| Abstrakt: |
Simple Summary: ATM and PALB2 are two of the main breast cancer susceptibility genes. In this research, we studied a crucial biological process called splicing that is responsible for removing introns and consecutively assembling exons, which contain the protein-coding sequence. We focused on mapping the splicing regulatory elements involved in the recognition of ATM exon 50, BRIP1 exon 1, and PALB2 exon 12, which present atypical GC-5′ splice sites. For this purpose, we used the biotechnological tool of hybrid minigenes, which mimic the splicing process of the gene of interest. Thus, we discovered one regulatory interval in ATM exon 50 and two in PALB2 exon 12 that constitute hotspots where splicing-disrupting variants can be placed. Then, 23 ATM and PALB2 candidate variants were tested. Nine ATM and three PALB2 variants impaired splicing, affecting the recognition of their corresponding exons. These variants may therefore be associated with increased breast cancer risk. Background/Objectives: The non-canonical GC-5′ splice sites (5′ss) are the most common exception (~1%) to the classical GT/AG splicing rule. They constitute weak 5′ss and can be regulated by splicing factors, so they are especially sensitive to genetic variations inducing the misrecognition of their respective exons. We aimed to investigate the GC-5′ss of the breast/ovarian cancer susceptibility genes, ATM (exon 50), BRIP1 (exon 1), and PALB2 (exon 12), and their dysregulation induced by DNA variants. Methods: Splicing assays of the minigenes, mgATM_49-52, mgBRIP1_1-2, and mgPALB2_5-12, were conducted to study the regulation of the indicated GC-5′ss. Results: A functional map of the splicing regulatory elements (SRE) formed by overlapping exonic microdeletions revealed three essential intervals, ATM c.7335_7344del, PALB2 c.3229_3258del, and c.3293_3322del, which are likely targets for spliceogenic SRE-variants. We then selected 14 ATM and 9 PALB2 variants (Hexplorer score < −40) located at these intervals that were assayed in MCF-7 cells. Nine ATM and three PALB2 variants affected splicing, impairing the recognition of exons 50 and 12, respectively. Therefore, these variants likely disrupt the active SREs involved in the inclusion of both exons in the mature mRNA. DeepCLIP predictions suggested the participation of several splicing factors in exon recognition, including SRSF1, SRSF2, and SRSF7, involved in the recognition of other GC sites. The ATM spliceogenic variants c.7336G>T (p.(Glu2446Ter)) and c.7340T>A (p.(Leu2447Ter)) produced significant amounts of full-length transcripts (55–59%), which include premature termination stop codons, so they would inactivate ATM through both splicing disruption and protein truncation mechanisms. Conclusions: ATM exon 50 and PALB2 exon 12 require specific sequences for efficient recognition by the splicing machinery. The mapping of SRE-rich intervals in minigenes is a valuable approach for the identification of spliceogenic variants that outperforms any prediction software. Indeed, 12 spliceogenic SRE-variants were identified in the critical intervals. [ABSTRACT FROM AUTHOR] |
