Systematic Minigene-Based Splicing Analysis and Tentative Clinical Classification of 52 CHEK2 Splice-Site Variants

Abstract

Background Disrupted pre-mRNA splicing is a frequent deleterious mechanism in hereditary cancer. We aimed to functionally analyze candidate spliceogenic variants of the breast cancer susceptibility gene CHEK2 by splicing reporter minigenes. Methods A total of 128 CHEK2 splice-site variants identified in the Breast Cancer After Diagnostic Gene Sequencing (BRIDGES) project (https://cordis.europa.eu/project/id/634935) were analyzed with MaxEntScan and subsetted to 52 variants predicted to impact splicing. Three CHEK2 minigenes, which span all 15 exons, were constructed and validated. The 52 selected variants were then genetically engineered into the minigenes and assayed in MCF-7 (human breast adenocarcinoma) cells. Results Of 52 variants, 46 (88.5%) impaired splicing. Some of them led to complex splicing patterns with up to 11 different transcripts. Thirty-four variants induced splicing anomalies without any trace or negligible amounts of the full-length transcript. A total of 89 different transcripts were annotated, which derived from different events: single- or multi-exon skipping, alternative site-usage, mutually exclusive exon inclusion, intron retention or combinations of the abovementioned events. Fifty-nine transcripts were predicted to introduce premature termination codons, 7 kept the original open-reading frame, 5 removed the translation start codon, 6 affected the 5′UTR (Untranslated Region), and 2 included missense variations. Analysis of variant c.684-2A > G revealed the activation of a non-canonical TG-acceptor site and exon 6 sequences critical for its recognition. Conclusions Incorporation of minigene read-outs into an ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme allowed us to classify 32 CHEK2 variants (27 pathogenic/likely pathogenic and 5 likely benign). However, 20 variants (38%) remained of uncertain significance, reflecting in part the complex splicing patterns of this gene.

Comprehensive splicing analysis of the alternatively spliced CHEK2 exons 8 and 10 reveals three enhancer/silencer‐rich regions and 38 spliceogenic variants

“…Moreover, the CHEK2 splicing patterns in breast tumour cells are extraordinarily complex as about 90 alternative transcripts were described [30]. In keeping with this view, we previously identified 89 different variant-induced transcripts of the CHEK2 gene [31].…”

“…Hybrid minigenes are simple versatile tools to initially assess the impact of any sequence variation on splicing, as we have previously shown in the main BC susceptibility genes [19][20][21][22][23][24]31,49]. In this regard, the maintenance of the genomic context, including intron length, is essential for exon recognition and, therefore, for minigene design [25,27,46].…”

“…In this study, we used a previously published minigene with CHEK2 exons 6-10 (mgChk2_ex6-10; insert sequence in supplementary material, Figure S1) [31]. In brief, this clone came from another larger construct (exons 6-14) that did not work properly [31], so we had to divide it into two constructs with exons 6-10 and 11-14. Both produced the expected splicing patterns, including the physiological alternative transcripts Δ(E8), Δ(E10), and Δ(E12p33).…”

“…After mgChk2_ex6-10 analysis, we classified 87 CHEK2 variants according to a Bayesian ACMG-AMP point system [39] that incorporates in-house developed CHEK2 specifications [31]. The classification system incorporates minigene read-outs as PVS1(RNA)/BP7 (RNA) codes of variable strength depending on the actual experimental outcome, as recently recommended by the ClinGen SVI Splicing Subgroup [38].…”

“…For this purpose, we employed a previously published hybrid minigene including CHEK2 exons 6-10 where formerly we tested 14 splice-site variants [31]. In this study, we have now tested 12 exonic microdeletions to map critical SRE intervals and later we interrogated 87 variants located within them.…”