DLD-1 cells did not diminish beta-catenin/Tcf-4-mediated transcription as effectively as APC wild-type. N1026S did not decrease c-myc transcription in DLD1 cells and nuclear beta-catenin in SW480 cells as effectively as WT. CONCLUSIONS: These findings strongly support a pathogenic role of the APC N1026S variant in the AFAP phenotype, reinforcing the importance of functional characterization of APC variants for genetic counseling.

Opinion about reproductive decision making among individuals undergoing BRCA1/2 genetic testing in a multicentre Spanish cohort.

BACKGROUND: Mutations in breast cancer BRCA1/2 genes increase breast and ovarian cancer risk and are transmitted with an autosomal dominant pattern. Opinion about reproductive decisions among individuals undergoing BRCA1/2 testing in our institutions is unknown. MATERIALS AND METHODS: Individuals (n = 77) undergoing BRCA1/2 testing were included in a prospective multicentre study to assess the clinical impact of genetic testing. Demographic and clinical information, psychological status and opinion about reproductive decisions were collected in two questionnaires administered prior to testing. Opinion regarding the use of assisted reproduction techniques for hereditary cancer susceptibility among health care professionals was also collected. RESULTS: Twenty-eight individuals (36%) reported that they would decide to have children, regardless of their result. In case of a mutation, 9 (12%) believed that they would decide not to have children, 42 (55%) would consider prenatal diagnosis (PND), 37 (48%) would consider preimplantation genetic diagnosis (PGD) and 23 (30%) would consider adoption. Fifty-seven (74%) and 47 (61%) reported that they considered it ethical to offer PND or PGD, respectively, to BRCA+ patients. Individuals older than 40 years were more likely to consider PND or PGD than younger subjects (P = 0.02 and 0.05, respectively). Individuals with cancer compared with those without a diagnosis of malignancy were more likely to consider PGD (61 versus 30%, P = 0.02) and to consider that it was ethical to offer it (74 versus 44%, P = 0.02). Most health care professionals were in favour of PND and PGD for individuals with hereditary cancer susceptibility (58 and 61%, respectively). CONCLUSIONS: BRCA1/2 genetic results could influence an individual's decisions regarding reproduction. Health care professionals who serve individuals undergoing BRCA testing should incorporate patient education regarding the potential impact of such testing on family planning.

Analysis of PALB2 gene in BRCA1/BRCA2 negative Spanish hereditary breast/ovarian cancer families with pancreatic cancer cases.

BACKGROUND: 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. METHODS: 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. RESULTS: 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%. CONCLUSIONS: 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.

What factors may influence psychological well being at three months and one year post BRCA genetic result disclosure?

Genetic testing for breast cancer predisposition has been available in the clinical practice for more than a decade. How the result of genetic testing affects the psychological well-being of the individuals is an under-researched area in many populations. Follow-up analysis of psychological well-being via HADS scale was performed in 364 individuals at 3 months and 1 year after the disclosure of BRCA1/2 genetic result. We analyzed potential predictors for pathological anxiety and variables associated to the variation of HADS scores over time. At pre-test only 16% and 4% of individuals presented symptoms of anxiety and depression, respectively. Having a prior diagnosis of cancer and presenting a pathological HADS-A score at the baseline were associated with clinically significant anxiety scores at one year, but the genetic test result was not. Thus, BRCA genetic testing does not influence short and long term anxiety and depression levels among those identified as mutation carriers. It is our task to demystify the allegedly negative impact of BRCA testing on psychological well being to increase the uptake of genetic testing and benefit those who are at high risk of developing breast, ovarian and prostate cancer.

Risk factors associated with the occurrence of breast cancer after bilateral salpingo-oophorectomy in high-risk women.

INTRODUCTION: Bilateral salpingo-oophorectomy (BSO) is a common procedure for preventing breast and ovarian cancer in high-risk women. The goal of this study was to determine the incidence of subsequent breast cancer (BC) in a high-risk population and to identify clinical and epidemiological predictors of BC following BSO. MATERIALS AND METHODS: One hundred and thirty-three consecutive high-risk women, tested for BRCA1 and BRCA2 mutations due to family history, underwent preventive or therapeutic BSO at one of the study hospitals. One hundred and three patients had breast tissue at risk and were considered evaluable for the event-free survival analysis. Twenty-five women harbored a deleterious mutation in BRCA1 and 25 in BRCA2 genes. RESULTS: Fifteen cases of invasive BC were diagnosed with a median interval of 49 months after BSO. Multivariate analysis showed that a prior BC after 50 years of age (p=0.004), age over 50 years at the time of BSO (p=0.005), and prior replacement or contraceptive hormonal treatment (p=0.007) were significantly associated with a shorter event-free survival. CONCLUSION: In conclusion, age at prior diagnosis of cancer, age at BSO and prior hormonal treatment may be predictors of breast cancer after BSO.

Uptake of predictive testing among relatives of BRCA1 and BRCA2 families: a multicenter study in northeastern Spain.

Identifying a BRCA mutation among families with hereditary breast and ovarian cancer enables distinguishing those who may benefit from a specific medical management. This study aimed to evaluate the uptake of predictive testing among close relatives of a proband in Spanish families with a BRCA1 or BRCA2 mutation, and to determine the associated demographic and clinical predictors. A retrospective cohort of families undergoing clinical genetic testing at four university hospitals in northeastern Spain was considered. From 108 unrelated BRCA1/2 families, 765 close relatives of probands were analyzed. Sixty percent of the first-degree and 28% of the second-degree relatives underwent predictive testing within a median time of 2 and 6 months, respectively, since the mutation disclosure to the proband. Relatives undergoing genetic testing were more likely to be female, first-degree, and belong to a family with a proband who had a high educational level. Relatives were also more likely to have offspring, a previous cancer diagnosis, and to be aged between 30 and 64 years. Among second-degree relatives, having a first-degree relative with cancer was highly correlated with uptake. In conclusion, uptake of BRCA1/2 predictive testing among close relatives was notably high and within a short period of time after disclosure of the mutation to the proband. Being female, a high educational level of the proband, and having a close relative with cancer were associated with uptake among relatives. Further studies are warranted to determine whether information is disseminated properly by probands and to learn about the reasons for those not undergoing testing.

MLH1 founder mutations with moderate penetrance in Spanish Lynch syndrome families.

The variants c.306+5G>A and c.1865T>A (p.Leu622His) of the DNA repair gene MLH1 occur frequently in Spanish Lynch syndrome families. To understand their ancestral history and clinical effect, we performed functional assays and a penetrance analysis and studied their genetic and geographic origins. Detailed family histories were taken from 29 carrier families. Functional analysis included in silico and in vitro assays at the RNA and protein levels. Penetrance was calculated using a modified segregation analysis adjusted for ascertainment. Founder effects were evaluated by haplotype analysis. The identified MLH1 c.306+5G>A and c.1865T>A (p.Leu622His) variants are absent in control populations and segregate with the disease. Tumors from carriers of both variants show microsatellite instability and loss of expression of the MLH1 protein. The c.306+5G>A variant is a pathogenic mutation affecting mRNA processing. The c.1865T>A (p.Leu622His) variant causes defects in MLH1 expression and stability. For both mutations, the estimated penetrance is moderate (age-cumulative colorectal cancer risk by age 70 of 20.1% and 14.1% for c.306+5G>A and of 6.8% and 7.3% for c.1865T>A in men and women carriers, respectively) in the lower range of variability estimated for other pathogenic Spanish MLH1 mutations. A common haplotype was associated with each of the identified mutations, confirming their founder origin. The ages of c.306+5G>A and c.1865T>A mutations were estimated to be 53 to 122 and 12 to 22 generations, respectively. Our results confirm the pathogenicity, moderate penetrance, and founder origin of the MLH1 c.306+5G>A and c.1865T>A mutations. These findings have important implications for genetic counseling and molecular diagnosis of Lynch syndrome.

Identification of a novel pathogenic mutation in BRCA2 in a Spanish breast-ovarian cancer family.

Germline mutations in the BRCA1/2 genes contribute to most of inherited breast and ovarian cancers. We analyzed a family fulfilling classical criteria of hereditary breast/ovarian cancer. After complete sequencing of coding regions and splice junctions of both genes, a nonpreviously reported mutation in BRCA2 was detected in the index case. Direct mutation detection was performed with their relatives, and three of them were also mutation carriers, two healthy males and a patient afflicted with borderline ovarian cancer. The c.2999delCT, consists of a deletion of two bases in exon 11, in the limits of the ovarian cancer cluster region. This is a frameshift mutation that causes a disruption of the translational reading frame resulting in a stop codon 10 amino acids downstream in the 934 position of the BRCA2 protein, causing a truncation protein. This often causes a loss of function in the protein as critical parts of the amino acid chain are no longer created. Because of it, this mutation must be classified as pathogenic and can be regarded as the cause of the cancers in this family.