ABCA4 midigenes reveal the full splice spectrum of all reported noncanonical splice site variants in Stargardt disease

Stargardt disease is caused by variants in the ABCA4 gene, a significant part of which are noncanonical splice site (NCSS) variants. In case a gene of interest is not expressed in available somatic cells, small genomic fragments carrying potential disease-associated variants are tested for splice abnormalities using in vitro splice assays. We recently discovered that when using small minigenes lacking the proper genomic context, in vitro results do not correlate with splice defects observed in patient cells. We therefore devised a novel strategy in which a bacterial artificial chromosome was employed to generate midigenes, splice vectors of varying lengths (up to 11.7 kb) covering almost the entire ABCA4 gene. These midigenes were used to analyze the effect of all 44 reported and three novel NCSS variants on ABCA4 pre-mRNA splicing. Intriguingly, multi-exon skipping events were observed, as well as exon elongation and intron retention. The analysis of all reported NCSS variants in ABCA4 allowed us to reveal the nature of aberrant splicing events and to classify the severity of these mutations based on the residual fraction of wild-type mRNA. Our strategy to generate large overlapping splice vectors carrying multiple exons, creating a toolbox for robust and high-throughput analysis of splice variants, can be applied to all human genes.

[1]  E. M. Jones,et al.  Many rare genetic variants have unrecognized large-effect disruptions to exon recognition , 2018 .

[2]  E. M. Jones,et al.  Large-scale screening of rare genetic variants in humans reveals frequent splicing disruptions , 2017, bioRxiv.

[3]  G. Houge,et al.  The intronic ABCA4 c.5461‐10T>C variant, frequently seen in patients with Stargardt disease, causes splice defects and reduced ABCA4 protein level , 2017, Acta ophthalmologica.

[4]  F. Cremers,et al.  In Silico Functional Meta‐Analysis of 5,962 ABCA4 Variants in 3,928 Retinal Dystrophy Cases , 2017, Human mutation.

[5]  Sri V. V. Deevi,et al.  Biallelic Mutation of ARHGEF18, Involved in the Determination of Epithelial Apicobasal Polarity, Causes Adult-Onset Retinal Degeneration. , 2017, American journal of human genetics.

[6]  Dorothy A. Thompson,et al.  Comprehensive Rare Variant Analysis via Whole-Genome Sequencing to Determine the Molecular Pathology of Inherited Retinal Disease. , 2017, American journal of human genetics.

[7]  F. Grassmann,et al.  Mutation Spectrum of the ABCA4 Gene in 335 Stargardt Disease Patients From a Multicenter German Cohort—Impact of Selected Deep Intronic Variants and Common SNPs , 2017, Investigative ophthalmology & visual science.

[8]  L. Schöls,et al.  Antisense Oligonucleotide Mediated Splice Correction of a Deep Intronic Mutation in OPA1 , 2016, Molecular therapy. Nucleic acids.

[9]  B. Cieply,et al.  The Musashi 1 Controls the Splicing of Photoreceptor-Specific Exons in the Vertebrate Retina , 2016, PLoS genetics.

[10]  J. Bennett,et al.  In vitro and in vivo rescue of aberrant splicing in CEP290-associated LCA by antisense oligonucleotide delivery. , 2016, Human molecular genetics.

[11]  Peter M. G. Munro,et al.  Identification and Correction of Mechanisms Underlying Inherited Blindness in Human iPSC-Derived Optic Cups , 2016, Cell stem cell.

[12]  F. Cremers,et al.  Photoreceptor Progenitor mRNA Analysis Reveals Exon Skipping Resulting from the ABCA4 c.5461-10T→C Mutation in Stargardt Disease. , 2016, Ophthalmology.

[13]  James Y. Zou.  Analysis of protein-coding genetic variation in 60,706 humans , 2015, Nature.

[14]  L. Hetterschijt,et al.  Antisense Oligonucleotide-based Splice Correction for USH 2 A-associated Retinal Degeneration Caused by a Frequent Deep-intronic Mutation , 2016 .

[15]  Georg Seelig,et al.  Learning the Sequence Determinants of Alternative Splicing from Millions of Random Sequences , 2015, Cell.

[16]  E. Stone,et al.  Basal exon skipping and genetic pleiotropy: A predictive model of disease pathogenesis , 2015, Science Translational Medicine.

[17]  E. Velasco,et al.  Functional Classification of BRCA2 DNA Variants by Splicing Assays in a Large Minigene with 9 Exons , 2015, Human mutation.

[18]  R. Molday,et al.  Differential Phospholipid Substrates and Directional Transport by ATP-binding Cassette Proteins ABCA1, ABCA7, and ABCA4 and Disease-causing Mutants*♦ , 2013, The Journal of Biological Chemistry.

[19]  Adam P. DeLuca,et al.  Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease , 2013, Human molecular genetics.

[20]  Kevin W Eliceiri,et al.  NIH Image to ImageJ: 25 years of image analysis , 2012, Nature Methods.

[21]  D. Scherman,et al.  AON-mediated Exon Skipping Restores Ciliation in Fibroblasts Harboring the Common Leber Congenital Amaurosis CEP290 Mutation , 2012, Molecular therapy. Nucleic acids.

[22]  A. D. den Hollander,et al.  Antisense Oligonucleotide (AON)-based Therapy for Leber Congenital Amaurosis Caused by a Frequent Mutation in CEP290 , 2012, Molecular therapy. Nucleic acids.

[23]  Emanuele Buratti,et al.  DBASS3 and DBASS5: databases of aberrant 3′- and 5′-splice sites , 2010, Nucleic Acids Res..

[24]  C. Béroud,et al.  Human Splicing Finder: an online bioinformatics tool to predict splicing signals , 2009, Nucleic acids research.

[25]  Michael Q. Zhang,et al.  An increased specificity score matrix for the prediction of SF2/ASF-specific exonic splicing enhancers. , 2006, Human molecular genetics.

[26]  D. Baralle,et al.  NF1 mRNA biogenesis: Effect of the genomic milieu in splicing regulation of the NF1 exon 37 region , 2006, FEBS letters.

[27]  J. Lupski,et al.  ABCA4 mutations causing mislocalization are found frequently in patients with severe retinal dystrophies. , 2005, Human molecular genetics.

[28]  F. Clark,et al.  Understanding alternative splicing: towards a cellular code , 2005, Nature Reviews Molecular Cell Biology.

[29]  Doree Sitkoff,et al.  models homology modeling : From sequence alignments to structural A comparative study of available software for high-accuracy , 2005 .

[30]  Meena Kishore Sakharkar,et al.  Distributions of exons and introns in the human genome , 2004, Silico Biol..

[31]  K. Osoegawa,et al.  BAC library construction. , 2004, Methods in molecular biology.

[32]  Jinhua Wang,et al.  ESEfinder: a web resource to identify exonic splicing enhancers , 2003, Nucleic Acids Res..

[33]  Christopher B. Burge,et al.  Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals , 2003, RECOMB '03.

[34]  S. Biswas,et al.  Biochemical Defects in Retina-specific Human ATP Binding Cassette Transporter Nucleotide Binding Domain 1 Mutants Associated with Macular Degeneration* , 2002, The Journal of Biological Chemistry.

[35]  W. J. Kent,et al.  BLAT--the BLAST-like alignment tool. , 2002, Genome research.

[36]  J. Lupski,et al.  Null missense ABCR (ABCA4) mutations in a family with stargardt disease and retinitis pigmentosa. , 2001, Investigative ophthalmology & visual science.

[37]  S. Salzberg,et al.  GeneSplicer: a new computational method for splice site prediction. , 2001, Nucleic acids research.

[38]  B. J. Klevering,et al.  Mutations in the ABCA4 (ABCR) gene are the major cause of autosomal recessive cone-rod dystrophy. , 2000, American journal of human genetics.

[39]  B. Lorenz,et al.  A comprehensive survey of sequence variation in the ABCA4 (ABCR) gene in Stargardt disease and age-related macular degeneration. , 2000, American journal of human genetics.

[40]  Jeremy Nathans,et al.  Biochemical defects in ABCR protein variants associated with human retinopathies , 2000, Nature Genetics.

[41]  L. Molday,et al.  ABCR expression in foveal cone photoreceptors and its role in Stargardt macular dystrophy , 2000, Nature Genetics.

[42]  K Rohrschneider,et al.  The 2588G-->C mutation in the ABCR gene is a mild frequent founder mutation in the Western European population and allows the classification of ABCR mutations in patients with Stargardt disease. , 1999, American journal of human genetics.

[43]  K Rohrschneider,et al.  Autosomal recessive retinitis pigmentosa and cone-rod dystrophy caused by splice site mutations in the Stargardt's disease gene ABCR. , 1998, Human molecular genetics.

[44]  J. Nathans,et al.  Stargardt's ABCR is localized to the disc membrane of retinal rod outer segments , 1997, Nature Genetics.

[45]  J. Lupski,et al.  A photoreceptor cell-specific ATP-binding transporter gene (ABCR) is mutated in recessive Starqardt macular dystrophy , 1997, Nature Genetics.

[46]  David Haussler,et al.  Improved splice site detection in Genie , 1997, RECOMB '97.

[47]  Marvin B. Shapiro,et al.  RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. , 1987, Nucleic acids research.

[48]  A Scialfa,et al.  [Fundus flavimaculatus]. , 1964, Giornale italiano di oftalmologia.