Cilia Genes Research Articles

Genetic Insights Into Hypothalamic Hamartoma: Unraveling Somatic Variants.

Hypothalamic hamartomas (HHs) are rare developmental brain lesions associated with drug-resistant epilepsy and often subjected to epilepsy surgery. Brain somatic variants in genes affecting the Sonic hedgehog (Shh) and primary cilia signaling pathways have been implicated in approximately 50% of nonsyndromic HH cases. This study aims to characterize a new cohort of 9 HH cases and elucidate their genetic etiology. We recruited 9 HH cases including 8 nonsyndromic cases of which 4 were type IV HH. Genomic DNA was extracted from peripheral blood and surgical brain tissues, and somatic variants were investigated using high-depth whole-exome sequencing. Pathogenic somatic variants in known HH genes (GLI3, OFD1, and PRKACA) were identified in 7 of the 9 cases. In addition, a 2-hit mutational event comprising a germline variant (predicted to impair kinase activity) and a somatic loss-of-heterozygosity was identified in TNK2, a gene encoding a brain-expressed tyrosine kinase. Our findings reinforce the role of somatic variants in Shh and cilia genes in HH cases while also shedding light on TNK2 as a potential novel disease-causing gene. This study emphasizes the increasing importance of brain mosaicism in epilepsy disorders and underscores the critical role of genetic diagnosis derived from resected brain tissue.

Circadian cilia transcriptome in mouse brain across physiological and pathological states

Primary cilia are dynamic sensory organelles that continuously undergo structural modifications in response to environmental and cellular signals, many of which exhibit rhythmic patterns. Building on our previous findings of rhythmic cilia-related gene expression in diurnal primates (baboon), this study extends the investigation to the nocturnal mouse brain to identify circadian patterns of cilia gene expression across brain regions. We used computational techniques and transcriptomic data from four publicly available databases, to examine the circadian expression of cilia-associated genes within six brain areas: brainstem, cerebellum, hippocampus, hypothalamus, striatum, and suprachiasmatic nucleus. Our analysis reveals that a substantial proportion of cilia transcripts exhibit circadian rhythmicity across the examined regions, with notable overrepresentation in the striatum, hippocampus, and cerebellum. We also demonstrate region-specific variations in the abundance and timing of circadian cilia genes’ peaks, indicating an adaptation to the distinct physiological roles of each brain region. Additionally, we show that the rhythmic patterns of cilia transcripts are shifted under various physiological and pathological conditions, including modulation of the dopamine system, high-fat diet, and epileptic conditions, indicating the adaptable nature of cilia transcripts’ oscillation. While limited to a few mouse brain regions, our study provides initial insights into the distinct circadian patterns of cilia transcripts and highlights the need for future research to expand the mapping across wider brain areas to fully understand the role of cilia’s spatiotemporal dynamics in brain functions.

The role of cilia in the development, survival, and regeneration of hair cells.

Mutations impacting cilia genes lead to a class of human diseases known as ciliopathies. This is due to the role of cilia in the development, survival, and regeneration of many cell types. We investigated the extent to which disrupting cilia impacted these processes in lateral line hair cells of zebrafish. We found that mutations in two intraflagellar transport (IFT) genes, ift88 and dync2h1, which lead to the loss of kinocilia, caused increased hair cell apoptosis. IFT gene mutants also have a decreased mitochondrial membrane potential, and blocking the mitochondrial uniporter causes a loss of hair cells in wild-type zebrafish but not mutants, suggesting mitochondria dysfunction may contribute to the apoptosis seen in these mutants. These mutants also showed decreased proliferation during hair cell regeneration but did not show consistent changes in support cell number or proliferation during hair cell development. These results show that the loss of hair cells seen following disruption of cilia through either mutations in anterograde or retrograde IFT genes appears to be due to impacts on hair cell survival but not necessarily development in the zebrafish lateral line.

SMYD3 Controls Ciliogenesis by Regulating Distinct Centrosomal Proteins and Intraflagellar Transport Trafficking.

The primary cilium is a microtubule-based sensory organelle that plays a critical role in signaling pathways and cell cycle progression. Defects in the structure and/or function of the primary cilium result in developmental diseases collectively known as ciliopathies. However, the constituents and regulatory mechanisms of the primary cilium are not fully understood. In recent years, the activity of the epigenetic modifier SMYD3 has been shown to play a key role in the regulation of cell cycle progression. However, whether SMYD3, a histone/lysine methyltransferase, contributes to the regulation of ciliogenesis remains unknown. Here, we report that SMYD3 drives ciliogenesis via the direct and indirect regulation of cilia-associated components. We show that SMYD3 is a novel component of the distal appendage and is required for centriolar appendage assembly. The loss of SMYD3 decreased the percentage of ciliated cells and resulted in the formation of stumpy cilia. We demonstrated that SMYD3 modulated the recruitment of centrosome proteins (Cep164, Fbf1, Ninein, Ttbk2 and Cp110) and the trafficking of intraflagellar transport proteins (Ift54 and Ift140) important for cilia formation and maintenance, respectively. In addition, we showed that SMYD3 regulated the transcription of cilia genes and bound to the promoter regions of C2cd3, Cep164, Ttbk2, Dync2h1 and Cp110. This study provides insights into the role of SMYD3 in cilia biology and suggests that SMYD3-mediated cilia formation/function may be relevant for cilia-dependent signaling in ciliopathies.

Joubert syndrome causing mutation in C2 domain of CC2D2A affects structural integrity of cilia and cellular signaling molecules.

Cilia are organelles extend from cells to sense external signals for tuning intracellular signaling for optimal cellular functioning. They have evolved sensory and motor roles in various cells for tissue organization and homeostasis in development and post-development. More than a thousand genes are required for cilia function. Mutations in them cause multisystem disorders termed ciliopathies. The null mutations in CC2D2A result in Meckel syndrome (MKS), which is embryonic lethal, whereas patients who have missense mutations in the C2 domain of CC2D2A display Joubert syndrome (JBTS). They survive with blindness and mental retardation. How C2 domain defects cause disease conditions is not understood. To answer this question, C2 domain of Cc2d2a (mice gene) was knocked down (KD) in IMCD-3 cells by shRNA. This resulted in defective cilia morphology observed by immunofluorescence analysis. To further probe the cellular signaling alteration in affected cells, gene expression profiling was done by RNAseq and compared with the controls. Bioinformatics analysis revealed that the differentially expressed genes (DEGs) have functions in cilia. Among the 61 cilia DEGs identified, 50 genes were downregulated and 11 genes were upregulated. These cilia genes are involved in cilium assembly, protein trafficking to the cilium, intraflagellar transport (IFT), cellular signaling like polarity patterning, and Hedgehog signaling pathway. This suggests that the C2 domain of CC2D2A plays a critical role in cilia assembly and molecular signaling hosted in cilia for cellular homeostasis. Taken together, the missense mutations in the C2 domain of CC2D2A seen in JBTS might have affected cilia-mediated signaling in neurons of the retina and brain.

Primary cilia-mediated regulation of microglial secretion in Alzheimer's disease.

Alzheimer's disease (AD) is a brain disorder manifested by a gradual decline in cognitive function due to the accumulation of extracellular amyloid plaques, disruptions in neuronal substance transport, and the degeneration of neurons. In affected neurons, incomplete clearance of toxic proteins by neighboring microglia leads to irreversible brain inflammation, for which cellular signaling is poorly understood. Through single-cell transcriptomic analysis, we discovered distinct regional differences in the ability of microglia to clear damaged neurites. Specifically, microglia in the septal region of wild type mice exhibited a transcriptomic signature resembling disease-associated microglia (DAM). These lateral septum (LS)-enriched microglia were associated with dense axonal bundles originating from the hippocampus. Further transcriptomic and proteomic approaches revealed that primary cilia, small hair-like structures found on cells, played a role in the regulation of microglial secretory function. Notably, primary cilia were transiently observed in microglia, and their presence was significantly reduced in microglia from AD mice. We observed significant changes in the secretion and proteomic profiles of the secretome after inhibiting the primary cilia gene intraflagellar transport particle 88 (Ift88) in microglia. Intriguingly, inhibiting primary cilia in the septal microglia of AD mice resulted in the expansion of extracellular amyloid plaques and damage to adjacent neurites. These results indicate that DAM-like microglia are present in the LS, a critical target region for hippocampal nerve bundles, and that the primary ciliary signaling system regulates microglial secretion, affecting extracellular proteostasis. Age-related primary ciliopathy probably contributes to the selective sensitivity of microglia, thereby exacerbating AD. Targeting the primary ciliary signaling system could therefore be a viable strategy for modulating neuroimmune responses in AD treatments.

Cilia Disorders in the Genomics Era: Historical Overview and Commentary on Ciliopathy Diagnostics

Introduction: Motile and sensory (primary) cilia are organelles that are found on the surface of almost all cells. Defects in cilia cause a number of multi-organ diseases known as ciliopathies, which have clinically heterogeneous symptoms. This heterogeneity makes diagnosing cilia disorders challenging and clinicians often rely on genetic sequencing to delineate ciliopathies from other diseases. However, there is not a consensus on which sequencing tools are most optimal for ciliopathy diagnosis and research. Methods: Here I review the implications of next-generation sequencing tools for ciliopathy diagnostics. I describe landmark studies that showed ciliopathies as genetic conditions and transition to the advantages and challenges of using next-generation sequencing techniques. In particular, I compare studies that utilized targeted sequencing with those that used whole-exome and/or whole-genome sequencing. Discussion: High throughput screens can identify novel cilia genes and show promise as a robust diagnostic tool in clinical settings. Moreover, I compare the effectiveness of whole-exome and whole-genome sequencing both for basic science research and clinical applications, arguing that whole-exome sequencing is a sufficient first pass in clinical settings. I also acknowledge that ciliopathies are associated with many, both significant and insignificant, genetic variants making interpreting next-generation sequencing data an ongoing challenge for scientists and clinicians. Conclusion: This review demonstrates the increasing body of knowledge on cilia genomics and highlights that next-generation sequencing will be integral towards optimizing diagnostics for these heterogeneous and debilitating group of disorders.

A TGFβ-ECM-integrin signaling axis drives structural reconfiguration of the bile duct to promote polycystic liver disease.

The formation of multiple cysts in the liver occurs in a number of isolated monogenic diseases or multisystemic syndromes, during which bile ducts develop into fluid-filled biliary cysts. For patients with polycystic liver disease (PCLD), nonsurgical treatments are limited, and managing life-long abdominal swelling, pain, and increasing risk of cyst rupture and infection is common. We demonstrate here that loss of the primary cilium on postnatal biliary epithelial cells (via the deletion of the cilia gene Wdr35) drives ongoing pathological remodeling of the biliary tree, resulting in progressive cyst formation and growth. The development of cystic tissue requires the activation of transforming growth factor-β (TGFβ) signaling, which promotes the expression of a procystic, fibronectin-rich extracellular matrix and which itself is perceived by a changing profile of integrin receptors on the cystic epithelium. This signaling axis is conserved in liver cysts from patients with either autosomal dominant polycystic kidney disease or autosomal dominant polycystic liver disease, indicating that there are common cellular mechanisms for liver cyst growth regardless of the underlying genetic cause. Cyst number and size can be reduced by inhibiting TGFβ signaling or integrin signaling in vivo. We suggest that our findings represent a therapeutic route for patients with polycystic liver disease, most of whom would not be amenable to surgery.

Recruitment of transcription factor ETS1 to activated accessible regions promotes the transcriptional program of cilia genes.

Defects in cilia genes, which are critical for cilia formation and function, can cause complicated ciliopathy syndromes involving multiple organs and tissues; however, the underlying regulatory mechanisms of the networks of cilia genes in ciliopathies remain enigmatic. Herein, we have uncovered the genome-wide redistribution of accessible chromatin regions and extensive alterations of expression of cilia genes during Ellis-van Creveld syndrome (EVC) ciliopathy pathogenesis. Mechanistically, the distinct EVC ciliopathy-activated accessible regions (CAAs) are shown to positively regulate robust changes in flanking cilia genes, which are a key requirement for cilia transcription in response to developmental signals. Moreover, a single transcription factor, ETS1, can be recruited to CAAs, leading to prominent chromatin accessibility reconstruction in EVC ciliopathy patients. In zebrafish, the collapse of CAAs driven by ets1 suppression subsequently causes defective cilia proteins, resulting in body curvature and pericardial oedema. Our results depict a dynamic landscape of chromatin accessibility in EVC ciliopathy patients, and uncover an insightful role for ETS1 in controlling the global transcriptional program of cilia genes by reprogramming the widespread chromatin state.

Early postnatal microglial ablation in the Ccdc39 mouse model reveals adverse effects on brain development and in neonatal hydrocephalus

BackgroundNeonatal hydrocephalus is a congenital abnormality resulting in an inflammatory response and microglial cell activation both clinically and in animal models. Previously, we reported a mutation in a motile cilia gene, Ccdc39 that develops neonatal progressive hydrocephalus (prh) with inflammatory microglia. We discovered significantly increased amoeboid-shaped activated microglia in periventricular white matter edema, reduced mature homeostatic microglia in grey matter, and reduced myelination in the prh model. Recently, the role of microglia in animal models of adult brain disorders was examined using cell type-specific ablation by colony-stimulating factor-1 receptor (CSF1R) inhibitor, however, little information exists regarding the role of microglia in neonatal brain disorders such as hydrocephalus. Therefore, we aim to see if ablating pro-inflammatory microglia, and thus suppressing the inflammatory response, in a neonatal hydrocephalic mouse line could have beneficial effects.MethodsIn this study, Plexxikon 5622 (PLX5622), a CSF1R inhibitor, was subcutaneously administered to wild-type (WT) and prh mutant mice daily from postnatal day (P) 3 to P7. MRI-estimated brain volume was compared with untreated WT and prh mutants P7-9 and immunohistochemistry of the brain sections was performed at P8 and P18-21.ResultsPLX5622 injections successfully ablated IBA1-positive microglia in both the WT and prh mutants at P8. Of the microglia that are resistant to PLX5622 treatment, there was a higher percentage of amoeboid-shaped microglia, identified by morphology with retracted processes. In PLX-treated prh mutants, there was increased ventriculomegaly and no change in the total brain volume was observed. Also, the PLX5622 treatment significantly reduced myelination in WT mice at P8, although this was recovered after full microglia repopulation by P20. Microglia repopulation in the mutants worsened hypomyelination at P20.ConclusionsMicroglia ablation in the neonatal hydrocephalic brain does not improve white matter edema, and actually worsens ventricular enlargement and hypomyelination, suggesting critical functions of homeostatic ramified microglia to better improve brain development with neonatal hydrocephalus. Future studies with detailed examination of microglial development and status may provide a clarification of the need for microglia in neonatal brain development.

See[ing] elegance in sensory biology: an interview with Piali Sengupta.

See[ing] elegance in sensory biology: an interview with Piali Sengupta.

Rab11a Is Essential for the Development and Integrity of the Stereocilia and Kinocilia in the Mammalian Organ of Corti.

The cochlea hair cells transform mechanic sounds to neural signals with a remarkable sensitivity and resolution. This is achieved via the precisely sculpted mechanotransduction apparatus of the hair cells and the supporting structure of the cochlea. The shaping of the mechanotransduction apparatus, the staircased stereocilia bundles on the apical surface of the hair cells, requires an intricate regulatory network including planar cell polarity (PCP) and primary cilia genes in orienting stereocilia bundles and building molecular machinery of the apical protrusions. The mechanism linking these regulatory components is unknown. Here, we show that a small GTPase known for its role in protein trafficking, Rab11a, is required for ciliogenesis in hair cells during development in mice. In addition, in the absence of Rab11a, stereocilia bundles lost their cohesion and integrity, and mice are deaf. These data indicate an essential role of protein trafficking in the formation of hair cell mechanotransduction apparatus, implicating a role of Rab11a or protein trafficking in linking the cilia and polarity regulatory components with the molecular machinery in building the cohesive and precisely shaped stereocilia bundles.

Analysis of genome-wide knockout mouse database identifies candidate ciliopathy genes

We searched a database of single-gene knockout (KO) mice produced by the International Mouse Phenotyping Consortium (IMPC) to identify candidate ciliopathy genes. We first screened for phenotypes in mouse lines with both ocular and renal or reproductive trait abnormalities. The STRING protein interaction tool was used to identify interactions between known cilia gene products and those encoded by the genes in individual knockout mouse strains in order to generate a list of “candidate ciliopathy genes.” From this list, 32 genes encoded proteins predicted to interact with known ciliopathy proteins. Of these, 25 had no previously described roles in ciliary pathobiology. Histological and morphological evidence of phenotypes found in ciliopathies in knockout mouse lines are presented as examples (genes Abi2, Wdr62, Ap4e1, Dync1li1, and Prkab1). Phenotyping data and descriptions generated on IMPC mouse line are useful for mechanistic studies, target discovery, rare disease diagnosis, and preclinical therapeutic development trials. Here we demonstrate the effective use of the IMPC phenotype data to uncover genes with no previous role in ciliary biology, which may be clinically relevant for identification of novel disease genes implicated in ciliopathies.

Islet primary cilia motility controls insulin secretion.

Primary cilia are specialized cell-surface organelles that mediate sensory perception and, in contrast to motile cilia and flagella, are thought to lack motility function. Here, we show that primary cilia in human and mouse pancreatic islets exhibit movement that is required for glucose-dependent insulin secretion. Islet primary cilia contain motor proteins conserved from those found in classic motile cilia, and their three-dimensional motion is dynein-driven and dependent on adenosine 5′-triphosphate and glucose metabolism. Inhibition of cilia motion blocks beta cell calcium influx and insulin secretion. Human beta cells have enriched ciliary gene expression, and motile cilia genes are altered in type 2 diabetes. Our findings redefine primary cilia as dynamic structures having both sensory and motile function and establish that pancreatic islet cilia movement plays a regulatory role in insulin secretion.

Clustering of Genetic Anomalies of Cilia Outer Dynein Arm and Central Apparatus in Patients with Transposition of the Great Arteries.

Transposition of the great arteries (TGA) is a congenital heart defect with a complex pathogenesis that has not been fully elucidated. In this study, we performed whole-exome sequencing (WES) in isolated TGA-diagnosed patients and analyzed genes of motile and non-motile cilia ciliogenesis and ciliary trafficking, as well as genes previously associated with this heart malformation. Deleterious missense and splicing variants of genes DNAH9, DNAH11, and ODAD4 of cilia outer dynein arm and central apparatus, HYDIN, were found in our TGA patients. Remarkable, there is a clustering of deleterious genetic variants in cilia genes, suggesting it could be an oligogenic disease. Our data evidence the genetic diversity and etiological complexity of TGA and point out that population allele determination and genetic aggregation studies are required to improve genetic counseling.

Endometrial gland specific progestagen-associated endometrial protein and cilia gene splicing changes in recurrent pregnancy loss.

Endometrial glands are essential for fertility, consisting of ciliated and secretory cells that facilitate a suitable uterine environment for embryo implantation. This study sought to determine whether an endometrial gland-specific transcriptome and splicing profile are altered in women with recurrent pregnancy loss. Our data provide a comprehensive catalogue of cilia and progestagen-associated endometrial protein (PAEP) gene isoforms and relative exon usage in endometrial glands. We report a previously unannotated endometrial gland cilia transcript GALNT11 and its susceptibility to exon skipping. Key endometrial receptivity gene transcripts are also reported to change in endometrial glands of women with recurrent pregnancy loss. The endometrial gland cilia and PAEP targets identified in this study could be used to identify a perturbed endometrium, isolate causes of recurrent pregnancy loss and develop targeted therapies in personalised medicine.Lay summarySuccessful embryo implantation is a trade-off between the lining of the womb which receives an implanting embryo, termed the endometrium, and a good quality embryo. For days 21–24 of the menstrual cycle, the endometrium undergoes changes into a receptive state in which it can receive an implanting embryo. Inappropriate endometrial receptivity is thought to underlie recurrent pregnancy loss. Improving pregnancy success in women with recurrent pregnancy loss requires an increased understanding of the endometrium at the molecular level. Genes contain the instructions for the cell and which genes are turned on or off determine how well it can do its role. We sought to determine a gene expression pattern of human endometrial glands in women with recurrent pregnancy loss (n = 5) vs a control group (n = 5). We identify target genes altered in women with recurrent pregnancy loss. Endometrial gland markers could be used to identify inappropriate endometrial receptivity.

Exploring the Mutational Landscape of Isolated Congenital Heart Defects: An Exome Sequencing Study Using Cardiac DNA.

Congenital heart defects (CHD) are the most common congenital anomalies in liveborn children. In contrast to syndromic CHD (SCHD), the genetic basis of isolated CHD (ICHD) is complex, and the underlying pathogenic mechanisms appear intricate and are incompletely understood. Next to rare Mendelian conditions, somatic mosaicism or a complex multifactorial genetic architecture are assumed for most ICHD. We performed exome sequencing (ES) in 73 parent–offspring ICHD trios using proband DNA extracted from cardiac tissue. We identified six germline de novo variants and 625 germline rare inherited variants with ‘damaging’ in silico predictions in cardiac-relevant genes expressed in the developing human heart. There were no CHD-relevant somatic variants. Transmission disequilibrium testing (TDT) and association testing (AT) yielded no statistically significant results, except for the AT of missense variants in cilia genes. Somatic mutations are not a common cause of ICHD. Rare de novo and inherited protein-damaging variants may contribute to ICHD, possibly as part of an oligogenic or polygenic disease model. TDT and AT failed to provide informative results, likely due to the lack of power, but provided a framework for future studies in larger cohorts. Overall, the diagnostic value of ES on cardiac tissue is limited in individual ICHD cases.

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.

Sporadic hypothalamic hamartoma is a ciliopathy with somatic and bi-allelic contributions.

Hypothalamic hamartoma with gelastic seizures is a well-established cause of drug-resistant epilepsy in early life. The development of novel surgical techniques has permitted the genomic interrogation of hypothalamic hamartoma tissue. This has revealed causative mosaic variants within GLI3, OFD1 and other key regulators of the sonic-hedgehog pathway in a minority of cases. Sonic-hedgehog signalling proteins localize to the cellular organelle primary cilia. We therefore explored the hypothesis that cilia gene variants may underlie hitherto unsolved cases of sporadic hypothalamic hamartoma. We performed high-depth exome sequencing and chromosomal microarray on surgically resected hypothalamic hamartoma tissue and paired leukocyte-derived DNA from 27 patients. We searched for both germline and somatic variants under both dominant and bi-allelic genetic models. In hamartoma-derived DNA of seven patients we identified bi-allelic (one germline, one somatic) variants within one of four cilia genes-DYNC2I1, DYNC2H1, IFT140 or SMO. In eight patients, we identified single somatic variants in the previously established hypothalamic hamartoma disease genes GLI3 or OFD1. Overall, we established a plausible molecular cause for 15/27 (56%) patients. Here, we expand the genetic architecture beyond single variants within dominant disease genes that cause sporadic hypothalamic hamartoma to bi-allelic (one germline/one somatic) variants, implicate three novel cilia genes and reconceptualize the disorder as a ciliopathy.

Interpreting ciliopathy-associated missense variants of uncertain significance (VUS) in Caenorhabditis elegans.

Better methods are required to interpret the pathogenicity of disease-associated variants of uncertain significance (VUS), which cannot be actioned clinically. In this study, we explore the use of an animal model (Caenorhabditis elegans) for in vivo interpretation of missense VUS alleles of TMEM67, a cilia gene associated with ciliopathies. CRISPR/Cas9 gene editing was used to generate homozygous knock-in C. elegans worm strains carrying TMEM67 patient variants engineered into the orthologous gene (mks-3). Quantitative phenotypic assays of sensory cilia structure and function (neuronal dye filling, roaming and chemotaxis assays) measured how the variants impacted mks-3 gene function. Effects of the variants on mks-3 function were further investigated by looking at MKS-3::GFP localization and cilia ultrastructure. The quantitative assays in C. elegans accurately distinguished between known benign (Asp359Glu, Thr360Ala) and known pathogenic (Glu361Ter, Gln376Pro) variants. Analysis of eight missense VUS generated evidence that three are benign (Cys173Arg, Thr176Ile and Gly979Arg) and five are pathogenic (Cys170Tyr, His782Arg, Gly786Glu, His790Arg and Ser961Tyr). Results from worms were validated by a genetic complementation assay in a human TMEM67 knock-out hTERT-RPE1 cell line that tests a TMEM67 signalling function. We conclude that efficient genome editing and quantitative functional assays in C. elegans make it a tractable in vivo animal model for rapid, cost-effective interpretation of ciliopathy-associated missense VUS alleles.