Apparent regional differences in the spectrum of BARD1 pathogenic variants in Spanish population and importance of copy number variants.

Only up to 25% of the cases in which there is a familial aggregation of breast and/or ovarian cancer are explained by germline mutations in the well-known BRCA1 and BRCA2 high-risk genes. Recently, the BRCA1-associated ring domain (BARD1), that partners BRCA1 in DNA repair, has been confirmed as a moderate-risk breast cancer susceptibility gene. Taking advantage of next-generation sequencing techniques, and with the purpose of defining the whole spectrum of possible pathogenic variants (PVs) in this gene, here we have performed a comprehensive mutational analysis of BARD1 in a cohort of 1946 Spanish patients who fulfilled criteria to be tested for germline pathogenic mutations in BRCA1 and BRCA2. We identified 22 different rare germline variants, being 5 of them clearly pathogenic or likely pathogenic large deletions, which account for 0.26% of the patients tested. Our results show that the prevalence and spectrum of mutations in the BARD1 gene might vary between different regions of Spain and expose the relevance to test for copy number variations.

Novel clinical and molecular findings in Spanish patients with naevoid basal cell carcinoma syndrome.

BACKGROUND: Naevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by developmental alterations and multiple basal cell carcinomas. Mutations in PTCH1, which encodes a membrane receptor for Sonic Hedgehog, are associated with the development of the disease. Most of them produce a truncated protein, which is unable to suppress Smoothened protein and continuously activates the downstream pathway. OBJECTIVES: We aimed to characterize 22 unrelated Spanish patients with NBCCS, the largest cohort with Gorlin syndrome reported to date in Spain. METHODS: Genomic analysis of PTCH1 was performed in patients with NBCCS and controls, and mutations were analysed using bioinformatics tools. RESULTS: We report for the first time two young patients, one each with uterus didelphys and ganglioneuroma, within the context of NBCCS. One patient showing a severe phenotype of the disease had developed basal cell carcinomas since childhood. Sanger sequencing of PTCH1 in this cohort identified 17 novel truncating mutations (11 frameshift, five nonsense and one mutation affecting an exon-intron splice site) and two novel missense mutations that were predicted to be pathogenic. The patients showed great clinical variability and inconsistent genotype-phenotype correlation, as seen in relatives carrying similar mutations. CONCLUSIONS: This study contributes to increase the pool of clinical manifestations of NBCCS, as well as increasing the number of pathogenic mutations identified in PTCH1 predisposing to the condition. The inconsistencies found between phenotype and genotype suggest the involvement of other modifying factors, genetic, epigenetic or environmental.

Almost 2% of Spanish breast cancer families are associated to germline pathogenic mutations in the ATM gene.

PURPOSE: There is still a considerable percentage of hereditary breast and ovarian cancer (HBOC) cases not explained by BRCA1 and BRCA2 genes. In this report, next-generation sequencing (NGS) techniques were applied to identify novel variants and/or genes involved in HBOC susceptibility. METHODS: Using whole exome sequencing, we identified a novel germline mutation in the moderate-risk gene ATM (c.5441delT; p.Leu1814Trpfs*14) in a family negative for mutations in BRCA1/2 (BRCAX). A case-control association study was performed to establish its prevalence in Spanish population, in a series of 1477 BRCAX families and 589 controls further screened, and NGS panels were used for ATM mutational screening in a cohort of 392 HBOC Spanish BRCAX families and 350 patients affected with diseases not related to breast cancer. RESULTS: Although the interrogated mutation was not prevalent in case-control association study, a comprehensive mutational analysis of the ATM gene revealed 1.78% prevalence of mutations in the ATM gene in HBOC and 1.94% in breast cancer-only BRCAX families in Spanish population, where data about ATM mutations were very limited. CONCLUSION: ATM mutation prevalence in Spanish population highlights the importance of considering ATM pathogenic variants linked to breast cancer susceptibility.

Genome-wide linkage analysis and tumoral characterization reveal heterogeneity in familial colorectal cancer type X.

BACKGROUND: Familial colorectal cancer type X (FCCTX) fulfils clinical criteria defining Lynch syndrome (LS), but is not related to germline mutations in DNA mismatch-repair genes. Its aetiology remains unexplained and there is little evidence of involvement of the common colorectal carcinogenetic pathways. We aimed to identify susceptibility loci and gain insights into carcinogenic pathways involved FCCTX tumour development. METHODS: We performed a linkage analysis in 22 FCCTX families. We also constructed a tissue microarray in order to define an immunohistochemical (IHC) profile for FCCTX tumours (N = 27) by comparing them to three other types of colorectal tumors: LS (N = 18), stable early-onset (N = 31) and other sporadic disease (N = 80). Additionally, we screened for BRAF/KRAS mutations and determined CpG island methylator phenotype (CIMP) status for all FCCTX tumours. RESULTS: We found suggestive evidence of linkage at four chromosomal regions; 2p24.3, 4q13.1, 4q31.21 and 12q21.2-q21.31. We screened genes in 12q21 and ruled out the implication of RASSF9 and NTS, good candidates due to their potential involvement in carcinogenesis and colorectal epithelium development. Based on IHC profiles FCCTX tumours did not form a single, exclusive cluster. They were clearly different from LS, but very similar to stable early onset tumours. The CIMP and chromosomal instability pathways were implicated in one-third and one-quarter of FCCTX cases, respectively. The remaining cases did not have alterations in any known carcinogenic pathways. CONCLUSIONS: Our results highlight the heterogeneity of FCCTX tumours and call into question the utility of using only clinical criteria to identify FCCTX cases.

Impact of chemotherapy on telomere length in sporadic and familial breast cancer patients.

Recently, we observed that telomeres of BRCA1/2 mutation carriers were shorter than those of controls or sporadic breast cancer patients, suggesting that mutations in these genes might be responsible for this event. Given the contradictory results reported in the literature, we tested whether other parameters, such as chemotherapy, could be modifying telomere length (TL). We performed a cross-sectional study measuring leukocyte TL of 266 sporadic breasts cancer patients treated with first-line chemotherapy, with a median follow-up of 240 days. Additionally, we performed both cross-sectional and longitudinal studies in a series of 236 familial breast cancer patients that included affected and non-affected BRCA1/2 mutation carriers. We have measured in leukocytes from peripheral blood: the TL, percentage of short telomeres (<3 kb), telomerase activity levels and the annual telomere shortening speed. In sporadic cases we found that chemotherapy exerts a transient telomere shortening effect (around 2 years) that varies depending on the drug combination. In familial cases, only patients receiving treatment were associated with telomere shortening but they recovered normal TL after a period of 2 years. Chemotherapy affects TL and should be considered in the studies that correlate TL with disease susceptibility.

A novel AXIN2 germline variant associated with attenuated FAP without signs of oligondontia or ectodermal dysplasia.

Truncating mutations in the AXIN2 gene, a key regulator of ß-catenin degradation in the Wnt pathway, have been reported in three families with gastrointestinal adenomatous polyposis and features of ectodermal dysplasia. However, the role of AXIN2 in familial adenomatous polyposis (FAP) syndrome is not completely understood. We performed an in-depth study of APC and MUTYH, and ruled out their implication in 23 FAP families. We then investigated the role of other genes involved in the Wnt pathway, including AXIN2, and identified a novel missense variant in AXIN2 in one family with attenuated FAP. Carriers of the variant exhibited a variable number of polyps but none showed any sign of ectodermal dysplasia. We have demonstrated the pathogenicity of this novel variant by establishing its low frequency in controls as well as by LOH analysis, a segregation study, and immunofluorescent staining of AXIN2 and ß-catenin proteins. This report expands the phenotype known to be related to AXIN2 alterations and raises the question of whether to screen AXIN2 in FAP cases negative for alterations in APC and MUTYH.

DNA copy number profiling reveals extensive genomic loss in hereditary BRCA1 and BRCA2 ovarian carcinomas.

BACKGROUND: Few studies have attempted to characterise genomic changes occurring in hereditary epithelial ovarian carcinomas (EOCs) and inconsistent results have been obtained. Given the relevance of DNA copy number alterations in ovarian oncogenesis and growing clinical implications of the BRCA-gene status, we aimed to characterise the genomic profiles of hereditary and sporadic ovarian tumours. METHODS: High-resolution array Comparative Genomic Hybridisation profiling of 53 familial (21 BRCA1, 6 BRCA2 and 26 non-BRCA1/2) and 15 sporadic tumours in combination with supervised and unsupervised analysis was used to define common and/or specific copy number features. RESULTS: Unsupervised hierarchical clustering did not stratify tumours according to their familial or sporadic condition or to their BRCA1/2 mutation status. Common recurrent changes, spanning genes potentially fundamental for ovarian carcinogenesis, regardless of BRCA mutations, and several candidate subtype-specific events were defined. Despite similarities, greater contribution of losses was revealed to be a hallmark of BRCA1 and BRCA2 tumours. CONCLUSION: Somatic alterations occurring in the development of familial EOCs do not differ substantially from the ones occurring in sporadic carcinomas. However, some specific features like extensive genomic loss observed in BRCA1/2 tumours may be of clinical relevance helping to identify BRCA-related patients likely to respond to PARP inhibitors.

Clinical and genetic characterization of classical forms of familial adenomatous polyposis: a Spanish population study.

BACKGROUND: Classical familial adenomatous polyposis (FAP) is characterized by the appearance of >100 colorectal adenomas. PATIENTS AND METHODS: We screened the APC and MUTYH genes for mutations and evaluated the genotype-phenotype correlation in 136 Spanish classical FAP families. RESULTS: APC/MUTYH mutations were detected in 107 families. Sixty-four distinct APC point mutations were detected in 95 families of which all were truncating mutations. A significant proportion (39.6%) had not been previously reported. Mutations were spread over the entire coding region and great rearrangements were identified in six families. Another six families exhibited biallelic MUTYH mutations. No APC or MUTYH mutations were detected in 29 families. These APC/MUTYH-negative families showed clinical differences with the APC-positive families. A poor correlation between phenotype and mutation site was observed. CONCLUSIONS: Our results highlight that a broad approach in the genetic study must be considered for classical FAP due to involvement of both APC and MUTYH and the heterogeneous spectrum of APC mutations observed in this Spanish population. The scarcely consistent genotype-phenotype correlation does not allow making specific recommendations regarding screening and management. Differences observed in APC/MUTYH-negative families may reflect a genetic basis other than mutations in APC and MUTYH genes for FAP predisposition.

Surgical management of hereditary colorectal cancer: surgery based on molecular analysis and family history.

The importance of colorectal cancer (CRC) is increasing. A proportion show a hereditary component, as in Lynch syndrome and Familial Adenomatous Polyposis, and a recently defined entity as well, namely, Familial Colorectal Cancer type X. The high probability to develop CRC in these groups may, at the time of recognition, change surgical management, including its timing or even the surgical technique. In some cases prophylactic surgery can play an important role. The possibility of using tools that allow recognition of the aforementioned syndromes, including microsatellite instability, immunohistochemistry for DNA mismatch repair system proteins, and especially their mutations, is on the basis of therapeutic strategies that differ from those employed in sporadic CRC cases.

Surgical management of hereditary colorectal cancer: surgery based on molecular analysis and family history

The importance of colorectal cancer (CRC) is increasing. Aproportion show a hereditary component, as in Lynch syndromeand Familial Adenomatous Polyposis, and a recently defined entityas well, namely, Familial Colorectal Cancer type X. The highprobability to develop CRC in these groups may, at the time ofrecognition, change surgical management, including its timing oreven the surgical technique. In some cases prophylactic surgerycan play an important role. The possibility of using tools that allowrecognition of the aforementioned syndromes, including microsatelliteinstability, immunohistochemistry for DNA mismatchrepair system proteins, and especially their mutations, is on thebasis of therapeutic strategies that differ from those employed insporadic CRC cases(AU)

MLH1 germline epimutations in selected patients with early-onset non-polyposis colorectal cancer.

Recently, the germline epigenetic inactivation of MLH1 has been reported in a number of patients with early-onset colorectal cancer among other characteristics. The aim of the present study is to evaluate the presence of MLH1 germline epimutations in selected colorectal cancer patients suspected of hereditary non-polyposis colorectal cancer (HNPCC) in order to determine in which patients the MLH1 epigenetic test should be performed. From a total of 109 microsatellite instability (MSI)-positive HNPCC-suspected patients, 11 showed a lack of MLH1 expression in tumor tissue and no germline mutations in the mismatch repair (MMR) genes. In nine of these cases and in three additional patients with multiple tumors, the study of the germline MLH1 promoter hypermethylation was performed by means of methylation-specific PCR and combined bisulfite-restriction analysis techniques. One of the selected patients resulted positive for the MLH1 epimutation, which was confirmed in the DNA extracted from buccal lavage. The patient with the epimutation had developed an epidermoid lip carcinoma and an early-onset colorectal tumor with MSI, no MLH1 expression, and loss of heterozygosity of the gene. Parents and siblings did not carry the epigenetic alteration, suggesting a de novo mechanism. Although germline MLH1 epimutations seem to be mostly uncommon, when the cases are well selected, the probability of finding them increases. Thus, taking into account ours and previous reports, we propose that screening for MLH1 epimutations in blood DNA could be performed in early-onset colorectal cancer patients with MSI, lack of MLH1 expression in the tumor, and no germline mutations in the MMR genes.

A haplotype containing the p53 polymorphisms Ins16bp and Arg72Pro modifies cancer risk in BRCA2 mutation carriers.

Germline mutations in the BRCA1 and BRCA2 genes confer a high lifetime risk of developing breast and other cancers; however, remarkable differences exist regarding disease manifestation in mutation carriers. It has been suggested that other genetic and/or environmental factors modify not only the appearance but also the age of onset and type of tumor in BRCA1/2-associated cases. The aim of the present study was to investigate the role of two p53 polymorphisms (c.97-147ins16bp and c.215c>g, p.Arg72Pro) as potential modifiers. For this purpose we investigated the possible association between the two polymorphisms and disease status in 447 BRCA1/2 mutation carriers belonging to 170 Spanish breast and/or ovarian cancer families. Genotype and haplotype analyses revealed that the presence of a specific haplotype carrying the allele without the 16-bp insertion and the variant allele for the Arg72Pro (No Ins-72Pro haplotype) was associated with an earlier age of onset in BRCA2 mutation carriers. We found an increased risk of developing a first primary tumor (breast or ovarian) before 35 years of age for individuals who carried at least one No Ins-72Pro haplotype (OR: 2.69; 95% CI: 1.15-6.29; P=0.022). We confirmed these data by a functional study in which we compared different p53 genotypes in relation to their apoptotic response after cell treatment with a cytotoxic drug (AraC). Our results revealed a decrease in p53 apoptotic rate associated with the No Ins-72Pro haplotype.

Lhermitte-Duclos disease and Cowden disease: clinical and genetic study in five patients with Lhermitte-Duclos disease and literature review.

BACKGROUND: Lhermitte-Duclos Disease (LDD) is an infrequent cerebellar disorder characterized by focal or diffuse enlargement of cerebellar folia presenting as a slowly growing mass in the posterior fossa. Over the past decade its association with Cowden disease (CD) has been recognized with increasing frequency. This latter disease is a genetic condition leading to the presence of multiple hamartomas and neoplasias which affect mainly the skin, thyroid, breast and genito-urinary and gastro-intestinal tracts. It has even been hypothesized that LDD and CD constitute a single entity. This work is aimed to analyse to what extent this association was present in patients treated for LDD at our institution. METHOD: We reviewed the medical records of five patients and performed clinical studies for CD manifestations, among them, genetic investigation for PTEN mutations. The International Cowden Consortium Criteria were applied for the diagnosis of CD. FINDINGS: Four of the five patients treated for LDD were also diagnosed of CD. The genetic study found PTEN mutations in two of them. Interpretation. LDD has been found to be closely related to CD in this series, in accordance with previous literature. However, the absence of CD diagnosis in one of the patients led us to suggest that, despite the strong association between these two diseases, LDD can also appear as an isolated condition.

Characterization of the A673 cell line (Ewing tumor) by molecular cytogenetic techniques.

The A673 cell line was established from a patient with a primary rhabdomyosarcoma (RMS), which is referred to in the literature either as a Ewing tumor (ET) or as RMS. Although the two tumoral types are associated with specific and well-characterized translocations, no cytogenetic report on this cell line has been published. We characterized the A673 cell line using a combination of spectral karyotyping (SKY), fluorescence in situ hybridization (FISH), and reverse transcriptase polymerase chain reaction (RT-PCR), which revealed the presence of a complex karyotype and a translocation involving chromosomes 11 and 22 and the fusion of EWS and FLI1 genes, both events being specific to ET. Neither cytogenetics nor molecular alterations specific to RMS were found.

Complex cytogenetic abnormalities including telomeric associations and MEN1 mutation in a pediatric ependymoma.

Ependymomas are neuroectodermal tumors of the brain and spinal cord. Some recurrent cytogenetic aberrations have been reported in these tumors, including alterations involving chromosomes 22, 6, and 11. However, consistent molecular alterations have not been identified in ependymal tumors. We studied a recurrent ependymoma in a 3-year-old patient by standard cytogenetic and molecular analysis of TP53 and MEN1 genes. In the present case, we found many of the cytogenetic features previously described as being recurrent in ependymomas, including unstable telomeric alterations. Furthermore, we detected a novel acquired heterozygous mutation in the MEN1 gene. The chromosomal instability produced by the telomeric alterations and the mutation in the MEN1 gene could be important events in the tumorigenesis of ependymomas.