Original articlePhotoreceptor Progenitor mRNA Analysis Reveals Exon Skipping Resulting from the ABCA4 c.5461-10T→C Mutation in Stargardt Disease
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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2022, Experimental Eye ResearchCitation Excerpt :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.
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
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Both authors contributed equally as senior authors.