Mutation ofPOC1Bin a Severe Syndromic Retinal Ciliopathy

We describe a consanguineous Iraqi family with Leber congenital amaurosis (LCA), Joubert syndrome (JBTS), and polycystic kidney disease (PKD). Targeted next-generation sequencing for excluding mutations in known LCA and JBTS genes, homozygosity mapping, and whole-exome sequencing identified a homozygous missense variant, c.317G>C (p.Arg106Pro), in POC1B, a gene essential for ciliogenesis, basal body, and centrosome integrity. In silico modeling suggested a requirement of p.Arg106 for the formation of the third WD40 repeat and a protein interaction interface. In human and mouse retina, POC1B localized to the basal body and centriole adjacent to the connecting cilium of photoreceptors and in …

Tackling the Limitations of Copolymeric Small Interfering RNA Delivery Agents by a Combined Experimental–Computational Approach

Despite the first successful applications of nonviral delivery vectors for small interfering RNA in the treatment of illnesses, such as the respiratory syncytial virus infection, the preparation of a clinically suitable, safe, and efficient delivery system still remains a challenge. In this study, we tackle the drawbacks of the existing systems by a combined experimental-computational in-depth investigation of the influence of the polymer architecture over the binding and transfection efficiency. For that purpose, a library of diblock copolymers with a molar mass of 30 kDa and a narrow dispersity (Đ1.12) was synthesized. We studied in detail the impact of an altered block size and/or compos…

KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome

Background Joubert syndrome (JBTS) and related disorders are defined by cerebellar malformation (molar tooth sign), together with neurological symptoms of variable expressivity. The ciliary basis of Joubert syndrome related disorders frequently extends the phenotype to tissues such as the eye, kidney, skeleton and craniofacial structures. Results Using autozygome and exome analyses, we identified a null mutation in KIAA0556 in a multiplex consanguineous family with hallmark features of mild Joubert syndrome. Patient-derived fibroblasts displayed reduced ciliogenesis potential and abnormally elongated cilia. Investigation of disease pathophysiology revealed that Kiaa0556-/- null mice possess…

A novel function of Huntingtin in the cilium and retinal ciliopathy in Huntington's disease mice

Huntington's disease (HD) is a neurodegenerative disorder caused by the toxic expansion of polyglutamine in the Huntingtin (HTT) protein. The pathomechanism is complex and not fully understood. Increasing evidence indicates that the loss of normal protein function also contributes to the pathogenesis, pointing out the importance of understanding the physiological roles of HTT. We provide evidence for a novel function of HTT in the cilium. HTT localizes in diverse types of cilia — including 9 + 0 non-motile sensory cilia of neurons and 9 + 2 motile multicilia of trachea and ependymal cells — which exert various functions during tissue development and homeostasis. In the photoreceptor cilium,…

Deep sequencing of the human retinae reveals the expression of odorant receptors

Several studies have demonstrated that the expression of odorant receptors (ORs) occurs in various tissues. These findings have served as a basis for functional studies that demonstrate the potential of ORs as drug targets for a clinical application. To the best of our knowledge, this report describes the first evaluation of the mRNA expression of ORs and the localization of OR proteins in the human retina that set a stage for subsequent functional analyses. RNA-Sequencing datasets of three individual neural retinae were generated using Next-generation sequencing and were compared to previously published but reanalyzed datasets of the peripheral and the macular human retina and to reference…

TALPID3 controls centrosome and cell polarity and the human ortholog KIAA0586 is mutated in Joubert syndrome (JBTS23)

Joubert syndrome (JBTS) is a severe recessive neurodevelopmental ciliopathy which can affect several organ systems. Mutations in known JBTS genes account for approximately half of the cases. By homozygosity mapping and whole-exome sequencing, we identified a novel locus, JBTS23, with a homozygous splice site mutation in KIAA0586 (alias TALPID3), a known lethal ciliopathy locus in model organisms. Truncating KIAA0586 mutations were identified in two additional patients with JBTS. One mutation, c.428delG (p.Arg143Lysfs*4), is unexpectedly common in the general population and may be a major contributor to JBTS. We demonstrate KIAA0586 protein localization at the basal body in human and mouse p…

Author response: TALPID3 controls centrosome and cell polarity and the human ortholog KIAA0586 is mutated in Joubert syndrome (JBTS23)

Additional file 1: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome

The phylogenetic distribution and sequence conservation of KIAA0556 orthologs in eukaryotes. Presence and sequence conservation of KIAA0556 are projected on the eukaryotic species tree to visualise the phylogenetic distribution of KIAA0556 orthologues as well as the distribution of the triple-repeat and quadruple-repeat configurations of the DUF4457 domains of unknown function. The black circles and white circles indicate which eukaryotic species contain or lack cilia/flagella. Recent KIAA0556 duplicates in Branchiostoma floridae and Paramecium tetraurelia are denoted by x2. *Dictyostelium discoideum protein sequence contains many â Nâ s (uncalled bases) in the N-terminal part of the sequen…

Additional file 1: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome

The phylogenetic distribution and sequence conservation of KIAA0556 orthologs in eukaryotes. Presence and sequence conservation of KIAA0556 are projected on the eukaryotic species tree to visualise the phylogenetic distribution of KIAA0556 orthologues as well as the distribution of the triple-repeat and quadruple-repeat configurations of the DUF4457 domains of unknown function. The black circles and white circles indicate which eukaryotic species contain or lack cilia/flagella. Recent KIAA0556 duplicates in Branchiostoma floridae and Paramecium tetraurelia are denoted by x2. *Dictyostelium discoideum protein sequence contains many â Nâ s (uncalled bases) in the N-terminal part of the sequen…

Additional file 6: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome

Results of the SF-TAP analysis with over-expressed N-terminally SF-TAP-tagged KIAA0556 in HEK293T cells. Shown is the number of unique identified peptides as well as the sequence coverage for each protein detected by mass spectrometry. Proteins identified in >1 out 17 SF-TAP control experiments (empty vector) were removed. (XLSX 28 kb)

Additional file 4: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome

IFT analysis in C. elegans K04F10.2( tm1830 ) mutants. a Intraflagellar transport rates in wild-type and K04F10.2(tm1830) mutant worms. Shown are the anterograde and retrograde velocities (μm.s-1/standard deviation (SD)) of GFP-tagged IFT proteins along amphid and phasmid channel cilia (combined; top rows), or phasmid cilia only (bottom rows). t-test pairwise comparison with wild-type controls, n number of particles, N measured number of amphids and phasmids. OSM-3 is the worm orthologue of KIF17; CHE-11 is the worm orthologue of IFT140; OSM-6 is the worm orthologue of IFT52. b Representative fluorescence images of phasmid cilia showing normal IFT protein localisations and distributions in …

Additional file 8: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome

Post-embryonic tissue expression of C. elegans katanin genes mei-1 , mei-2 and F47G4.4. Shown are fluorescence images of worms expressing a transcriptional GFP reporter under the control of the indicated geneâ s promoter, which stains the entire cell in which it is expressed. DiI (red) co-stain identifies six pairs of ciliated amphid neurons and both pairs of ciliated phasmid neurons. Arrowheads denote cells with both red and green signals. Other ciliated head cells are identifiable by long dendritic processes (arrows) extending to the anterior end of the worm. Scale bars, 20Â Îźm (all images similarly scaled). (JPG 611 kb)

Additional file 2: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome

Alignment of IFT25 with permutated KIAA0556 repeat sequences. When aligned using HHpred, a significant part of the Chlamydomonas IFT25 N-terminus was unmatched with human KIAA0556 and significant sequence remained at the C-terminus of the repeats, suggesting a circular permutation relationship between the repeats and IFT25. Shown is a HHpred alignment of IFT25 orthologues with permutated repeat sequences (r1–4) from KIAA0556 orthologues, which results in improved sequence matches. In each permutated repeat sequence, 30–40 amino acids from the beginning of each repeat have been added to the end of the same repeat (denoted by red box) using manual editing. The precise number of amino acids tr…

Additional file 3: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome

Ciliary phenotypes that are unaffected in C. elegans K04F10.2( tm1830 ) mutants. a K04F10.2 mutants possess normal fluorescent dye (DiI) filling in amphid (head) and phasmid (tail) neurons. Scale bars, 15 μm. b The lengths and morphologies of various sensory neuronal cilia are normal in K04F10.2 mutants. Shown are fluorescence images of cilia from worms expressing str-1p::GFP (AWB neuron), gcy-5p::GFP (ASER neuron) and OSM-6::GFP (PHA/B neurons) transgenes. Numbers (± standard error of the mean) refer to cilium lengths. Scale bars, 2 μm. c–e K04F10.2 mutants possess normal sensory benzaldehyde chemoattraction (n = 10), osmotic avoidance (n = 10), and foraging/roaming (n = 34) behaviours. ch…

Additional file 7: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome

Supplementary information to the data in Fig. 8 . a Schematic representation of all the different KIAA0556 fragments used to screen our selection of 200+ ciliary proteins. The predicted protein repeat domains, shown in Additional files 1 and 2, are depicted as d1 to d4. Constructs were generated containing isolated domains as well as a combination of domains. b Single transfections of PalMyr-KIAA0556 and mRFP-KATNBL1, showing that membrane localisation of the mRFP tagged protein is indeed dependent on the interaction with the PalMyr-tagged protein. (JPG 491 kb)

Additional file 5: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome

Data supplementary to the nocodazole destabilization assay shown in Fig. 7 . a, b Replicate images of DMSO or nocodazole-treated hTERT-RPE1 cells. Cells were transfected with SF-TAP-tagged KIAA0556 (detected with anti-FLAG immunostaining; green) or GFP-KIAA0556 and counterstained with anti-acetylated tubulin (red) and DAPI (blue). Cells with high KIAA0556 expression are characterised by a filamentous staining pattern and spots of accumulated KIAA0556 signal. In non-transfected cells, 10 minute nocodazole treatment resulted in the loss of a stabilised MT network (see especially the high exposure images), as judged by loss of (almost) all cytoplasmic acetylated tubulin staining and/or the abs…