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The identification of structural variant (SV) breakpoints plays a crucial role in understanding the genetic variants, mutagenic mechanisms, and functional consequences that drive various genetic diseases. While next-generation sequencing (NGS) has become a cornerstone in single nucleotide variant (SNP) discovery and characterization, short-read NGS technology faces significant challenges in resolving large genomic rearrangements such as duplications, deletions, inversions, and translocations. Nanopore sequencing offers a promising alternative by enabling precise mapping of chromosomal rearrangement breakpoints, and characterization of chromosomal alterations, thereby improving the genetic diagnosis of such conditions. Using long-read whole-genome sequencing, we examined the breakpoints of a cytogenetically balanced chromosomal translocation, t(8;22)(q13.3;q11.23), initially detected during prenatal diagnosis and later confirmed as de novo in a patient who developed NF2-associated schwannomatosis in late infancy. Nanopore sequencing revealed that the translocation disrupted the NF2 gene. This case highlights the power of nanopore long-read sequencing in detecting the exact consequences of de novo , apparently balanced translocations and in uncovering the genetic underpinnings of abnormal phenotypes. Given its ability to resolve complex SVs with high precision, nanopore sequencing might be considered a valuable complement to conventional genetic diagnostic methods, enhancing our understanding of genetic diseases and potentially improving diagnostic yield and risk assessment.

Background Emerging evidence suggests that breast microbiota dysbiosis contributes to cancer initiation, progression, prognosis and treatment efficacy. Anyway, available data are referred only to female patients, and studies on males are completely missing. Male breast cancer (MBC) is 70–100 times less frequent, but the mortality rate adjusted to incidence is higher in men than in females. Currently, MBC diagnostic approaches and treatments have generally been extrapolated from the clinical experience gained in women, while few studies focus on characterizing male cancer biology. Taking into account the rising importance of the oncobiome field and the need of MBC targeted studies, we explored the breast cancer oncobiome of male and female patients. Methods 16S rRNA gene sequencing was performed in 20 tumor and 20 non-pathological adjacent FFPE breast tissues from male and female patients. Results We documented, for the first time, the presence of a sexually dimorphic breast-associated microbiota, here defined as “breast microgenderome”. Moreover, the paired analysis of tumor and non-pathological adjacent tissues suggests the presence of a cancer-associated dysbiosis in male patients, with surrounding tissue conserving a healthier microbiome, whereas in female patients, the entire breast tissue is predisposed to cancer development. Finally, the phylum Tenericutes, especially the genera Mesoplasma and Mycobacterium , could to be involved in breast carcinogenesis, in both sexes, deserving further investigation, not only for its role in cancer development but even as potential prognostic biomarker. Conclusions Breast microbiota characterization can enhance the understanding of male breast cancer pathogenesis, being useful for detection of new prognostic biomarkers and development of innovative personalized therapies, remarking the relevant gender differences. Plain Language Summary Breast tissue can become inhabited by microbes through different pathways, and an uneven distribution of these microorganisms could potentially contribute to the development, prognosis, and treatment response of breast cancer. However, the current available data primarily focus on female patients, with a significant dearth of studies on males. To address this gap, the present study investigates the microbiota composition of both tumorous and healthy breast tissue samples from both male and female patients. The findings of this research highlight a disparity in the types of bacteria present in male and female breast tissue. Specifically, it shows that male patients with breast cancer have a higher imbalance of bacteria in the cancerous area compared to the surrounding healthy tissue. In contrast, in females the dysbiosis extend to the whole breast tissue. Moreover, the study identifies specific strains of bacteria that might potentially be involved in the development of breast cancer in both males and females. In conclusion, this study underscores the significance of microbial colonization in breast tissue and its potential influence on breast cancer in both males and females. By expanding our understanding of the microbial composition in breast cancer, we can pave the way for innovative diagnostic methods and treatment approaches for male breast cancer, while simultaneously advancing our knowledge of this complex disease. Highlights Few studies have been conducted to describe male cancer biology in comparison with female breast cancer. For the first time, we discovered a sexually dimorphic breast-associated microbiome, termed “breast microgenderome”. In females, the dysbiosis extend to the whole breast tissue, while in men, it appears to be confined to the tumor site. Tenericutes, particularly the genera Mesoplasma and Mycobacterium , may be implicated in breast carcinogenesis in both sexes.

Schwannomatosis is characterized by the development of multiple non-vestibular, non-intradermal schwannomas. Constitutional inactivating variants in two genes, SMARCB1 and, very recently, LZTR1, have been reported. We performed exome sequencing of 13 schwannomatosis patients from 11 families without SMARCB1 deleterious variants. We identified four individuals with heterozygous loss-of-function variants in LZTR1. Sequencing of the germline of 60 additional patients identified 18 additional heterozygous variants in LZTR1. We identified LZTR1 variants in 43% and 30% of familial (three of the seven families) and sporadic patients, respectively. In addition, we tested LZTR1 protein immunostaining in 22 tumors from nine unrelated patients with and without LZTR1 deleterious variants. Tumors from individuals with LZTR1 variants lost the protein expression in at least a subset of tumor cells, consistent with a tumor suppressor mechanism. In conclusion, our study demonstrates that molecular analysis of LZTR1 may contribute to the molecular characterization of schwannomatosis patients, in addition to NF2 mutational analysis and the detection of chromosome 22 losses in tumor tissue. It will be especially useful in differentiating schwannomatosis from mosaic Neurofibromatosis type 2 (NF2). However, the role of LZTR1 in the pathogenesis of schwannomatosis needs further elucidation.

The accurate detection of low-allelic variants is still challenging, particularly for the identification of somatic mosaicism, where matched control sample is not available. High throughput sequencing, by the simultaneous and independent analysis of thousands of different DNA fragments, might overcome many of the limits of traditional methods, greatly increasing the sensitivity. However, it is necessary to take into account the high number of false positives that may arise due to the lack of matched control samples. Here, we applied deep amplicon sequencing to the analysis of samples with known genotype and variant allele fraction (VAF) followed by a tailored statistical analysis. This method allowed to define a minimum value of VAF for detecting mosaic variants with high accuracy. Then, we exploited the estimated VAF to select candidate alterations in NF2 gene in 34 samples with unknown genotype (30 blood and 4 tumor DNAs), demonstrating the suitability of our method. The strategy we propose optimizes the use of deep amplicon sequencing for the identification of low abundance variants. Moreover, our method can be applied to different high throughput sequencing approaches to estimate the background noise and define the accuracy of the experimental design.

Rhabdoid tumors are aggressive malignancies that show loss-of-function mutations of SMARCB1 gene, a member of the SWI/SNF chromatin-remodeling complex controlling gene transcription. One-third of patients affected by rhabdoid tumor harbor a germ-line mutation of SMARCB1 defining a rhabdoid tumor predisposition syndrome. The occurrence of a second somatic mutation determines the development of neoplasia in a two-hit model. Most germ-line mutations occur de novo, and few cases of recurrence in a sibship have been described. Here we report on a new Italian family with recurrence of SMARCB1 germ-line deletion in two siblings due to gonadal mosaicism. The deletion was identified in the 9-month-old proband with malignant rhabdoid tumor of the right kidney and disseminated metastases. Testing of both parents confirmed the de novo origin of the mutation, but recurrence was then detected prenatally in a new pregnancy. This is the sixth family with malignant rhabdoid tumor predisposition syndrome with the recurrence of the same germ-line SMARCB1 mutation in the sibship but not in healthy parents, suggesting that gonadal mosaicism is a less rare event than supposed. The clinical outcome in our patient confirms previous data of poorer outcome in patients with rhabdoid tumor predisposition syndrome.

The identification of founder mutations in cancer predisposing genes is important to improve risk assessment in geographically defined populations, since it may provide specific targets resulting in cost-effective genetic testing. Here, we report the characterization of the BRCA1 c.190T>C (p.Cys64Arg) mutation, mapped to the RING-finger domain coding region, that we detected in 43 hereditary breast/ovarian cancer (HBOC) families, for the large part originating from the province of Bergamo (Northern Italy). Haplotype analysis was performed in 21 families, and led to the identification of a shared haplotype extending over three BRCA1-associated marker loci (0.4 cM). Using the DMLE+2.2 software program and regional population demographic data, we were able to estimate the age of the mutation to vary between 3,100 and 3,350 years old. Functional characterization of the mutation was carried out at both transcript and protein level. Reverse transcriptase-PCR analysis on lymphoblastoid cells revealed expression of full length mRNA from the mutant allele. A green fluorescent protein (GFP)-fragment reassembly assay showed that the p.Cys64Arg substitution prevents the binding of the BRCA1 protein to the interacting protein BARD1, in a similar way as proven deleterious mutations in the RING-domain. Overall, 55 of 83 (66%) female mutation carriers had a diagnosis of breast and/or ovarian cancer. Our observations indicate that the BRCA1 c.190T>C is a pathogenic founder mutation present in the Italian population. Further analyses will evaluate whether screening for this mutation can be suggested as an effective strategy for the rapid identification of at-risk individuals in the Bergamo area.

By incorporating major developments in genetics, ophthalmology, dermatology, and neuroimaging, to revise the diagnostic criteria for neurofibromatosis type 1 (NF1) and to establish diagnostic criteria for Legius syndrome (LGSS). We used a multistep process, beginning with a Delphi method involving global experts and subsequently involving non-NF experts, patients, and foundations/patient advocacy groups. We reached consensus on the minimal clinical and genetic criteria for diagnosing and differentiating NF1 and LGSS, which have phenotypic overlap in young patients with pigmentary findings. Criteria for the mosaic forms of these conditions are also recommended. The revised criteria for NF1 incorporate new clinical features and genetic testing, whereas the criteria for LGSS were created to differentiate the two conditions. It is likely that continued refinement of these new criteria will be necessary as investigators (1) study the diagnostic properties of the revised criteria, (2) reconsider criteria not included in this process, and (3) identify new clinical and other features of these conditions. For this reason, we propose an initiative to update periodically the diagnostic criteria for NF1 and LGSS. [Display omitted]

MULTIPLE endocrine neoplasia type 2A (MEN 2A) is a dominantly inherited cancer syndrome that affects tissues derived from neural ectoderm. It is characterized by medullary thyroid carcinoma (MTC) and phaeochromocytoma l . The MEN2A gene has recently been localized by a combination of genetic and physical mapping techniques to a 480-kilobase region in chromosome 10qll.2 (refs 2,3). The DNA segment encompasses the RET proto-oncogene, a receptor tyrosine kinase gene expressed in MTC and phaeochromocytoma and at lower levels in normal human thyroid 4 . This suggested RET as a candidate for the MEN2A gene. We have identified missense mutations of the RET proto-oncogene in 20 of 23 apparently distinct MEN 2A families, but not in 23 normal controls. Further, 19 of these 20 mutations affect the same conserved cysteine residue at the boundary of the RET extracellular and transmembrane domains.

The extensive use of next‐generation sequencing (NGS) multi‐gene panels and advanced analysis algorithms have led to the identification of numerous genetic variants associated with breast, ovarian, and pancreatic cancer. Copynumber variations (CNVs), defined as deletions and duplications of specific DNA regions, account for up to 10% of pathogenic variants and can affect any of the cancer‐predisposing genes. Despite this, CNVs’ contribution beyond BRCA1 and BRCA2 remains underexplored. This observational study analyzed data from 2949 patients, primarily affected by breast or ovarian cancer, who underwent NGS testing with a 22‐gene hereditary cancer panel between 2018 and 2023, with a focus on CNV results. In line with comparison studies, a total diagnostic yield of 14.8% was observed with pathogenic variants in BRCA1, BRCA2, CHEK2, ATM, and PALB2 accounting for most of positive findings. In contrast, CNVs were found in 1.4% of patients, displaying a peculiar distribution pattern. PALB2 exhibited the highest frequency of pathogenic CNVs (66.7%), representing 62.2% of all PALB2 pathogenic variants. Notably, 24 out of 28 PALB2 CNV carriers shared the deletion of Exon 11. Further investigations revealed identical breakpoints and common geographical origins, and moreover, the same haplotype for some of the families suggests a relatively recent founder effect. Simultaneous sequence and copy number analyses resulted in likely higher positive predictive value of the test and, more interestingly, disclosed an unforeseen single contribution of CNVs in PALB2 gene, confirming geography as a key factor in shaping human genetic variations.

Background/Objective: Large genomic rearrangements of PALB2 gene, particularly deletions and duplications, have been linked to hereditary breast–ovarian cancer. Our research specifically focuses on delineating the intronic breakpoints associated with rearrangements of PALB2 exon 11, which is crucial for understanding the mechanisms underlying these genomic changes in patients with hereditary breast and ovarian syndrome. Methods: By using next-generation sequencing, we identified one duplication and three deletions of PALB2 exon 11, confirmed by Multiplex Ligation-Dependent Probe Amplification analysis. To assess the impact on transcription and potential splicing issues, reverse-transcription PCR was performed on patients’ RNA. For the detailed characterization of intronic breakpoints, the primer walking approach and long-range PCR were implemented, followed by Sanger sequencing. Results: Our analysis revealed a tandem duplication of 5134 base pairs (bp) mediated by AluY repeats located in introns 10 and 11, respectively. Moreover, identical deletions were identified in three unrelated patients, encompassing an approximate 8050 bp region mediated by AluSx elements. Both genomic alterations resulted in a truncated PALB2 protein due to the introduction of a premature stop codon. Conclusions: This study underscores the remarkable instability of intronic regions flanking exon 11 of PALB2 and identifies a previously unreported hotspot involving Alu repeats with very high sequence homology in introns 10 and 11 of the gene. Our findings suggest avenues for further research, such as investigating the prevalence of similar genomic rearrangements in larger cohorts and exploring functional studies to understand how these alterations contribute to hereditary breast cancer pathogenesis.