Using the ACMG/AMP framework to capture evidence related to predicted and observed impact on splicing: Recommendations from the ClinGen SVI Splicing Subgroup

Walker, Logan C.; Hoya, Miguel de la; Wiggins, George A. R.; Lindy, Amanda; Vincent, Lisa M.; Parsons, Michael T.; Canson, Daffodil M.; Bis-Brewer, Dana M.; Cass, Ashley; Tchourbanov, Alexander; Zimmermann, Heather; Byrne, Alicia B.; Karam, Rachid; Spurdle, Amanda B.; Biesecker, Leslie G.; Harrison, Steven M.; Tayoun, Ahmad Abou; Berg, Jonathan S.; Brenner, Steven E.; Cutting, Garry R.; Ellard, Sian; Greenblatt, Marc S.; Kang, Peter B.; Karbassi, Izabela; Karchin, Rachel; Mester, Jessica L.; O’Donnell-Luria, Anne H.; Pesaran, Tina; Plon, Sharon E.; Rehm, Heidi L.; Strande, Natasha T.; Tavtigian, Sean V.

doi:10.1016/j.ajhg.2023.06.002

Cited by 90 publications

(43 citation statements)

References 64 publications

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“…SpliceAI is widely used in the clinical setting, including variant curation activities conducted by ClinGen Variant Curation Expert Panels. As far as we know, SpliceAI has been calibrated mostly against intronic and exonic variants targeting consensus splice sites (or creating de novo sites) [57,58], but little is known about its specific performance against variants overlapping functional SREs. For that reason, we decided to test SpliceAI against SRE variants located in CHEK2 exons 8 and 10.…”

Section: Discussionmentioning

confidence: 99%

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

Sanoguera‐Miralles,

Llinares‐Burguet,

Bueno‐Martínez

et al. 2024

The Journal of Pathology

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Splicing is controlled by a large set of regulatory elements (SREs) including splicing enhancers and silencers, which are involved in exon recognition. Variants at these motifs may dysregulate splicing and trigger loss‐of‐function transcripts associated with disease. Our goal here was to study the alternatively spliced exons 8 and 10 of the breast cancer susceptibility gene CHEK2. For this purpose, we used a previously published minigene with exons 6–10 that produced the expected minigene full‐length transcript and replicated the naturally occurring events of exon 8 [Δ(E8)] and exon 10 [Δ(E10)] skipping. We then introduced 12 internal microdeletions of exons 8 and 10 by mutagenesis in order to map SRE‐rich intervals by splicing assays in MCF‐7 cells. We identified three minimal (10‐, 11‐, 15‐nt) regions essential for exon recognition: c.863_877del [ex8, Δ(E8): 75%] and c.1073_1083del and c.1083_1092del [ex10, Δ(E10): 97% and 62%, respectively]. Then 87 variants found within these intervals were introduced into the wild‐type minigene and tested functionally. Thirty‐eight of them (44%) impaired splicing, four of which (c.883G>A, c.883G>T, c.884A>T, and c.1080G>T) induced negligible amounts (<5%) of the minigene full‐length transcript. Another six variants (c.886G>A, c.886G>T, c.1075G>A, c.1075G>T, c.1076A>T, and c.1078G>T) showed significantly strong impacts (20–50% of the minigene full‐length transcript). Thirty‐three of the 38 spliceogenic variants were annotated as missense, three as nonsense, and two as synonymous, underlying the fact that any exonic change is capable of disrupting splicing. Moreover, c.883G>A, c.883G>T, and c.884A>T were classified as pathogenic/likely pathogenic variants according to ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)‐based criteria. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

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Section: Discussionmentioning

confidence: 99%

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

Sanoguera‐Miralles,

Llinares‐Burguet,

Bueno‐Martínez

et al. 2024

The Journal of Pathology

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“…Based on this splicing event, we replace the PP3 criterion originally assigned to the variant with PVS1_ Strong, as recommended by ClinGen splicing subgroup (Walker et al, 2023). Consequently, the KMT2A NM_001197104.2:c.5664+6T>C variant was reclassified as "Likely pathogenic" with the following ACMG/AMP/ ClinGen criteria: PM2_Supporting, PM6, PVS1_Strong.…”

Section: Rna Splicing Analysis and Variant Reclassificationmentioning

confidence: 99%

Aberrant splicing caused by a novel KMT2A variant in Wiedemann–Steiner syndrome

Niu,

Teng,

Zhang

2024

Molec Gen & Gen Med

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IntroductionWiedemann–Steiner syndrome (WSS) is a rare autosomal‐dominant disorder caused by KMT2A variants. The aim of this study was to characterize a novel KMT2A variant in a child with WSS and demonstrate integrated diagnostic approaches.MethodsA 3‐year‐old female with developmental delay, distinctive facial features, and anal fistula underwent whole exome sequencing (WES). RNA analysis was performed to assess splicing effects caused by a novel variant.ResultsWES identified novel heterozygous KMT2A c.5664+6T>C variant initially classified as a variant of uncertain significance. RNA analysis provided evidence of aberrant splicing (exon 20 skipping), allowing reclassification to likely pathogenic. The patient exhibited typical WSS features along with a potential novel finding of anal fistula.ConclusionThis report describes a novel non‐canonical splice site variant in KMT2A associated with WSS. RNA analysis was critical for variant reclassification. Detailed phenotypic evaluation revealed common and expanded WSS manifestations. This case highlights the importance of combining clinical assessment, DNA testing, and RNA functional assays for the diagnosis of rare genetic disorders.

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“…10 Pathogenicity of the detected genomic DMD variants was determined according to the standard guidelines. 11,12 3 | RE SULTS…”

Section: In Silico Bioinformatic Analysismentioning

confidence: 99%

Cryptic exon activation caused by a novel deep‐intronic splice‐altering variant in Becker muscular dystrophy

Xie,

Lu,

Liu

et al. 2023

Clinical Laboratory Analysis

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BackgroundAn accurate genetic diagnosis of Becker muscular dystrophy (BMD) can be sometimes challenging due to deep intronic DMD variants. Here, we report on the genetic diagnosis of a BMD patient with a novel deep‐intronic splice‐altering variant in DMD.MethodsThe index case was a 3.8‐year‐old boy who was suspected of having a diagnosis of BMD based on his clinical, muscle imaging, and pathological features. Routine genomic detection approaches did not detect any disease‐causing variants in him. Muscle‐derived DMD mRNA studies, followed by genomic Sanger sequencing and in silico bioinformatic analyses, were performed in the patient.ResultsDMD mRNA studies detected a cryptic exon‐containing transcript and normally spliced DMD transcript in the patient. The cryptic exon‐containing transcript encoded a frameshift and premature termination codon (NP_003997.1:p.[=,Asp2740Valfs*52]). Further genomic Sanger sequencing and bioinformatic analysis identified a novel deep‐intronic splice‐altering variant in DMD (c.8217 + 23338A > G). The novel variant strengthened a cryptic donor splice site and activated a cryptic acceptor splice site in the deep‐intronic region of DMD intron 55, resulting in the activation of a new dystrophin cryptic exon found in the patient.ConclusionOur case report expands the genetic spectrum of BMD and highlights the essential role of deep‐intronic cryptic exon‐activating variants in genetically unsolved BMD patients.

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Using the ACMG/AMP framework to capture evidence related to predicted and observed impact on splicing: Recommendations from the ClinGen SVI Splicing Subgroup

Cited by 90 publications

References 64 publications

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

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

Aberrant splicing caused by a novel KMT2A variant in Wiedemann–Steiner syndrome

Cryptic exon activation caused by a novel deep‐intronic splice‐altering variant in Becker muscular dystrophy

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