A Splicing Variant in RDH8 Is Associated with Autosomal Recessive Stargardt Macular Dystrophy

Abstract

Stargardt macular dystrophy is a genetic disorder, but in many cases, the causative gene remains unrevealed. Through a combined approach (whole-exome sequencing and phenotype/family-driven filtering algorithm) and a multilevel validation (international database searching, prediction scores calculation, splicing analysis assay, segregation analyses), a biallelic mutation in the RDH8 gene was identified to be responsible for Stargardt macular dystrophy in a consanguineous Italian family. This paper is a report on the first family in which a biallelic deleterious mutation in RDH8 is detected. The disease phenotype is consistent with the expected phenotype hypothesized in previous studies on murine models. The application of the combined approach to genetic data and the multilevel validation allowed the identification of a splicing mutation in a gene that has never been reported before in human disorders.

Keywords: RDH8; Stargardt disease; all-trans-retinol dehydrogenase; macular dystrophy; retinal disease.

Figure 1

Pedigree of the family. Genotypes for c.262+1G>A in RDH8 gene are reported below each tested individual. The affected sisters (II:2 and II:4) received a clinical diagnosis of Stargardt macular dystrophy. Their parents (I:1 and I:2) were reported as sighted but never evaluated. The healthy brother (II:5), son (III:4), and daughters (III:1, III:2, and III:3) of the affected sisters were evaluated by ophthalmologists who confirmed the absence of signs of macular disease.

Figure 2

(A,B) OCT imaging of the macular region showing subretinal deposits in the foveal region and thinning of the outer nuclear layers ((A): Right eye; (B): Left eye). (C) OCT imaging of the normal macular region.

Figure 3

Fundus autofluorescence showing perifoveal and peripheral flecks as well retinal pigment epithelium atrophy in the foveal and perifoveal region. RE: Right Eye, LE: Left Eye.

Figure 4

(A) Ganzfeld mixed rod–cone electroretinograms in both eyes. (B) Ganzfeld cone electroretinograms in both eyes. RE: Right eye, LE: left eye.

Figure 5

Minigene assay, Sanger sequencing of RHD8. (A) Agarose gel shows RT-PCR results of minigene assay for variant c.262+1G>A. Lane 1 shows an amplicon of 423 bp corresponding to a wild-type genotype (159 bp of normal splicing of exons 2 + 264 bp of pSPL3 exon); lane 2 shows an amplicon of 492 bp corresponding to abnormal splicing produced by mutation c.262+1G>A (492 bp [159 bp of normal splicing (exon 2) + 69 bp of partial retention of intron 2] + 264 bp of pSPL3 exon); lane 3 shows the amplification of pSPL3 without RHD8 cloning; lane 4 shows the amplification of HEK 293 T cDNA without transfection of pSPL3; and lane 5 shows negative control of PCR amplification. (B) Sanger sequence shows the normal sequence and the partial retention of intron 2. (C) Genomic sequence of RHD8 exon 2 is in blue capital letters, underlined in yellow is c.262+1G>A variant, and underlined in green is the partial retention of intron 2.