PDZD7 is a modifier of retinal disease and a contributor to digenic Usher syndrome
Inga Ebermann, Jennifer B. Phillips, Max C. Liebau, Robert K. Koenekoop, Bernhard Schermer, Irma Lopez, Ellen Schäfer, Anne-Francoise Roux, Claudia Dafinger, Antje Bernd, Eberhart Zrenner, Mireille Claustres, Bernardo Blanco, Gudrun Nürnberg, Peter Nürnberg, Rebecca Ruland, Monte Westerfield, Thomas Benzing, Hanno J. Bolz
Inga Ebermann, Jennifer B. Phillips, Max C. Liebau, Robert K. Koenekoop, Bernhard Schermer, Irma Lopez, Ellen Schäfer, Anne-Francoise Roux, Claudia Dafinger, Antje Bernd, Eberhart Zrenner, Mireille Claustres, Bernardo Blanco, Gudrun Nürnberg, Peter Nürnberg, Rebecca Ruland, Monte Westerfield, Thomas Benzing, Hanno J. Bolz
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Research Article Ophthalmology

PDZD7 is a modifier of retinal disease and a contributor to digenic Usher syndrome

Abstract

Usher syndrome is a genetically heterogeneous recessive disease characterized by hearing loss and retinitis pigmentosa (RP). It frequently presents with unexplained, often intrafamilial, variability of the visual phenotype. Although 9 genes have been linked with Usher syndrome, many patients do not have mutations in any of these genes, suggesting that there are still unidentified genes involved in the syndrome. Here, we have determined that mutations in PDZ domain–containing 7 (PDZD7), which encodes a homolog of proteins mutated in Usher syndrome subtype 1C (USH1C) and USH2D, contribute to Usher syndrome. Mutations in PDZD7 were identified only in patients with mutations in other known Usher genes. In a set of sisters, each with a homozygous mutation in USH2A, a frame-shift mutation in PDZD7 was present in the sister with more severe RP and earlier disease onset. Further, heterozygous PDZD7 mutations were present in patients with truncating mutations in USH2A, G protein–coupled receptor 98 (GPR98; also known as USH2C), and an unidentified locus. We validated the human genotypes using zebrafish, and our findings were consistent with digenic inheritance of PDZD7 and GPR98, and with PDZD7 as a retinal disease modifier in patients with USH2A. Pdzd7 knockdown produced an Usher-like phenotype in zebrafish, exacerbated retinal cell death in combination with ush2a or gpr98, and reduced Gpr98 localization in the region of the photoreceptor connecting cilium. Our data challenge the view of Usher syndrome as a traditional Mendelian disorder and support the reclassification of Usher syndrome as an oligogenic disease.

Authors

Inga Ebermann, Jennifer B. Phillips, Max C. Liebau, Robert K. Koenekoop, Bernhard Schermer, Irma Lopez, Ellen Schäfer, Anne-Francoise Roux, Claudia Dafinger, Antje Bernd, Eberhart Zrenner, Mireille Claustres, Bernardo Blanco, Gudrun Nürnberg, Peter Nürnberg, Rebecca Ruland, Monte Westerfield, Thomas Benzing, Hanno J. Bolz

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Figure 1

PDZD7 encodes a homolog of harmonin and whirlin localizing to the ciliary base.

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PDZD7 encodes a homolog of harmonin and whirlin localizing to the cilia...
(A) Genomic structure of PDZD7. Mutations are in red. Gray-shaded triangles illustrate relations of exons to protein regions in B. (B) PDZD7 protein isoforms. Intron 8 contains 2 Alu retroelement insertions that modify splicing and produce a short N-terminal transcript (GenBank AK026862) (48). Exons 9 and 10 are alternatively spliced; exclusion from the mature mRNA shifts the reading frame and results in a 561 aa isoform. Ab, Position of epitope for antibody generation. (C) Overlapping PCR amplicons from human retinal cDNA. A, c.1–763 (exon 1 versus exon 5); B, c.512–1899 (exon 3 versus exon 12); C, c.1753–3102+41 (exon 11 versus 3′ UTR); M, 1 kb length standard (Alu-specific amplicon not shown). Arrows indicate primers in relation to the protein in B. The larger band in B represents full-length PDZD7; the smaller one results from exclusion of exons 9 and 10. Blue arrows in B depict primers used to amplify the sequence resulting from Alu-derived splicing that is specific for the first PDZD7 isoform shown (c.1391–Alu in intron 8). The sequences for all primers shown are given in Supplemental Table 2. (D) PDZD7 localizes to the ciliary base. In hTERT-RPE1 cells, costaining with acetylated tubulin revealed PDZD7 localization at the ciliary base and the nucleus. (E) Ciliary base staining was confirmed in costainings of PDZD7 and γ tubulin. Preincubation of the PDZD7 antibody with the immunizing peptide abolished this signal. To exclude a remaining weak signal after blocking peptide preincubation, exposure time was prolonged more than 3-fold (lower panel). Arrowheads indicate PDZD7 staining at the ciliary base. Scale bars: 5 μm.