Material and Methods

Subjects and Clinical Evaluation
A nonconsanguineous Turkish family (family A) with three siblings affected by COD and CRD and an isolated Dutch individual (family B) with ACHM that evolved into a progressive retinal dystrophy were included in this study (Figure 1A). These families belong to a large cohort of individuals affected by ACHM (n = 21), COD (n = 110), or CRD (n = 112). Most of the probands are the only affected persons in their family, and they were ascertained in various ophthalmic centers in the Netherlands, Belgium, the United Kingdom, and Canada. The individuals diagnosed with ACHM had a history of congenital pendular nystagmus, reduced visual acuity, photophobia, and poor or absent color vision in early infancy and had absent cone function (but normal rod responses) on ERG. Individuals in our cohort were classified as having COD when they presented with a childhood or early-adult-onset progressive deterioration of visual acuity and color vision, reduced cone responses on ERG, and normal rod responses for ≥5 years.6 Inclusion criteria for CRD were progressive loss of central vision, color-vision disturbances, and a reduction of both cone (equally or more severely reduced) and rod responses on ERG.6
After identification of the genetic defect, the medical records of the affected individuals of families A and B were evaluated. Ophthalmologic examinations were performed on several occasions and included best-corrected visual acuity (Snellen chart), slit-lamp biomicroscopy, ophthalmoscopy, color-vision testing (Hardy-Rand-Rittler color-vision test and Lanthony panel D-15 tests), and visual-field testing using Goldmann kinetic perimetry (targets V-4e and I-4e to I-1e). Fundus photographs of both the macular area and the four peripheral quadrants were available for two individuals. ERG was performed according to the protocol of the International Society for Clinical Electrophysiology of Vision.16 Time-domain optical coherence tomography (OCT; Stratus OCT 3000, Carl Zeiss Meditec) was obtained in one affected individual (A-II:4), and spectral-domain OCT (SD-OCT; Heidelberg Spectralis HRA+OCT, Heidelberg Engineering; 30° single-line scans, ten frames per line) was obtained in proband B-II:1. The acquisition of SD-OCT was limited as a result of nystagmus. This study was approved by the institutional review boards of the participating centers and adhered to the tenets of the Declaration of Helsinki. All subjects provided written informed consent prior to participation in the study.

Exome Sequencing and Variant Identification
A SOLiD4 sequencing platform (Life Technologies) was utilized for WES in 12 CRD- or COD-affected probands from families with suspected autosomal-recessive inheritance, and the exomes were enriched according to the manufacturer’s protocol with the use of the SureSelect Human All Exon v.2 Kit (50 Mb), containing the exonic sequences of approximately 21,000 genes (Agilent Technologies). LifeScope software v.2.1 (Life Technologies) was used for mapping color space reads along the hg19 reference genome assembly (UCSC Genome Browser). Single-nucleotide variants were called by high-stringency calling with the DiBayes algorithm. Small insertions and deletions were detected with the SOLiD Small Indel Tool (Life Technologies). For individual A-II:1, 69,686,646 reads were uniquely mapped to the gene-coding regions; median coverage was 58.2×, and there were 44,784 sequence variants. For proband B-II:1, 75,493,298 reads were uniquely mapped to the gene-coding regions; median coverage was 66.7×, and there were 47,304 sequence variants. For validating the WES sequence variants and excluding the presence of other POC1B mutations, all coding exons and exon-intron boundaries of POC1B were amplified with primers designed with Primer 3 software (Table S1, available online).

mRNA Analysis by RT-PCR
For assessing the effect of c.810+1G>T on the POC1B transcript, total RNA was isolated from peripheral-blood cells from the affected person B-II:1 and three control individuals according to the manufacturer’s (QIAGEN) protocol. The peripheral-blood cells of B-II:1 and the control individuals were cultured according to standard procedures with the use of phytohaemagglutinine. The leukocytes of the affected individual were grown for 4–6 hr with or without cycloheximide for visualization of the effect of the mutation and possible degradation of nonsense-containing mRNAs by nonsense-mediated decay.17 With the use of a hypotone osmotic shock and centrifugation, the leucocytes were separated from the erythrocytes. Reverse transcription with iScript (Biorad) was performed on 1 μg of total RNA. RT-PCR experiments were performed with 2.5 μl cDNA with primers in exons 5 and 9 (Table S2) (35 cycles) and were followed by Sanger sequencing using a 3100 or 3730 DNA Analyzer (Applied Biosystems).

Zebrafish Morpholino Knockdown
Tupfel long fin zebrafish were bred and raised under standard conditions.18 All experiments were carried out in accordance with European guidelines on animal experiments (2010/63/EU). Zebrafish eggs were obtained from natural spawning. Antisense morpholino oligonucleotides (MOs) blocking the translation (5′-GATCCTCCATTACAGACGCCATGAT-3′) or the 5′ splice site of exon 2 (5′-AAGTTCTCTGTCTTATTCAGGAGGA-3′) of poc1b15 were obtained from GeneTools. A MO directed against a human β-globin intronic mutation (5′-CCTCTTACCTCAGTTACAATTTATA-3′) was used as a standard negative control. For MO knockdown, 1 nl of diluted MO (ranging from 2 to 10 ng) was injected into the yolk of 1- to 2-cell-stage embryos with a Pneumatic PicoPump pv280 (World Precision Instruments). A minimum sample size of 80 larvae was used in each injection experiment. After injection, embryos were raised at 28.5°C in E3 embryo medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4) supplemented with 0.1% (w/v) methylene blue. At 4 days postfertilization (dpf), embryos were divided into two groups on the basis of the presence of the ocular phenotype described by Pearson et al.15 (<15 arbitrary units on a stereomicroscope’s ocular scale bar; Zeiss). For both groups of embryos, as well as injected and uninjected controls, optokinetic responses (OKRs) were measured and histological analysis of the retina was performed.
cDNAs encoding human wild-type and variant (p.Gln67del and p.Arg106Pro) proteome of centriole 1B (POC1B, previously Pix1) were cloned in pCS2+/DEST with the use of Gateway Technology (Life Technologies), linearized, and used as templates for in vitro transcription. mRNAs were prepared with the mMESSAGE mMACHINE Kit (Life Technologies) according to the manufacturer’s instructions. A dose of 100 pg mRNA was injected with the MOs as described above.

Zebrafish OKR Assay
The OKR was measured by a previously described method.19 Zebrafish larvae were mounted in an upright position in 3% methylcellulose in a small Petri dish. The Petri dish was placed on a platform surrounded by a rotating drum 8 cm in diameter. A pattern of alternating black and white vertical stripes was displayed on the drum interior (each stripe subtended an angle of 36°). Larvae (4 dpf) were visualized through a stereomicroscope positioned over the drum and illuminated with fiberoptic lights. Eye movements were recorded while larvae were optically stimulated by the rotating stripes. Larvae were subjected to a protocol of a 30 s counterclockwise rotation, a 10 s rest, and a 30 s clockwise rotation. Thereafter, the larvae were washed out of the methylcellulose and fixed for histological analysis.

Staining of Photoreceptor Outer Segments in Zebrafish Embryos
Larvae (4 dpf) tested for OKR were fixed in 4% formaldehyde in PBS for 24 hr, dehydrated through graded ethanol steps from 70% to 100%, and embedded via a standard protocol in glycol methacrylate. The eyes were sectioned (2 μm), stained with boron-dipyrromethene (1:10,000 in PBS) for membranes and with DAPI (1:8,000) for nuclei, and imaged with a Zeiss Axio Imager Z1 fluorescence microscope.

Immunostaining and Microscopy
Zebrafish and rat samples for immunohistochemistry (unfixed and fixed in 4% paraformaldehyde) were rinsed in 30% sucrose in PBS and directly frozen in Tissue Tek in melting isopentane. Cryosections of unfixed adult zebrafish and rat eyes (7 μm) were stained for Poc1b with anti-hsPOC1B (1:50; generously provided by Chad Pearson and Mary Pinter, University of Colorado) as described for cultured cells by Pearson et al.15 Sections were counterstained with GT335 (1:100; mouse monoclonal antibody against polyglutamylated tubulin, kindly provided by Carsten Janke, Centre National de la Recherche Scientifique Centre de Recherches en Biochimie Macromoleculaire). Sections of fixed zebrafish morphants were stained for green and red double cones with a mouse monoclonal antibody (zpr-1, raised against Arr3a, 1:500; ZIRC) and for rods with anti-rhodopsin (1:500; Novus Biologicals). Sections were washed with PBS, permeabilized for 20 min in 0.01% (v/v) Tween-20 in PBS, and washed again. Next, sections were blocked with 10% normal goat serum and 2% BSA in PBS, and primary antibodies were incubated overnight at 4°C in blocking buffer. After washing with PBS, secondary antibodies were incubated in blocking buffer for 45 min at room temperature. Samples were counterstained with DAPI and mounted with Prolong Gold (Life Technologies). For all sections, goat anti-mouse or goat anti-rabbit (Alexa 488 or 568, respectively, 1:500; Life Technologies) secondary antibody was used.

cDNA Constructs
All expression constructs were created with Gateway Technology (Life Technologies) according to the manufacturer’s instructions. These constructs encoded 3×HA-POC1B wild-type and variants (p.Arg106Pro and p.Gln67del) and 3×FLAG-FAM161A for coimmunoprecipitation and encoded monomeric red fluorescent protein (mRFP)-POC1B wild-type and variants and PalmMyr-CFP-FAM161A for colocalization studies. cDNA constructs encoding the full-length POC1B of 478 amino acids (POC1B; RefSeq accession numbers NM_172240.2 [gene] and NP_758440.1 [protein]) or different fragments thereof were generated by Gateway-adapted PCR and subsequently cloned.20 The first fragment (POC1B-WD40) spanned amino acids 1–297 and contained the WD40 domain. The second fragment (POC1B-SR) spanned amino acids 298–426 and did not hold any known domains. The third fragment (POC1B-CC) spanned amino acids 427–478 and contained a single coiled-coil domain (Figure S1). Constructs encoding POC1B variants p.Gln67del and p.Arg106Pro were generated by site-directed mutagenesis PCR. Constructs encoding the full-length FAM161A were generated from a full-length FAM161A clone.21 The sequence of all entry clones was verified by Sanger sequencing.

Yeast Two-Hybrid Assay
The GAL4-based yeast two-hybrid system (HybriZAP, Stratagene) was used for identifying binary protein-protein interaction partners of POC1B. The construct encoding the full-length POC1B and the three constructs encoding fragments of POC1B were fused to a DNA binding domain (GAL4-BD) and used as bait for screening a human oligo-dT-primed retinal cDNA library. The yeast strain PJ69-4A, which carries the HIS3 (histidine), ADE2 (adenine), and LacZ (β-galactosidase) reporter genes, was used as a host. Interactions were analyzed by assessment of reporter gene (HIS3 and ADE2) activation via growth on selective media and β-galactosidase colorimetric filter lift assays (LacZ reporter gene). cDNA inserts of clones containing putative interaction partners were confirmed by Sanger sequencing.

Localization in hTERT-RPE1 Cells
Human TERT-immortalized retinal pigment epithelium 1 (hTERT-RPE1) cells were cultured as previously described.22 Cells were seeded on coverslips, grown to 80% confluency, and subsequently serum starved for 24 hr in medium containing only 0.2% fetal calf serum for inducing cilium growth. The cells were then cotransfected with constructs encoding mRFP-POC1B (wild-type or variants) and PalmMyr-CFP-FAM161A (wild-type) with the use of Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. Cells were fixed in 4% paraformaldehyde for 20 min, treated with 1% Triton X-100 in PBS for 5 min, and blocked in 2% BSA in PBS for 20 min. Cells were incubated with the primary antibody GT335 (cilium and basal body marker, 1:500) and α-RPGRIP1L (SNC039 + SNC040,23 transition zone marker, 1:500), diluted in 2% BSA in PBS, for 1 hr. After washing in PBS, the cells were incubated with the secondary antibody for 45 min. Secondary antibodies goat anti-mouse, goat anti-guinea pig, and goat anti-rabbit (Alexa 488, 568, and 647, respectively, 1:500; Life Technologies) were diluted in 2% BSA in PBS. Cells were washed with PBS and briefly with milliQ before being mounted in Vectashield containing DAPI (Vector Laboratories). The cellular localization of wild-type and variant POC1B proteins was analyzed with a Zeiss Axio Imager Z1 fluorescence microscope equipped with a 63× objective lens. Optical sections were generated through structured illumination by the insertion of an ApoTome slider into the illumination path and subsequent processing with AxioVision (Zeiss) and Photoshop CS4 (Adobe Systems) software.

Coimmunoprecipitation
3×HA-POC1B (wild-type and variants) and 3×FLAG-FAM161A were cosynthesized in human embryonic kidney 293T (HEK293T) cells. As a negative control, the functionally unrelated p63 was cosynthesized with both POC1B and FAM161A. As positive controls, the previously described interactions between nephrocystin-4 (encoded by NPHP4 [MIM 607215]) and RPGRIP124 and between lebercilin (encoded by LCA5 [MIM 611408]) and FAM161A were used.21 After 48 hr of expression of these genes, cells were lysed on ice in lysis buffer (50 mM Tris-HCL [pH 7.5], 150 mM NaCl, and 0.5% Triton X-100) supplemented with complete protease inhibitor cocktail (Roche). Lysates were incubated with anti-HA affinity matrix (Roche) or with anti-FLAG M2 agarose from mouse (Sigma-Aldrich) for 5 hr at 4°C. After incubation, beads with bound protein complexes were washed in lysis buffer and subsequently taken up in 4× NuPAGE Sample Buffer and heated for 10 min at 70°C. Beads were precipitated by centrifugation, and supernatant was run on a NuPAGE Novex 4%–12% Bis-Tris SDS-PAGE gel. The interaction between 3×HA-POC1B and 3×FLAG-FAM161A was assessed by immunoblotting, followed by staining with either monoclonal mouse anti-HA or monoclonal mouse anti-FLAG (1:1,000; Sigma-Aldrich) as a primary antibody and goat anti-mouse IRDye800 (1:20,000; Li-Cor) as a secondary antibody. Fluorescence was analyzed on a Li-Cor Odyssey 2.1 infrared scanner.