A Deep Exon Cryptic Splice Site Promotes Aberrant Intron Retention in a Von Willebrand Disease Patient

A translationally silent single nucleotide mutation in exon 44 (E44) of the von Willebrand factor (VWF) gene is associated with inefficient removal of intron 44 in a von Willebrand disease (VWD) patient. This intron retention (IR) event was previously attributed to reordered E44 secondary structure that sequesters the normal splice donor site. We propose an alternative mechanism: the mutation introduces a cryptic splice donor site that interferes with the function of the annotated site to favor IR. We evaluated both models using minigene splicing reporters engineered to vary in secondary structure and/or cryptic splice site content. Analysis of splicing efficiency in transfected K562 cells suggested that the mutation-generated cryptic splice site in E44 was sufficient to induce substantial IR. Mutations predicted to vary secondary structure at the annotated site also had modest effects on IR and shifted the balance of residual splicing between the cryptic site and annotated site, supporting competition among the sites. Further studies demonstrated that introduction of cryptic splice donor motifs at other positions in E44 did not promote IR, indicating that interference with the annotated site is context dependent. We conclude that mutant deep exon splice sites can interfere with proper splicing by inducing IR.

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Cryptic splice site activation by a splice donor site mutation of dystrophin intron 64 is determined by intronic splicing regulatory elements

Neuromuscular Disorders ◽

10.1016/j.nmd.2016.06.042 ◽

2016 ◽

Vol 26 ◽

pp. S96

Author(s):

E. Niba ◽

A. Nishuda ◽

V. Tran ◽

D. Vu ◽

M. Matsumoto ◽

...

Keyword(s):

Splice Site ◽

Donor Site ◽

Regulatory Elements ◽

Splice Donor Site ◽

Cryptic Splice Site ◽

Splice Donor ◽

Site Mutation ◽

Splicing Regulatory Elements ◽

Splice Donor Site Mutation

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A 6-bp deletion at the splice donor site of the first intron resulted in aberrant splicing using a cryptic splice site within exon 1 in a patient with succinyl-CoA: 3-Ketoacid CoA transferase (SCOT) deficiency

Molecular Genetics and Metabolism ◽

10.1016/j.ymgme.2006.04.014 ◽

2006 ◽

Vol 89 (3) ◽

pp. 280-282 ◽

Cited By ~ 14

Author(s):

Toshiyuki Fukao ◽

Satomi Sakurai ◽

Marie-Odile Rolland ◽

Marie-Therese Zabot ◽

Andreas Schulze ◽

...

Keyword(s):

Splice Site ◽

Donor Site ◽

Splice Donor Site ◽

Cryptic Splice Site ◽

Splice Donor ◽

Aberrant Splicing ◽

First Intron ◽

Coa Transferase ◽

Exon 1

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A 5′ splice site mutation affecting the pre-mRNA splicing of two upstream exons in the collagen COL1A1 gene. Exon 8 skipping and altered definition of exon 7 generates truncated proα1(I) chains with a non-collagenous insertion destabilizing the triple helix

Biochemical Journal ◽

10.1042/bj3020729 ◽

1994 ◽

Vol 302 (3) ◽

pp. 729-735 ◽

Cited By ~ 21

Author(s):

J F Bateman ◽

D Chan ◽

I Moeller ◽

M Hannagan ◽

W G Cole

Keyword(s):

Splice Site ◽

De Novo ◽

Donor Site ◽

Mrna Splicing ◽

Splice Donor Site ◽

Sequence Motif ◽

Type I ◽

Splice Donor ◽

Site Mutation ◽

Definition Of

A heterozygous de novo G to A point mutation in intron 8 at the +5 position of the splice donor site of the gene for the pro alpha 1(I) chain of type I procollagen, COL1A1, was defined in a patient with type IV osteogenesis imperfecta. The splice donor site mutation resulted not only in the skipping of the upstream exon 8 but also unexpectedly had the secondary effect of activating a cryptic splice site in the next upstream intron, intron 7, leading to re-definition of the 3′ limit of exon 7. These pre-mRNA splicing aberrations cause the deletion of exon 8 sequences from the mature mRNA and the inclusion of 96 bp of intron 7 sequence. Since the mis-splicing of the mutant allele product resulted in the maintenance of the correct codon reading frame, the resultant pro alpha 1(I) chain contained a short non-collagenous 32-amino-acid sequence insertion within the repetitive Gly-Xaa-Yaa collagen sequence motif. At the protein level, the mutant alpha 1(I) chain was revealed by digestion with pepsin, which cleaved the mutant procollagen within the protease-sensitive non-collagenous insertion, producing a truncated alpha 1(I). This protease sensitivity demonstrated the structural distortion to the helical structure caused by the insertion. In long-term culture with ascorbic acid, which stimulates the formation of a mature crosslinked collagen matrix, and in tissues, there was no evidence of the mutant chain, suggesting that during matrix formation the mutant chain was unable to stably incorporated into the matrix and was degraded proteolytically.

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Comparison of in vitro and in vivo splice site selection in kappa-immunoglobulin precursor mRNA.

Molecular and Cellular Biology ◽

10.1128/mcb.8.6.2610 ◽

1988 ◽

Vol 8 (6) ◽

pp. 2610-2619 ◽

Cited By ~ 7

Author(s):

D E Lowery ◽

B G Van Ness

Keyword(s):

Splice Site ◽

Site Selection ◽

Donor Site ◽

Splice Donor Site ◽

Splice Sites ◽

Splice Donor ◽

Splice Site Selection ◽

Nuclear Extracts

The processing of a number of kappa-immunoglobulin primary mRNA (pre-mRNA) constructs has been examined both in vitro and in vivo. When a kappa-immunoglobulin pre-mRNA containing multiple J segment splice sites is processed in vitro, the splice sites are used with equal frequency. The presence of signal exon, S-V intron, or variable (V) region has no effect on splice site selection in vitro. Nuclear extracts prepared from a lymphoid cell line do not restore correct splice site selection. Splice site selection in vitro can be altered by changing the position or sequence of J splice donor sites. These results differ from the processing of similar pre-mRNAs expressed in vivo by transient transfection. The 5'-most J splice donor site was exclusively selected in vivo, even in nonlymphoid cells, and even in transcripts where in vitro splicing favored a 3' J splice site. The in vitro results are consistent with a model proposing that splice site selection is influenced by splice site strength and proximity; however, our in vivo results demonstrate a number of discrepancies with such a model and suggest that splice site selection may be coupled to transcription or a higher-order nuclear structure.

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Combined partial exon skipping and cryptic splice site activation as a new molecular mechanism for recessive type 1 von Willebrand disease

Thrombosis and Haemostasis ◽

10.1160/th06-07-0417 ◽

2006 ◽

Vol 96 (12) ◽

pp. 711-716 ◽

Cited By ~ 13

Author(s):

Lisa Gallinaro ◽

Francesca Sartorello ◽

Elena Pontara ◽

Maria Cattini ◽

Antonella Bertomoro ◽

...

Keyword(s):

Splice Site ◽

Stop Codon ◽

Consensus Sequence ◽

Premature Stop Codon ◽

Exon Skipping ◽

Von Willebrand Disease ◽

Cryptic Splice Site ◽

Recessive Type ◽

Von Willebrand

SummaryWe describe the complex picture associated with a mutated splice junction in intron 13 of von Willebrand factor (VWF) gene. The proband, characterized by a marked decrease in plasma and platelet VWF and near normal multimer organization, was classified as recessive type 1 von Willebrand disease (VWD). Genetic analysis demonstrated that he was homozygous for the 1534–3C>A mutation in the consensus sequence of the acceptor splicing site of intron 13 of the VWF gene. Platelet mRNA analysis documented three VWF transcripts: a wild type generated by the correct recognition of the mutated splice site, a smaller transcript not containing exon 14, and a longer one that, in addition to exons 13 and 14, included a 62bp fragment corresponding to the end of intron 13. The small transcript derives from the skipping of exon 14, the long one from the activation of a cryptic splice site in intron 13; both show a premature stop codon inVWF propeptide, so the probandVWF derives entirely from the correct splice site recognition. Combined incomplete exon skipping and cryptic splice site activation are first recognized in VWD. Since the 1534–3C>A mutation does not abolish the normal processing of mRNA, it is unlikely to be found in type 3VWD. This mutation therefore appears to be peculiar to type 1 VWD.

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Comparison of in vitro and in vivo splice site selection in kappa-immunoglobulin precursor mRNA

Molecular and Cellular Biology ◽

10.1128/mcb.8.6.2610-2619.1988 ◽

1988 ◽

Vol 8 (6) ◽

pp. 2610-2619

Author(s):

D E Lowery ◽

B G Van Ness

Keyword(s):

Splice Site ◽

Site Selection ◽

Donor Site ◽

Splice Donor Site ◽

Splice Sites ◽

Splice Donor ◽

Splice Site Selection ◽

Nuclear Extracts

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Abstract 3412: Molecular and Cellular Characterization of Splice Site Mutations in MYBPC3 -encoded Myosin Binding Protein C

Circulation ◽

10.1161/circ.118.suppl_18.s_419-d ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Jeanne L Theis ◽

J M Bos ◽

Steve R Ommen ◽

Michael J Ackerman

Keyword(s):

Splice Site ◽

Protein C ◽

Binding Protein ◽

Donor Site ◽

Splice Donor Site ◽

Splice Donor ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Alternatively Spliced ◽

Myosin Binding

Background: Mutations in MYBPC3 -encoded myosin binding protein C underlie the most common genotype in hypertrophic cardiomyopathy (HCM). Compared to most HCM-associated mutations which are primarily missense mutations, MYBPC3-HCM is often caused by insertions, deletions, nonsense mutations or mutations involving the canonical splice site. However, the effects of most mutations that prematurely truncate MYBPC3 are unknown. Access to cardiac specific mRNA and tissue has permitted a molecular and cellular elucidation of the consequences of two splice site mutations. Methods: Mutational analysis of MYBPC3 revealed 2 HCM-susceptibility mutations involving the splice donor sites of exons 7 and 30. Reverse transcription was performed on cDNA generated from RNA extracted from cardiac tissue obtained following surgical myectomy. Western blot analysis and immunohistochemistry (IHC) of the myofilaments were performed to further assess the impact of the splice-site mutations. Results: Both splice donor mutations produced abnormal RNA splicing of MYBPC3. The c.821+1 g>a mutation in the splice donor site of exon 7 generated two alternatively-spliced mutant transcripts resulting in two frame-shifted, premature truncations (H257 fs/37 and H257 fs/15). The c.3330+2 t>g mutation in exon 30’s splice donor site produced a single alternatively-spliced mutant transcript that translated into a frame-shifted premature truncation (V1063 fs/37). Expression levels of wild type myosin binding protein C were decreased in the myectomy specimen from the patient with the exon 7 splice donor site mutation. By IHC, the spatial organization of myosin binding protein C was disrupted severely in both patients. Conclusions: This study provides the first molecular and cellular characterization of HCM-causing mutations involving canonical splice-site motifs within the intron. Loss of function of the splice site resulted in exon skipping and generation of frame-shifted and prematurely truncated myosin binding protein C and disruption of the distribution and organization of myosin binding protein C in the heart tissue. This research has received full or partial funding support from the American Heart Association, AHA Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).

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Biallelic variants in COPB1 cause a novel, severe intellectual disability syndrome with cataracts and variable microcephaly

Genome Medicine ◽

10.1186/s13073-021-00850-w ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

William L. Macken ◽

Annie Godwin ◽

Gabrielle Wheway ◽

Karen Stals ◽

Liliya Nazlamova ◽

...

Keyword(s):

Genome Sequencing ◽

Donor Site ◽

Xenopus Tropicalis ◽

Splice Donor Site ◽

Homologous Region ◽

Disease Genes ◽

Coat Proteins ◽

Splice Donor ◽

Severe Intellectual Disability

Abstract Background Coat protein complex 1 (COPI) is integral in the sorting and retrograde trafficking of proteins and lipids from the Golgi apparatus to the endoplasmic reticulum (ER). In recent years, coat proteins have been implicated in human diseases known collectively as “coatopathies”. Methods Whole exome or genome sequencing of two families with a neuro-developmental syndrome, variable microcephaly and cataracts revealed biallelic variants in COPB1, which encodes the beta-subunit of COPI (β-COP). To investigate Family 1’s splice donor site variant, we undertook patient blood RNA studies and CRISPR/Cas9 modelling of this variant in a homologous region of the Xenopus tropicalis genome. To investigate Family 2’s missense variant, we studied cellular phenotypes of human retinal epithelium and embryonic kidney cell lines transfected with a COPB1 expression vector into which we had introduced Family 2’s mutation. Results We present a new recessive coatopathy typified by severe developmental delay and cataracts and variable microcephaly. A homozygous splice donor site variant in Family 1 results in two aberrant transcripts, one of which causes skipping of exon 8 in COPB1 pre-mRNA, and a 36 amino acid in-frame deletion, resulting in the loss of a motif at a small interaction interface between β-COP and β’-COP. Xenopus tropicalis animals with a homologous mutation, introduced by CRISPR/Cas9 genome editing, recapitulate features of the human syndrome including microcephaly and cataracts. In vitro modelling of the COPB1 c.1651T>G p.Phe551Val variant in Family 2 identifies defective Golgi to ER recycling of this mutant β-COP, with the mutant protein being retarded in the Golgi. Conclusions This adds to the growing body of evidence that COPI subunits are essential in brain development and human health and underlines the utility of exome and genome sequencing coupled with Xenopus tropicalis CRISPR/Cas modelling for the identification and characterisation of novel rare disease genes.

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