Elsevier

Ophthalmology

Volume 123, Issue 6, June 2016, Pages 1375-1385
Ophthalmology

Original article
Photoreceptor Progenitor mRNA Analysis Reveals Exon Skipping Resulting from the ABCA4 c.5461-10T→C Mutation in Stargardt Disease

https://doi.org/10.1016/j.ophtha.2016.01.053Get rights and content

Purpose

To elucidate the functional effect of the ABCA4 variant c.5461-10T→C, one of the most frequent variants associated with Stargardt disease (STGD1).

Design

Case series.

Participants

Seventeen persons with STGD1 carrying ABCA4 variants and 1 control participant.

Methods

Haplotype analysis of 4 homozygotes and 11 heterozygotes for c.5461-10T→C and sequence analysis of the ABCA4 gene for a homozygous proband. Fibroblasts were reprogrammed from 3 persons with STGD1 into induced pluripotent stem cells, which were differentiated into photoreceptor progenitor cells (PPCs). The effect of the c.5461-10T→C variant on RNA splicing by reverse-transcription polymerase chain reaction was analyzed using PPC mRNA. In vitro assays were performed with minigene constructs containing ABCA4 exon 39. We analyzed the natural history and ophthalmologic characteristics of 4 persons homozygous for c.5461-10T→C.

Main Outcome Measures

Haplotype and rare variant data for ABCA4, RNA splice defects, age at diagnosis, visual acuity, fundus appearance, visual field, electroretinography (ERG) results, fluorescein angiography results, and fundus autofluorescence findings.

Results

The frequent ABCA4 variant c.5461-10T→C has a subtle effect on splicing based on prediction programs. A founder haplotype containing c.5461-10T→C was found to span approximately 96 kb of ABCA4 and did not contain other rare sequence variants. Patient-derived PPCs showed skipping of exon 39 or exons 39 and 40 in the mRNA. HEK293T cell transduction with minigenes carrying exon 39 showed that the splice defects were the result of the c.5461-10T→C variant. All 4 subjects carrying the c.5461-10T→C variant in a homozygous state showed a young age of STGD1 onset, with low visual acuity at presentation and abnormal cone ERG results. All 4 demonstrated severe cone–rod dystrophy before 20 years of age and were legally blind by 25 years of age.

Conclusions

The ABCA4 variant c.5461-10T→C is located on a founder haplotype lacking other disease-causing rare sequence variants. In vitro studies revealed that it leads to mRNA exon skipping and ABCA4 protein truncation. Given the severe phenotype in persons homozygous for this variant, we conclude that this variant results in the absence of ABCA4 activity.

Section snippets

Subjects and Clinical Evaluation

We ascertained 15 persons with STGD1 carrying the c.5461-10T→C variant in a homozygous state (patients 1–4) or compound heterozygous state (patients 5–15) from the Netherlands and Germany. In addition, we studied 2 individuals with STGD1 with a single ABCA4 allele (patients 16 and 17) and a control. Genotype data for patients 1 through 17 can be found in Supplemental Table 1 (available at www.aaojournal.org). This study was approved by the institutional review board and adhered to the tenets of

Clinical Characteristics of Patients Carrying the Homozygous c.5461-10T→C ABCA4 Mutation

Clinical characteristics of patients with the homozygous c.5461-10T→C variant are shown in Table 1. All patients had loss of central vision early in life and were diagnosed with STGD1 between 6 and 14 years of age. On follow-up, visual acuity showed a sharp decline to severe visual impairment within 2 or 3 years from first symptoms (Fig 1), leading to legal blindness (visual acuity, 20/500 or lower) in early adulthood. Figure 2 depicts fundus, fundus angiography, fundus autofluorescence, and

Discussion

By using stem cell technology and in vitro splice assays, we showed that the frequent ABCA4 c.5461-10T→C variant is associated with exon 39 or exons 39 and 40 skipping, which results in truncated ABCA4 proteins. Haplotype and sequence analysis in 15 persons with STGD1 strongly suggested that this variant is part of a shared founder haplotype spanning a sizeable part of the ABCA4 gene. In addition, no other rare single nucleotide variants were detected in this shared haplotype, which could

Acknowledgments

We thank Erwin van Wijk for providing the Gateway-adapted minigene vector.

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      Our finding on this splicing defect is also in line with the results of Sangermano et al. (2018) who reported exon 40 skipping in HEK293T cells expressing the c.5714+5G>A variant from a midigene construct. However, in contrast to our results, the RT-PCR products amplified from the COS-7 cell expressing this variant yielded a larger aberrantly spliced product that included 150-nt from intron 40Rivera et al, 2000. This divergence may be attributable to the relatively strong vector splice sites (Sangermano et al., 2018), the regulatory sequence near the specific exons investigated and differences in splicing machinery in the cell lines tested.

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    Supplemental material is available at www.aaojournal.org.

    Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

    Supported by the FP7-PEOPLE-2012-ITN programme EyeTN, Brussels, Belgium (agreement no.: 317472 [F.P.M.C.]); the Macula Vision Research Foundation, Philadelphia, PA (F.P.M.C.); the Nijmeegse Oogonderzoek Stichting, Nijmegen, the Netherlands (F.P.M.C. and C.B.H.); the MD Fonds, Utrecht, the Netherlands (C.B.H.); the Stichting A.F. Deutman Researchfonds Oogheelkunde, Nijmegen, the Netherlands (C.B.H.); the Foundation Fighting Blindness, Columbia, MD, (grant no.: TA-GT-0912-0582-RAD [R.W.J.C.]); the Ghent University Special Research Fund, Ghent, Belgium (grant no.: BOF15/GOA/011 [E.D.B.]); the Research Foundation-Flanders, Brussels, Belgium (grant no.: G0C6715N [E.D.B.], Doctoral Fellowship [M.B.], and Senior Clinical Investigator award [E.D.B.]); and the Funds for Research in Ophthalmology, Brussels, Belgium (M.B.).

    Author Contributions:

    Conception and design: Cremers, Albert

    Analysis and interpretation: Sangermano, Bax, Bauwens, van den Born, De Baere, Garanto, Collin, Goercharn-Ramlal, Cremers, Albert

    Data collection: Sangermano, Bax, Bauwens, van den Born, De Baere, Garanto, Collin, den Engelsman-van Dijk, Rohrschneider, Hoyng

    Obtained funding: Cremers

    Overall responsibility: Sangermano, Bax, Bauwens, van den Born, De Baere, Garanto, Collin, Goercharn-Ramlal, den Engelsman-van Dijk, Rohrschneider, Hoyng, Cremers, Albert

    Both authors contributed equally as senior authors.

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