LAUSR

Simple Summary PALB2 pathogenic variants confer high risk of breast cancer. Here, we have analyzed the impact of PALB2 variants on splicing, a gene expression step that removes introns to form the mature messenger RNA. This process is performed by the splicing machinery through the recognition of specific sequences, namely the 3′ and 5′ splice sites, which determine the exon ends. Variants at these sequences may trigger anomalous splicing and aberrant transcripts that may be associated with a disease. To test the impact of variants on splicing, we used a biotechnological tool called minigene, which replicates, at small-scale, the human gene of interest. Thus, we checked 16 PALB2 variants at the intron/exon boundaries using the minigene mgPALB2_ex1-3. We found that twelve variants disrupted splicing, six of which could be classified as likely pathogenic. These results facilitate the clinical management of carrier patients and families since they may benefit from tailored prevention protocols and therapies. Abstract PALB2 loss-of-function variants are associated with significant increased risk of breast cancer as well as other types of tumors. Likewise, splicing disruptions are a common mechanism of disease susceptibility. Indeed, we previously showed, by minigene assays, that 35 out of 42 PALB2 variants impaired splicing. Taking advantage of one of these constructs (mgPALB2_ex1-3), we proceeded to analyze other variants at exons 1 to 3 reported at the ClinVar database. Thirty-one variants were bioinformatically analyzed with MaxEntScan and SpliceAI. Then, 16 variants were selected for subsequent RNA assays. We identified a total of 12 spliceogenic variants, 11 of which did not produce any trace of the expected minigene full-length transcript. Interestingly, variant c.49-1G > A mimicked previous outcomes in patient RNA (transcript ∆(E2p6)), supporting the reproducibility of the minigene approach. A total of eight variant-induced transcripts were characterized, three of which (∆(E1q17), ∆(E3p11), and ∆(E3)) were predicted to introduce a premature termination codon and to undergo nonsense-mediated decay, and five (▼(E1q9), ∆(E2p6), ∆(E2), ▼(E3q48)-a, and ▼(E3q48)-b) maintained the reading frame. According to an ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme, which integrates mgPALB2 data, six PALB2 variants were classified as pathogenic/likely pathogenic, five as VUS, and five as likely benign. Furthermore, five ±1,2 variants were catalogued as VUS because they produced significant proportions of in-frame transcripts of unknown impact on protein function.