- 作者：Riccardo Sangermano ; MSc<sup>1sup><sup> ; sup><sup>2sup> ; Nathalie M. Bax ; MD<sup>3sup> ; Miriam Bauwens ; MSc<sup>4sup> ; L. Ingeborgh van den Born ; MD ; PhD<sup>5sup> ; Elfride De Baere ; MD ; PhD<sup>4sup> ; Alejandro Garanto ; PhD<sup>1sup><sup> ; sup><sup>2sup> ; Rob W.J. Collin ; PhD<sup>1sup><sup> ; sup><sup>2sup> ; Angelique S.A. Goercharn-Ramlal ; PhD<sup>1sup> ; Anke H.A. den Engelsman-van Dijk ; BSc<sup>1sup> ; Klaus Rohrschneider ; MD ; PhD<sup>6sup> ; Carel B. Hoyng ; MD ; PhD<sup>3sup> ; Frans P.M. Cremers ; PhD<sup>1sup><sup> ; sup><sup>2sup><sup> ; sup><sup>&lowast ; sup><sup> ; sup><sup>Frans.Cremers@radboudumc.nl" class="auth_mail" title="E-mail the corresponding authorsup> ; Silvia Albert ; PhD<sup>1sup><sup> ; sup><sup>2sup><sup> ; sup><sup>&lowast ; sup><sup> ; sup><sup>Silvia.Albert@radboudumc.nl" class="auth_mail" title="E-mail the corresponding authorsup>
- 关键词：BCVA ; best-corrected visual acuity ; CRD ; cone&ndash ; rod dystrophy ; DAPI ; 4&prime ; 6-diamidino-2-phenylindole ; DMEM/F12 ; Dulbecco's Modified Eagle Medium ; Nutrient Mixture F-12 ; dNTPs ; deoxynucleotide triphosphates ; ERG ; electroretinography ; iPSC ; induced pluripotent stem cell ; PCR ; polymerase chain reaction ; PPC ; photoreceptor progenitor cell ; RT-PCR ; reverse-transcription polymerase chain reaction ; SNP ; single nucleotide polymorphism ; STGD1 ; autosomal recessive Stargardt disease
- 刊名：Ophthalmology
- 出版年：2016
- 出版时间：June 2016
- 年：2016
- 卷：123
- 期：6
- 页码：1375-1385
- 全文大小：1609 K
sSec_2">Design
spara0015">Case series.
sSec_3">Participants
spara0020">Seventeen persons with STGD1 carrying ABCA4 variants and 1 control participant.
sSec_4">Methods
spara0025">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.
sSec_5">Main Outcome Measures
spara0030">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.
sSec_6">Results
spara0035">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.
sSec_7">Conclusions
spara0040">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.