Characterising splicing defects of ABCA4 variants within exons 13–50 in patient-derived fibroblasts

Huang, Di; Thompson, Jennifer A.; Chen, Shang-Chih; Adams, A.M.; Pitout, Ianthe; Lima, Alanis; Zhang, Dan; Jeffery, Rachael C. Heath; Attia, Mary S.; McLaren, Terri L.; Lamey, Tina M.; Roach, John N. De; McLenachan, Samuel; Aung-Htut, May T.; Fletcher, Sue; Wilton, Steve; Chen, Fred K.

doi:10.1016/j.exer.2022.109276

Cited by 4 publications

(3 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As for the c.4469G>A variant, two similarly expressed transcripts were identified with RT-PCR. The first showed a 73-nt shortened exon 30 at the 3′ end, which is in line with the previously determined splicing prediction and previous research ( 21 ). This led to a shift in the open reading frame and a premature stop codon (p.Cys1490Glufs*12).…”

Section: Resultssupporting

confidence: 92%

Stargardt disease-associated missense and synonymous ABCA4 variants result in aberrant splicing

Kaltak

Corradi

Collin

et al. 2023

Human Molecular Genetics

View full text Add to dashboard Cite

Missense variants in ABCA4 constitute approximately 50% of causal variants in Stargardt disease (STGD1). Their pathogenicity is attributed to their direct effect on protein function, whilst their potential impact on pre-mRNA splicing disruption remains poorly understood. Interestingly, synonymous ABCA4 variants have previously been classified as ‘severe’ variants based on in silico analyses. Here, we systemically investigated the role of synonymous and missense variants in ABCA4 splicing by combining computational predictions and experimental assays. To identify variants of interest, we used SpliceAI to ascribe defective splice predictions on a dataset of 5579 biallelic STGD1 probands. We selected those variants with predicted delta scores for acceptor/donor gain > 0.20, and no previous reports on their effect on splicing. Fifteen ABCA4 variants were selected, four of which were predicted to create a new splice acceptor site and eleven to create a new splice donor site. In addition, three variants of interest with delta scores < 0.20 were included. The variants were introduced in wild-type midigenes that contained 4 to 12 kb of ABCA4 genomic sequence, which were subsequently expressed in HEK293T cells. By using RT-PCR and Sanger sequencing, we identified splice aberrations for 16 of 18 analyzed variants. SpliceAI correctly predicted the outcomes for 16 out of 18 variants, illustrating its reliability in predicting the impact of coding ABCA4 variants on splicing. Our findings highlight a causal role for coding ABCA4 variants in splicing aberrations, improving the severity assessment of missense and synonymous ABCA4 variants, and guiding to new treatment strategies for STGD1.

show abstract

Section: Resultssupporting

confidence: 92%

Stargardt disease-associated missense and synonymous ABCA4 variants result in aberrant splicing

Kaltak

Corradi

Collin

et al. 2023

Human Molecular Genetics

View full text Add to dashboard Cite

show abstract

“…Previous studies also revealed common intronic variants of the ABCA4 gene, including the mutations c.5461-10T>C and c.4773+3A>G 3,4 . Subsequent in vitro studies showed that both variants cause aberrant alternative splicing of the ABCA4 gene; c.5461-10T>C causes skipping of exon 39 and 40 5,6 , while c.4773+3A>G causes skipping of exon 33 and 34 7,8 .…”

Section: Introductionmentioning

confidence: 99%

“…Subsequent in vitro studies showed that both variants cause aberrant alternative splicing of the ABCA4 gene; c.5461-10T>C causes skipping of exon 39 and 40 5,6 , while c.4773+3A>G causes skipping of exon 33 and 34 7,8 .…”

Section: Introductionmentioning

confidence: 99%

Using RNA-targeting CRISPR-Cas13 and engineered U1 systems to reduceABCA4splice variants in Stargardt disease

Liou,

Urrutia-Cabrera,

Westin

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

Dysregulation of the alternative splicing process results in aberrant mRNA transcripts, leading to dysfunctional proteins or nonsense-mediated decay that cause a wide range of mis-splicing diseases. Development of therapeutic strategies to target the alternative splicing process could potentially shift the mRNA splicing from disease isoforms to a normal isoform and restore functional protein. As a proof of concept, we focus on Stargardt disease (STGD1), an autosomal recessive inherited retinal disease caused by biallelic genetic variants in the ABCA4 gene. The splicing variants c.5461-10T>C and c.4773+3A>G in ABCA4 cause the skipping of exon 39-40 and exon 33-34 respectively. In this study, we compared the efficacy of different RNA-targeting systems to modulate these ABCA4 splicing defects, including four CRISPR-Cas13 systems (CASFx-1, CASFx-3, RBFOX1N-dCas13e-C and RBFOX1N-dPspCas13b-C) as well as an engineered U1 system (ExSpeU1). Using a minigene system containing ABCA4 variants in the human retinal pigment epithelium ARPE19, our results show that RBFOX1N-dPspCas13b-C is the best performing CRISPR-Cas system, which enabled up to 80% reduction of the mis-spliced ABCA4 c.5461-10T>C variants and up to 78% reduction of the ABCA4 c.4773+3A>G variants. In comparison, delivery of a single ExSpeU1 was able to effectively reduce the mis-spliced ABCA4 c.4773+3A>G variants by up to 84%. We observed that the effectiveness of CRISPR-based and U1 splicing regulation is strongly dependent on the sgRNA/snRNA targeting sequences, highlighting that optimal sgRNA/snRNA designing is crucial for efficient targeting of mis-spliced transcripts. Overall, our study demonstrated the potential of using RNA-targeting CRISPR-Cas technology and engineered U1 to reduce mis-spliced transcripts for ABCA4, providing an important step to advance the development of gene therapy to treat STGD1.

show abstract

Advances towards personalized therapies for Stargardt disease

Huang,

McLenachan,

Chen

2023

Expert Review of Ophthalmology

View full text Add to dashboard Cite

Characterising splicing defects of ABCA4 variants within exons 13–50 in patient-derived fibroblasts

Cited by 4 publications

References 48 publications

Stargardt disease-associated missense and synonymous ABCA4 variants result in aberrant splicing

Stargardt disease-associated missense and synonymous ABCA4 variants result in aberrant splicing

Using RNA-targeting CRISPR-Cas13 and engineered U1 systems to reduceABCA4splice variants in Stargardt disease

Advances towards personalized therapies for Stargardt disease

Contact Info

Product

Resources

About