CCDC38 is required for sperm flagellum biogenesis and male fertility in mice.

The sperm flagellum is essential for male fertility, and defects in flagellum biogenesis are associated with male infertility. Deficiency of coiled-coil domain-containing (CCDC) 42 (CCDC42) is specifically associated with malformation of mouse sperm flagella. Here, we find that the testis-specific protein CCDC38 interacts with CCDC42, localizing on the manchette and sperm tail during spermiogenesis. Inactivation of CCDC38 in male mice results in a distorted manchette, multiple morphological abnormalities of the flagella of spermatozoa and eventually male sterility. Furthermore, we find that CCDC38 interacts with intraflagellar transport protein 88 (IFT88), as well as outer dense fibrous 2 (ODF2), and the knockout of Ccdc38 reduces transport of ODF2 to the flagellum. Altogether, our results uncover the essential role of CCDC38 in sperm flagellum biogenesis, and suggest that some mutations of these genes might be associated with male infertility in humans.

Primary cilia are critical for tracheoesophageal septation.

INTRODUCTION: Primary cilia play pivotal roles in the patterning and morphogenesis of a wide variety of organs during mammalian development. Here we examined murine foregut septation in the cobblestone mutant, a hypomorphic allele of the gene encoding the intraflagellar transport protein IFT88, a protein essential for normal cilia function. RESULTS: We reveal a crucial role for primary cilia in foregut division, since their dramatic decrease in cilia in both the foregut endoderm and mesenchyme of mutant embryos resulted in a proximal tracheoesophageal septation defects and in the formation of distal tracheo(broncho)esophageal fistulae similar to the most common congenital tracheoesophageal malformations in humans. Interestingly, the dorsoventral patterning determining the dorsal digestive and the ventral respiratory endoderm remained intact, whereas Hedgehog signaling was aberrantly activated. CONCLUSIONS: Our results demonstrate the cobblestone mutant to represent one of the very few mouse models that display both correct endodermal dorsoventral specification but defective compartmentalization of the proximal foregut. It stands exemplary for a tracheoesophageal ciliopathy, offering the possibility to elucidate the molecular mechanisms how primary cilia orchestrate the septation process. The plethora of malformations observed in the cobblestone embryo allow for a deeper insight into a putative link between primary cilia and human VATER/VACTERL syndromes.

MEIG1 determines the manchette localization of IFT20 and IFT88, two intraflagellar transport components in male germ cells.

Sperm flagella formation is a complex process that requires cargo transport systems to deliver structural proteins for sperm flagella assembly. Two cargo transport systems, the intramanchette transport (IMT) and intraflagellar transport (IFT), have been shown to play critical roles in spermatogenesis and sperm flagella formation. IMT exists only in elongating spermatids, while IFT is responsible for delivering cargo proteins in the developing cilia/flagella. Our laboratory discovered that mouse meiosis expressed gene 1 (MEIG1), a gene essential for sperm flagella formation, is present in the manchette of elongating spermatids. IFT complex components, IFT20 and IFT88, are also present in the manchette of the elongating spermatids. Given that the three proteins have the same localization in elongating spermatids and are essential for normal spermatogenesis and sperm flagella formation, we hypothesize that they are in the same complex, which is supported by co-immunoprecipitation assay using mouse testis extracts. In the Meig1 knockout mice, neither IFT20 nor IFT88 was present in the manchette in the elongating spermatids even though their localizations were normal in spermatocytes and round spermatids. However, MEIG1 was still present in the manchette in elongating spermatids of the conditional Ift20 knockout mice. In the sucrose gradient assay, both IFT20 and IFT88 proteins drifted from higher density fractions to lighter ones in the Meig1 knockout mice. MEIG1 distribution was not changed in the conditional Ift20 knockout mice. Finally, testicular IFT20 and IFT88 protein and mRNA levels were significantly reduced in Meig1 knockout mice. Our data suggests that MEIG1 is a key protein in determining the manchette localization of certain IFT components, including IFT20 and IFT88, in male germ cells.

Hedgehog signaling regulates Wolffian duct development through the primary cilium†.

Primary cilia play pivotal roles in embryonic patterning and organogenesis through transduction of the Hedgehog signaling pathway (Hh). Although mutations in Hh morphogens impair the development of the gonads and trigger male infertility, the contribution of Hh and primary cilia in the development of male reproductive ductules, including the epididymis, remains unknown. From a Pax2Cre; IFT88fl/fl knock-out mouse model, we found that primary cilia deletion is associated with imbalanced Hh signaling and morphometric changes in the Wolffian duct (WD), the embryonic precursor of the epididymis. Similar effects were observed following pharmacological blockade of primary cilia formation and Hh modulation on WD organotypic cultures. The expression of genes involved in extracellular matrix, mesenchymal-epithelial transition, canonical Hh and WD development was significantly altered after treatments. Altogether, we identified the primary cilia-dependent Hh signaling as a master regulator of genes involved in WD development. This provides new insights regarding the etiology of sexual differentiation and male infertility issues.

Ift88 regulates enamel formation via involving Shh signaling.

OBJECTIVES: The ciliopathies are a wide spectrum of human diseases, which are caused by perturbations in the function of primary cilia. Tooth enamel anomalies are often seen in ciliopathy patients; however, the role of primary cilia in enamel formation remains unclear. MATERIALS AND METHODS: We examined mice with epithelial conditional deletion of the ciliary protein, Ift88, (Ift88fl / fl ;K14Cre). RESULTS: Ift88fl / fl ;K14Cre mice showed premature abrasion in molars. A pattern of enamel rods which is determined at secretory stage, was disorganized in Ift88 mutant molars. Many amelogenesis-related molecules expressing at the secretory stage, including amelogenin and ameloblastin, enamelin, showed significant downregulation in Ift88 mutant molar tooth germs. Shh signaling is essential for amelogenesis, which was found to be downregulated in Ift88 mutant molar at the secretory stage. Application of Shh signaling agonist at the secretory stage partially rescued enamel anomalies in Ift88 mutant mice. CONCLUSION: Findings in the present study indicate that the function of the primary cilia via Ift88 is critical for the secretory stage of amelogenesis through involving Shh signaling.

Aberrant Multiciliogenesis in Idiopathic Pulmonary Fibrosis.

We previously identified a novel molecular subtype of idiopathic pulmonary fibrosis (IPF) defined by increased expression of cilium-associated genes, airway mucin gene MUC5B, and KRT5 marker of basal cell airway progenitors. Here we show the association of MUC5B and cilia gene expression in human IPF airway epithelial cells, providing further rationale for examining the role of cilium genes in the pathogenesis of IPF. We demonstrate increased multiciliogenesis and changes in motile cilia structure of multiciliated cells both in IPF and bleomycin lung fibrosis models. Importantly, conditional deletion of a cilium gene, Ift88 (intraflagellar transport 88), in Krt5 basal cells reduces Krt5 pod formation and lung fibrosis, whereas no changes are observed in Ift88 conditional deletion in club cell progenitors. Our findings indicate that aberrant injury-activated primary ciliogenesis and Hedgehog signaling may play a causative role in Krt5 pod formation, which leads to aberrant multiciliogenesis and lung fibrosis. This implies that modulating cilium gene expression in Krt5 cell progenitors is a potential therapeutic target for IPF.

Ciliary proteins specify the cell inflammatory response by tuning NFκB signalling, independently of primary cilia.

Complex inflammatory signalling cascades define the response to tissue injury but also control development and homeostasis, limiting the potential for these pathways to be targeted therapeutically. Primary cilia are subcellular regulators of cellular signalling, controlling how signalling is organized, encoded and, in some instances, driving or influencing pathogenesis. Our previous research revealed that disruption of ciliary intraflagellar transport (IFT), altered the cell response to IL-1ß, supporting a putative link emerging between cilia and inflammation. Here, we show that IFT88 depletion affects specific cytokine-regulated behaviours, changing cytosolic NFκB translocation dynamics but leaving MAPK signalling unaffected. RNA-seq analysis indicates that IFT88 regulates one third of the genome-wide targets, including the pro-inflammatory genes Nos2, Il6 and Tnf Through microscopy, we find altered NFκB dynamics are independent of assembly of a ciliary axoneme. Indeed, depletion of IFT88 inhibits inflammatory responses in the non-ciliated macrophage. We propose that ciliary proteins, including IFT88, KIF3A, TTBK2 and NPHP4, act outside of the ciliary axoneme to tune cytoplasmic NFκB signalling and specify the downstream cell response. This is thus a non-canonical function for ciliary proteins in shaping cellular inflammation.This article has an associated First Person interview with the first author of the paper.

Sex-Dependent Effects of Nephron Ift88 Disruption on BP, Renal Function, and Cystogenesis.

BACKGROUND: Primary cilia regulation of renal function and BP in health and disease is incompletely understood. This study investigated the effect of nephron ciliary loss on renal physiology, BP, and ensuing cystogenesis. METHODS: Mice underwent doxycycline (DOX)-inducible nephron-specific knockout (KO) of the Ift88 gene at 2 months of age using a Cre-LoxP strategy. BP, kidney function, and renal pathology were studied 2 and 9 months after DOX (Ift88 KO) or vehicle (control). RESULTS: At 2 months post-DOX, male, but not female, Ift88 KO, compared with sex-matched control, mice had reduced BP, enhanced salt-induced natriuresis, increased urinary nitrite and nitrate (NOx) excretion, and increased kidney NOS3 levels, which localized to the outer medulla; the reductions in BP in male mice were prevented by L-NAME. At 9 months post-DOX, male, but not female, Ift88 KO mice had polycystic kidneys, elevated BP, and reduced urinary NOx excretion. No differences were observed in plasma renin concentration, plasma aldosterone, urine vasopressin, or urine PGE2 between Ift88 KO and control mice at 2 or 9 months post-DOX. CONCLUSIONS: Nephron cilia disruption in male, but not female, mice (1) reduces BP prior to cyst formation, (2) increases NOx production that may account for the lower BP prior to cyst formation, and (3) induces polycystic kidneys that are associated with hypertension and reduced renal NO production.

Ift88, but not Kif3a, is required for establishment of the periciliary membrane compartment.

The primary cilium is a sensory organelle at the cell surface with integral functions in cell signaling. It contains a microtubular axoneme that is rooted in the basal body (BB) and serves as a scaffold for the movement of intraflagellar transport (IFT) particles by Kinesin-2 along the cilium. Ift88, a member of the anterograde moving IFT-B1 complex, as well as the Kinesin-2 subunit Kif3a are required for cilia formation. To facilitate signaling, the cilium restricts the access of molecules to its membrane ("ciliary gate"). This is thought to be mediated by cytoskeletal barriers ("subciliary domains") originating from the BB subdistal/distal appendages, the periciliary membrane compartment (PCMC) as well as the transition fibers and zone (TF/TZ). The PCMC is a poorly characterized membrane domain surrounding the ciliary base with exclusion of certain apical membrane proteins. Here we describe that Ift88, but not Kinesin-2, is required for the establishment of the PCMC in MDCK cells. Likewise, in C. elegans mutants of the Ift88 ortholog osm-5 fail to establish the PCMC, while Kinesin-2 deficient osm-3 mutants form PCMCs normally. Furthermore, disruption of IFT-B1 into two subcomplexes, while disrupting ciliogenesis, does not interfere with PCMC formation. Our findings suggest that cilia are not a prerequisite for the formation of the PCMC, and that separate machineries with partially overlapping functions are required for the establishment of each.

Increase in primary cilia in the epidermis of patients with atopic dermatitis and psoriasis.

Primary cilia influence cell activity, and thus have a unique role in maintaining cell proliferation and differentiation. In atopic dermatitis (AD) and psoriasis, areas of skin inflammation exhibit dysregulated keratinocyte homeostasis. The role of primary cilia in these conditions remains unclear. The objectives of this study is to elucidate the incidence of primary cilia in skin inflammation and the potential mechanism underlying the dysregulation of keratinocytes. Primary cilia were observed using immunofluorescence staining. Normal skin samples were compared with skin samples from patients with AD or psoriasis in terms of cilia numbers and length. The effect of cytokine stimulation on ciliogenesis in keratinocytes was analysed using a primary keratinocyte culture. IFT88, an important ciliary intraflagellar protein, was blocked in Th2 and Th17 cytokines-stimulated keratinocytes. These effects were analysed with quantitative polymerase chain reaction and Western blot. Significant increases in ciliated cells were observed in AD and psoriasis skin samples compared with normal skin samples. The stimulation of keratinocytes using Th2 and Th17 cytokines modulated the formation of primary cilia. The amount of IFT88 in the primary cilia associated with the phosphorylation of JNK, but not p38, in keratinocytes stimulated with interleukin-13, 17A and 22. An increase of ciliated cells in the epidermis may impair keratinocyte differentiation under stress conditions caused by inflammation in both AD and psoriasis patients.

Integrin-ß1 is required for the renal cystogenesis caused by ciliary defects.

Defects in the function of primary cilia are commonly associated with the development of renal cysts. On the other hand, the intact cilium appears to contribute a cystogenic signal whose effectors remain unclear. As integrin-ß1 is required for the cystogenesis caused by the deletion of the polycystin 1 gene, we asked whether it would be similarly important in the cystogenetic process caused by other ciliary defects. We addressed this question by investigating the effect of integrin-ß1 deletion in a ciliopathy genetic model in which the Ift88 gene, a component of complex B of intraflagellar transport that is required for the proper assembly of cilia, is specifically ablated in principal cells of the collecting ducts. We showed that the renal cystogenesis caused by loss of Ift88 is prevented when integrin-ß1 is simultaneously depleted. In parallel, pathogenetic manifestations of the disease, such as increased inflammatory infiltrate and fibrosis, were also significantly reduced. Overall, our data indicate that integrin-ß1 is also required for the renal cystogenesis caused by ciliary defects and point to integrin-ß1-controlled pathways as common drivers of the disease and as possible targets to interfere with the cystogenesis caused by ciliary defects.

Genetic reduction of cilium length by targeting intraflagellar transport 88 protein impedes kidney and liver cyst formation in mouse models of autosomal polycystic kidney disease.

Polycystin-1 (PC1) and -2 (PC2), products of the PKD1 and PKD2 genes, are mutated in autosomal dominant polycystic kidney disease (ADPKD). They localize to the primary cilia; however, their ciliary function is in dispute. Loss of either the primary cilia or PC1 or PC2 causes cyst formation. However, loss of both cilia and PC1 or PC2 inhibits cyst growth via an unknown pathway. To help define a pathway, we studied cilium length in human and mouse kidneys. We found cilia are elongated in kidneys from patients with ADPKD and from both Pkd1 and Pkd2 knockout mice. Cilia elongate following polycystin inactivation. The role of intraflagellar transport proteins in Pkd1-deficient mice is also unknown. We found that inactivation of Ift88 (a gene expressing a core component of intraflagellar transport) in Pkd1 knockout mice, as well as in a new Pkd2 knockout mouse, shortened the elongated cilia, impeded kidney and liver cystogenesis, and reduced cell proliferation. Multi-stage in vivo analysis of signaling pathways revealed ß-catenin activation as a prominent, early, and sustained event in disease onset and progression in Pkd2 single knockout but not in Pkd2.Ift88 double knockout mouse kidneys. Additionally, AMPK, mTOR and ERK pathways were altered in Pkd2 single knockout mice but only AMPK and mTOR pathway alteration were rescued in Pkd2.Ift88 double knockout mice. Thus, our findings advocate an essential role of polycystins in the structure and function of the primary cilia and implicate ß-catenin as a key inducer of cystogenesis downstream of the primary cilia. Our data suggest that modulating cilium length and/or its associated signaling events may offer novel therapeutic approaches for ADPKD.

Ift88 is involved in mandibular development.

The mandible is a crucial organ in both clinical and biological fields due to the high frequency of congenital anomalies and the significant morphological changes during evolution. Primary cilia play a critical role in many biological processes, including the determination of left/right axis patterning, the regulation of signaling pathways, and the formation of bone and cartilage. Perturbations in the function of primary cilia are known to cause a wide spectrum of human diseases: the ciliopathies. Craniofacial dysmorphologies, including mandibular deformity, are often seen in patients with ciliopathies. Mandibular development is characterized by chondrogenesis and osteogenesis; however, the role of primary cilia in mandibular development is not fully understood. To address this question, we generated mice with mesenchymal deletions of the ciliary protein, Ift88 (Ift88fl/fl ;Wnt1Cre). Ift88fl/fl ;Wnt1Cre mice showed ectopic mandibular bone formation, whereas Ift88 mutant mandible was slightly shortened. Meckel's cartilage was modestly expanded in Ift88fl/fl ;Wnt1Cre mice. The downregulation of Hh signaling was found in most of the mesenchyme of Ift88 mutant mandible. However, mice with a mesenchymal deletion of an essential molecule for Hh signaling activity, Smo (Smofl/fl ;Wnt1Cre), showed only ectopic mandibular formation, whereas Smo mutant mandible was significantly shortened. Ift88 is thus involved in chondrogenesis and osteogenesis during mandibular development, partially through regulating Sonic hedgehog (Shh) signaling.

Primary cilia are critical for Sonic hedgehog-mediated dopaminergic neurogenesis in the embryonic midbrain.

Midbrain dopaminergic (mDA) neurons modulate various motor and cognitive functions, and their dysfunction or degeneration has been implicated in several psychiatric diseases. Both Sonic Hedgehog (Shh) and Wnt signaling pathways have been shown to be essential for normal development of mDA neurons. Primary cilia are critical for the development of a number of structures in the brain by serving as a hub for essential developmental signaling cascades, but their role in the generation of mDA neurons has not been examined. We analyzed mutant mouse lines deficient in the intraflagellar transport protein IFT88, which is critical for primary cilia function. Conditional inactivation of Ift88 in the midbrain after E9.0 results in progressive loss of primary cilia, a decreased size of the mDA progenitor domain, and a reduction in mDA neurons. We identified Shh signaling as the primary cause of these defects, since conditional inactivation of the Shh signaling pathway after E9.0, through genetic ablation of Gli2 and Gli3 in the midbrain, results in a phenotype basically identical to the one seen in Ift88 conditional mutants. Moreover, the expansion of the mDA progenitor domain observed when Shh signaling is constitutively activated does not occur in absence of Ift88. In contrast, clusters of Shh-responding progenitors are maintained in the ventral midbrain of the hypomorphic Ift88 mouse mutant, cobblestone. Despite the residual Shh signaling, the integrity of the mDA progenitor domain is severely disturbed, and consequently very few mDA neurons are generated in cobblestone mutants. Our results identify for the first time a crucial role of primary cilia in the induction of mDA progenitors, define a narrow time window in which Shh-mediated signaling is dependent upon normal primary cilia function for this purpose, and suggest that later Wnt signaling-dependent events act independently of primary cilia.

Heterotrimeric kinesin-2 (KIF3) mediates transition zone and axoneme formation of mouse photoreceptors.

Anterograde intraflagellar transport (IFT) employing kinesin-2 molecular motors has been implicated in trafficking of photoreceptor outer segment proteins. We generated embryonic retina-specific (prefix "emb") and adult tamoxifen-induced (prefix "tam") deletions of KIF3a and IFT88 in adult mice to study photoreceptor ciliogenesis and protein trafficking. In (emb)Kif3a(-/-) and in (emb)Ift88(-/-) mice, basal bodies failed to extend transition zones (connecting cilia) with outer segments, and visual pigments mistrafficked. In contrast, (tam)Kif3a(-/-) and (tam)Ift88(-/-) photoreceptor axonemes disintegrated slowly post-induction, starting distally, but rhodopsin and cone pigments trafficked normally for more than 2 weeks, a time interval during which the outer segment is completely renewed. The results demonstrate that visual pigments transport to the retinal outer segment despite removal of KIF3 and IFT88, and KIF3-mediated anterograde IFT is responsible for photoreceptor transition zone and axoneme formation.

Myosin heavy chain 10 (MYH10) is required for centriole migration during the biogenesis of primary cilia.

The actin cytoskeleton has been implicated in the assembly of cilia, but roles of actin-dependent motor proteins in ciliogenesis remain unclear. Myosin heavy chain 10 (MYH10), one of the isoforms of non-muscle myosin II, is known to mediate centrosome reorientation during cell migration. Here we show that MYH10 is required for centriole migration to the apical plasma membrane, which occurs at the onset of ciliogenesis. Knockdown of MYH10 in RPE1 cells caused a reduction in the levels of cortical filamentous actin (F-actin) and its binding protein EZRIN. Moreover, both centriole migration and subsequent cilium assembly were defective in MYH10 depleted cells. We further found that MYH10 influences centrosomal recruitment of IFT88, which is required for the transport of building blocks to the ciliary tip. The role of MYH10 in IFT88 recruitment appears to be indirect in that there is a correlation between centriolar IFT88 levels and centriolar positions along the apical-basal axis during ciliogenesis. Our results indicate that MYH10 contributes to ciliogenesis in RPE1 cells by promoting cortical actin-dependent centriole migration.

Hypoxia-Inducible Factor-2alpha Affects the MEK/ERK Signaling Pathway via Primary Cilia in Connection with the Intraflagellar Transport Protein 88 Homolog.

The ability of cells to communicate with their surrounding is a prerequisite for essential processes such as proliferation, apoptosis, migration, and differentiation. To this purpose, primary cilia serve as antennae-like structures on the surface of most mammalian cell types. Cilia allow signaling via hedgehog, Wnt or TGF-beta pathways. Their length, in part controlled by the activity of intraflagellar transport (IFT), is a parameter for adequate function of primary cilia. Here we show, in murine neuronal cells, that intraflagellar transport protein 88 homolog (IFT88) directly interacts with the hypoxia-inducible factor-2α (HIF-2α), hitherto known as an oxygen-regulated transcription factor. Furthermore, HIF-2α accumulates in the ciliary axoneme and promotes ciliary elongation under hypoxia. Loss of HIF-2α affected ciliary signaling in neuronal cells by decreasing transcription of Mek1/2 and Erk1/2. Targets of the MEK/ERK signaling pathway, such as Fos and Jun, were significantly decreased. Our results suggest that HIF-2α influences ciliary signaling by interacting with IFT88 under hypoxic conditions. This implies an unexpected and far more extensive function of HIF-2α than described before.

Maternal protein deficiency alters primary cilia length in renal tubular and impairs kidney development in fetal rat.

Introduction: Intrauterine malnutrition impairs embryo kidney development and leads to kidney disease and hypertension in adulthood, yet the underlying mechanism remains unclear. Methods: With a maternal protein restriction (MPR) rat model, we investigated the critical ciliogenesis factors and ß-catenin pathway in FGR fetal kidneys and analyzed the impact of aberrant primary cilia on renal tubular epithelium. Results: The data showed decreased nephron number and renal tubular dysgenesis in FGR fetus. FGR fetus showed deregulated expression of ciliogenesis factors including upregulation of IFT88 and downregulation of DYNLT1, accompanied with cilia elongation in renal tubular epithelial cells. Wnt7b, the key ligand for Wnt/ß-catenin signaling, was downregulated and nuclear translocation of ß-catenin was decreased. The proapoptotic protein was upregulated. In vitro study with HK-2 cells showed that overexpression of IFT88 lengthened the cilia, inhibited ß-catenin signaling. Besides, IFT88 overexpression suppressed cell proliferation, activated autophagy, and induced cell apoptosis. Inhibition of autophagy partly restored the cilia length and cell viability. Likewise, knockdown of DYNLT1 led to cilia elongation, suppressed cell proliferation, and promoted apoptosis in HK-2 cell. However, the cilia elongation induced by DYNLT1 knockdown was not autophagy-dependent, but associated with reactive oxygen species (ROS) accumulation. Discussion: We elucidated that intrauterine protein malnutrition led to deregulation of ciliogenesis factors and cilia elongation in renal tubular epithelial, inhibited ß-catenin signaling, and induced cell apoptosis and ultimately, compromised kidney development.

Increasing Ciliary ARL13B Expression Drives Active and Inhibitor-Resistant Smoothened and GLI into Glioma Primary Cilia.

ADP-ribosylation factor-like protein 13B (ARL13B), a regulatory GTPase and guanine exchange factor (GEF), enriches in primary cilia and promotes tumorigenesis in part by regulating Smoothened (SMO), GLI, and Sonic Hedgehog (SHH) signaling. Gliomas with increased ARL13B, SMO, and GLI2 expression are more aggressive, but the relationship to cilia is unclear. Previous studies have showed that increasing ARL13B in glioblastoma cells promoted ciliary SMO accumulation, independent of exogenous SHH addition. Here, we show that SMO accumulation is due to increased ciliary, but not extraciliary, ARL13B. Increasing ARL13B expression promotes the accumulation of both activated SMO and GLI2 in glioma cilia. ARL13B-driven increases in ciliary SMO and GLI2 are resistant to SMO inhibitors, GDC-0449, and cyclopamine. Surprisingly, ARL13B-induced changes in ciliary SMO/GLI2 did not correlate with canonical changes in downstream SHH pathway genes. However, glioma cell lines whose cilia overexpress WT but not guanine exchange factor-deficient ARL13B, display reduced INPP5e, a ciliary membrane component whose depletion may favor SMO/GLI2 enrichment. Glioma cells overexpressing ARL13B also display reduced ciliary intraflagellar transport 88 (IFT88), suggesting that altered retrograde transport could further promote SMO/GLI accumulation. Collectively, our data suggest that factors increasing ARL13B expression in glioma cells may promote both changes in ciliary membrane characteristics and IFT proteins, leading to the accumulation of drug-resistant SMO and GLI. The downstream targets and consequences of these ciliary changes require further investigation.

A Novel 13q12 Microdeletion Associated with Familial Syndromic Corneal Opacification.

Progressive corneal opacification can result from multiple etiologies, including corneal dystrophies or systemic and genetic diseases. We describe a novel syndrome featuring progressive epithelial and anterior stromal opacification in a brother and sister and their mildly affected father, with all three family members having sensorineural hearing loss and two also with tracheomalacia/laryngomalacia. All carried a 1.2 Mb deletion at chromosome 13q12.11, with no other noteworthy co-segregating variants identified on clinical exome or chromosomal microarray. RNAseq analysis from an affected corneal epithelial sample from the proband's brother revealed downregulation of XPO4, IFT88, ZDHHC20, LATS2, SAP18, and EEF1AKMT1 within the microdeletion interval, with no notable effect on the expression of nearby genes. Pathway analysis showed upregulation of collagen metabolism and extracellular matrix (ECM) formation/maintenance, with no significantly down-regulated pathways. Analysis of overlapping deletions/variants demonstrated that deleterious variants in XPO4 were found in patients with laryngomalacia and sensorineural hearing loss, with the latter phenotype also being a feature of variants in the partially overlapping DFNB1 locus, yet none of these had reported corneal phenotypes. Together, these data define a novel microdeletion-associated syndromic progressive corneal opacification and suggest that a combination of genes within the microdeletion may contribute to ECM dysregulation leading to pathogenesis.