148: CHEK2 splice-site variants: minigene dissection

Sanoguera-Miralles L et al., Clinical Chemistry - This episode examines a minigene-based functional study of 52 CHEK2 splice-site variants from the BRIDGES project, reporting widespread splice disruption, characterization of 89 transcripts, and an ACMG/AMP-informed tentative clinical classification. Key terms: CHEK2, splicing, minigene assay, variant classification, breast cancer. Study Highlights:The authors selected 52 candidate splice-site variants from 128 intron–exon boundary changes and tested them in three validated CHEK2 minigenes in MCF-7 cells. Forty-six variants (88.5%) impaired splicing and 34 produced negligible full-length transcript, generating a total of 89 different transcripts of which 59 are predicted to introduce premature termination codons. They identified complex outcomes including multi-exon skipping, cryptic site usage and a rare noncanonical TG acceptor activated by c.684-2A>G, and incorporated minigene read-outs into an ACMG/AMP-based point system to tentatively classify variants. The workflow allowed classification of 27 pathogenic/likely pathogenic and 5 likely benign variants, while 20 remained variants of uncertain significance. Conclusion:Minigene assays revealed high spliceogenicity among selected CHEK2 variants and, when combined with a modified ACMG/AMP framework, provided tentative clinical classifications but left a substantial fraction (38%) as VUSs, highlighting the need for complementary case-control, family, or patient RNA data. Music:Enjoy the music based on this article at the end of the episode. Article title:Systematic Minigene-Based Splicing Analysis and Tentative Clinical Classification of 52 CHEK2 Splice-Site Variants First author:Sanoguera-Miralles L Journal:Clinical Chemistry DOI:10.1093/clinchem/hvad125 Reference:Sanoguera-Miralles L, Valenzuela-Palomo A, Bueno-Martínez E, Esteban-Sánchez A, Lorca V, Llinares-Burguet I, García-Álvarez A, Pérez-Segura P, Infante M, Easton DF, Devilee P, Vreeswijk MPG, de la Hoya M, Velasco-Sampedro EA. Systematic Minigene-Based Splicing Analysis and Tentative Clinical Classification of 52 CHEK2 Splice-Site Variants. Clinical Chemistry. 2024;70(1):319–338. doi:10.1093/clinchem/hvad125 License:This episode is based on an open-access article published under the Creative Commons Attribution 4.0 International License (CC BY 4.0) – https://creativecommons.org/licenses/by/4.0/ Support:Base by Base – Stripe donations: https://donate.stripe.com/7sY4gz71B2sN3RWac5gEg00 Official website https://basebybase.com On PaperCast Base by Base you'll discover the latest in genomics, functional genomics, structural genomics, and proteomics. Episode link: https://basebybase.com/episodes/systematic-minigene-based-splicing-analysis-and-tentative-clinical-classification-of-52-chek2-splice-site-variants QC:This episode was checked against the original article PDF and publication metadata for the episode release published on 2025-09-25. QC Scope:- article metadata and core scientific claims from the narration- excludes analogies, intro/outro, and music- transcript coverage: Audited the spoken content for accuracy of core methods, results, and clinical implications of the CHEK2 minigene splicing analysis, including variant selection, three-minigene design, splicing outcomes, TG acceptor discovery, ACMG/AMP classification, and clinical impact; noted minor terminology variations.- transcript topics: CHEK2 splicing and cancer risk; Minigene methodology and experimental design; Bioinformatic filtering and variant prioritization; Splicing outcomes and transcript diversity (89 transcripts, 59 with PTCs); ACMG/AMP-based tentative classification (32 variants; 27 P/LP, 5 LB; 20 VUS); Notable c.684-2A>G TG acceptor activation and exon 6 enhancers QC Summary: Chapters (00:00:14) - First look at the genetic uncertainty in cancer screening(00:02:23) - Breast Cancer genetic screening, the QIQUE2 gene(00:04:19) - Breast cancer: the minigene approach(00:06:06) - Flip-flopping in splicing(00:08:46) - The pathogenicity of C6842AG