Two novel splicing mutations in the SLC45A2 gene cause Oculocutaneous Albinism Type IV by unmasking cryptic splice sites

AbstractPrevious studies studying mis-splicing mutations were based on exome data and thus our current knowledge is largely limited to exons and the canonical splice sites. To comprehensively characterise intronic mis-splicing mutations, we analysed 1134 pan-cancer whole genomes and transcriptomes together with 3022 normal control samples. The ratio-based splicing analysis resulted in 678 somatic intronic mutations, with 46% residing in deep introns. Among the 309 deep intronic single nucleotide variants, 245 altered core splicing codes, with 38% activating cryptic splice sites, 12% activating cryptic polypyrimidine tracts, and 36% and 12% disrupting authentic polypyrimidine tracts and branchpoints, respectively. All the intronic cryptic splice sites were created at pre-existing GT/AG dinucleotides or by GC-to-GT conversion. Notably, 85 deep intronic mutations indicated gain of splicing enhancers or loss of splicing silencers. We found that 64 tumour suppressors were affected by intronic mutations and blood cancers showed higher proportion of deep intronic mutations. In particular, a telomere maintenance gene, POT1, was recurrently mis-spliced by deep intronic mutations in blood cancers. We validated a pseudoexon activation involving a splicing silencer in POT1 by CRISPR/Cas9. Our results shed light on previously unappreciated mechanisms by which noncoding mutations acting on splicing codes in deep introns contribute to tumourigenesis.

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Induction of cryptic pre-mRNA splice-switching by antisense oligonucleotides

Scientific Reports ◽

10.1038/s41598-021-94639-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kristin A. Ham ◽

Niall P. Keegan ◽

Craig S. McIntosh ◽

May T. Aung-Htut ◽

Khine Zaw ◽

...

Keyword(s):

Rna Splicing ◽

Antisense Oligonucleotides ◽

Live Cells ◽

Splice Sites ◽

Cryptic Splice Site ◽

Exon Definition ◽

Rna Targets ◽

Cryptic Splice Sites ◽

Six Genes ◽

Intended Effect

AbstractAntisense oligomers (AOs) are increasingly being used to modulate RNA splicing in live cells, both for research and for the development of therapeutics. While the most common intended effect of these AOs is to induce skipping of whole exons, rare examples are emerging of AOs that induce skipping of only part of an exon, through activation of an internal cryptic splice site. In this report, we examined seven AO-induced cryptic splice sites in six genes. Five of these cryptic splice sites were discovered through our own experiments, and two originated from other published reports. We modelled the predicted effects of AO binding on the secondary structure of each of the RNA targets, and how these alterations would in turn affect the accessibility of the RNA to splice factors. We observed that a common predicted effect of AO binding was disruption of the exon definition signal within the exon’s excluded segment.

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Control of adenovirus E1B mRNA synthesis by a shift in the activities of RNA splice sites

Molecular and Cellular Biology ◽

10.1128/mcb.4.5.966-972.1984 ◽

1984 ◽

Vol 4 (5) ◽

pp. 966-972

Author(s):

C Montell ◽

E F Fisher ◽

M H Caruthers ◽

A J Berk

Keyword(s):

Splice Site ◽

Rna Splicing ◽

Early Phase ◽

Late Phase ◽

Productive Infection ◽

Splice Sites ◽

Mrna Synthesis ◽

Adenovirus E1b ◽

Cryptic Splice Sites ◽

Rna Splice Sites

The primary transcript from adenovirus 2 early region 1B (E1B) is processed by differential RNA splicing into two overlapping mRNAs, 13S and 22S. The 22S mRNA is the major E1B mRNA during the early phase of infection, whereas the 13S mRNA predominates during the late phase. In previous work, it has been shown that this shift in proportions of the E1B mRNAs is influenced by increased cytoplasmic stability of the 13S mRNA at late times in infection. Two observations presented here demonstrate that the increase in proportion of the 13S mRNA at late times is also regulated by a change in the specificity of RNA splicing. First, the relative concentrations of the 13S to 22S nuclear RNAs were not constant throughout infection but increased at late times. Secondly, studies with the mutant, adenovirus 2 pm2250 , provided evidence that there was an increased propensity to utilize a 5' splice in the region of the 13S 5' splice site at late times in infection. Adenovirus 2 pm2250 has a G----C transversion in the first base of E1B 13S mRNA intron preventing splicing of the 13S mRNA but not of the 22S mRNA. During the early phase of a pm2250 infection, the E1B primary transcripts were processed into the 22S mRNA only. However, during the late phase, when the 13S mRNA normally predominates, E1B primary transcripts were also processed by RNA splicing at two formerly unused or cryptic 5' splice sites. Both cryptic splice sites were located much closer to the disrupted 13S 5' splice site than to the 22S 5' splice site. Thus, the temporal increase in proportion of the 13S mRNA to the 22S mRNA is regulated by two processes, an increase in cytoplasmic stability of the 13S mRNA and an increased propensity to utilize the 13S 5' splice site during the late phase of infection. Adenovirus 2 pm2250 was not defective for productive infection of HeLa cells or for transformation of rat cells.

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Multiple cryptic splice sites can be activated by IDS point mutations generating misspliced transcripts

Journal of Molecular Medicine ◽

10.1007/s00109-006-0057-1 ◽

2006 ◽

Vol 84 (8) ◽

pp. 692-700 ◽

Cited By ~ 15

Author(s):

Susanna Lualdi ◽

Maria G. Pittis ◽

Stefano Regis ◽

Rossella Parini ◽

Anna E. Allegri ◽

...

Keyword(s):

Point Mutations ◽

Splice Sites ◽

Cryptic Splice Sites

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Spontaneous splicing mutations at the dihydrofolate reductase locus in Chinese hamster ovary cells

Molecular and Cellular Biology ◽

10.1128/mcb.6.6.1926-1935.1986 ◽

1986 ◽

Vol 6 (6) ◽

pp. 1926-1935

Author(s):

P J Mitchell ◽

G Urlaub ◽

L Chasin

Keyword(s):

Amino Acid ◽

Dihydrofolate Reductase ◽

Splice Site ◽

Chinese Hamster Ovary ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Splice Sites ◽

Ovary Cells ◽

Splicing Mutations ◽

Intron 4

We isolated and characterized three spontaneous mutants of Chinese hamster ovary cells that were deficient in dihydrofolate reductase activity. All three mutants contained no detectable enzyme activity and produced dihydrofolate reductase mRNA species that were shorter than those of the wild type by about 120 bases. Six exons are normally represented in this mRNA; exon 5 was missing in all three mutant mRNAs. Nuclease S1 analysis of the three mutants indicated that during the processing of the mutant RNA, exon 4 was spliced to exon 6. The three mutant genes were cloned, and the regions around exons 4 and 5 were sequenced. In one mutant, the GT dinucleotide at the 5' end of intron 5 had changed to CT. In a second mutant, the first base in exon 5 had changed from G to T. In a revertant of this mutant, this base was further mutated to A, a return to a purine. Approximately 25% of the mRNA molecules in the revertant were spliced correctly to produce an enzyme with one presumed amino acid change. In the third mutant, the AG at the 3' end of intron 4 had changed to AA. A mutation that partially reversed the mutant phenotype had changed the dinucleotide at the 5' end of intron 4 from GT to AT. The splicing pattern in this revertant was consistent with the use of cryptic donor and acceptor splice sites close to the original sites to produce an mRNA with three base changes and a protein with two amino acid changes. These mutations argue against a scanning model for the selection of splice site pairs and suggest that only a single splice site need be inactivated to bring about efficient exon skipping (a regulatory mechanism for some genes). The fact that all three mutants analyzed exhibited exon 5 splicing mutations indicates that these splice sites are hot spots for spontaneous mutation.

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Activation of cryptic splice sites in three patients with chronic granulomatous disease

Molecular Genetics & Genomic Medicine ◽

10.1002/mgg3.854 ◽

2019 ◽

Vol 7 (9) ◽

Author(s):

Martin de Boer ◽

Karin van Leeuwen ◽

Mathias Hauri‐Hohl ◽

Dirk Roos

Keyword(s):

Chronic Granulomatous Disease ◽

Granulomatous Disease ◽

Splice Sites ◽

Cryptic Splice Sites

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Synonymous Mutations inRNASEH2ACreate Cryptic Splice Sites Impairing RNase H2 Enzyme Function in Aicardi-Goutières Syndrome

Human Mutation ◽

10.1002/humu.22336 ◽

2013 ◽

Vol 34 (8) ◽

pp. 1066-1070 ◽

Cited By ~ 13

Author(s):

Gillian I. Rice ◽

Martin A.M. Reijns ◽

Stephanie R. Coffin ◽

Gabriella M.A. Forte ◽

Beverley H. Anderson ◽

...

Keyword(s):

Enzyme Function ◽

Splice Sites ◽

Synonymous Mutations ◽

Rnase H2 ◽

Cryptic Splice Sites

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Generation of a Novel Mouse Model of Cooley’s Anemia.

Blood ◽

10.1182/blood.v106.11.3654.3654 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3654-3654

Author(s):

Yongliang Huo ◽

Sean McConnell ◽

Chiao-Wang Sun ◽

Li-Chen Wu ◽

Thomas M. Ryan

Keyword(s):

Blood Transfusion ◽

Mouse Model ◽

Adult Mouse ◽

Globin Gene ◽

Marker Gene ◽

Gene Replacement ◽

Globin Genes ◽

Splice Sites ◽

Cooley's Anemia ◽

Cryptic Splice Sites

Abstract A novel mouse model of Cooley’s Anemia (CA) has been generated by targeted gene replacement of the adult murine α-globin genes with human α-globin and the adult mouse β-globin genes with a human γ- to β-globin gene switching cassette containing a β0 thalassemic allele. A positive-negative gene replacement construct was designed to simultaneously delete both of the adult mouse α-globin genes by inserting a 3.8kb human α1-globin gene and a hygromycin marker gene flanked by loxP sites in murine embryonic stem (ES) cells. Both adult murine β-globin genes were deleted by insertion of an Hprt marker gene that was later replaced by a 5.6kb human Aγ-globin gene, 4.1kb human β0-globin gene, and a loxP flanked hygromycin marker gene by a “tag and exchange” strategy. The human β0-globin knock-in allele contains a single G to A nucleotide mutation in the first base of intervening sequence 1 [β0-IVS1(GtoA)-globin]. This single base change destroys the splice donor site of IVS-1 resulting in the recruitment of several cryptic splice sites. Use of these cryptic splice sites produces a frameshift in the mRNA that results in no functional β-globin polypeptide synthesis from this allele. This β0-IVS1(GtoA)-globin gene mutation is a naturally occurring β0 thalassemia allele found in Mediterranean populations. Chimeric mice were generated from both the α- and β-globin targeted cells lines. After germline transmission the α- and β-globin targeted mice were bred to cre recombinase transgenic mice to remove the marker genes. Heterozygous CA mice exhibit β thalassemia intermedia. The α- and β-globin targeted mice were interbred to produce animals homozygous for the human α1- and γβ0-globin knock-in alleles. Instead of dying early in fetal life as all current homozygous β0 thalassemia mouse models, these novel homozygous CA mice survive solely on high levels of human fetal hemoglobin (α2γ2) throughout fetal development. Newborn homozygous CA mice are blood transfusion dependent similar to β thalassemia major infants. This novel model of CA has multiple improvements over existing models of β thalassemia. Namely, CA mice express 100% human hemoglobin in their RBCs, mimic the human γ- to β-globin gene switch, synthesize no functional β-globin chains after birth, have a single mutant human β0-globin knock-in allele at each β-globin locus, and are blood transfusion dependent for life after birth.

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