Splicing analysis for exonic and intronic mismatch repair gene variants associated with Lynch syndrome confirms high concordance between minigene assays and patient RNA analyses

Abstract

A subset of DNA variants causes genetic disease through aberrant splicing. Experimental splicing assays, either RT-PCR analyses of patient RNA or functional splicing reporter minigene assays, are required to evaluate the molecular nature of the splice defect. Here, we present minigene assays performed for 17 variants in the consensus splice site regions, 14 exonic variants outside these regions, and two deep intronic variants, all in the DNA mismatch-repair (MMR) genes MLH1, MSH2, MSH6, and PMS2, associated with Lynch syndrome. We also included two deep intronic variants in APC and PKD2. For one variant (MLH1 c.122A>G), our minigene assay and patient RNA analysis could not confirm the previously reported aberrant splicing. The aim of our study was to further investigate the concordance between minigene splicing assays and patient RNA analyses. For 30 variants results from patient RNA analyses were available, either performed by our laboratory or presented in literature. Some variants were deliberately included in this study because they resulted in multiple aberrant transcripts in patient RNA analysis, or caused a splice effect other than the prevalent exon skip. While both methods were completely concordant in the assessment of splice effects, four variants exhibited major differences in aberrant splice patterns. Based on the present and earlier studies, together showing an almost 100% concordance of minigene assays with patient RNA analyses, we discuss the weight given to minigene splicing assays in the current criteria proposed by InSiGHT for clinical classification of MMR variants.

Biochemical and structural characterization of two variants of uncertain significance in the PMS2 gene

“…For nonsynonymous variants designated as a VUS, activity assays using either extracts from human cells or a cell‐free system have been used to evaluate the functional consequences of the variant on MMR (Corrette‐Bennett & Lahue, ; Drost et al., ; Drost et al., ; Geng et al., ; Heinen & Juel Rasmussen, ; Hinrichsen et al., ; Wang & Hays, ). For variants in other areas of the gene such as splice sites, in silico predictions and splicing assays have been used to help understand the role of the variant (van der Klift et al., ).…”

Functional classification of DNA variants by hybrid minigenes: Identification of 30 spliceogenic variants of BRCA2 exons 17 and 18

“…Thus, DNA variants: c.7806-9T>G (exon 17 skipping+ex17-del69+ivs16-ins8) [35], c.7975A>G (partial exon 17 skipping) [36], c.7976G>A and c.7976G>C (total exon 17 skipping) [37,38], c.7988A>T (partial exon 18 skipping) [38], c.7992A>T (partial exon 18 skipping) [39], c.8023A>G (ex18-del309) [21] and c.8168A>G (partial ex18-del163) [40] displayed the same splicing patterns in patient RNA and minigene MGBR2_ex14-20, lending further support to the reproducibility of minigene results. Furthermore, a recent splicing study of 30 variants of MLH1 , MSH2 , MSH6 , and PMS2 genes (Lynch syndrome) showed that outcomes of patient RNA and minigene assays were almost identical [41]. Thus, we can conclude that the minigene strategy is sensitive and specific, so its use is suitable for the initial characterization of the splicing anomalies [21].…”

The changing landscape of Lynch syndrome due to PMS2 mutations

“…Other aspects of MMR function that can be hindered as a result of a VUS include protein localization and mRNA splicing that are essential for maintaining genomic integrity. 91,92 Both PMS2 and MLH1 possess nuclear localization signals that are required for import into this organelle where nuclear import appears to be dependent upon heterodimerization. 93 A VUS that disrupts heterodimerization will also likely interrupt nuclear localization thereby causing an MMR defect.…”

“…For the identification of pathogenic variants that impact mRNA splicing, minigene reporter assays can be performed where the splicing patterns of transcripts generated after transient transfection of PCR amplified genomic DNA from the WT and variant constructs are compared. 91,94 Sixteen PMS2 variants were previously tested using this assay, where several of the variants displayed aberrant splicing patterns. 91 Taken together, these assays are essential to the understanding of the regulation of MMR proteins and are required for the proper designation of a particular VUS as pathogenic or benign.…”

“…91,94 Sixteen PMS2 variants were previously tested using this assay, where several of the variants displayed aberrant splicing patterns. 91 Taken together, these assays are essential to the understanding of the regulation of MMR proteins and are required for the proper designation of a particular VUS as pathogenic or benign.…”