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The ciliopathies are a category of diseases caused by disruption of the physiological functions of cilia. Ciliary dysfunction results in a broad range of phenotypes, including renal, hepatic, and pancreatic cyst formation; situs abnormalities; retinal degeneration; anosmia; cerebellar or other brain anomalies; postaxial polydactyly; bronchiectasis; and infertility. The specific clinical features are dictated by the subtype, structure, distribution, and function of the affected cilia. This review highlights the clinical variability caused by dysfunction of motile and nonmotile primary cilia and emphasizes the genetic heterogeneity and phenotypic overlap that are characteristics of these disorders. There is a need for additional research to understand the shared and unique functions of motile and nonmotile cilia and the pathophysiology resulting from mutations in cilia, basal bodies, or centrosomes. Increased understanding of ciliary biology will improve the diagnosis and management of primary ciliary dyskinesia, syndromic ciliopathies, and cilia-related cystic diseases.
Ciliopathies are phenotypically and genetically heterogeneous disorders that share ciliary dysfunction as a common pathological mechanism. Motile cilia are primarily found on epithelial cells that line the respiratory tract, brain ventricles, and oviducts. These cilia have nine microtubule doublets surrounding a central pair, a “9+2” arrangement that is visible by electron microscopy of cross-sectional views. Absence or dysfunction of motile cilia causes primary ciliary dyskinesia (PCD). In contrast, primary cilia lack the central microtubule doublet and have a “9+0” arrangement of microtubules. Primary cilia extend from the surface of almost all cell types in the human body, including epithelial cells such as those lining the kidney tubules and bile ducts as well as nonepithelial cells such as chondrocytes and neurons (1). Primary cilia are present as monocilia and exist as a single extension per cell. Initially, cilia were described as motile or nonmotile, with the latter group encompassing primary cilia or sensory cilia (Table 1). However, the discovery of motile primary cilia at the embryonic node and the identification of sensory functions for motile cilia has rendered these historic designations imperfect. Although these broad classifications have utility, as the complexities of cilia structure and function are better understood, further delineation of subtypes may occur. For example, the rod and cone cells of the retina possess a very unique type of primary cilium (connecting cilium) equipped with a specialized expanded tip that forms the outer segment of these photoreceptor cells (2). The node or organizer, a transient embryonic structure required for left-right patterning, also possesses a unique type of primary cilium that lacks a central pair of microtubules but is nevertheless motile. Dysfunction of cilia at the node can result in situs inversus or heterotaxy, a phenotype that is characteristic of PCD and primary, nonmotile ciliopathies. Understanding the biological function of motile and nonmotile cilia will provide insight into the underlying pathophysiology of ciliary disorders and the wide array of clinical phenotypes resulting from their disruption. The number of diseases known to be caused by ciliary pathology has increased dramatically in the last decade (summarized in Table 2). The diagnosis and management of patients with ciliary disorders will benefit from elucidating the shared and unique functions directed by motile and nonmotile cilia in a tissue- and developmental-stage–specific context.
Primary cilia, both nonmotile cilia and motile cilia at the node, function as sensory organelles and coordinators of signal transduction pathways such as hedgehog (Hh), wingless (WNT), platelet-derived growth factor-α, and fibroblast growth factor pathways (3, 4). In addition, primary cilia are important for the regulation of intracellular calcium. Nonmotile cilia are sensory antennas of the cell, and intact nonmotile cilia function is required for maintenance of the balance between the canonical and noncanonical (planar cell polarity) parts of the WNT pathway. For example, disruption of planar cell polarity contributes to the pathogenesis of polycystic kidney disease (PKD) by resulting in abnormalities in multicellular activities that maintain tubular diameter, such as mitotic spindle orientation and convergence and extension movements (5). Nonmotile cilia also sense mechanical stimuli such as fluid flow in the lumen of tubular structures (kidney tubules and bile ducts) and convert these to alterations in calcium fluxes within the cell (1). Proteins defective in human PKD are required for this mechanosensory function of nonmotile cilia. Hence, during embryologic development, primary cilia regulate fundamental processes, such as determination of asymmetry of internal organs and neural tube and limb patterning. Nonmotile cilia are also required for the maintenance of differentiated tissues, such as retina and renal tubules. In addition, they play a role in the pathogenesis of certain types of obesity, probably through their role in appetite control and adipocyte differentiation (6).
Heterotaxy, or situs ambiguus, is an abnormal arrangement of thoracic and/or abdominal viscera. Situs inversus is another type of laterality disorder in which all internal organs are reversed. Typically, patients with situs inversus do not have the congenital anomalies that occur in heterotaxy syndrome. Embryologically, heterotaxy and situs inversus fall into the category of disorders of laterality because they have their root in the abnormal development of the left–right body axis. Primary cilia present on the embryonic node are required for the generation of the left–right axis during embryonic development (7, 8). The ciliated embryonic node is unusual in that it contains motile primary cilia that generate fluid flow and nonmotile primary cilia that act as mechanosensors. Abnormal situs is a hallmark feature of a number of ciliopathies.
The nomenclature related to heterotaxy has traditionally been confusing, with both anatomic and clinical designations (e.g., right isomerism, asplenia syndrome) (9). Any internal organ that is asymmetrically positioned can be abnormal in individuals with heterotaxy. Improper symmetry, such as bilateral trilobed lungs, and failure to regress symmetric embryonic structures, such as persistent bilateral superior vena cava, are additional manifestations of heterotaxy. Midline defects occur in approximately 40% of patients (10). The wide phenotypic spectrum and range of congenital anomalies that can be seen in heterotaxy syndrome has hindered clinical care and research progress.
?Classic? heterotaxy, in which a characteristic cardiovascular malformation is identified in conjunction with visceral situs anomalies, comprises approximately 3% of congenital heart defect cases and has an estimated prevalence of 1 in 10,000 live births. However, this most likely underestimates the true incidence of heterotaxy spectrum disorders. Heterotaxy is a clinically and genetically heterogeneous condition, and patients with a combination of cardiovascular malformations and visceral situs anomalies represent the severe end of this spectrum. Isolated cardiovascular malformations may be caused by mutations in the same genes that cause heterotaxy, and therefore the true incidence of these defects of laterality is not known with certainty (11). Overall, male patients with heterotaxy outnumber female patients 2:1, although within the polysplenia subtype the gender distribution is equal. Known environmental risk factors for heterotaxy include twin gestation, maternal diabetes, and maternal cocaine use (12).
The clinical evaluation for patients with heterotaxy syndrome is focused on delineating the anatomy and managing the congenital anomalies present (13). Most frequently, patients with heterotaxy present in the newborn period with cyanotic congenital heart disease. Echocardiography is important for defining cardiac anatomy. In some cases, chest MRA is useful for better visualization of the vasculature. Abdominal ultrasound is necessary to determine the position of the liver and stomach, spleen position and number, and renal anatomy. Other imaging includes head ultrasound or brain MRI, upper gastrointestinal series to evaluate malrotation, chest radiograph and spine films, and consideration of hebatobiliaryscintigraphy. A blood smear for Howell-Jolly bodies is an initial evaluation for splenic function. Chromosome microarray analysis is indicated due to the association with chromosome abnormalities. In addition, molecular testing for ZIC3, NODAL, CFC1, and FOXH1, four genes known to cause heterotaxy, is available on a clinical basis.
Consideration should be given to evaluation for PCD in patients with heterotaxy. The prevalence of PCD in this population is unknown, but recent clinical research and animal models suggest that the underlying etiology may be identical for a subset of patients in these groups. Neonatal respiratory distress (not related to cardiovascular malformations) is a frequent manifestation of PCD. Chronic wet, productive cough; recurrent pneumonia; and persistent rhinitis are important features that should trigger evaluation. Bronchiectasis, chronic otitis media with conductive hearing loss, hydrocephalus, or retinitis pigmentosa are features of motile ciliary dysfunction that should be recognized as symptoms unrelated to the cardiovascular malformations in heterotaxy and should prompt evaluation by the appropriate specialists, including pulmonologists. Further clinical research is necessary to better define the degree of overlap between PCD and heterotaxy and to provide better management of comorbidities.
Heterotaxy is highly heritable, indicating a strong genetic influence, although complex or multifactorial causation may predominate. X-linked heterotaxy is caused by mutations or deletions of the zinc-finger transcription factor ZIC3. We have investigated the mechanistic basis of X-linked heterotaxy in mouse and Xenopus models (14–16). The results indicate that Zic3 acts early in development upstream of Nodal expression at the node. Further identification of the developmental pathways disrupted by Zic3 loss of function will elucidate important candidate genes for human heterotaxy and will further delineate shared and divergent functions with genes causing PCD and other ciliopathies.
Mutations in genes important in the generation of left–right patterning, such as NODAL and CFC1, have been shown to cause heterotaxy (17–19). Gene mutations identified in human laterality disorders have been reviewed recently (13). The majority of the mutations identified have reduced penetrance and variable expressivity. Although most cases of heterotaxy are single occurrences in a pedigree, approximately 10% of infants with heterotaxy have a family history of a close relative with a congenital heart defect. Epidemiologic data strongly indicate the important role of genetic factors in heterotaxy, but there is a fundamental lack of information about the genetic basis of disease in the vast majority of cases.
Diseases of the nonmotile cilia are commonly associated with a spectrum of fibrocystic changes in the kidneys and the liver, including PKD, nephronophthisis (NPHP) and renal cystic dysplasia, congenital hepatic fibrosis, Caroli's disease, and polycystic liver disease (20, 21). Other features include developmental anomalies of the cerebellum and midbrain, retinal degeneration, colobomas of the retina and iris, polydactyly, abnormal bone growth, obesity, anosmia and other sensory defects, situs defects, and pancreatic cysts or dysplasia (Table 2). Autosomal dominant (ADPKD) and recessive (ARPKD) polycystic kidney diseases and NPHP are the most common hepatorenal fibrocystic diseases. Pleiotropic syndromic ciliopathies with multisystem involvement include Joubert syndrome (JS) and related cerebellar disorders, Bardet-Biedl (BBS), Alstrom syndrome, Meckel-Gruber (MKS), Senior-Loken syndrome, and oral-facial-digital type 1 (OFD1) syndromes. Skeletal ciliopathies include Jeune chondrodysplasia, cranioectodermal dysplasia, short-rib polydactyly, and Ellis-Van Creveld (EVC) syndromes.
Liver disease in ciliopathies is not a primary disease of the hepatocytes but rather is a developmental defect of the portobiliary system, termed ductal plate malformation (DPM) (21, 22). DPM is defective remodeling of the biliary system characterized by retention of excessive numbers of embryonic bile duct remnants, abnormal portal veins, and periportal fibrosis. DPM of the peripheral biliary system causes congenital hepatic fibrosis, whereas DPM of the central biliary tree results in Caroli's disease. The combination of the two is referred to as Caroli's syndrome. The characteristics of liver disease in ARPKD and ADPKD are different. ARPKD is characterized by congenital hepatic fibrosis and Caroli's disease and is often complicated by portal hypertension, whereas ADPKD patients develop polycystic liver disease and typically do not have portal hypertension (20, 21). In contrast to Caroli's disease/Caroli's syndrome, in which liver cysts are dilated parts of the bile tree itself, polycystic liver disease is characterized by isolated cysts that are not in continuity with the bile tree.
JS and related cerebellar disorders are a heterogeneous group of disorders in which the defining common pathology is the so-called “molar tooth sign” on axial brain MRI that is caused by a combination of mid- and hindbrain anomalies, including cerebellar vermis hypoplasia and abnormal superior cerebellar peduncles (23). Oculomotor apraxia and speech apraxia are typical. Variable features in JS include retinal degeneration, colobomas, renal disease (commonly in the form of NPHP), congenital hepatic fibrosis/Caroli's syndrome, and polydactyly. BBS is characterized by retinal degeneration, cognitive impairment, obesity, and hypothalamic hypogonadism in male patients; variable features include renal disease, congenital hepatic fibrosis, polydactyly, cardiac defects, situs inversus, and abnormalities of internal genital organs in female patients (24). Älstrom syndrome is also characterized by retinal dystrophy, obesity, cardiomyopathy, progressive sensorineural hearing impairment, and insulin resistance. MKS represents the most severe end of the nonmotile ciliopathy spectrum, with the typical triad of occipital encephalocele, cystic dysplastic kidneys, and postaxial polydactyly (25). Congenital hepatic fibrosis is an invariable feature in MKS. OFD1 is an X-linked dominant, male-lethal, nonmotile ciliopathy characterized by prominent external features, including oral clefts, hamartomas, or cysts of the tongue, and digital anomalies and visceral involvement, including PKD and biliary and pancreatic cystic disease (26). Jeune chondrodysplasia, which is also referred to as asphyxiating thoracic dysplasia, is characterized by small thorax due to short ribs, short stature that is mainly caused by short limbs, and polydactyly (27). Jeune patients who survive the small thorax-related respiratory distress develop NPHP, retinopathy, and fibrocystic disease of the pancreas. EVC is characterized by short stature associated with short limbs, congenital cardiac defects, teeth abnormalities, congenital hepatic fibrosis, and situs anomalies in some cases (28). Cranioectodermal dysplasia is characterized by frontal predominance and widely spaced eyes; teeth, hair, and nail abnormalities; congenital hepatic fibrosis; and a NPHP-like renal pathology (29).
Genetic heterogeneity (multiple genes causing similar clinical phenotype) and genotypic and phenotypic overlap are two characteristics of syndromic ciliopathies (30, 31). There are multiple genes identified for JS and related cerebellar disorders, BBS, and MKS, still not accounting for all patients. These disorders were originally defined based on their most prominent clinical features. As we understand these disorders better, we realize that the borders between them are blurred at the phenotypic and genotypic levels.
Nephronophthisis (NPHP), an autosomal recessive cystic kidney disease, represents the most common genetic cause of terminal renal failure occurring in the first three decades of life. It may be associated with retinitis pigmentosa (RP) in the renal-retinal disorder Senior-Loken syndrome or with cerebellar vermis aplasia and mental retardation in JS. Ten different recessive genes have been identified as mutated in NPHP-related ciliopathies (NPHP-RC) with and without extrarenal manifestations.
NPHP1 is mutated in NPHP type 1 (32). Its gene product, nephrocystin-1, localizes to transition fibers of cilia and acts in focal adhesion signaling. Mutations in the NPHP2/inversin gene cause infantile NPHP (type 2) with the rare association of situs inversus and RP. Demonstration of expression of NPHP1 and NPHP2 in primary cilia of renal epithelial cells (33) supported the “ciliopathy theory” for the pathogenesis of cystic kidney diseases (34). This theory states that the products (“cystoproteins”) of all genes mutated in cystic kidney disease in humans, mice, or zebrafish are expressed in primary cilia, basal bodies, or centrosomes of renal epithelial cells (35). Identification of NPHP3 as mutated in NPHP type 3 supported this theory and revealed an Nphp3 mutation as causing the renal cystic disease in pcy mice (36). For this animal model, efficient treatment was demonstrated (37).
Some of the general features of renal cystic diseases are exemplified by studies on the NPHP gene products nephrocystins. Positional cloning of NPHP4 as mutated in Senior-Loken syndrome (38) led to the demonstration that its gene product, nephrocystin-4, is conserved in Caenorhabditis elegans and expressed together with nephrocystin-1 in ciliated head and tail neurons of the nematode (39). The role of primary cilia function for retinal–renal syndromes was confirmed by identification of the novel NPHP5 gene (40), demonstrating interaction of nephrocystin-5 with calmodulin and the retinitis pigmentosa GTPase regulator (RPGR). Recently, several signaling pathways have been implicated in the downstream signaling pathways that connect ciliary and basal body function to the renal cystic phenotype. These include the Wnt signaling and planar cell polarity pathways (41–43) and the hedgehog signaling pathway (44).
Recessive mutation of NPHP6/CEP290 was found to cause JS (45). NPHP6 encodes a centrosomal protein, nephrocystin-6, which modulates the activity of ATF4/CREB2, a transcription factor implicated in cAMP-dependent renal cyst formation. We demonstrated that abrogation of nphp6 function in zebrafish causes planar cell polarity (convergent extension) defects and recapitulates the human phenotype of JS. Hypomorphic mutations in NPHP6/CEP290 were shown to cause retinal degeneration (rds16) in mice (46) and represent a major cause of Leber's congenital amaurosis in humans (47). Identification of the NPHP6 gene, therefore, established a link between centrosome function and tissue architecture in the pathogenesis of cystic kidney disease and in central nervous system development. Mutation of GLIS2/NPHP7 causes NPHP type 7 in humans and mice (48). Differential gene expression studies on Glis2 mutant kidneys demonstrated that genes promoting epithelial-to-mesenchymal transition and fibrosis are up-regulated in the absence of Glis2, demonstrating that Glis2 is essential for the maintenance of renal tissue architecture through prevention of apoptosis and fibrosis. Recently, mutations of RPGRIP1L/NPHP8 were shown to cause MKS and JS (49), and it has been demonstrated that NEK8 mutations may rarely cause NPHP (50).
Ciliary dysfunction leads to a wide range of phenotypes, many of which overlap. In recessive ciliopathies, the nature of the two recessive mutations determines the severity and extent of organ involvement, leading to seemingly different disorders. In this context, loss-of-function mutations cause severe, early-onset, dysplastic, multiorgan disease, whereas reduced-function mutations cause mild, late-onset, degenerative disease with limited organ involvement. For example, recent data indicate that mutations in TTC21B, which encodes the intraflagellar transport protein IFT139, cause isolated NPHP or Jeune asphyxiating thoracic dystrophy depending on mutation type (51). Causal genes can also act as modifier alleles in clinically distinct disorders. In the case of TTC21B, sequence-based interrogation of a large ciliopathy cohort identified pathogenic rare variants in approximately 5% of cases. These alleles are proposed to act in trans with other disease causing genes to influence the range of ciliopathy phenotypes.
In human ciliary disorders, motor and sensory functions have been considered distinct properties of motile and nonmotile cilia, respectively. However, there are recent data that indicate that motile cilia of the airway are important for sensory reception (52, 53). It is theoretically possible that defects in certain ciliary proteins whose functions are important for the motile and nonmotile cilia can result in dysfunction of both types of cilia, thereby causing diverse phenotypes. For example, recently seven patients with Leber's congenital amarosis due to CEP290 mutations were shown to have defective respiratory cilia (54). In addition, patients with autosomal dominant polycystic kidney disease have been shown to have increased prevalence of radiographic bronchiectasis (55). Additional examples of subtle or subclinical phenotypic overlap may become apparent with the growing clinical appreciation of the spectrum of ciliopathy features.
Heterotaxy, PCD, and other ciliopathies that occasionally have abnormal laterality as features share a similar underlying pathogenesis centering on the role of cilia. Heterotaxy may result from defects in any one of a series of signaling or morphogenetic processes that are required for the establishment of the left-right axis. In contrast, situs inversus caused by PCD usually results from specific defects in the structure and function of cilia at the node. Recent data indicate that not less than 6% of patients with PCD have heterotaxy, highlighting the overlap of these conditions (56). Situs defects are also observed in nonmotile ciliopathies, including RHPD, EVC, JC, and rare cases of BBS, JS, and OFD1. There is a lack of clarity regarding the mechanisms underlying the development of situs inversus versus heterotaxy as phenotypes. An improved mechanistic understanding of the steps leading to the development of abnormal laterality is critical to delineate the interrelatedness of these disorders and to direct proper management and therapy.
Within the last 5 years, the improved technology available for genetic analyses has had a dramatic impact on the investigation of ciliopathies. This has facilitated a more rapid identification of causative genes, investigation of modifier alleles, expansion and refinement of genotype–phentoype correlations, and improvement of clinical diagnostics.
The use of genome-wide copy number analysis coupled with in vitro and in vivo analyses of gene function is a powerful technique for the identification of novel genetic causes of ciliopathies. Imbalances in chromosomal copy number resulting in deletion or duplication of a small number of genes are a known cause of congenital heart defects in a small number of patients with genetic syndromes. Until recently, it has not been possible to evaluate the entire genome for these genetic alterations. Recently, whole–genome, single-nucleotide polymorphism genotyping was used to evaluate chromosomal copy number imbalances in a well characterized cohort of patients with complex cardiovascular malformations and heterotaxy syndrome. The results indicate that 15 to 30% of patients have pathogenic copy number variation. Efforts are ongoing to identify dosage-sensitive genes within these regions of chromosomal imbalance that will allow for identification of novel genes required for cardiac development and patterning (57). Identification of specific genes underlying heterotaxy spectrum congenital heart disease will lead to the development of novel diagnostics and therapeutics and improved management.
Sequence-based analysis is another strategy that has been enhanced by the development of novel genomic technology and subsequently has proven tremendously valuable for the study of ciliopathies. Massively parallel resequencing improves the ability to interrogate large numbers of disease-causing genes in well phenotyped cohorts, and as a result there is a wider appreciation of the genetic architecture and phenotypic spectrum of the ciliopathies (58, 59). These methods are also translating into improved availability of testing on a clinical basis. However, challenges remain in the interpretation of rare variants in a clinical setting. Exome capture technology allows unbiased interrogation of all coding region of the genome, allowing identification of novel disease-causing genes. This approach is particularly useful for autosomal recessive conditions. Recently, exome capture identified 12 different truncating mutations of SDCCAG8 (serologically defined colon cancer antigen 8) in 20 individuals with Senior-Loken syndrome of 10 different families (60). SDCCAG8 is localized at both centrioles and interacts directly with OFD1. Depletion of sdccag8 causes kidney cysts and a body axis defect in zebrafish and induces cell polarity defects in three-dimensional renal cell cultures. Further gene identification in NPHP will result in the definition of functional networks of primary cilia, centrosomes, and planar cell polarity as they pertain to the pathogenic mechanisms of NPHP-associated syndromes and other cystic kidney diseases.
Primary cilia are ubiquitous organelles that serve a sensory function. Disruption of the function of primary cilia results in clinically heterogeneous disorders that may affect a single organ or result in a syndromic phenotype. There is substantial clinical and genetic heterogeneity. There is some evidence suggesting that certain ciliary defects might affect primary and motile cilia associated with PCD. Additional research is needed to better understand the biological functions of these diverse classes of cilia, the genetic basis of disease, and the management of clinical symptoms caused by functional disruption.
The authors gratefully acknowledge the sponsorship and meeting organization provided by the Primary Ciliary Dyskinesia Foundation.
The conference was supported by a grant from the National Institutes of Health (R13HL105073–01) and by the Primary Ciliary Dyskinesia Foundation. The authors’ research is supported by the National Institutes of Health grants HL088639 (S.M.W.) and DK068305 and DK064614 (F.H.) and Burroughs-Wellcome Fund #1008496 (S.M.W.).
Author Disclosure: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.