AG‐exclusion zone revisited: Lessons to learn from 91 intronic NF1 3′ splice site mutations outside the canonical AG‐dinucleotides

Wimmer, Katharina; Schamschula, Esther; Wernstedt, Annekatrin; Traunfellner, Pia; Amberger, Albert; Zschocke, Johannes; Kroisel, Peter M.; Chen, Yunjia; Callens, Tom; Messiaen, Ludwine

doi:10.1002/humu.24005

Cited by 24 publications

(27 citation statements)

References 26 publications

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“…In particular, we observed that one of these variants, namely the c.5206-11C > G substitution, introduced a novel AG in the exclusion zone (AGEZ) between the authentic 3’ss AG and the branch point. The creation of a novel 3′ss causes intronic retention (type 3 splice effect) in accordance with previously published data [ 51 ].…”

Section: Discussionsupporting

confidence: 91%

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Hybrid Minigene Assay: An Efficient Tool to Characterize mRNA Splicing Profiles of NF1 Variants

Morbidoni

Baschiera

Forzan

et al. 2021

Cancers

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Neurofibromatosis type 1 (NF1) is caused by heterozygous loss of function mutations in the NF1 gene. Although patients are diagnosed according to clinical criteria and few genotype-phenotype correlations are known, molecular analysis remains important. NF1 displays allelic heterogeneity, with a high proportion of variants affecting splicing, including deep intronic alleles and changes outside the canonical splice sites, making validation problematic. Next Generation Sequencing (NGS) technologies integrated with multiplex ligation-dependent probe amplification (MLPA) have largely overcome RNA-based techniques but do not detect splicing defects. A rapid minigene-based system was set up to test the effects of NF1 variants on splicing. We investigated 29 intronic and exonic NF1 variants identified in patients during the diagnostic process. The minigene assay showed the coexistence of multiple mechanisms of splicing alterations for seven variants. A leaky effect on splicing was documented in one de novo substitution detected in a sporadic patient with a specific phenotype without neurofibromas. Our splicing assay proved to be a reliable and fast method to validate novel NF1 variants potentially affecting splicing and to detect hypomorphic effects that might have phenotypic consequences, avoiding the requirement of patient’s RNA.

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Section: Discussionsupporting

confidence: 91%

“… 1 RNA analysis performed in our laboratory; 2 E, gnomAD exomes; G, gnomAD genomes; 3 P, pathogenic; LP, likely pathogenic; VUS, variant of uncertain significance; LB; likely benign; B, benign [ 44 ]; 4 AG creation in the AGEZ zone [ 51 ]. …”

Section: Figurementioning

confidence: 99%

Hybrid Minigene Assay: An Efficient Tool to Characterize mRNA Splicing Profiles of NF1 Variants

Morbidoni

Baschiera

Forzan

et al. 2021

Cancers

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“…The DMD c.3603+820G>T variant in intron 26 disrupts an AG, creating an AG-exclusion zone between an available consensus lariat branch point and 39 splice site. 27 Spliceosomal use of a naturally occurring consensus 59 splice site sequence and this strengthened 39 splice site result in the inclusion of a variantactivated pseudoexon into a majority of DMD transcripts, encoding 19 missense amino acids followed by a stop codon (figure 3C). RT-PCR confirmed abnormal inclusion of the variant-activated pseudoexon into DMD transcripts and residual, low levels of DMD transcripts with normal splicing of exons 25-26-27 (figure 2E).…”

Section: Data Availabilitymentioning

confidence: 99%

WGS and RNA Studies Diagnose Noncoding DMD Variants in Males With High Creatine Kinase

Waddell

Bryen²,

Cummings³

et al. 2021

Neurol Genet

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ObjectiveTo describe the diagnostic utility of whole-genome sequencing and RNA studies in boys with suspected dystrophinopathy, for whom multiplex ligation-dependent probe amplification and exomic parallel sequencing failed to yield a genetic diagnosis, and to use remnant normal DMD splicing in 3 families to define critical levels of wild-type dystrophin bridging clinical spectrums of Duchenne to myalgia.MethodsExome, genome, and/or muscle RNA sequencing was performed for 7 males with elevated creatine kinase. PCR of muscle-derived complementary DNA (cDNA) studied consequences for DMD premessenger RNA (pre-mRNA) splicing. Quantitative Western blot was used to determine levels of dystrophin, relative to control muscle.ResultsSplice-altering intronic single nucleotide variants or structural rearrangements in DMD were identified in all 7 families. Four individuals, with abnormal splicing causing a premature stop codon and nonsense-mediated decay, expressed remnant levels of normally spliced DMD mRNA. Quantitative Western blot enabled correlation of wild-type dystrophin and clinical severity, with 0%–5% dystrophin conferring a Duchenne phenotype, 10% ± 2% a Becker phenotype, and 15% ± 2% dystrophin associated with myalgia without manifesting weakness.ConclusionsWhole-genome sequencing relied heavily on RNA studies to identify DMD splice-altering variants. Short-read RNA sequencing was regularly confounded by the effectiveness of nonsense-mediated mRNA decay and low read depth of the giant DMD mRNA. PCR of muscle cDNA provided a simple, yet informative approach. Highly relevant to genetic therapies for dystrophinopathies, our data align strongly with previous studies of mutant dystrophin in Becker muscular dystrophy, with the collective conclusion that a fractional increase in levels of normal dystrophin between 5% and 20% is clinically significant.

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“…Suggestive evidence exists that non-canonical SAVs might be a more common cause of Mendelian disease than is commonly appreciated. 9,19,21 . Several previous approaches to prioritizing SAVs are based on information theory analysis, which compares wildtype and alternate sequences to a matrix of negative logarithms of the frequencies of nucleotides in the positions of wild type splice sites.…”

Section: Introductionmentioning

confidence: 99%

“…The sequence between the branch point and the 3’ splice site is generally devoid of AG dinucleotides and is called the AG□exclusion zone; variants that introduce an AG in this zone tend to be pathogenic. 19 Additionally, variants in introns or exons can activate cryptic splice sites to the extent that they are preferentially utilized compared to wildtype splice sites. We will use the term ‘canonical’ SAV to refer to variants at the ±1 or ±2 splice sites, and ‘non-canonical’ SAV to refer to any other SAV.…”

Section: Introductionmentioning

confidence: 99%

Interpretable prioritization of splice variants in diagnostic next-generation sequencing

Daniš

Jacobsen

Carmody

et al. 2021

Preprint

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A critical challenge in genetic diagnostics is the computational assessment of candidate splice variants, specifically the interpretation of nucleotide changes located outside of the highly conserved dinucleotide sequences at the 5′ and 3′ ends of introns. To address this gap, we developed the Super Quick Informationcontent Random-forest Learning of Splice variants (SQUIRLS) algorithm. SQUIRLS generates a small set of interpretable features for machine learning by calculating the information-content (IC) of wildtype and variant sequences of canonical and cryptic splice sites, assessing changes in candidate splicing regulatory sequences, and incorporating characteristics of the sequence such as exon length, disruptions of the AG exclusion zone, and conservation. We curated a comprehensive collection of disease-associated splicealtering variants at positions outside of the highly conserved AG/GT dinucleotides at the termini of introns. SQUIRLS trains two random-forest classifiers for the donor and for the acceptor and combines their outputs by logistic regression to yield a final score. We show that SQUIRLS transcends previous state of the art accuracy in classifying splice variants as assessed by rank analysis in simulated exomes and is significantly faster than competing methods. SQUIRLS provides tabular output files for incorporation into diagnostic pipelines for exome and genome analysis, as well as visualizations that contextualize predicted effects of variants on splicing to make it easier to interpret splice variants in diagnostic settings

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AG‐exclusion zone revisited: Lessons to learn from 91 intronic NF1 3′ splice site mutations outside the canonical AG‐dinucleotides

Cited by 24 publications

References 26 publications

Hybrid Minigene Assay: An Efficient Tool to Characterize mRNA Splicing Profiles of NF1 Variants

Hybrid Minigene Assay: An Efficient Tool to Characterize mRNA Splicing Profiles of NF1 Variants

WGS and RNA Studies Diagnose Noncoding DMD Variants in Males With High Creatine Kinase

Interpretable prioritization of splice variants in diagnostic next-generation sequencing

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