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BackgroundBRCA1 and BRCA2 are the two principal tumour suppressor genes associated with inherited high risk of breast and ovarian cancer. Genetic testing of BRCA1/2 will often reveal one or more sequence variants of uncertain clinical significance, some of which may affect normal splicing patterns and thereby disrupt gene function. mRNA analyses are therefore among the tests used to interpret the clinical significance of some genetic variants. However, these could be confounded by the appearance of naturally occurring alternative transcripts unrelated to germline sequence variation or defects in gene function. To understand which novel splicing events are associated with splicing mutations and which are part of the normal BRCA2 splicing repertoire, a study was undertaken by members of the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium to characterise the spectrum of naturally occurring BRCA2 mRNA alternate-splicing events.MethodsmRNA was prepared from several blood and breast tissue-derived cells and cell lines by contributing ENIGMA laboratories. cDNA representing BRCA2 alternate splice sites was amplified and visualised using capillary or agarose gel electrophoresis, followed by sequencing.ResultsWe demonstrate the existence of 24 different BRCA2 mRNA alternate-splicing events in lymphoblastoid cell lines and both breast cancer and non-cancerous breast cell lines.ConclusionsThese naturally occurring alternate-splicing events contribute to the array of cDNA fragments that may be seen in assays for mutation-associated splicing defects. Caution must be observed in assigning alternate-splicing events to potential splicing mutations.

Several unclassified variants (UVs) have been identified in splicing regions of disease-associated genes and their characterization as pathogenic mutations or benign polymorphisms is crucial for the understanding of their role in disease development. In this study, 24 UVs located at BRCA1 and BRCA2 splice sites were characterized by transcripts analysis. These results were used to evaluate the ability of nine bioinformatics programs in predicting genetic variants causing aberrant splicing (spliceogenic variants) and the nature of aberrant transcripts. Eleven variants in BRCA1 and 8 in BRCA2, including 8 not previously characterized at transcript level, were ascertained to affect mRNA splicing. Of these, 16 led to the synthesis of aberrant transcripts containing premature termination codons (PTCs), 2 to the up-regulation of naturally occurring alternative transcripts containing PTCs, and one to an in-frame deletion within the region coding for the DNA binding domain of BRCA2, causing the loss of the ability to bind the partner protein DSS1 and ssDNA. For each computational program, we evaluated the rate of non-informative analyses, i.e. those that did not recognize the natural splice sites in the wild-type sequence, and the rate of false positive predictions, i.e., variants incorrectly classified as spliceogenic, as a measure of their specificity, under conditions setting sensitivity of predictions to 100%. The programs that performed better were Human Splicing Finder and Automated Splice Site Analyses, both exhibiting 100% informativeness and specificity. For 10 mutations the activation of cryptic splice sites was observed, but we were unable to derive simple criteria to select, among the different cryptic sites predicted by the bioinformatics analyses, those actually used. Consistent with previous reports, our study provides evidences that in silico tools can be used for selecting splice site variants for in vitro analyses. However, the latter remain mandatory for the characterization of the nature of aberrant transcripts.

Li-Fraumeni syndrome (LFS) is an autosomal dominant disorder caused by mutations in the TP53 gene, predisposing to a wide spectrum of early-onset cancers, including brain tumors. In medulloblastoma patients, the role of TP53 has been extensively investigated, though the prevalence of de novo mutations has not been addressed. We characterized TP53 mutations in a monocentric cohort of consecutive Sonic Hedgehog (SHH)-activated medulloblastoma patients. Germline testing was offered based on tumor p53 immunostaining positivity. Among 24 patients, three (12.5%) showed tumor p53 overexpression, of whom two consented to undergo germline testing and resulted as carriers of TP53 mutations. In the first case, family history was uneventful and the mutation was not found in either of the parents. The second patient, with a family history suggestive of LFS, unexpectedly resulted as a carrier of the mosaic mutation c.742=/C>T p.(Arg248=/Trp). The allele frequency was 26% in normal tissues and 42–77% in tumor specimens. Loss of heterozygosity (LOH) in the tumor was also confirmed. Notably, the mosaic case has been in complete remission for more than one year, while the first patient, as most TP53-mutated medulloblastoma cases from other cohorts, showed a severe and rapidly progressive disease. Our study reported the first TP53 mosaic mutation in medulloblastoma patients and confirmed the importance of germline testing in p53 overexpressed SHH-medulloblastoma, regardless of family history.

Purpose Monoallelic germ-line mutations in the BRCA1 / FANCS , BRCA2 / FANCD1 and PALB2 / FANCN genes confer high risk of breast cancer. Biallelic mutations in these genes cause Fanconi anemia (FA), characterized by malformations, bone marrow failure, chromosome fragility, and cancer predisposition ( BRCA2 / FANCD1 and PALB2 / FANCN ), or an FA-like disease presenting a phenotype similar to FA but without bone marrow failure ( BRCA1 / FANCS ). FANCM monoallelic mutations have been reported as moderate risk factors for breast cancer, but there are no reports of any clinical phenotype observed in carriers of biallelic mutations. Methods Breast cancer probands were subjected to mutation analysis by sequencing gene panels or testing DNA damage response genes. Results Five cases homozygous for FANCM loss-of-function mutations were identified. They show a heterogeneous phenotype including cancer predisposition, toxicity to chemotherapy, early menopause, and possibly chromosome fragility. Phenotype severity might correlate with mutation position in the gene. Conclusion Our data indicate that biallelic FANCM mutations do not cause classical FA, providing proof that FANCM is not a canonical FA gene. Moreover, our observations support previous findings suggesting that FANCM is a breast cancer-predisposing gene. Mutation testing of FANCM might be considered for individuals with the above-described clinical features.

Primary ovarian neuroendocrine neoplasms (Ov-NENs) are infrequent and mainly represented by well-differentiated forms (neuroendocrine tumors — NETs — or carcinoids). Poorly differentiated neuroendocrine carcinomas (Ov-NECs) are exceedingly rare and only few cases have been reported in the literature. A subset of Ov-NECs are admixed with non-neuroendocrine carcinomas, as it occurs in other female genital organs, as well (mostly endometrium and uterine cervix), and may be assimilated to mixed neuroendocrine/non-neuroendocrine neoplasms (MiNENs) described in digestive and extra-digestive sites. Here, we present a case of large cell Ov-NEC admixed with an endometrioid carcinoma of the ovary, arising in the context of ovarian endometriosis, associated with a uterine endometrial atypical hyperplasia (EAH). We performed targeted next-generation sequencing analysis, along with a comprehensive immunohistochemical study and FISH analysis for TP53 locus, separately on the four morphologically distinct lesions (Ov-NEC, endometrioid carcinoma, endometriosis, and EAH). The results of our study identified molecular alterations of cancer-related genes ( PIK3CA , CTNNB1 , TP53 , RB1 , ARID1A , and p16 ), which were present with an increasing gradient from preneoplastic lesions to malignant proliferations, both neuroendocrine and non-neuroendocrine components. In conclusion, our findings underscored that the two neoplastic components of this Ov-MiNEN share a substantially identical molecular profile and they progress from a preexisting ovarian endometriotic lesion, in a patient with a coexisting preneoplastic proliferation of the endometrium, genotypically and phenotypically related to the ovarian neoplasm. Moreover, this study supports the inclusion of MiNEN in the spectrum ovarian and, possibly, of all gynecological NENs, among which they are currently not classified.

Background: Germline disease-causing mutations in BRCA1 and BRCA2 genes confer high risk of breast and ovarian cancer, but account approximately for only 15% of familial cases. Theoretical models and experimental observations have indicated that the remaining familial aggregations would be explained by low-penetrance alleles. Moreover, alleles acting as genetic modifiers would modulate the breast cancer risk in carriers of BRCA mutations. The Ins16bp and Arg72Pro polymorphisms of p53 were implicated in breast cancer and recently it has been shown that these polymorphisms could have an effect when combined as specific haplotypes. Here, we investigated the possible role of the Ins16bp and Arg72Pro polymorphisms and their haplotypes as low-penetrance alleles in familial breast cancer. Methods: The Ins16bp and Arg72Pro polymorphisms were genotyped in a total of 350 familial index cases affected with breast cancer and negative for mutations in BRCA genes, and 352 controls. The Ins16bp and Arg72Pro polymorphisms were studied separately, and as haplotypes and haplotypes combinations. Results: None of the performed analyses resulted statistically significant. Conclusions: These observations suggested that neither the Ins16bp or Arg72Pro polymorphisms considered separately, nor any related haplotype, were associated with breast cancer risk in BRCA-mutation negative familial cases.

Highly penetrant variants of BRCA1/2 genes are involved in hereditary predisposition to breast and ovarian cancer. The detection of pathogenic BRCA variants has a considerable clinical impact, allowing appropriate cancer-risk management. However, a major drawback is represented by the identification of variants of uncertain significance (VUS). Many VUS potentially affect mRNA splicing, making transcript analysis an essential step for the definition of their pathogenicity. Here, we characterize the impact on splicing of ten BRCA1/2 variants. Aberrant splicing patterns were demonstrated for eight variants whose alternative transcripts were fully characterized. Different events were observed, including exon skipping, intron retention, and usage of de novo and cryptic splice sites. Transcripts with premature stop codons or in-frame loss of functionally important residues were generated. Partial/complete splicing effect and quantitative contribution of different isoforms were assessed, leading to variant classification according to Evidence-based Network for the Interpretation of Mutant Alleles (ENIGMA) consortium guidelines. Two variants could be classified as pathogenic and two as likely benign, while due to a partial splicing effect, six variants remained of uncertain significance. The association with an undefined tumor risk justifies caution in recommending aggressive risk-reduction treatments, but prevents the possibility of receiving personalized therapies with potential beneficial effect. This indicates the need for applying additional approaches for the analysis of variants resistant to classification by gene transcript analyses.

Germline pathogenic variants (PVs) in the BRCA1 or BRCA2 genes cause high breast cancer risk. Recurrent or founder PVs have been described worldwide including some in the Bergamo province in Northern Italy. The aim of this study was to compare the BRCA1/2 PV spectra of the Bergamo and of the general Italian populations. We retrospectively identified at five Italian centers 1019 BRCA1/2 PVs carrier individuals affected with breast cancer and representative of the heterogeneous national population. Each individual was assigned to the Bergamo or non-Bergamo cohort based on self-reported birthplace. Our data indicate that the Bergamo BRCA1/2 PV spectrum shows less heterogeneity with fewer different variants and an average higher frequency compared to that of the rest of Italy. Consistently, four PVs explained about 60% of all carriers. The majority of the Bergamo PVs originated locally with only two PVs clearly imported. The Bergamo BRCA1/2 PV spectrum appears to be private. Hence, the Bergamo population would be ideal to study the disease risk associated with local PVs in breast cancer and other disease-causing genes. Finally, our data suggest that the Bergamo population is a genetic isolate and further analyses are warranted to prove this notion.