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

Heleen M Van Der Klift,Anne M L Jansen,Peter Devilee,Niki Van Der Steenstraten,Elsa C Bik,Juul T Wijnen,Carli M J Tops

doi:10.1002/mgg3.145

Heleen M Van Der Klift, Anne M L Jansen + Show 5 more

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https://doi.org/10.1002/mgg3.145

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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.

Highlights

An estimated 15–60% of pathogenic mutations cause genetic disease through disruption of constitutional premRNA splicing (Wang and Cooper 2007)
On the basis of these and previous results, we suggest that minigene assay results deserve a more prominent position amongst the criteria recently presented by the International Society for Gastrointestinal Hereditary Tumours (InSiGHT) for clinical classification of MMR gene variants (Thompson et al 2014)
Patient RNA analysis is the preferred approach to the evaluation of potentially spliceogenic variants in genes that are expressed in peripheral blood lymphocytes, such as the MMR and BRCA genes, splicing reporter minigene assays represent an interesting alternative in some situations

Summary

Introduction

An estimated 15–60% of pathogenic mutations cause genetic disease through disruption of constitutional premRNA splicing (Wang and Cooper 2007). Variants in the consensus donor (50) and acceptor (30) splice site regions – defined as the last three exonic to the first six intronic bases, and the last 12 intronic to the first two exonic bases, respectively (Cartegni et al 2002) – can abolish or diminish the strength of canonical splice sites. All types of variants, including nonsense mutations and frameshifting insertions and deletions, can disturb constitutional splicing, in molecular diagnostic practice often only variants of uncertain significance (VUS) are investigated for aberrant splicing. VUS, such as intronic variants, missense and silent variants, cannot be considered pathogenic without further supportive evidence.

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