BRCA1 and BRCA2 Testing through Next Generation Sequencing in a Small Cohort of Italian Breast/Ovarian Cancer Patients: Novel Pathogenic and Unknown Clinical Significance Variants.

The aim of this report is to describe results of BRCA1 and BRCA2 Next Generation Sequencing Analysis (NGS) analysis in 132 selected Italian patients with breast/ovarian cancer. A NGS pipeline with a reliable Copy Number Variation (CNV) prediction algorithm was applied. In addition, VarSome and Priors V2.0 Software were employed for in silico analysis of novel missense variants. A total of 37 BRCA1 and BRCA2 pathogenic variants were found in 34 unrelated subjects with a frequency of positive patients of 25.7% (34/132). Twenty-four deleterious variants were detected in BRCA1 (representing the 64.9% of all identified pathogenic defects) and thirteen (35.1% of all identified pathogenic variants) in BRCA2 gene. The percentage of patients carrying a variant of unknown significance (VUS) was 7.5% (10/132). In addition, seven novel variants (five in BRCA2 and two in BRCA1 gene), never previously reported, were identified. Our approach represents a robust and easy-to-use method for full BRCA1/2 screening. However, a consistent number of our high-risk families still remained without a satisfying answer. Necessarily, further collective efforts must be directed to a definitive classification of VUSs. The future auspice is that the use of multi-gene panel and more advanced screenings, such as whole exome sequencing and/or RNA seq, in routine diagnostics increases the detection rate.

Novel BRCA1 Large Genomic Rearrangements in Italian Breast/Ovarian Cancer Patients.

BACKGROUND: In recent years, the number of patients being offered BRCA1/2 testing has changed dramatically. Advances in high-throughput sequencing technology have led many diagnostic laboratories to test next-generation sequencing (NGS)-based platforms as the main technology for clinical testing. As a consequence, the proportion of novel BRCA1/2 variants detected has greatly increased. Here, we describe two novel BRCA1 large deletions detected in Italian patients affected by hereditary breast and ovarian cancer syndrome (HBOC). METHODS: We applied an NGS pipeline with a reliable copy number variation (CNV) prediction algorithm. Successively, samples were investigated using the Multiplex Amplicon Quantification (MAQ) assay and array comparative genomic hybridization (CGH). In a single case, long-range polymerase chain reaction (PCR) was employed for careful detection of the breakpoint region, while the RepeatMasker program was used to identify Alu sequences at the junction point. RESULTS: A 137.8 kb deletion, involving the first six exons of BRCA1 and the full NBR2, BRCA1P1, NBR1, and TMEM106a genes, was detected in an Italian woman diagnosed with high-grade serous ovarian carcinoma. A second rearrangement, involving the deletion of BRCA1 11-14 exons, was detected in a breast cancer patient and was fully characterized and reported according to recommended Human Genome Variation Society (HGVS) nomenclature: NG_005905.2: g.125038_143266del; NM_007294.3: c.2817_4716del; NP_009225: p.Lys862Metfs? CONCLUSION: Although it was not possible to perform a familial segregation analysis and more direct evidence of the relationship between genotype and phenotype is necessary, both of the novel reported rearrangements cause the loss of crucial functional domains of the BRCA1 protein and this event supports their pathogenicity.

The euchromatic 9p+ polymorphism is a locus-specific amplification caused by repeated copies of a small DNA segment mapping within 9p12.

A large duplication involving the proximal euchromatic region of chromosome 9p was detected by conventional cytogenetics in a healthy 33-year-old woman and in two unrelated foetuses; both of them received the rearrangement from their healthy father. The duplicated segment was R(RBG) and C(CBG)-negative and G(GTG)-positive and was also positive for a 9-specific painting probe. It was preliminarily interpreted as a pathological quantitative change of the genome in the foetuses. FISH analyses allowed us to characterise the chromosome boundaries of this polymorphism, being identified by the RP11-15E1 BAC clone, proximally, and by the RP11-402N8 clone, distally, both probes falling within the 9p12 region. The contiguous, distally, RP11-916H19 probe was not included in the amplification, and may represent the discriminating genetic locus between chromosome polymorphism and chromosome mutation. The 9p12 amplification was approximately 12, 7 and 8 Mb in the three different families and was stable through generations. Our observations confirm the already provided evidence that proximal 9p duplications represent a benign euchromatic polymorphism. However, we demonstrated that these variants are not a simple duplication of the region 9p11.2-p13.1, as already suggested, but that they result from a many-fold amplification of a segment mapping within 9p12. These results provide important insights both in the genetic counselling and in the prenatal diagnosis of rare euchromatic chromosome variants and in understanding the architecture of the human genome.

Screening of mutations in the CFTR gene in 1195 couples entering assisted reproduction technique programs.

Genetic testing of the cystic fibrosis transmembrane conductance (CFTR) gene is currently performed in couples undergoing assisted reproduction techniques (ART), because of the high prevalence of healthy carriers in the population and the pathogenic relationship with congenital bilateral absence of vas deferens (CBAVD). However, discordant data have been reported concerning the usefulness of this genetic test in couples with no family history of cystic fibrosis (CF). In this study, we report the results of CFTR molecular screening in 1195 couples entering ART. Genetic testing was initially carried out in a single partner of each couple. CFTR mutations were detected in 55 subjects (4.6%), a percentage that overlaps with the one reported in the general population. However, significantly higher frequencies of were found in CBAVD individuals (37.5%) and in males with nonobstructive azoospermia (6.6%). The 5T allele was found in 78 patients (6.5%). This figure was again significantly different in males with nonobstructive-azoospermia (9.9%) and in those with CBAVD (100%). All together, 139 subjects (11.6%) had either a CFTR mutation or the 5T allele. Subsequent molecular analysis of their partners disclosed a CFTR mutation or 5T allele in nine cases (6.5%). However, none of these couples had CFTR alterations in both members, a CFTR mutation being invariably present in one partner and the 5T allele in the other. In order to improve genetic counselling of these couples, the TG-M470V-5T association was analyzed, and a statistically significant relationship between 12TG-V470 and CBAVD was detected.