First estimate of the scale of canonical 5′ splice site GT&gt;GC variants capable of generating wild‐type transcripts

Lin, Jin‐Huan; Tang, Xin‐Ying; Boulling, Arnaud; Zou, Wenbin; Masson, Emmanuelle; Fichou, Yann; Raud, Loann; Tertre, Marlène Le; Deng, S.Z.; Berlivet, Isabelle; Ka, Chandran; Mort, Matthew; Hayden, Matthew J.; Leman, Raphaël; Houdayer, Claude; Gac, Gérald Le; Cooper, David Neil; Li, Zhao‐Shen; Férec, Claude; Liao, Zhuan; Chen, Jian‐Min

doi:10.1002/humu.23821

Cited by 32 publications

(80 citation statements)

References 102 publications

(126 reference statements)

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“…In both cases (i.e., HESX1 c.357+2T>C and SPINK1 c.194+2T>C), the FLGSA‐derived data were in perfect agreement with the in vivo data. Fourth, in terms of the rate of GT>GC variants generating wild‐type transcripts, the FLGSA‐derived data agreed well with data obtained not only from disease‐causing variants but also from BRCA1 variants analyzed in their natural genomic sequence contexts (Chen et al, 2020; Findlay et al, 2018; Lin et al, 2019). Fifth, the FLGSA‐derived data correlated well with predictions made by SpliceAI, a recently developed artificial intelligence‐based splicing prediction tool (Chen et al, 2020; Jaganathan et al, 2019).…”

Section: Gene Symbol Chr Hg38 Position Reference Allele Variant Allesupporting

confidence: 76%

“…However, we have recently provided evidence that such variants in human disease genes may not invariably be pathogenic. Specifically, combining data derived from a meta‐analysis of human disease‐causing +2T>C variants and a cell culture‐based Full‐Length Gene Splicing Assay (FLGSA) of engineered +2T>C substitutions, we estimated that ~15–18% of +2T>C variants generate up to 84% wild‐type transcripts (Lin et al, 2019). In another recent study, the functional effects of over 4,000 BRCA1 variants were analyzed by means of saturation genome editing (Findlay et al, 2018).…”

Section: Gene Symbol Chr Hg38 Position Reference Allele Variant Allementioning

confidence: 99%

“…Employing cell culture‐based FLGSA, we have previously analyzed the functional impact of 103 engineered +2T>C variants. About 82% of these variants resulted in the generation of only aberrantly spliced transcripts (Lin et al, 2019). The other 18% of variants retained some ability to generate wild‐type transcripts but none were capable of generating a wild‐type transcript level equal to or higher than its wild‐type counterpart (Lin et al, 2019).…”

Section: Gene Symbol Chr Hg38 Position Reference Allele Variant Allementioning

confidence: 99%

“…About 82% of these variants resulted in the generation of only aberrantly spliced transcripts (Lin et al, 2019). The other 18% of variants retained some ability to generate wild‐type transcripts but none were capable of generating a wild‐type transcript level equal to or higher than its wild‐type counterpart (Lin et al, 2019). The accuracy and reliability of the FLGSA‐derived data were supported by multiple lines of evidence.…”

Section: Gene Symbol Chr Hg38 Position Reference Allele Variant Allementioning

confidence: 99%

“…First, the findings have a strong biological basis; any +2T>C substitutions in the canonical 5′ splice site GT dinucleotides will decrease the complementarity between the 9‐bp consensus sequence for the U2‐type 5′ splice site (defined as sequence spanning nucleotide positions −3_+6) and the 3′‐GUCCAUUCA‐5′ sequence at the 5′ end of U1 small nuclear RNA (snRNA; see Lin et al, 2019 and references therein). Second, and consistent with this biological basis, the canonical 5′ splice sites in which substitution of GT by GC generated wild‐type transcripts exhibit a stronger complementarity to the 5′ end of U1 snRNA than those sites whose substitutions of GT by GC did not lead to the generation of wild‐type transcripts (Lin et al, 2019). Third, of the 103 engineered +2T>C substitutions, two had corresponding patient‐derived in vivo splicing data reported.…”

Section: Gene Symbol Chr Hg38 Position Reference Allele Variant Allementioning

confidence: 99%

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5′ splice site GC>GT and GT>GC variants differ markedly in terms of their functionality and pathogenicity

et al. 2020

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In the human genome, most 5′ splice sites (~99%) employ the canonical GT dinucleotide whereas a small minority (~1%) use the noncanonical GC dinucleotide. The functionality and pathogenicity of 5′ splice site GT>GC (+2T>C) variants have been extensively studied but we know very little about 5′ splice site GC>GT (+2C>T) variants. Herein, we have addressed this deficiency by performing a meta‐analysis of reported +2C>T “pathogenic” variants together with a functional analysis of engineered +2C>T substitutions using a cell culture‐based full‐length gene splicing assay. Our results establish proof of concept that +2C>T variants are qualitatively different from +2T>C variants in terms of their functionality and suggest that, in sharp contrast to +2T>C variants, most if not all +2C>T variants have no pathological relevance. Our findings have important implications for interpreting the clinical relevance of +2C>T variants and understanding the evolutionary switching between GT and GC 5′ splice sites in mammalian genomes.

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Section: Gene Symbol Chr Hg38 Position Reference Allele Variant Allesupporting

confidence: 76%

Section: Gene Symbol Chr Hg38 Position Reference Allele Variant Allementioning

confidence: 99%

Section: Gene Symbol Chr Hg38 Position Reference Allele Variant Allementioning

confidence: 99%

Section: Gene Symbol Chr Hg38 Position Reference Allele Variant Allementioning

confidence: 99%

Section: Gene Symbol Chr Hg38 Position Reference Allele Variant Allementioning

confidence: 99%

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5′ splice site GC>GT and GT>GC variants differ markedly in terms of their functionality and pathogenicity

et al. 2020

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Identification and analysis of short indels inducing exon extension/shrinkage events

Qu,

Sakaguchi,

Kikutake

et al. 2024

FEBS Open Bio

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The search for genetic variants that act as causative factors in human diseases by disrupting the normal splicing process has primarily focused on single nucleotide variants (SNVs). It is worth noting that insertions or deletions (indels) have also been sporadically reported as causative disease variants through their potential impact on the splicing process. In this study, to perform identification of indels inducing exon extension/shrinkage events, we used individual‐specific genomes and RNA sequencing (RNA‐seq) data pertaining to the corresponding individuals and identified 12 exon extension/shrinkage events that were potentially induced by indels that disrupted authentic splice sites or created novel splice sites in 235 normal individuals. By evaluating the impact of these abnormal splicing events on the resulting transcripts, we found that five events led to the generation of premature termination codons (PTCs), including those occurring within genes associated with genetic disorders. Our analysis revealed that the potential functions of indels have been underexamined, and it is worth considering the possibility that indels may affect splice site usage, using RNA‐seq data to discover novel potentially disease‐associated mutations.

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Splicing predictions, minigene analyses, and ACMG‐AMP clinical classification of 42 germline PALB2 splice‐site variants

Valenzuela-Palomo

Bueno-Martínez

Sanoguera-Miralles

et al. 2021

The Journal of Pathology

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PALB2 loss-of-function variants confer high risk of developing breast cancer. Here we present a systematic functional analysis of PALB2 splice-site variants detected in approximately 113,000 women in the large-scale sequencing project Breast Cancer After Diagnostic Gene Sequencing (BRIDGES; https://bridges-research.eu/). Eighty-two PALB2 variants at the intron-exon boundaries were analyzed with MaxEntScan. Forty-two variants were selected for the subsequent splicing functional assays. For this purpose, three splicing reporter minigenes comprising exons 1-12 were constructed. The 42 potential spliceogenic variants were introduced into the minigenes by sitedirected mutagenesis and assayed in MCF-7/MDA-MB-231 cells. Splicing anomalies were observed in 35 variants, 23 of which showed no traces or minimal amounts of the expected full-length transcripts of each minigene. More than 30 different variant-induced transcripts were characterized, 23 of which were predicted to truncate the PALB2 protein. The pathogenicity of all variants was interpreted according to an in-house adaptation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) variant classification scheme. Up to 23 variants were classified as pathogenic/likely pathogenic. Remarkably, three AE1,2 variants (c.49-2A>T, c.108+2T>C, and c.211+1G>A) were classified as variants of unknown significance, as they produced significant amounts of either in-frame transcripts of unknown impact on the PALB2 protein function or the minigene full-length transcripts. In conclusion, we have significantly contributed to the ongoing effort of identifying spliceogenic variants in the clinically relevant PALB2 cancer susceptibility gene. Moreover, we suggest some approaches to classify the findings in accordance with the ACMG-AMP rationale.

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First estimate of the scale of canonical 5′ splice site GT>GC variants capable of generating wild‐type transcripts

Cited by 32 publications

References 102 publications

5′ splice site GC>GT and GT>GC variants differ markedly in terms of their functionality and pathogenicity

5′ splice site GC>GT and GT>GC variants differ markedly in terms of their functionality and pathogenicity

Identification and analysis of short indels inducing exon extension/shrinkage events

Splicing predictions, minigene analyses, and ACMG‐AMP clinical classification of 42 germline PALB2 splice‐site variants

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