Cost‐effective molecular inversion probe‐based
            <i>ABCA4</i>
            sequencing reveals deep‐intronic variants in Stargardt disease

Khan, Mubeen; Cornelis, Stéphanie S.; Khan, Muhammad Imran; Elmelik, Duaa; Manders, Eline; Bakker, Sem; Derks, Ronny; Neveling, Kornelia; Vorst, Maartje van de; Gilissen, Christian; Meunier, Isabelle; Defoort, S.; Puech, Bernard; Devos, Aurore; Schulz, Heidi L.; Stöhr, Heidi; Graßmann, Felix; Weber, Bernhard H. F.; Dhaenens, Claire‐Marie; Cremers, Frans P.M.

doi:10.1002/humu.23787

Cited by 38 publications

(46 citation statements)

References 48 publications

(102 reference statements)

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“…Previously, we generated a library of 31 overlapping WT midigenes (BA1–BA31; Sangermano et al, ) (Khan et al, ). Through Gateway cloning and subsequent site‐directed mutagenesis, mutant constructs were generated for all 19 variants investigated in this study (Figure ).…”

Section: Methodsmentioning

confidence: 99%

Identification of splice defects due to noncanonical splice site or deep‐intronic variants in ABCA4

et al. 2019

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Pathogenic variants in the ATP‐binding cassette transporter A4 (ABCA4) gene cause a continuum of retinal disease phenotypes, including Stargardt disease. Noncanonical splice site (NCSS) and deep‐intronic variants constitute a large fraction of disease‐causing alleles, defining the functional consequences of which remains a challenge. We aimed to determine the effect on splicing of nine previously reported or unpublished NCSS variants, one near exon splice variant and nine deep‐intronic variants in ABCA4, using in vitro splice assays in human embryonic kidney 293T cells. Reverse transcription‐polymerase chain reaction and Sanger sequence analysis revealed splicing defects for 12 out of 19 variants. Four deep‐intronic variants create pseudoexons or elongate the upstream exon. Furthermore, eight NCSS variants cause a partial deletion or skipping of one or more exons in messenger RNAs. Among the 12 variants, nine lead to premature stop codons and predicted truncated ABCA4 proteins. At least two deep‐intronic variants affect splice enhancer and silencer motifs and, therefore, these conserved sequences should be carefully evaluated when predicting the outcome of NCSS and deep‐intronic variants.

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Section: Methodsmentioning

confidence: 99%

Identification of splice defects due to noncanonical splice site or deep‐intronic variants in ABCA4

et al. 2019

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“…A few deep intronic variants adjacent to other exons other than exon 2 have been reported as a cause of COL2A1 ‐associated diseases 45 . In fact, variants in deep intronic regions have been described as frequent causative variants in several other genes, such as CEP290 46 and ABCA4 47 . This kind of variants may not be detected by routine exome sequencing.…”

Section: Discussionmentioning

confidence: 99%

A novel deep intronic COL2A1 mutation in a family with early‐onset high myopia/ocular‐only Stickler syndrome

Sun

Xiao

et al. 2020

Ophthalmic Physiologic Optic

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Citation information: Sun W, Xiao X, Li S, Jia X, & Zhang Q. A novel deep intronic COL2A1 mutation in a family with early-onset high myopia/ ocular-only Stickler syndrome. Ophthalmic Physiol Opt 2020; 40: 281-288. https://doi. AbstractPurpose: To identify the genetic defect causing early-onset high myopia (eoHM)/ ocular-only Stickler syndrome (ocular-STL) in a large Chinese family. Methods: Genomic DNA and clinical data from a four-generation family with eoHM/ocular-STL were collected. Whole-exome sequencing was performed on one affected member in initial screening. Linkage scan based on microsatellite markers was carried out initially from candidate loci associated with autosomal dominant eoHM and Stickler syndrome. Sanger sequencing was used to detect potential variants. The pathogenicity of candidate variants was evaluated using mini genes ex vivo. Results: Eight patients and five unaffected members in the family participated in the study, in which the patients had high myopia with other variable ocular phenotypes but without extraocular abnormalities. Whole exome sequencing did not detect any potential pathogenic variant in all genes known to associate with the disease. The eoHM/ocular-STL in the family was mapped to markers around COL2A1 by candidate loci linkage scan, with a maximum lod score of 3.31 for D12S1590 at θ = 0. A novel deep intronic variant, c.86-50C > G in intron 1 of COL2A1, was detected by Sanger sequencing and co-segregated with eoHM/ocular-STL in the family. Ex vivo splicing test using mini genes confirmed that the variant created a new splicing acceptor 49 bp before the canonical splicing site of exon 2, resulted in addition of 49 bp fragment in the transcript (from c.86-49 to c.86-1) and premature termination. Conclusions: Linkage study, bioinformatics prediction, and ex vivo transcript analysis suggest a novel deep intronic variant adjacent to 5-prime of exon 2 of COL2A1, affecting exon 2 splicing, as a potential cause of ocular-STL in a large family. To our knowledge, this is the first report of an intronic variant around exon 2 as a cause of ocular-STL while a series of variants in the coding region of exon 2, a dispensable alternative-splicing exon for extraocular tissues, in COL2A1 have been reported to cause Stickler syndrome-related ocular phenotype alone.

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“…by listing all 165 variants previously tested using in vitro splice assays (Braun et al 2013;Bauwens et al 2015;Sangermano et al 2016;Sangermano et al 2018;Bauwens et al 2019;Fadaie et al 2019;Khan et al 2019;Sangermano et al 2019) or RT-PCR analysis of patient-derived photoreceptor progenitor cells (PPCs) (Sangermano et al 2016;Albert et al 2018).…”

Section: Similarly We Detected Previously Reported Causal Splice Varmentioning

confidence: 99%

“…Thus far, 1,180 unique ABCA4 variants have been reported in 8,777 alleles of 6,684 cases (Cornelis et al 2017) (www.lovd.nl/ABCA4). A large proportion of the variants affect non-canonical splice site (NCSS) sequences, with variable effects on mRNA processing (Schulz et al 2017;Sangermano et al 2018;Khan et al 2019). In addition, several deep-intronic (DI) variants have been identified (Braun et al 2013;Zernant et al 2014;Bauwens et al 2015;Bax et al 2015;Bauwens et al 2019;Fadaie et al 2019;Khan et al 2019;Sangermano et al 2019).…”

Section: Introductionmentioning

confidence: 99%

“…In Supplemental Table S16, we listed all published 349 DI variant alleles (Braun et al 2013;Zernant et al 2014;Bauwens et al 2015;Bax et al 2015;Schulz et al 2017;Albert et al 2018;Zernant et al 2018;Bauwens et al 2019;Fadaie et al 2019;Khan et al 2019;Nassisi et al 2019;Sangermano et al 2019). The three most frequent are c.4253+43G>A (n=100), c.4539+2001G>A (n=64) and c.5196+1137G>A (n=47).…”

Section: Current State Of Knowledge On Deep-intronic Variants In Abca4mentioning

confidence: 99%

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Resolving the dark matter of ABCA4 for 1,054 Stargardt disease probands through integrated genomics and transcriptomics

Khan¹,

Cornelis²,

Pozo‐Valero³

et al. 2019

Preprint

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Missing heritability in human diseases represents a major challenge. Although whole-genome sequencing enables the analysis of coding and non-coding sequences, substantial costs and data storage requirements hamper its large-scale use to (re)sequence genes in genetically unsolved cases.The ABCA4 gene implicated in Stargardt disease (STGD1) has been studied extensively for 22 years, but thousands of cases remained unsolved. Therefore, single molecule molecular inversion probes were designed that enabled an automated and cost-effective sequence analysis of the complete 128-kb ABCA4 gene. Analysis of 1,054 unsolved STGD and STGD-like probands resulted in bi-allelic variations in 448 probands. Twenty-seven different causal deep-intronic variants were identified in 117 alleles. Based on in vitro splice assays, the 13 novel causal deep-intronic variants were found to result in pseudo-exon (PE) insertions (n=10) or exon elongations (n=3). Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions that were accompanied by flanking exon deletions. Structural variant analysis revealed 11 distinct deletions, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. Integrated complete gene sequencing combined with transcript analysis, identified pathogenic deep-intronic and structural variants in 26% of bi-allelic cases not solved previously by sequencing of coding regions. This strategy serves as a model study that can be applied to other inherited diseases in which only one or a few genes are involved in the majority of cases.

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Cost‐effective molecular inversion probe‐based ABCA4 sequencing reveals deep‐intronic variants in Stargardt disease

Cited by 38 publications

References 48 publications

Identification of splice defects due to noncanonical splice site or deep‐intronic variants in ABCA4

Identification of splice defects due to noncanonical splice site or deep‐intronic variants in ABCA4

A novel deep intronic COL2A1 mutation in a family with early‐onset high myopia/ocular‐only Stickler syndrome

Resolving the dark matter of ABCA4 for 1,054 Stargardt disease probands through integrated genomics and transcriptomics

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