A Novel Pathogenic BRCA1 Splicing Variant Produces Partial Intron Retention in the Mature Messenger RNA

Abstract

About 10% of all breast cancers arise from hereditary mutations that increase the risk of breast and ovarian cancers; and about 25% of these are associated with the BRCA1 or BRCA2 genes. The identification of BRCA1/BRCA2 mutations can enable physicians to better tailor the clinical management of patients; and to initiate preventive measures in healthy carriers. The pathophysiological significance of newly identified variants poses challenges for genetic counseling. We characterized a new BRCA1 variant discovered in a breast cancer patient during BRCA1/2 screening by next-generation sequencing. Bioinformatic predictions; indicating that the variant is probably pathogenetic; were verified using retro-transcription of the patient’s RNA followed by PCR amplifications performed on the resulting cDNA. The variant causes the loss of a canonic donor splice site at position +2 in BRCA1 intron 21; and consequently the partial retention of 156 bp of intron 21 in the patient’s transcript; which demonstrates that this novel BRCA1 mutation plays a pathogenetic role in breast cancer. These findings enabled us to initiate appropriate counseling and to tailor the clinical management of this family. Lastly; these data reinforce the importance of studying the effects of sequence variants at the RNA level to verify their potential role in disease onset.

A multi-gene panel beyond BRCA1/BRCA2 to identify new breast cancer-predisposing mutations by a picodroplet PCR followed by a next-generation sequencing strategy: a pilot study

“…Obviously, this issue will be amplified, and will receive much more critical evaluation, consequent to the widespread use of gene panels that include genes whose role in BC onset is not well established, as in the cases reported herein. Functional in vitro evaluations combined with molecular analysis and genotype/phenotype correlations, within the same family could shed light on the pathogenetic effects of novel variants [18,19,38,39].…”

The High-Throughput Analyses Era: Are We Ready for the Data Struggle?

“…These techniques have impacted every field of molecular research, escalating previously used sequencing technologies [ 11 ], and opening the way to the -omic sciences foundation [ 1 , 2 ]. Indeed, NGS methods allow the sequencing of entire genomes [ 12 , 13 , 14 , 15 ], of exomes [ 16 , 17 , 18 ], of panels of genes related to a disease of interest [ 19 , 20 , 21 ], or of a single gene [ 22 , 23 , 24 , 25 , 26 ], but can also be used to explore the entire transcriptome [ 27 , 28 , 29 ], small RNAs [ 30 , 31 , 32 ], the epigenome [ 33 , 34 ], and the microbiome [ 35 , 36 , 37 , 38 ].…”

“…In this view, it is not surprising that NGS is also becoming a reference method for molecular diagnostics [ 10 ]. In particular, NGS allows not only the analysis, in more patients simultaneously, of disease-related genes in less time and at lower costs than traditional approaches, but also the sequencing of panels of genes up to the complete exome [ 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 ]. In this way, it is possible to increase the diagnostic sensitivity, to discover novel disease-related genes and also obtain data regarding other genes that were potentially acting as disease-phenotype modifiers [ 19 , 40 , 41 , 42 ].…”

A Functional Analysis of the Unclassified Pro2767Ser BRCA2 Variant Reveals Its Potential Pathogenicity that Acts by Hampering DNA Binding and Homology-Mediated DNA Repair

“…The identification of novel variants and VUSs, particularly at an early age, in relatives of patients and during the screening of non-affected subjects, is a relevant clinical challenge. Indeed, the correct interpretation of the significance of the BRCA1 and BRCA2 variants is crucial for genetic counseling and for the clinical management of HBOC patients and their relatives [6,7,8]. Therefore, much effort is being made to determine the role of BRCA1 and BRCA2 VUSs and of novel variants [9,10,11,12].…”