PDZD7 is a modifier of retinal disease and a contributor to digenic Usher syndrome - PubMed (original) (raw)

. 2010 Jun;120(6):1812-23.

doi: 10.1172/JCI39715. Epub 2010 May 3.

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

Affiliations

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

Inga Ebermann et al. J Clin Invest. 2010 Jun.

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.

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Figures

Figure 1

Figure 1. PDZD7 encodes a homolog of harmonin and whirlin localizing to the ciliary base.

(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.

Figure 2

Figure 2. PDZD7 mutations in Usher syndrome type 2 families.

(A) Homozygosity for p.C1447QfsX29_USH2A_ in FCa and FCb. The retinal phenotype is more severe in FCa, who carries a de novo PDZD7 mutation, p.R56PfsX24. (B) GER1 is double heterozygous for p.R1505SfsX7_USH2A_ and a PDZD7 splice site alteration (causing in-frame inclusion of either 16 or 68 unrelated amino acids). The latter may represent a benign variant that does not contribute to the phenotype. (C) Evidence for digenic inheritance due to double heterozygosity for truncating mutations in GPR98 (USH2C), p.A5713LfsX3, and PDZD7, p.C732LfsX18, in GER2. The healthy sister carries the PDZD7 deletion but not the GPR98 mutation. (D) In family U329, 4 siblings carry the PDZD7 mutation p.R56PfsX24 in heterozygous state. Microsatellite marker and sequence analyses largely excluded a second mutation in PDZD7. The index patient, U329-1, carries a heterozygous missense change, p.L3160F, in MYO15A (not shown). Different PDZD7 alleles are indicated by colors. Samples from the parents (first generation cousins) were not available. U329-2 has high frequency hearing loss and a history of long-term working in a noisy environment. U329-4 has asymmetric hearing loss (mild/severe) that is probably unrelated to the hearing impairment in U329-1. IVS1 and IVS10 denote polymorphic CA-repeats (not annotated).

Figure 3

Figure 3. PDZD7 interacts with USH2C (GPR98) and USH2A.

(A) Schematic representation of the truncations used for coimmunoprecipitation experiments. (B and C) V5-tagged USH2C6158–6306 or USH2A4984–5202 was coexpressed with the 3 N-terminally FLAG-tagged PDZ domain truncations of PDZD7 or FLAG-tagged Eps1–225 as a negative control. Expression of proteins in cellular lysates was confirmed by immunoblotting with V5 and FLAG antibodies, respectively. After immunoprecipitation with FLAG antibody, coprecipitated V5-tagged USH2C6158–6306 or USH2A4984–5202 was detected with V5-specific antibodies. (D) In the reverse experiment, a V5-tagged PDZD7 truncation coprecipitated with FLAG-tagged USH2C6158–6306 or USH2A4984–5202. Deletion of the PDZ-binding motif (aa DTHL) in the cytoplasmic tails of USH2C and USH2A weakened the interaction with the PDZ2 domain of PDZD7.

Figure 4

Figure 4. The pdzd7 gene is duplicated in zebrafish and expressed in sensory cells.

(A) Genomic structure of pdzd7a and pdzd7b. Exon 8 of pdzd7a (purple) is alternatively spliced. The pdzd7a morpholino (red diamond) causes aberrant splicing. The Pdzd7 antibody recognizes 100 aa of the Pdzd7a PDZ1 domain (red bar). (B–E) RNA in situ hybridization. pdzd7a and pdzd7b are expressed in the retina (B, C) and in mechanosensory hair cells (am, anterior macula, shown in D, E). (F–J) Pdzd7a protein localization in sensory cells. (F) At 5dpf, the Pdzd7 antibody (red) labels the region near connecting cilia (short arrows). A subset of cells in the INL and GCL (long arrows) are also labeled. Synaptic layers, neurites, and cilia are visualized with anti-acetylated tubulin (green). Pdzd7a (red) localizes to the apical borders of macular cells (G) beneath the kinocilia (green, arrows) and in ciliated neuromast cells (H). High magnification views of Pdzd7a (red, brackets) at the base of the connecting cilium (green, arrows) in retinas of adult fish (I) and young mouse (J). Pdzd7a is also observed at lower levels in the outer segments of adult zebrafish photoreceptors (arrowhead in I). on, optic nerve; l, lens; onl, outer nuclear layer. (K) Western blot analysis of Pdzd7a protein from morpholino and control larvae. β-actin was used as a loading control. Scale bars: 50 μm (B–E); 20 μm (F); 10 μm (G, H); 20 μm (I); 5 μm (J).

Figure 5

Figure 5. Loss of Pdzd7a results in structural defects, retinal cell death, and protein mislocalization.

(A–D) Phalloidin-stained hair bundles of the anterior crista in 5dpf control (A, B) and _pdzd7a_MO-injected (C, D) larvae. Stereocilia in controls are upright and tapered (asterisks), whereas bundles are bent and disorganized after morpholino injection. (E) Graph of average number of dying photoreceptor cells, assayed by anti-active caspase-3 labeling, in control and morpholino-injected fish. Morpholino combinations and dosages (either full: 1.0; or half: 0.5) are indicated for each group. n = 20 for each category. *P ≤ 10–6 derived from Student’s t test comparing retinal cell death rates in morphant larvae to those of control animals. SEM is indicated by error bars for each group. (F–I) High magnification scans of photoreceptor cells in 5dpf larvae showing localization of Pdzd7 (F, G) and Gpr98 (H, I) antibodies (red) in control and _pdzd7a_MO injected larvae; acetylated tubulin in green in all panels. In the control, Pdzd7a (red, F) and Gpr98 (red, H) are present at the base of the connecting cilia (small arrows). Images taken from a single focal plane. Gpr98 is also present at the photoreceptor synapse (large arrows), where Pdzd7a is not detected. Labeling by both antibodies is reduced at the connecting cilium after _pdzd7a_MO injection (G, I), whereas synaptic localization is unaffected by pdzd7 knockdown. Scale bars: 10 μm (A, C); 5 μm (B, D); 5 μm (F–I).

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