Full-length transcript amplification and sequencing as universal method to test mRNA integrity and biallelic expression in mismatch repair genes

Morak, Monika; Schaefer, K L; Steinke‐Lange, Verena; Koehler, Udo; Keinath, Susanne; Massdorf, Trisari; Mauracher, Brigitte; Rahner, Nils; Bailey, Jessica; Kling, Christiane; Haeusser, Tanja; Laner, Andreas; Holinski‐Feder, Elke

doi:10.1038/s41431-019-0472-8

Cited by 15 publications

(23 citation statements)

References 49 publications

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“…It has been shown that a BRCA1 spliceogenic variant resulting in 70–80% expression of a non-functional transcript ( de la Hoya et al, 2016 ) is not risk-associated. There is some evidence to suggest that the tolerable level of expression may be similar for MSH2 ; MSH2 c.1275A > G, reported to be associated with 70% expression of aberrant transcript r.[1229_1276del, 1275a > g] ( Morak et al, 2019 ), is currently classified as a VUS but with accumulating clinical evidence trending toward likely benign. While, evidence from a knock-down study assessing correlation between total mRNA expression levels and MMR protein relative repair activity in human fibroblast cell lines ( Kansikas et al, 2014 ) indicates that ∼25% MLH1 or MSH2 mRNA expression results in abrogated repair activity.…”

Section: Resultsmentioning

confidence: 99%

Contribution of mRNA Splicing to Mismatch Repair Gene Sequence Variant Interpretation

et al. 2020

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Functional assays that assess mRNA splicing can be used in interpretation of the clinical significance of sequence variants, including the Lynch syndrome-associated mismatch repair (MMR) genes. The purpose of this study was to investigate the contribution of splicing assay data to the classification of MMR gene sequence variants. We assayed mRNA splicing for 24 sequence variants in MLH1, MSH2, and MSH6, including 12 missense variants that were also assessed using a cell-free in vitro MMR activity (CIMRA) assay. Multifactorial likelihood analysis was conducted for each variant, combining CIMRA outputs and clinical data where available. We collated these results with existing public data to provide a dataset of splicing assay results for a total of 671 MMR gene sequence variants (328 missense/in-frame indel), and published and unpublished repair activity measurements for 154 of these variants. There were 241 variants for which a splicing aberration was detected: 92 complete impact, 33 incomplete impact, and 116 where it was not possible to determine complete versus incomplete splicing impact. Splicing results mostly aided in the interpretation of intronic (72%) and silent (92%) variants and were the least useful for missense substitutions/in-frame indels (10%). MMR protein functional activity assays were more useful in the analysis of these exonic variants but by design they were not able to detect clinically important splicing aberrations identified by parallel mRNA assays. The development of high throughput assays that can quantitatively assess impact on mRNA transcript expression and protein function in parallel will streamline classification of MMR gene sequence variants.

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Section: Resultsmentioning

confidence: 99%

Contribution of mRNA Splicing to Mismatch Repair Gene Sequence Variant Interpretation

et al. 2020

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“…Morak et al investigated the prevalence in the German population of five founder CNV-neutral structural variants (SV) and identified only one patient with an insertion in PMS2 intron 7, concluding that these SV are not frequent [ 199 ]. The detection of CNV-neutral SV requires specific methods, such as full-length cDNA or deep-intronic sequencing [ 190 , 200 ], and the optimal strategy is different depending on the type of SV. As the paracentric inversion involving MSH2 exons 1 to 7 can be a frequent cause of unexplained LS in some populations [ 201 ], two specific probes have been added to the commercialized MLPA-kit MLH1/MSH2-P003 (MRC Holland) to detect the rearrangement breakpoint reported in intron 7 [ 194 , 195 ].…”

Section: Diagnosis Of Lynch Syndromementioning

confidence: 99%

Diagnosis of Lynch Syndrome and Strategies to Distinguish Lynch-Related Tumors from Sporadic MSI/dMMR Tumors

Leclerc

Vermaut²,

Buisine

2021

Cancers

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Microsatellite instability (MSI) is a hallmark of Lynch syndrome (LS)-related tumors but is not specific to it, as approximately 80% of MSI/mismatch repair-deficient (dMMR) tumors are sporadic. Methods leading to the diagnosis of LS have considerably evolved in recent years and so have tumoral tests for LS screening and for the discrimination of LS-related to MSI-sporadic tumors. In this review, we address the hallmarks of LS, including the clinical, histopathological, and molecular features. We present recent advances in diagnostic and screening strategies to identify LS patients. We also discuss the pitfalls associated with the current strategies, which should be taken into account to improve the diagnosis of LS and avoid inappropriate clinical management.

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“…In contrast to MLH1 and MSH2, only a small fraction of MSH6 variants have been studied with RNA analyses, and few variants have been reported to affect splicing (Table S1). MSH6 splicing analyses have been performed by examining RNA in whole blood or other tissues from variant carriers, by reverse transcription PCR (RT-PCR) and long-range RT-PCR analysis [37,51,71,[74][75][76][77][78][79][80][81][82][83][84], and, more recently, by targeted RNA sequencing [71,75]. Moreover, in cases where RNA is not available, minigene splicing assays have been performed [76,77].…”

Section: Rna Splicing Analysismentioning

confidence: 99%

“…This points to the fact that when interpreting RNA results, it is important to know the contribution of naturally occurring isoforms. Recent studies have identified several different MSH6 transcript isoforms [74,75,85,86], including isoforms with cassette events, such as the skipping of exons 2 (resulting in frameshift), exon 3 (frameshift), or exon 4 (frameshift); multicasette events, such as the skipping of exons 2 and 3 (frameshift), or skipping of exons 3 and 4 (in-frame); or transcript isoforms using cryptic splice sites in various exons. The skipping of exon 4 is the most common naturally occurring transcript and is observed in up to 25% of the reference transcript expression.…”

Section: Rna Splicing Analysismentioning

confidence: 99%

Classification of MSH6 Variants of Uncertain Significance Using Functional Assays

Frederiksen

Jensen

Tümer

et al. 2021

IJMS

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Lynch syndrome (LS) is one of the most common hereditary cancer predisposition syndromes worldwide. Individuals with LS have a high risk of developing colorectal or endometrial cancer, as well as several other cancers. LS is caused by autosomal dominant pathogenic variants in one of the DNA mismatch repair (MMR) genes MLH1, MSH2, PMS2 or MSH6, and typically include truncating variants, such as frameshift, nonsense or splicing variants. However, a significant number of missense, intronic, or silent variants, or small in-frame insertions/deletions, are detected during genetic screening of the MMR genes. The clinical effects of these variants are often more difficult to predict, and a large fraction of these variants are classified as variants of uncertain significance (VUS). It is pivotal for the clinical management of LS patients to have a clear genetic diagnosis, since patients benefit widely from screening, preventive and personal therapeutic measures. Moreover, in families where a pathogenic variant is identified, testing can be offered to family members, where non-carriers can be spared frequent surveillance, while carriers can be included in cancer surveillance programs. It is therefore important to reclassify VUSs, and, in this regard, functional assays can provide insight into the effect of a variant on the protein or mRNA level. Here, we briefly describe the disorders that are related to MMR deficiency, as well as the structure and function of MSH6. Moreover, we review the functional assays that are used to examine VUS identified in MSH6 and discuss the results obtained in relation to the ACMG/AMP PS3/BS3 criterion. We also provide a compiled list of the MSH6 variants examined by these assays. Finally, we provide a future perspective on high-throughput functional analyses with specific emphasis on the MMR genes.

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Full-length transcript amplification and sequencing as universal method to test mRNA integrity and biallelic expression in mismatch repair genes

Cited by 15 publications

References 49 publications

Contribution of mRNA Splicing to Mismatch Repair Gene Sequence Variant Interpretation

Contribution of mRNA Splicing to Mismatch Repair Gene Sequence Variant Interpretation

Diagnosis of Lynch Syndrome and Strategies to Distinguish Lynch-Related Tumors from Sporadic MSI/dMMR Tumors

Classification of MSH6 Variants of Uncertain Significance Using Functional Assays

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