Basal Body Protein Research Articles

Primary cilia formation requires the Leigh syndrome-associated mitochondrial protein NDUFAF2.

Mitochondria-related neurodegenerative diseases have been implicated in the disruption of primary cilia function. Mutation in an intrinsic mitochondrial complex I component NDUFAF2 has been identified in Leigh syndrome, a severe inherited mitochondriopathy. Mutations in ARMC9, which encodes a basal body protein, cause Joubert syndrome, a ciliopathy with defects in the brain, kidney, and eye. Here, we report a mechanistic link between mitochondria metabolism and primary cilia signaling. We discovered that loss of NDUFAF2 caused both mitochondrial and ciliary defects in vitro and in vivo and identified NDUFAF2 as a binding partner for ARMC9. We also found that NDUFAF2 was both necessary and sufficient for cilia formation and that exogenous expression of NDUFAF2 rescued the ciliary and mitochondrial defects observed in cells from patients with known ARMC9 deficiency. NAD+ supplementation restored mitochondrial and ciliary dysfunction in ARMC9-deficient cells and zebrafish and ameliorated the ocular motility and motor deficits of a patient with ARMC9 deficiency. The present results provide a compelling mechanistic link, supported by evidence from human studies, between primary cilia and mitochondrial signaling. Importantly, our findings have significant implications for the development of therapeutic approaches targeting ciliopathies.

The ARPKD Protein DZIP1L Regulates Ciliary Protein Entry by Modulating the Architecture and Function of Ciliary Transition Fibers.

Serving as the cell's sensory antennae, primary cilia are linked to numerous human genetic diseases when they malfunction. DZIP1L, identified as one of the genetic causes of human autosomal recessive polycystic kidney disease (ARPKD), is an evolutionarily conserved ciliary basal body protein. Although it has been reported that DZIP1L is involved in the ciliary entry of PKD proteins, the underlying mechanism remains elusive. Here, an uncharacterized role of DZIP1L is reported in modulating the architecture and function of transition fibers (TFs), striking ciliary base structures essential for selective cilia gating. Using C. elegans as a model, C01G5.7 (hereafter termed DZIP-1) is identified as the sole homolog of DZIP1L, which specifically localizes to TFs. While DZIP-1 or ANKR-26 (the ortholog of ANKRD26) deficiency shows subtle impact on TFs, co-depletion of DZIP-1 and ANKR-26 disrupts TF assembly and cilia gating for soluble and membrane proteins, including the ortholog of ADPKD protein polycystin-2. Notably, the synergistic role for DZIP1L and ANKRD26 in the formation and function of TFs is highly conserved in mammalian cilia. Hence, the findings illuminate an evolutionarily conserved role of DZIP1L in TFs architecture and function, highlighting TFs as a vital part of the ciliary gate implicated in ciliopathies ARPKD.

Abstract 15752: Differential IgG Repertoire in Individuals With Chagas Cardiomyopathy

Chagas cardiomyopathy (CC) is caused by the infection of Trypanosoma cruzi ( T. cruzi ) and represents a major etiology for heart failure in Latin America. Trying fill gaps related to humoral response in CC, we used a PhIP-Seq technology to describe differential IgG profile between individuals with CC and individuals infected by T. cruzi , but without symptoms (Indeterminate - IND). We built a library comprising all T. cruzi proteome (12766 proteins) and incubated against 1293 serum samples: 396 T. cruzi seronegative samples (98 controls and 298 individuals with cardiomyopathy caused by other causes than Chagas disease) and 897 T. cruzi seropositive samples (684 CC and 213 IND). 1870 peptides were increased in T. cruzi seropositive IgG compared to T. cruzi seronegative samples. Peptides from Nucleoporin NUP149, a flagellar attachment zone protein, basal body protein, surface protein TolT and cytoskeleton associated protein were the peptides with highest IgG response. Using as little as 5 peptides, I derived a classifier model for T. cruzi infection with 97% accuracy. Then, I compared CC and IND samples using logistic regression models after splitting our sample into 2 cohort groups. From these, 316 peptides were significantly different between CC and IND: 289 peptides were increased in CC (27% of them belong to the trans-sialidase class) and 27 peptides were increased in IND (FDR>0.01). Aiming to develop a classifier to separate CCC from IND, I analyzed our data using machine learning approaches. LASSO returned the best model using 48 peptides, with 70% of accuracy, 74% of sensitivity and 69% of specificity. The peptides selected for the model belong to 38 proteins, 21% being unknow function proteins, 18% trans-sialidases, 8% flagellar associated proteins, and 60S ribosomal protein, Nucleoporins and R27-2 proteins classes representing 5% each. Ninety (90%) of selected peptides in LASSO were identified in the previous results using the logistic regression model. I showed for the first time that Chagas Cardiomyopathy individuals have a differential IgG profile against T. cruzi proteins compared to IND individuals. I described a differential IgG response against 84 sequences (62 increased in CCC and 22 increased in IND) that can be related to disease development.

Mutations in SCNM1 cause orofaciodigital syndrome due to minor intron splicing defects affecting primary cilia

Orofaciodigital syndrome (OFD) is a genetically heterogeneous ciliopathy characterized by anomalies of the oral cavity, face, and digits. We describe individuals with OFD from three unrelated families having bi-allelic loss-of-function variants in SCNM1 as the cause of their condition. SCNM1 encodes a protein recently shown to be a component of the human minor spliceosome. However, so far the effect of loss of SCNM1 function on human cells had not been assessed. Using a comparative transcriptome analysis between fibroblasts derived from an OFD-affected individual harboring SCNM1 mutations and control fibroblasts, we identified a set of genes with defective minor intron (U12) processing in the fibroblasts of the affected subject. These results were reproduced in SCNM1 knockout hTERT RPE-1 (RPE-1) cells engineered by CRISPR-Cas9-mediated editing and in SCNM1 siRNA-treated RPE-1 cultures. Notably, expression of TMEM107 and FAM92A encoding primary cilia and basal body proteins, respectively, and that of DERL2, ZC3H8, and C17orf75, were severely reduced in SCNM1-deficient cells. Primary fibroblasts containing SCNM1 mutations, as well as SCNM1 knockout and SCNM1 knockdown RPE-1 cells, were also found with abnormally elongated cilia. Conversely, cilia length and expression of SCNM1-regulated genes were restored in SCNM1-deficient fibroblasts following reintroduction of SCNM1 via retroviral delivery. Additionally, functional analysis in SCNM1-retrotransduced fibroblasts showed that SCNM1 is a positive mediator of Hedgehog (Hh) signaling. Our findings demonstrate that defective U12 intron splicing can lead to a typical ciliopathy such as OFD and reveal that primary cilia length and Hh signaling are regulated by the minor spliceosome through SCNM1 activity.

Hippocampals neurogenesis is impaired in mice with a deletion in the coiled coil domain of Talpid3-implications for Joubert syndrome.

Mutations in Talpid3, a basal body protein essential for the assembly of primary cilia, have been reported to be causative for Joubert Syndrome (JS). Herein, we report prominent developmental defects in the hippocampus of a conditional knockout mouse lacking the conserved exons 11 and 12 of Talpid3. At early postnatal stages, the Talpid3 mutants exhibit a reduction in proliferation in the dentate gyrus and a disrupted glial scaffold. The occurrence of mis-localized progenitors in the granule cell layer suggests a role for the disrupted glial scaffold in cell migration resulting in defective subpial neurogenic zone-to-hilar transition. Neurospheres derived from the hippocampus of Talpid3fl/flUbcCre mouse, in which Talpid3 was conditionally deleted, lacked primary cilia and were smaller in size. In addition, neurosphere cells showed a disrupted actin cytoskeleton and defective migration. Our findings suggest a link between the hippocampal defects and the learning/memory deficits seen in JS patients.

Detection of DZIP1L mutations by whole-exome sequencing in consanguineous families with polycystic kidney disease

BackgroundAutosomal recessive polycystic kidney disease is a cystic kidney disease with early onset and clinically characterized by enlarged echogenic kidneys, hypertension, varying degrees of kidney dysfunction, and liver fibrosis. It is most frequently caused by sequence variants in the PKHD1 gene, encoding fibrocystin. In more rare cases, sequence variants in DZIP1L are seen, encoding the basal body protein DAZ interacting protein 1-like protein (DZIP1L). So far, only four different DZIP1L variants have been reported.MethodsFour children from three consanguineous families presenting with polycystic kidney disease were selected for targeted or untargeted exome sequencing.ResultsWe identified two different, previously not reported homozygous DZIP1L sequence variants: c.193 T > C; p.(Cys65Arg), and c.216C > G; p.(Cys72Trp). Functional analyses of the c.216C > G; p.(Cys72Trp) variant indicated mislocalization of mutant DZIP1L.ConclusionsIn line with published data, our results suggest a critical role of the N-terminal domain for proper protein function. Although patients with PKHD1-associated autosomal recessive polycystic kidney disease often have liver abnormalities, none of the present four patients showed any clinically relevant liver involvement. Our data demonstrate the power and efficiency of next-generation sequencing-based approaches. While DZIP1L-related polycystic kidney disease certainly represents a rare form of the disease, our results emphasize the importance of including DZIP1L in multigene panels and in the data analysis of whole-exome sequencing for cystic kidney diseases.Graphical abstractA higher resolution version of the Graphical abstract is available as Supplementary information

Isolation of the Buchnera aphidicola flagellum basal body complexes from the Buchnera membrane.

Buchnera aphidicola is an intracellular bacterial symbiont of aphids and maintains a small genome of only 600 kbps. Buchnera is thought to maintain only genes relevant to the symbiosis with its aphid host. Curiously, the Buchnera genome contains gene clusters coding for flagellum basal body structural proteins and for flagellum type III export machinery. These structures have been shown to be highly expressed and present in large numbers on Buchnera cells. No recognizable pathogenicity factors or secreted proteins have been identified in the Buchnera genome, and the relevance of this protein complex to the symbiosis is unknown. Here, we show isolation of Buchnera flagellum basal body proteins from the cellular membrane of Buchnera, confirming the enrichment of flagellum basal body proteins relative to other proteins in the Buchnera proteome. This will facilitate studies of the structure and function of the Buchnera flagellum structure, and its role in this model symbiosis.

Molecular Targets and Strategies for Inhibition of the Bacterial Type III Secretion System (T3SS); Inhibitors Directly Binding to T3SS Components.

The type III secretion system (T3SS) is a virulence apparatus used by many Gram-negative pathogenic bacteria to cause infections. Pathogens utilizing a T3SS are responsible for millions of infections yearly. Since many T3SS knockout strains are incapable of causing systemic infection, the T3SS has emerged as an attractive anti-virulence target for therapeutic design. The T3SS is a multiprotein molecular syringe that enables pathogens to inject effector proteins into host cells. These effectors modify host cell mechanisms in a variety of ways beneficial to the pathogen. Due to the T3SS’s complex nature, there are numerous ways in which it can be targeted. This review will be focused on the direct targeting of components of the T3SS, including the needle, translocon, basal body, sorting platform, and effector proteins. Inhibitors will be considered a direct inhibitor if they have a binding partner that is a T3SS component, regardless of the inhibitory effect being structural or functional.

Super-Resolution Microscopy and FIB-SEM Imaging Reveal Parental Centriole-Derived, Hybrid Cilium in Mammalian Multiciliated Cells.

Motile cilia are cellular beating machines that play a critical role in mucociliary clearance, cerebrospinal fluid movement, and fertility. In the airways, hundreds of motile cilia present on the surface of a multiciliated epithelia cell beat coordinately to protect the epithelium from bacteria, viruses, and harmful particulates. During multiciliated cell differentiation, motile cilia are templated from basal bodies, each extending a basal foot-an appendage linking motile cilia together to ensure coordinated beating. Here, we demonstrate that among the many motile cilia of a multiciliated cell, a hybrid cilium with structural features of both primary and motile cilia is harbored. The hybrid cilium is conserved in mammalian multiciliated cells, originates from parental centrioles, and its cellular position is biased and dependent on ciliary beating. Furthermore, we show that the hybrid cilium emerges independently of other motile cilia and functions in regulating basal body alignment.

Basal Body Protein TbSAF1 Is Required for Microtubule Quartet Anchorage to the Basal Bodies in Trypanosoma brucei

Sperm flagellar protein 1 (Spef1, also known as CLAMP) is a microtubule-associated protein involved in various microtubule-related functions from ciliary motility to polarized cell movement and planar cell polarity. In Trypanosoma brucei, the causative agent of trypanosomiasis, a single Spef1 ortholog (TbSpef1) is associated with a microtubule quartet (MtQ), which is in close association with several single-copied organelles and is required for their coordinated biogenesis during the cell cycle. Here, we investigated the interaction network of TbSpef1 using BioID, a proximity-dependent protein-protein interaction screening method. Characterization of selected candidates provided a molecular description of TbSpef1-MtQ interactions with nearby cytoskeletal structures. Of particular interest, we identified a new basal body protein TbSAF1, which is required for TbSpef1-MtQ anchorage to the basal bodies. The results demonstrate that MtQ-basal body anchorage is critical for the spatial organization of cytoskeletal organelles, as well as the morphology of the membrane-bound flagellar pocket where endocytosis takes place in this parasite.IMPORTANCE Trypanosoma brucei contains a large array of single-copied organelles and structures. Through extensive interorganelle connections, these structures replicate and divide following a strict temporal and spatial order. A microtubule quartet (MtQ) originates from the basal bodies and extends toward the anterior end of the cell, stringing several cytoskeletal structures together along its path. In this study, we examined the interaction network of TbSpef1, the only protein specifically located to the MtQ. We identified an interaction between TbSpef1 and a basal body protein TbSAF1, which is required for MtQ anchorage to the basal bodies. This study thus provides the first molecular description of MtQ association with the basal bodies, since the discovery of this association ∼30 years ago. The results also reveal a general mechanism of the evolutionarily conserved Spef1/CLAMP, which achieves specific cellular functions via their conserved microtubule functions and their diverse molecular interaction networks.

Functional Analysis of Hydrolethalus Syndrome Protein HYLS1 in Ciliogenesis and Spermatogenesis in Drosophila.

Cilia and flagella are conserved subcellular organelles, which arise from centrioles and play critical roles in development and reproduction of eukaryotes. Dysfunction of cilia leads to life-threatening ciliopathies. HYLS1 is an evolutionarily conserved centriole protein, which is critical for ciliogenesis, and its mutation causes ciliopathy–hydrolethalus syndrome. However, the molecular function of HYLS1 remains elusive. Here, we investigated the function of HYLS1 in cilia formation using the Drosophila model. We demonstrated that Drosophila HYLS1 is a conserved centriole and basal body protein. Deletion of HYLS1 led to sensory cilia dysfunction and spermatogenesis abnormality. Importantly, we found that Drosophila HYLS1 is essential for giant centriole/basal body elongation in spermatocytes and is required for spermatocyte centriole to efficiently recruit pericentriolar material and for spermatids to assemble the proximal centriole-like structure (the precursor of the second centriole for zygote division). Hence, by taking advantage of the giant centriole/basal body of Drosophila spermatocyte, we uncover previously uncharacterized roles of HYLS1 in centriole elongation and assembly.

Structural and functional variations in human bronchial epithelial cells cultured in air-liquid interface using different growth media.

The human bronchial epithelium is an important barrier tissue that is damaged or pathologically altered in various acute and chronic respiratory conditions. To represent the epithelial component of respiratory disease, it is essential to use a physiologically relevant model of this tissue. The human bronchial epithelium is a highly organized tissue consisting of a number of specialized cell types. Primary human bronchial epithelial cells (HBEC) can be differentiated into a mucociliated tissue in air-liquid interface (ALI) cultures using appropriately supplemented media under optimized growth conditions. We compared the histology, ciliary length, and function, diffusion, and barrier properties of HBEC from donors with no respiratory disease grown in two different media, PneumaCult-ALI or Bronchial Epithelial Differentiation Medium (BEDM). In the former group, HBEC have a more physiological pseudostratified morphology and mucociliary differentiation, including increased epithelial thickness, intracellular expression of airway-specific mucin protein MUC5AC, and total expression of cilia basal-body protein compared with cells from the same donor grown in the other medium. Baseline expression levels of inflammatory mediators, thymic stromal lymphopoietin (TSLP), soluble ST2, and eotaxin-3 were lower in PneumaCult-ALI. Additionally, the physiological cilia beat frequency and electrical barrier properties with transepithelial electrical resistance were significantly different between the two groups. Our study has shown that these primary cell cultures from the same donor grown in the two media possess variable structural and functional characteristics. Therefore, it is important to objectively validate primary epithelial cell cultures before experimentation to ensure they are appropriate to answer a specific scientific question.

Babo1, formerly Vop1 and Cop1/2, is no eyespot photoreceptor but a basal body protein illuminating cell division in Volvox carteri.

In photosynthetic organisms many processes are light dependent and sensing of light requires light-sensitive proteins. The supposed eyespot photoreceptor protein Babo1 (formerly Vop1) has previously been classified as an opsin due to the capacity for binding retinal. Here, we analyze Babo1 and provide evidence that it is no opsin. Due to the localization at the basal bodies, the former Vop1 and Cop1/2 proteins were renamed V.c. Babo1 and C.r. Babo1. We reveal a large family of more than 60 Babo1-related proteins from a wide range of species. The detailed subcellular localization of fluorescence-tagged Babo1 shows that it accumulates at the basal apparatus. More precisely, it is located predominantly at the basal bodies and to a lesser extent at the four strands of rootlet microtubules. We trace Babo1 during basal body separation and cell division. Dynamic structural rearrangements of Babo1 particularly occur right before the first cell division. In four-celled embryos Babo1 was exclusively found at the oldest basal bodies of the embryo and on the corresponding d-roots. The unequal distribution of Babo1 in four-celled embryos could be an integral part of a geometrical system in early embryogenesis, which establishes the anterior-posterior polarity and influences the spatial arrangement of all embryonic structures and characteristics. Due to its retinal-binding capacity, Babo1 could also be responsible for the unequal distribution of retinoids, knowing that such concentration gradients of retinoids can be essential for the correct patterning during embryogenesis of more complex organisms. Thus, our findings push the Babo1 research in another direction.

\u03b3-Carboxymuconolactone decarboxylase: a novel cell cycle-related basal body protein in the early branching eukaryote Trichomonas vaginalis

Backgroundγ-Carboxymuconolactone decarboxylase (CMD) participates in the β-ketoadipate pathway, which catalyzes aromatic compounds to produce acetyl- or succinyl-CoA, in prokaryotes and yeast. Our previous study demonstrated that expression of a CMD homologue that contains two signatures (dualCMD) is negatively regulated by iron in Trichomonas vaginalis. However, we were not able to identify the components of the β-ketoadipate pathway in the parasite’s genome. These observations prompted us to investigate the biological functions of this novel CMD homologue in T. vaginalis.MethodsThe specific anti-TvCMD1 antibody was generated, and the expression of TvCMD1 in T. vaginalis cultured under iron-rich and iron-deficient were evaluated. Phylogenetic, metabolomic and substrate induction (protocatechuate and benzoate) analysis were conducted to clarify the function of dualCMD in trichomonad cells. Subcellular localization of TvCMD1 was observed by confocal microscopy. The cell cycle-related role of TvCMD1 was assessed by treating cells with G2/M inhibitor nocodazole.ResultsWe confirmed that T. vaginalis is not able to catabolize the aromatic compounds benzoate and protocatechuate, which are known substrates of the β-ketoadipate pathway. Using immunofluorescence microscopy, we found that TvCMD1 is spatially associated with the basal body, a part of the cytoskeletal organizing center in T. vaginalis. TvCMD1 accumulated upon treatment with the G2/M inhibitor nocodazole. Additionally, TvCMD1 was expressed and transported to/from the basal body during cytokinesis, suggesting that TvCMD1 plays a role in cell division.ConclusionWe demonstrated that TvCMD1 is unlikely to participate in the β-ketoadipate pathway and demonstrated that it is a novel basal body-localizing (associated) protein. This model sheds light on the importance of genes that are acquired laterally in the coevolution of ancient protists, which surprisingly functions in cell cycle regulation of T. vaginalis.

Variation in Basal Body Localisation and Targeting of Trypanosome RP2 and FOR20 Proteins

TOF-LisH-PLL motifs define FOP family proteins; some members are involved in flagellum assembly. The critical role of FOP family protein FOR20 is poorly understood. Here, we report relative localisations of the four FOP family proteins in parasitic Trypanosoma brucei: TbRP2, TbOFD1 and TbFOP/FOP1-like are mature basal body proteins whereas TbFOR20 is present on pro- and mature basal bodies - on the latter it localises distal to TbRP2. We discuss how the data, together with published work for another protist Giardia intestinalis, informs on likely FOR20 function. Moreover, our localisation study provides convincing evidence that the antigen recognised by monoclonal antibody YL1/2 at trypanosome mature basal bodies is FOP family protein TbRP2, not tyrosinated α-tubulin as widely stated in the literature. Curiously, FOR20 proteins from T. brucei and closely related African trypanosomes possess short, negatively-charged N-terminal extensions absent from FOR20 in other trypanosomatids and other eukaryotes. The extension is necessary for protein targeting, but insufficient to re-direct TbRP2 to probasal bodies. Yet, FOR20 from the American trypanosome T. cruzi, which lacks any extension, localises to pro- and mature basal bodies when expressed in T. brucei. This identifies unexpected variation in FOR20 architecture that is presently unique to one clade of trypanosomatids.

Mutations in ARMC9, which Encodes a Basal Body Protein, Cause Joubert Syndrome in Humans and Ciliopathy Phenotypes in Zebrafish

Joubert syndrome (JS) is a recessive neurodevelopmental disorder characterized by hypotonia, ataxia, abnormal eye movements, and variable cognitive impairment. It is defined by a distinctive brain malformation known as the "molar tooth sign" on axial MRI. Subsets of affected individuals have malformations such as coloboma, polydactyly, and encephalocele, as well as progressive retinal dystrophy, fibrocystic kidney disease, and liver fibrosis. More than 35 genes have been associated with JS, but in a subset of families the genetic cause remains unknown. All of the gene products localize in and around the primary cilium, making JS a canonical ciliopathy. Ciliopathies are unified by their overlapping clinical features and underlying mechanisms involving ciliary dysfunction. In this work, we identify biallelic rare, predicted-deleterious ARMC9 variants (stop-gain, missense, splice-site, and single-exon deletion) in 11 individuals with JS from 8 families, accounting for approximately 1% of the disorder. The associated phenotypes range from isolated neurological involvement to JS with retinal dystrophy, additional brain abnormalities (e.g., heterotopia, Dandy-Walker malformation), pituitary insufficiency, and/or synpolydactyly. We show that ARMC9 localizes to the basal body of the cilium and is upregulated during ciliogenesis. Typical ciliopathy phenotypes (curved body shape, retinal dystrophy, coloboma, and decreased cilia) in a CRISPR/Cas9-engineered zebrafish mutant model provide additional support for ARMC9 as a ciliopathy-associated gene. Identifying ARMC9 mutations as a cause of JS takes us one step closer to a full genetic understanding of this important disorder and enables future functional work to define the central biological mechanisms underlying JS and other ciliopathies.

Proximity Interactions among Basal Body Components in Trypanosoma brucei Identify Novel Regulators of Basal Body Biogenesis and Inheritance.

ABSTRACTThe basal body shares similar architecture with centrioles in animals and is involved in nucleating flagellar axonemal microtubules in flagellated eukaryotes. The early-branching Trypanosoma brucei possesses a motile flagellum nucleated from the basal body that consists of a mature basal body and an adjacent pro-basal body. Little is known about the basal body proteome and its roles in basal body biogenesis and flagellar axoneme assembly in T. brucei. Here, we report the identification of 14 conserved centriole/basal body protein homologs and 25 trypanosome-specific basal body proteins. These proteins localize to distinct subdomains of the basal body, and several of them form a ring-like structure surrounding the basal body barrel. Functional characterization of representative basal body proteins revealed distinct roles in basal body duplication/separation and flagellar axoneme assembly. Overall, this work identified novel proteins required for basal body duplication and separation and uncovered new functions of conserved basal body proteins in basal body duplication and separation, highlighting an unusual mechanism of basal body biogenesis and inheritance in this early divergent eukaryote.

Using iPS cells to uncover cilia protein function and model disease

SummaryPrimary cilia are small, hair‐like protrusions on most cells and the photoreceptor outer segment is a highly specialised light sensing primary cilium. Retinitis pigmentosa 2 (RP2) is a cilia and basal body protein that is involved in regulating ciliary protein traffic. Mutations in the RP2 gene lead to a severe form of X‐linked RP. Patient‐derived induced pluripotent stem cells (iPSC) provide a potent new technology, which allows the directed differentiation of most cell types to model disease. Recent advances have included the differentiation of iPSC into retinal pigmented epithelium (RPE) cells and three‐dimensional optic cups, which are stratified and express photoreceptor progenitor cells. We have reprogrammed skin fibroblasts from an RP2 patient, carrying the most common RP2 stop mutation R120X, into iPSC and differentiated them into RPE cells, allowing us to study RP2 mutation disease mechanisms in vitro. Using translational read‐through drugs (PTC124 and G418) we have successfully restored full‐length, functional RP2 in these cells. Therefore, using iPSC to model disease in relevant cell types and tissues provides an important tool for the evaluation of potential therapies.

The hydrolethalus syndrome protein HYLS-1 regulates formation of the ciliary gate.

Transition fibres (TFs), together with the transition zone (TZ), are basal ciliary structures thought to be crucial for cilium biogenesis and function by acting as a ciliary gate to regulate selective protein entry and exit. Here we demonstrate that the centriolar and basal body protein HYLS-1, the C. elegans orthologue of hydrolethalus syndrome protein 1, is required for TF formation, TZ organization and ciliary gating. Loss of HYLS-1 compromises the docking and entry of intraflagellar transport (IFT) particles, ciliary gating for both membrane and soluble proteins, and axoneme assembly. Additional depletion of the TF component DYF-19 in hyls-1 mutants further exacerbates TZ anomalies and completely abrogates ciliogenesis. Our data support an important role for HYLS-1 and TFs in establishment of the ciliary gate and underline the importance of selective protein entry for cilia assembly.

Genome-Wide Evaluation of Epistasis with APOL1 Risk Variants in Sickle Cell Disease Nephropathy

BACKGROUND: Two functional variants (G1 and G2) in the APOL1 gene have been implicated as contributing to the high rate of chronic kidney disease in African Americans (Genovese et al., 2010; Kopp et al., 2010). We subsequently demonstrated that the APOL1 G1 and G2 variants, as well as non-coding genetic variation in the neighboring gene MYH9, were associated with proteinuria in a cohort of sickle cell disease (SCD) patients (Ashley-Koch et al., 2011). When controlling for the effect of the functional variants in APOL1, the effect of MYH9 on proteinuria remained, suggesting that both genes were independent risk factors in SCD nephropathy. A statistical interaction between the two genes predicting glomerular filtration rate (GFR) was also observed, suggesting a possible role of epistasis. In addition, we have demonstrated that myh9 and apol1 functionally interact in an in vivo zebrafish model, particularly in the context of anemic stress and more specifically that the APOL1 G2 risk allele modulates myh9 expression in the zebrafish (Anderson et al., 2015). In an effort to identify additional genes that may interact with APOL1 to influence risk for SCD nephropathy, we have performed a genome-wide evaluation of epistasis with APOL1 G1 and G2 variants.METHODS: Adults with SCD were recruited from sickle cell centers at Duke University, University of North Carolina at Chapel Hill, Emory University, and East Carolina University. Medical history, laboratory values and DNA for genotyping were collected. GFR was estimated using the definition from the 'Modification of Diet in Renal Disease' study (Levey et al., 1999). Genotyping was performed using the Illumina Human610-Quad BeadChip (Illumina, San Diego, CA) and missing genotypes were imputed using IMPUTE2 (Howie et al., 2009) with a global reference panel from the 1000 Genomes project. Principal component (PC) analysis was conducted using EIGENSOFT (Patterson et al., 2006) to obtain measures of population substructure. APOL1 coding variants G1 (rs73885319) and G2 (rs71785313) were genotyped using Taqman assays (Applied Biosystems, Foster City, CA). To test for interactions between each SNP and the G1 or G2 alleles, linear regression was used to predict GFR and logistic regression for proteinuria. All models included main and interaction effects of SNPs (coded additively), as well as two genome-wide PCs to control for population stratification. False discovery rate (FDR) q-values were generated using PROC MULTTEST in SAS (SAS Systems, Cary, NC).RESULTS: A region on chromosome 6 significantly interacted with the APOL1 G2 allele to predict GFR (FDR q's < 0.036); the most significant SNP was rs6916880 (p=6.4x10-8). We also identified SNPs in a region on chromosome 4 which significantly interacted with APOL1 G2 to predict GFR (FDR q's=0.075). Finally, rs10790180 in CEP164 showed a significant interaction with the APOL1 G2 allele to predict GFR (FDR q=0.0932). No significant interactions were observed with the APOL1 G1 allele to predict GFR, or with either APOL1 risk allele to predict proteinuria (FDR q's > 0.9).DISCUSSION: We have identified several genome-wide significant gene-gene interactions involving the APOL1 G2 allele influencing GFR in an adult SCD cohort. Although the most significant interactions were observed with intergenic regions on chromosomes 6 and 4, both regions are characterized by histone marks in ENSEMBL, indicating that they may have important regulatory function. We also observed an interaction between a SNP in CEP164 and the APOL1 G2 allele. CEP164 is a basal body protein required for assembly of the primary cilium (Graser et al., 2007), and mutations in CEP164 have been associated with nephronophthisis-related ciliopathies (Chaki et al., 2012). Moreover, suppression of cep164 in the developing zebrafish resulted in a ciliopathy phenotype that included pronephric tubule cysts (Chaki et al., 2012). We did not replicate previous reports of epistasis with APOL1 in ESRD (Divers et al., 2014; Bostrom et al., 2012), but our work has shown that SCD nephropathy may be a unique form of kidney disease with distinct genetic drivers, due to the context of anemic stress (Anderson et al., 2015; Ashley-Koch et al., 2011). DisclosuresNo relevant conflicts of interest to declare.