Publications by authors named "Stephanie S Cornelis"

12 Publications

  • Page 1 of 1

Association of Sex With Frequent and Mild ABCA4 Alleles in Stargardt Disease.

JAMA Ophthalmol 2020 10;138(10):1035-1042

Department of Human Genetics, Radboud University Medical Center, Nijmegen, the Netherlands.

Importance: The mechanisms behind the phenotypic variability and reduced penetrance in autosomal recessive Stargardt disease (STGD1), often a blinding disease, are poorly understood. Identification of the unknown disease modifiers can improve patient and family counseling and provide valuable information for disease management.

Objective: To assess the association of incompletely penetrant ABCA4 alleles with sex in STGD1.

Design, Setting, And Participants: Genetic data for this cross-sectional study were obtained from 2 multicenter genetic studies of 1162 patients with clinically suspected STGD1. Unrelated patients with genetically confirmed STGD1 were selected. The data were collected from June 2016 to June 2019, and post hoc analysis was performed between July 2019 and January 2020.

Main Outcomes And Measures: Penetrance of reported mild ABCA4 variants was calculated by comparing the allele frequencies in the general population (obtained from the Genome Aggregation Database) with the genotyping data in the patient population (obtained from the ABCA4 Leiden Open Variation Database). The sex ratio among patients with and patients without an ABCA4 allele with incomplete penetrance was assessed.

Results: A total of 550 patients were included in the study, among which the mean (SD) age was 45.7 (18.0) years and most patients were women (311 [57%]). Five of the 5 mild ABCA4 alleles, including c.5603A>T and c.5882G>A, were calculated to have incomplete penetrance. The women to men ratio in the subgroup carrying c.5603A>T was 1.7 to 1; the proportion of women in this group was higher compared with the subgroup not carrying a mild allele (difference, 13%; 95% CI, 3%-23%; P = .02). The women to men ratio in the c.5882G>A subgroup was 2.1 to 1, and the women were overrepresented compared with the group carrying no mild allele (difference, 18%; 95% CI, 6%-30%; P = .005).

Conclusions And Relevance: This study found an imbalance in observed sex ratio among patients harboring a mild ABCA4 allele, which concerns approximately 25% of all patients with STGD1, suggesting that STGD1 should be considered a polygenic or multifactorial disease rather than a disease caused by ABCA4 gene mutations alone. The findings suggest that sex should be considered as a potential disease-modifying variable in both basic research and clinical trials on STGD1.
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http://dx.doi.org/10.1001/jamaophthalmol.2020.2990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7441467PMC
October 2020

Resolving the dark matter of ABCA4 for 1054 Stargardt disease probands through integrated genomics and transcriptomics.

Genet Med 2020 07 20;22(7):1235-1246. Epub 2020 Apr 20.

Bartiméus Diagnostic Center for Complex Visual Disorders, Zeist, The Netherlands.

Purpose: Missing heritability in human diseases represents a major challenge, and this is particularly true for ABCA4-associated Stargardt disease (STGD1). We aimed to elucidate the genomic and transcriptomic variation in 1054 unsolved STGD and STGD-like probands.

Methods: Sequencing of the complete 128-kb ABCA4 gene was performed using single-molecule molecular inversion probes (smMIPs), based on a semiautomated and cost-effective method. Structural variants (SVs) were identified using relative read coverage analyses and putative splice defects were studied using in vitro assays.

Results: In 448 biallelic probands 14 known and 13 novel deep-intronic variants were found, resulting in pseudoexon (PE) insertions or exon elongations in 105 alleles. 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 and flanking exon deletions. Eleven distinct large deletions were found, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband.

Conclusion: Deep sequencing of ABCA4 and midigene-based splice assays allowed the identification of SVs and causal deep-intronic variants in 25% of biallelic STGD1 cases, which represents a model study that can be applied to other inherited diseases.
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http://dx.doi.org/10.1038/s41436-020-0787-4DOI Listing
July 2020

In or Out? New Insights on Exon Recognition through Splice-Site Interdependency.

Int J Mol Sci 2020 03 26;21(7). Epub 2020 Mar 26.

Department of Human Genetics, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands.

Noncanonical splice-site mutations are an important cause of inherited diseases. Based on in vitro and stem-cell-based studies, some splice-site variants show a stronger splice defect than expected based on their predicted effects, suggesting that other sequence motifs influence the outcome. We investigated whether splice defects due to human-inherited-disease-associated variants in noncanonical splice-site sequences in , , and could be rescued by strengthening the splice site on the other side of the exon. Noncanonical 5'- and 3'-splice-site variants were selected. Rescue variants were introduced based on an increase in predicted splice-site strength, and the effects of these variants were analyzed using in vitro splice assays in HEK293T cells. Exon skipping due to five variants in noncanonical splice sites of exons in , , and could be partially or completely rescued by increasing the predicted strengths of the other splice site of the same exon. We named this mechanism "splicing interdependency", and it is likely based on exon recognition by splicing machinery. Awareness of this interdependency is of importance in the classification of noncanonical splice-site variants associated with disease and may open new opportunities for treatments.
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http://dx.doi.org/10.3390/ijms21072300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7177576PMC
March 2020

Late-Onset Stargardt Disease Due to Mild, Deep-Intronic ABCA4 Alleles.

Invest Ophthalmol Vis Sci 2019 10;60(13):4249-4256

Department of Ophthalmology, Radboud University Medical Center, Nijmegen, The Netherlands.

Purpose: To investigate the role of two deep-intronic ABCA4 variants, that showed a mild splice defect in vitro and can occur on the same allele as the low penetrant c.5603A>T, in Stargardt disease (STGD1).

Methods: Ophthalmic data were assessed of 18 STGD1 patients who harbored c.769-784C>T or c.4253+43G>A in combination with a severe ABCA4 variant. Subjects carrying c.[769-784C>T; 5603A>T] were clinically compared with a STGD1 cohort previously published carrying c.5603A>T noncomplex. We calculated the penetrances of the intronic variants using ABCA4 allele frequency data of the general population and investigated the effect of c.769-784C>T on splicing in photoreceptor progenitor cells (PPCs).

Results: Mostly, late-onset, foveal-sparing STGD1 was observed among subjects harboring c.769-784C>T or c.4253+43G>A (median age of onset, 54.5 and 52.0 years, respectively). However, ages of onset, phenotypes in fundo, and visual acuity courses varied widely. No significant clinical differences were observed between the c.[769-784C>T; 5603A>T] cohort and the c.4253+43G>A or the c.5603A>T cohort. The penetrances of c.769-784C>T (20.5%-39.6%) and c.4253+43G>A (35.8%-43.1%) were reduced, when not considering the effect of yet unidentified or known factors in cis, such as c.5603A>T (identified in 7/7 probands with c.769-784C>T; 1/8 probands with c.4253+43G>A). Variant c.769-784C>T resulted in a pseudo-exon insertion in 15% of the total mRNA (i.e., ∼30% of the c.769-784C>T allele alone).

Conclusions: Two mild intronic ABCA4 variants could further explain missing heritability in late-onset STGD1, distinguishing it from AMD. The observed clinical variability and calculated reduced penetrance urge research into modifiers within and outside of the ABCA4 gene.
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http://dx.doi.org/10.1167/iovs.19-27524DOI Listing
October 2019

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

Hum Mutat 2019 12 3;40(12):2365-2376. Epub 2019 Sep 3.

Department of Human Genetics, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, Nijmegen, The Netherlands.

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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http://dx.doi.org/10.1002/humu.23890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899986PMC
December 2019

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

Hum Mutat 2019 10 18;40(10):1749-1759. Epub 2019 Jun 18.

Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.

Purpose: Stargardt disease (STGD1) is caused by biallelic mutations in ABCA4, but many patients are genetically unsolved due to insensitive mutation-scanning methods. We aimed to develop a cost-effective sequencing method for ABCA4 exons and regions carrying known causal deep-intronic variants.

Methods: Fifty exons and 12 regions containing 14 deep-intronic variants of ABCA4 were sequenced using double-tiled single molecule Molecular Inversion Probe (smMIP)-based next-generation sequencing. DNAs of 16 STGD1 cases carrying 29 ABCA4 alleles and of four healthy persons were sequenced using 483 smMIPs. Thereafter, DNAs of 411 STGD1 cases with one or no ABCA4 variant were sequenced. The effect of novel noncoding variants on splicing was analyzed using in vitro splice assays.

Results: Thirty-four ABCA4 variants previously identified in 16 STGD1 cases were reliably identified. In 155/411 probands (38%), two causal variants were identified. We identified 11 deep-intronic variants present in 62 alleles. Two known and two new noncanonical splice site variants showed splice defects, and one novel deep-intronic variant (c.4539+2065C>G) resulted in a 170-nt mRNA pseudoexon insertion (p.[Arg1514Lysfs*35,=]).

Conclusions: smMIPs-based sequence analysis of coding and selected noncoding regions of ABCA4 enabled cost-effective mutation detection in STGD1 cases in previously unsolved cases.
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http://dx.doi.org/10.1002/humu.23787DOI Listing
October 2019

Deep-intronic ABCA4 variants explain missing heritability in Stargardt disease and allow correction of splice defects by antisense oligonucleotides.

Genet Med 2019 08 15;21(8):1751-1760. Epub 2019 Jan 15.

Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.

Purpose: Using exome sequencing, the underlying variants in many persons with autosomal recessive diseases remain undetected. We explored autosomal recessive Stargardt disease (STGD1) as a model to identify the missing heritability.

Methods: Sequencing of ABCA4 was performed in 8 STGD1 cases with one variant and p.Asn1868Ile in trans, 25 cases with one variant, and 3 cases with no ABCA4 variant. The effect of intronic variants was analyzed using in vitro splice assays in HEK293T cells and patient-derived fibroblasts. Antisense oligonucleotides were used to correct splice defects.

Results: In 24 of the probands (67%), one known and five novel deep-intronic variants were found. The five novel variants resulted in messenger RNA pseudoexon inclusions, due to strengthening of cryptic splice sites or by disrupting a splicing silencer motif. Variant c.769-784C>T showed partial insertion of a pseudoexon and was found in cis with c.5603A>T (p.Asn1868Ile), so its causal role could not be fully established. Variant c.4253+43G>A resulted in partial skipping of exon 28. Remarkably, antisense oligonucleotides targeting the aberrant splice processes resulted in (partial) correction of all splicing defects.

Conclusion: Our data demonstrate the importance of assessing noncoding variants in genetic diseases, and show the great potential of splice modulation therapy for deep-intronic variants.
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http://dx.doi.org/10.1038/s41436-018-0414-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6752325PMC
August 2019

Author Response: Penetrance of the ABCA4 p.Asn1868Ile Allele in Stargardt Disease.

Invest Ophthalmol Vis Sci 2018 11;59(13):5566-5568

Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.

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http://dx.doi.org/10.1167/iovs.18-25944DOI Listing
November 2018

Identification and Analysis of Genes Associated with Inherited Retinal Diseases.

Methods Mol Biol 2019 ;1834:3-27

Department of Human Genetics, Donders Institute for Brain Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands.

Inherited retinal diseases (IRDs) display a very high degree of clinical and genetic heterogeneity, which poses challenges in finding the underlying defects in known IRD-associated genes and in identifying novel IRD-associated genes. Knowledge on the molecular and clinical aspects of IRDs has increased tremendously in the last decade. Here, we outline the state-of-the-art techniques to find the causative genetic variants, with special attention for next-generation sequencing which can combine molecular diagnostics and retinal disease gene identification. An important aspect is the functional assessment of rare variants with RNA and protein effects which can only be predicted in silico. We therefore describe the in vitro assessment of putative splice defects in human embryonic kidney cells. In addition, we outline the use of stem cell technology to generate photoreceptor precursor cells from patients' somatic cells which can subsequently be used for RNA and protein studies. Finally, we outline the in silico methods to interpret the causality of variants associated with inherited retinal disease and the registry of these variants.
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http://dx.doi.org/10.1007/978-1-4939-8669-9_1DOI Listing
April 2019

The Common ABCA4 Variant p.Asn1868Ile Shows Nonpenetrance and Variable Expression of Stargardt Disease When Present in trans With Severe Variants.

Invest Ophthalmol Vis Sci 2018 07;59(8):3220-3231

Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands.

Purpose: To assess the occurrence and the disease expression of the common p.Asn1868Ile variant in patients with Stargardt disease (STGD1) harboring known, monoallelic causal ABCA4 variants.

Methods: The coding and noncoding regions of ABCA4 were sequenced in 67 and 63 STGD1 probands respectively, harboring monoallelic ABCA4 variants. In case p.Asn1868Ile was detected, segregation analysis was performed whenever possible. Probands and affected siblings harboring p.Asn1868Ile without additional variants in cis were clinically evaluated retrospectively. Two asymptomatic siblings carrying the same ABCA4 variants as their probands were clinically examined. The penetrance of p.Asn1868Ile was calculated using allele frequency data of ABCA4 variants in non-Finnish European individuals.

Results: The p.Asn1868Ile variant was found in cis with known variants in 14/67 probands. In 27/67 probands, we identified p.Asn1868Ile without additional variants in cis, in combination with known, mainly severe ABCA4 variants. In 23/27 probands, the trans configuration was established. Among 27 probands and 6/7 STGD1 siblings carrying p.Asn1868Ile, 42% manifested late-onset disease (>44 years). We additionally identified four asymptomatic relatives carrying a combination of a severe variant and p.Asn1868Ile; ophthalmologic examination in two persons did not reveal STGD1. Based on ABCA4 allele frequency data, we conservatively estimated the penetrance of p.Asn1868Ile, when present in trans with a severe variant, to be below 5%.

Conclusions: A significant fraction of genetically unexplained STGD1 cases carries p.Asn1868Ile as a second variant. Our findings suggest exceptional differences in disease expression or even nonpenetrance of this ABCA4 variant, pointing toward an important role for genetic or environmental modifiers in STGD1.
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http://dx.doi.org/10.1167/iovs.18-23881DOI Listing
July 2018

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

Genome Res 2018 01 21;28(1):100-110. Epub 2017 Nov 21.

Department of Human Genetics and Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands.

Stargardt disease is caused by variants in the 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 gene. These midigenes were used to analyze the effect of all 44 reported and three novel NCSS variants on 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 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.
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http://dx.doi.org/10.1101/gr.226621.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749174PMC
January 2018

In Silico Functional Meta-Analysis of 5,962 ABCA4 Variants in 3,928 Retinal Dystrophy Cases.

Hum Mutat 2017 04 3;38(4):400-408. Epub 2017 Feb 3.

Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.

Variants in the ABCA4 gene are associated with a spectrum of inherited retinal diseases (IRDs), most prominently with autosomal recessive (ar) Stargardt disease (STGD1) and ar cone-rod dystrophy. The clinical outcome to a large degree depends on the severity of the variants. To provide an accurate prognosis and to select patients for novel treatments, functional significance assessment of nontruncating ABCA4 variants is important. We collected all published ABCA4 variants from 3,928 retinal dystrophy cases in a Leiden Open Variation Database, and compared their frequency in 3,270 Caucasian IRD cases with 33,370 non-Finnish European control individuals. Next to the presence of 270 protein-truncating variants, 191 nontruncating variants were significantly enriched in the patient cohort. Furthermore, 30 variants were deemed benign. Assessing the homozygous occurrence of frequent variants in IRD cases based on the allele frequencies in control individuals confirmed the mild nature of the p.[Gly863Ala, Gly863del] variant and identified three additional mild variants (p.(Ala1038Val), c.5714+5G>A, and p.(Arg2030Gln)). The p.(Gly1961Glu) variant was predicted to act as a mild variant in most cases. Based on these data, in silico analyses, and American College of Medical Genetics and Genomics guidelines, we provide pathogenicity classifications on a five-tier scale from benign to pathogenic for all variants in the ABCA4-LOVD database.
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http://dx.doi.org/10.1002/humu.23165DOI Listing
April 2017
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