Clinical and genetic heterogeneity of primary ciliopathies (Review).

Ciliopathies comprise a group of complex disorders, with involvement of the majority of organs and systems. In total, >180 causal genes have been identified and, in addition to Mendelian inheritance, oligogenicity, genetic modifications, epistatic interactions and retrotransposon insertions have all been described when defining the ciliopathic phenotype. It is remarkable how the structural and functional impairment of a single, minuscule organelle may lead to the pathogenesis of highly pleiotropic diseases. Thus, combined efforts have been made to identify the genetic substratum and to determine the pathophysiological mechanism underlying the clinical presentation, in order to diagnose and classify ciliopathies. Yet, predicting the phenotype, given the intricacy of the genetic cause and overlapping clinical characteristics, represents a major challenge. In the future, advances in proteomics, cell biology and model organisms may provide new insights that could remodel the field of ciliopathies.

1. Introduction

Ciliopathies comprise a heterogeneous group of genetic disorders caused by structural or functional disruption of cilia, or by abnormal cilia biogenesis (1,2). The two main subcategories, namely motile and immotile/primary ciliopathies, both involve disruption of the cilium, and also share several causal genes (3-5). However, clinically, they are quite different; while motile ciliopathies (Kartagener syndrome and primary ciliary dyskinesia) are characterized by pulmonary disease, infertility, situs inversus or reversal of organ laterality (6), primary ciliopathies include a wide class of diseases that range from organ-specific disorders to pleiotropic syndromes with multiorgan involvement. These distinct phenotypes may be explained through the structural differences between primary and motile cilia, as well as their distinct functions (7).

The aim of the present review was to comprehensively describe the primary ciliopathies, focusing on genetic heterogeneity, diagnosis and clinical aspects, with a brief overview of their biological basis.

2. Cilium

Structure

Motile cilia have been observed in protozoa since the early microscopy era (8). Unlike motile cilia, which are concentrated in clusters and line the respiratory tract, fallopian tubes, the efferent ductules of the testis and brain ventricles (9), the primary cilium is a single hair-like organelle, with variable length (1-9 μm) (10), projecting from the apical surface of almost all types of cells, with certain exceptions (lymphocytes, granulocytes, hepatocytes and acinar cells) (11). Primary cilia are dynamic organelles that are assembled in the G0/G1 cell cycle stage and become disassembled with the onset of cell division (12).

Both types of cilia are structurally composed of a microtubule backbone, termed the axoneme, surrounded by matrix and covered by the ciliary membrane, which is continuous with the plasma membrane (Fig. 1). At the base of this ensemble, a specialized centriole, referred to as the basal body (BB), docks the cilium to the cell (13). The axoneme of the primary cilium consists of 9 outer doublet microtubules, (9 + 0 type), while motile cilia possess an extra inner pair of microtubules, reinforced by nexin bridges (9 + 2 type) and an accessory structure involved in motility, formed of dynein arms and radial spokes (14). Each doublet contains a complete microtubule (A tubule) and one incomplete microtubule (B tubule), which are composed of tubulin protofilaments and are attached to each other through tektins and Ca-binding ribbon proteins (15). The BB structure contains 9 radially arranged microtubule triplets (A, B and C) and no central pair. The A and B tubules expand into the proximal segment of the cilium and are connected to the ciliary membrane by Y fibers, constituting a distinctive subcompartment known as the transition zone. Proximal to the Y fibers are the transitional fibers, which help to anchor the BB to the plasma membrane (16). The BB is responsible for the configuration of the microtubule scaffold and coordinates the ciliary trafficking pathway; thus, it is involved, together with transitional fibers, in ciliogenesis (17). Surrounding the transitional fibers are numerous strings of particles, acting as a selective filter for intraflagellar transport (IFT) molecules, known as the ciliary necklace (18,19). In the distal region of the cilium, the backbone contains a single microtubule fiber (A tubule), which delimits the ciliary tip, a proteic zone with cell type-specific structure and function (20,21). In addition to this classical structure of the cilia, there is evidence showing the existence of motile 9 + 0 cilia, covering the node, which are responsible for left-right asymmetry, or sensory 9 + 2 cilia, which are observed in the inner ear cells (22-26).

Figure 1

Schematic representation of the cilium structure. (A) Longitudinal section, (B-a) transversal view of the motile cilium and (B-b) transversal view of the primary cilium. IFT, intraflagellar transport; Wnt, wingless.

Function of the primary cilium

Since their discovery in the kidneys and the thyroid gland by Zimmermann in 1898, primary cilia have been considered as vestigial organelles, without a specific function, due to their lack of motility and their absence in several cells during mitosis (27). In 1975, Webber and Lee (28) raised the hypothesis of a possible sensory role of mammalian nephron cilia, by comparing them to those in sensory tissues. This hypothesis was confirmed in 2000 in a study by Pazour et al (29), which presented experimental evidence showing the physiological function of the primary cilium. Once the implication of primary cilia in human diseases was demonstrated (30), the awareness of the significance of this organelle increased. Subsequently, a number of studies demonstrated the complex roles of primary cilia as mechanoreceptors, chemoreceptors and osmosensors (31-34).

A highly specialized process occurring in the ciliary compartment is the IFT: A bidirectional movement during which a protein complex (IFT particle) is shuttled along the microtubule backbone from the BB to the tip of the cilium (through kinesin-anterograde transport) and back (facilitated by dynein-retrograde transport) (35). As the synthesis of proteins essential for the development of cilia is not possible inside the ciliary compartment and proteins are carried through IFT, the importance of IFT in ciliogenesis must be emphasized, as well as its involvement in the delivery of signals from the cilium to the cell, higlighting its significant role in cilia-mediated signaling pathways (36).

Furthermore, >25 receptors and ion channels have been localized to the ciliary membrane, where a growing number of extracellular signals are received and transduced by the ciliary ensemble, facilitating certain signaling pathways that control the development of organs, as well as behavioral processes. Particularly important primary cilia-related signaling pathways include the following: Wingless (Wnt), Hedgehog (Hh), receptor tyrosine kinase (RTK), G-protein coupled receptors (GPCRs), Notch, transforming growth factor-β (TGF-β), mechanistic target of rapamycin (mTOR) and Salvador-Warts-Hippo (SWH) signaling. In addition, other signaling pathways that have been linked to primary cilia include extracellular matrix protein-mediated signaling, transient receptor potential channel-mediated signaling, vasopressin signaling in renal epithelial cells, somatostatin, serotonin and melanin-concentrating hormone signaling (37,38).

The Wnt signaling pathway comprises a large family of secreted, cysteine-rich proteins, acting as a network of signal transduction pathways that are responsible for embryonic development, as well as tissue homeostasis and regeneration in adults (39). At least three signaling pathways have been described: The canonical Wnt pathway (or Wnt/β-catenin pathway), and the non-canonical planar cell polarity (PCP) and Wnt/Ca2+ pathways. The primary cilium and BB were found to be required for the regulation of both canonical and non-canonical Wnt signaling pathways. Canonical Wnt signaling acts through its end effectors as co-transcriptional factors, together with the T-cell factor/lymphoid enhancer factor 1 family of proteins, and co-activates the expression of Wnt target genes to modulate the cell cycle, leading to cell differentiation, proliferation, adhesion and migration, and tissue development (40). Canonical Wnt signaling appears to be directly or indirectly implicated in the formation of almost all organ systems during embryogenesis; it has been shown to be involved in anterior head fold formation and neuroectodermal patterning, in controlling further posterior patterning, as well as in the genesis and development of the heart, lungs, kidney, eyes, skin, blood cells and bone (41,42). In addition, the essential role of the Wnt pathway in stem cell renewal has been highlighted (42,43). The non-canonical PCP pathway appears to act independently on transcription and plays a key role in the modification and rearrangement of the actin cytoskeleton. Moreover, the molecular constituents of this pathway were shown to randomize the orientation of polarized epithelial cells and to coordinate the morphology and convergent extension of dorsal mesodermal and ectodermal cells during gastrulation and neural tube closure (44). Yet, the role of cilia in canonical Wnt signal transduction is controversial, although several studies have suggested the importance of primary cilia in the decrease of canonical Wnt signaling (45-47). By contrast, the contribution of the integrity of primary cilia to the non-canonical PCP Wnt pathway is well established. Movement of the BB to the apical cell surface and centriolar position are essential for the establishment of cell polarity; thus, defects in ciliary proteins implicated in ciliogenesis and BB migration lead to various PCP errors (48). The Wnt/Ca2+ pathway shares a number of components with the PCP, but has been described as a separate pathway, which stimulates intracellular Ca2+ release from the endoplasmic reticulum. Ca2+ waves are hypothesized to serve as a key modulator in early pattern formation during embryo gastrulation. The Wnt/Ca2+ pathway regulates embryogenesis in a complex manner, including promoting ventral cell fate, negative regulation of dorsal axis development, regulation of tissue separation and convergent extension movements during gastrulation, as well as heart formation. The Wnt/Ca2+ pathway also functions as a critical modulator of both the canonical and PCP pathways (44). Wnt signaling also regulates a number of other signaling pathways that have not been yet completely elucidated, but appear to be linked with myogenesis, axonal guidance, neuronal migration and synaptogenesis (49,50).

Another key signaling pathway that was demonstrated to be essential for a variety of developmental processes is the Hh signaling pathway, described through its three Hh homologues: Desert Hh, Indian Hh (Ihh) and Sonic Hh (Shh). The Shh pathway, the most extensively investigated signaling pathway, functions due to the synergy of several molecule/proteins acting as transmembrane receptors, namely Patched homolog 1, Smoothened (SMO) and GLI transcription factors, leading to the transcription of Hh target genes (51). The role of Shh proteins emerges during embryonic development and morphogenesis, controlling left-right asymmetry, dorso-ventral axes and distal limb patterning. Moreover, proliferation of hematopoietic, retinal and neural stem cells, as well as development of epithelial tissues during organogenesis, appear to be modulated by Shh (52). Primary cilia are essential for the transduction of Hh signaling, playing a dual role through positive and negative regulation. It has been shown that abnormal cilia may lead to either loss-of-function Hh phenotypes in the neural tube, or gain-of-function Hh phenotypes in the limbs, indicating that the Hh pathway may play an important role in primary cilia biogenesis (38).

The migration, proliferation, differentiation and apoptosis of cells are also controlled by another cilia-related pathway, the platelet-derived growth factor receptor-α (PDGFRα) pathway (53,54). PDGFRα belongs to the large family of RTK transmembrane receptors and is required for activation of the Ras-Mek1/2-Erk1/2 pathway, thus causing axonemal reestablishment, cell cycle progression and chemotaxis (55). The development of numerous cells and tissues, including neurons, oligodendrocytes, astrocytes, alveolar smooth muscle cells, cardiac fibroblasts and bone cells, relies on PDGFα signaling (56). PDGFRα, which is bound to the membrane of the primary cilium, regulates cytoskeletal reorganization to drive directional migration of fibroblasts in wound healing. Defects in primary cilia lead to abnormal wound healing. Moreover, disassembly of cilia, which allows the centriole to participate in mitosis during cell cycle progression, is modulated by PGFRα signaling (57). Along with this signaling, other RTK signaling pathways have recently been described, including EGFR, which plays an important role in mechanosensation and the migration of kidney epithelial cells or airway smooth muscle cells, and insulin-like growth factor receptor, which is involved in preadipocyte differentiation (37).

GPCRs comprise a large family of transmembrane receptors divided into six classes (A-F), for which >30 receptors belonging to the A (rhodopsin-like receptors), B (secretin receptor family) and F (frizzled/SMO-component of Shh signaling) classes are found on the ciliary membrane. Among these receptors, opsin, olfactory, serotonin (HTR6), somatostatin (SSTR3), vasopressin (V2R), dopamine (D1R, D2R and D5R) and prostaglandin (EP4) receptors are involved in a wide spectrum of cellular and physiological processes, including photoreception, olfactory sensation, feeding behavior, pain sensation, osmotic function in kidney cells, physiological function in cardiac myocytes, neuronal processes and energy homeostasis (58,59). GPCRs are involved in neuronal or retinal cilia function and control the length of primary cilia or ciliogenesis. Conversely, absence or shortening of primary cilia may interfere with normal brain development, interneuron connectivity, gonadotropin hormone release at the nerve terminals or the sensory potential of cells (48).

In addition to these main signaling cascades, an increasing number of pathways have been associated with primary cilia. It has been concluded that the Notch signaling is involved in the physiology of primary cilia. Notch3 receptor, which is localized in the ciliary membrane, is activated by presenilin 2, a ciliary BB enzyme, thereby regulating epidermal cell proliferation and differentiation (60). Loss of primary cilia or knockdown of IFT molecules may result in diminished Notch activation, leading to decreased cell proliferation and differentiation defects. In the neuroepithelia of the developing neural tube, activation of Notch signaling leads to increased primary cilium length, as well as accumulation of Smo molecules within the primary cilium. This interplay between the Notch and Shh pathways in primary cilia may specify ventral cell fate in the developing neural tube (38).

TGF-β signaling has been recently associated with cilia, whereas TGF-β1 and TGF-β2 receptors are located on the ciliary tip. Primary cilia use diverse methods to regulate TGF-β pathways, through SMAD2/3 and ERK1/2 activation by TGF stimulation, modulating various cellular processes, such as the differentiation of cardiomyocytes, osteocytes and myofibroblasts. Moreover, endothelial primary cilia, which act as flow sensors in the blood vessels, inhibit the endothelial-to-mesenchymal transition, and this process is related to attenuation of the TGF-β signaling. There is also strong evidence regarding the impairment of mechanosensation and maturation in human osteoblasts due to shortening of primary ciliary length through TGF-β signaling (61,62).

The SWH pathway controls organ size and cell proliferation through a core of serine/threonine-kinases that interact with nephrocystin 4 or Crumbs 3 receptors located in the cilium. One of the major components of SWH signaling, MST1/2, which is localized to the BB, has been found to be crucial for primary cilia biogenesis, with loss of MST1/2 leading to defects in ciliogenesis (63).

It has also been demonstrated that the primary cilium regulates mTOR signaling, which plays a pivotal role in metabolism and cell proliferation, thereby determining cell size, through the Lkb1 tumor suppressor, AMP-activated protein kinase and folliculin. In epithelial primary cilia, the mTOR pathway is upregulated by polycystin-1 through the tuberin protein, thus being involved in cyst formation (64,65).

Brain-derived neurotrophic factor signaling, which is involved in neuronal development, synaptic plasticity, satiety and weight control, has recently been proposed to be linked with the BBS4 protein and primary cilia (66).

3. Ciliopathies

Given the notable complexity of interconnected signaling pathways in cilia, the role of the primary cilium as a cellular hub is becoming increasingly obvious; its clinical importance emerges from the consequences of its structural or functional defects, which lead to a broad category of disorders, collectively termed as ciliopathies.

The term 'ciliopathy' is most likely attributed to immotile or primary cilia-related disorders, and it has been recently allocated to certain conditions that have long been known as separate clinical entities (67). The first ciliophathy ever defined was Bardet-Biedl syndrome (BBS) in 2003 (68), although this disease had been known since 1866, when Laurence and Moon (69) first described the phenotype, including retinitis pigmentosa, mental retardation, hypogonadism and spastic paraplegia, in four cases. Decades later, a similar phenotype, consisting of obesity, retinal dystrophy, polydactyly and cognitive problems with learning difficulties, was reported in 1920 by Bardet and in 1922 by Biedl (70).

Clinical overlaps in primary ciliopathies

As a result of the presence of primary cilia in nearly all tissues and organs, impairment of their structure or function may result in a vast group of phenotypes, ranging from single organ impairment to complex systemic disorders (Fig. 2). In addition to their isolated involvement, the kidney and the eyes (retina) are also implicated in defining the heterogeneous pattern of primary ciliopathies, with the participation of other organs, including the brain, skeletal system and liver (71). Additional system contributions in defining the ciliopathic clinical picture are summarized in Table I.

Figure 2

Organ and system involvement in primary ciliopathies.

Table I

Additional clinical features of ciliopathiesa.

Type of system	Clinical feature
Cardiovascular	Atrial or/and ventricular septal defects, dilated cardiomyopathy, hypertrophic cardiomyopathy and valvular defects
Respiratory	Breathing abnormalities, respiratory insufficiency, pulmonary hypoplasia, atelectatic lungs and interstitial fibrosis
Endocrine	Panhypopituitarism, growth hormone deficiency, hypothyroidism, diabetes mellitus and hypogonadism
Genital	Genital hypoplasia, micropenis and ambiguous genitalia
Pancreatic	Pancreatic dysgenesis, pancreatic fibrosis and cystic pancreas
Aural	Sensorial hearing loss

Features displayed in this table were collected after an overview analysis of OMIM clinical synopsis (www.omim.org). OMIM, Online Mendelian Inheritance in Man.

Renal manifestations

Renal impairment is the most common sign in primary ciliopathies, histologically characterized by renal cysts, a thickened and irregular tubular basement membrane, and interstitial fibrosis. Clinically, two frequently observed categories have been defined: Polycystic kidney disease (PKD) and nephronophthisis (NPHP). Both entities are characterized by a progressive decline in renal function, eventually leading to renal failure (72,73). The onset of the diseases varies. Some signs could be detected prenatally due to the presence of oligohydramnios and enlarged kidneys, or shortly after birth due to the occurrence of severe hypertension or respiratory insufficiency (74,75). During childhood, the symptoms of renal disease are unspecific and may include polydipsia, polyuria, secondary enuresis and urinary concentration defects. Poor growth may occur due to chronic dehydration. As a result of renal insufficiency and its progression to end-stage renal failure, new complications may develop, including anemia, metabolic acidosis, anorexia and/or hypertension (76). Renal ultrasound examination shows large, normal-sized or small kidneys, with increased echogenicity, loss of corticomedullary differentiation and the presence of renal cysts (76,77). Dysplastic, lobulated or horseshoe kidneys, kidney malrotation and renal agenesis are less frequently encountered in ciliopathic disorders (78).

Liver manifestations

Liver cysts, liver fibrosis and ductal plate malformation with abnormal bile ducts may be summarized as liver fibrocystic diseases, and they are often found, in addition to PKD, in primary ciliopathies (79). The liver disease can remain asymptomatic, or it can lead to complications, particularly portal hypertension and esophageal varices, cholangitis or cholestasis (80). End-stage hepatic disease requiring transplantation has also been reported in some patients (81,82). The cardinal symptom is hepatomegaly, which can be associated with elevated serum levels of hepatic enzymes, or liver hyperechogenicity on abdominal ultrasound (83).

Ocular manifestations

Retinal dystrophy (with both rod and cone photoreceptor involvement) is commonly encountered in primary ciliopathic disorders. The clinical manifestations of visual impairment range from night blindness, color blindness and loss of peripheral vision, to progressive visual loss and complete blindness (84). Disruptions of ocular motility, such as oculomotor apraxia and nystagmus, are also frequently described (85,86). Additional ocular defects include strabismus, amblyopia, astigmatism, congenital cataracts and coloboma (78).

Central nervous system (CNS) manifestations

The major neuroimaging finding, which characterizes a distinct group of diseases referred to as Joubert syndrome (JS) and related disorders, is the 'molar tooth sign' (MTS), comprising cerebellar vermis hypoplasia or aplasia, with enlargement of the fourth ventricle, thickened and horizontalized superior cerebellar peduncles and a deepened interpeduncular fossa (87,88). Neurological abnormalities may also include Dandy-Walker malformation (DWM), ventriculomegaly, periventricular nodular heterotopia, hydrocephalus, encephalocele/meningocele, polymicrogyria, absence of the pituitary gland, corpus callosum defects and morphological brainstem abnormalities (83,89-91). A wide range of clinical signs may be observed, such as hypotonia, ataxia, developmental delay, intellectual disability (ID), impaired or absent speech, behavioral disturbances such as hyperactivity and aggressiveness, and self-mutilation (92,93).

Skeletal manifestations

Clinical manifestations of the skeletal system may vary from mild phenotypes, such as polydactyly, to severe deformities, possibly leading to death. Polydactyly of the hands and/or feet, which is usually post-axial, but may also be pre-axial and, in some cases, central or mesoaxial, is present in most individuals with cilia-related disorders (94-96). In addition to polydactyly, the hands and feet may be affected to various degrees by oligodactyly, syndactyly, camptodactyly, brachydactyly, carpal and tarsal shortening, short long bones, rhizomelic micromelia, fibular aplasia or limb agenesis (97,98). Truncal skeletal defects may include a constrictive thoracic cage, with shortened and horizontalized ribs, which may be life-threatening in some cases; abnormal or absent clavicles, small scapulae and scoliosis may also be observed (99). Cranioskeletal characteristics include craniosynostosis, macro- or microcephaly, head shape anomalies, frontal bossing, a prominent forehead, bitemporal narrowing, cleft palate, zygomatic arch hypoplasia, maxillary hypoplasia and micrognathia (100).

Diagnosis of primary ciliopathies Clinical diagnosis

Given the numerous overlapping features and marked genetic heterogeneity, considerable efforts have been made to diagnose and classify ciliopathies, in order to optimize clinical management of the patients and improve the accuracy of genetic counseling.

For some of these diseases with severe phenotypes leading to a high mortality rate in utero or during the perinatal period, a prenatal diagnosis is possible in the presence of pathognomonic ultrasonographic signs in conjunction with α-fetoprotein testing of the amniotic fluid and DNA testing of the fetus (101). Early in the pregnancy (weeks 8-11), ultrasonographic screening can detect certain fetal malformations, such as an enlarged cisterna magna or encephalocele (102). Between 11 and 14 weeks of pregnancy, enlarged polycystic kidneys or polydactyly may be detected (103), while later in the second trimester, other brain anomalies (e.g., DWM and hydrocephalus) (104) and severe skeletal anomalies (e.g., rhizomelic shortening of the long bones and hypoplastic thoracic cage) may be identified (105). Fetal MRI can detect MTS at 27 weeks of pregnancy (106).

Postnatally, Beales and Kenny proposed a clinical diagnosis algorithm starting with the presence of renal and retinal involvement and/or polydactyly (Fig. 3). Adding limb or rib abnormalities to this core of clinical manifestations may easily direct the diagnosis to ciliary skeletal dysplasias. Furthermore, identification of ectodermal defects in this group suggests the diagnosis of oral-facial-digital syndrome (OFDS) or cranioectodermal dysplasia (CED), while their absence indicates the diagnosis of short-rib polydactyly syndrome (SRPS). The detection of MTS and other CNS abnormalities should raise the suspicion of JS or JS-related disorders, whereas the presence of obesity points towards the diagnosis of BBS or Alström syndrome (ALMS) (107).

Figure 3

Diagram of the clinical diagnosis algorithm of primary ciliopathies. Adapted with permission from (107). MTS, molar tooth sign; PKD, polycystic kydney disease; RP, retinitis pigmentosa.

Genetics and molecular diagnosis

Since the description of the first ciliopathy gene, BBS6, by two distinct research groups in 2000, due to the advances in genomic sequencing technologies, a number of genes have been associated with ciliary phenotypes (108,109). Only in the last 5 years, through the intensive use of specific gene panels, whole exome and whole genome sequencing >100 new ciliary genes have been identified. At present, there are >190 known genes associated with recognized ciliopathies, of which >140 genes (Table SI) are implicated in primary ciliopathies. Other candidate genes (>240), of which the protein products have been shown to be associated with cilia function or structure, may be involved in either new or confirmed primary or motile ciliopathies (110). Ciliopathies are considered to be Mendelian disorders (111), although a plethora of evidence has also indicated a non-Mendelian pattern of inheritance, or even environmental contribution to defining the phenotype. Genetic locus heterogeneity, copy number variants (112,113), oligogenicity (114,115), multiple allelism (116-119) and transposon-mediated mutagenesis (120) have been described, highlighting the marked complexity of the genetic mechanisms responsible for ciliopathic phenotypes. Moreover, the severity or variability of the phenotypes is suggested to be modulated by the pattern of ciliary gene expression and its effect on protein function (null, truncating or hypomorphic) (121), by epistatic interactions (122,123) and by genetic modifiers or stochastic effects (45,111,124-128).

Classification of primary ciliopathies

Several types of ciliopathies have been recognized, considering the level to which an organ is affected for defining their phenotype.

Retinal ciliopathies

Retinal ciliopathies include clinical entities manifesting as retinal degeneration, and they are caused by defective morphogenesis or dysfunction of specialized sensory cilia from the retina that form the outer segment of photoreceptors. Proteins, such as rhodopsin or ambient lighting-dependent proteins, are trafficked along these specialized primary cilia by means of IFT particles. Impairment of IFT leads to the accumulation of rhodopsin, defects in outer segment development and cell death, which result in the phenotype of retinal degeneration (129,130). Among non-syndromic retinal ciliopathies, the ocular phenotype ranges from the most common retinitis pigmentosa [Mendelian Inheritance in Man (MIM), 26800] (47), which initially manifests as night blindness, followed by loss of peripheral vision, due to the impairment of rod photoreceptor function, and can progress to complete blindness (84,131) to the most severe congenital retinal dystrophy, Leber congenital amaurosis (LCA; MIM, 204000), which frequently results in blindness within the first year of life. Visual loss is usually accompanied by sensory nystagmus, amaurotic pupillary response and absent electroretinogram signs. Photophobia, high refractive errors, keratoconus and enophthalmos are often seen in LCA. Involvement of the retina may range from normal, to retinal degeneration, retinal aplasia or biochemical dysfunction (dysplasia) (132). Overlapping with these two disorders, other ocular dystrophies have also been described as retinal ciliopathies: Cone dystrophy (MIM, 304020), characterized by visual loss and color vision defects, cone-rod dystrophy (MIM, 120970), characterized by photophobia, abnormal color vision, night and peripheral vision loss, and macular dystrophy (MIM, 300834), characterized by loss of color and sharp vision (133). Progressive retinal degeneration and sensorineural hearing loss are the first symptoms found in ALMS (MIM, 203800); these are accompanied later in childhood by obesity and diabetes mellitus. Additional features, such as cardiomyopathy, epilepsy, respiratory disturbance and renal or endocrine dysfunction, support the classification of these disorders as syndromic retinal ciliopathies (134). A rare combination of retinal and renal ciliopathies characterizes Senior-Løken syndrome (SLSN; MIM, 266900), with a specific clinical presentation consisting of retinal dystrophy and NPHP. Consequently, SLSN is considered by some studies as a syndromic retino-ciliopathy or, by others, as a renal (NPHP-related) ciliopathy (135,136).

Renal ciliopathies

Renal ciliopathies encompass a group of disorders, the hallmark of which is kidney disease, including autosomal dominant polycystic kidney disease (ADPKD; MIM, 173900), autosomal recessive polycystic kidney disease (ARPKD; MIM, 263200) and NPHP (MIM, 256100). In the kidney, epithelial primary cilia lining the nephron tubules and collecting ducts act as sensory antennae sensitive to urine composition, osmolarity and flow. Defects in several signaling pathways, such as G-protein signaling, mTOR or Wnt, induced by decreased of flow-mediated intracellular calcium concentration, may lead to cyst formation. Moreover, disruption of the balance between canonical and non-canonical Wnt signaling may affect the polarity of epithelial tubular cells, also resulting in cyst formation (137).

ADPKD and ARPKD are different, not only due to the inheritance pattern, but also based on the microscopic and ultrasonographic appearance of the cysts, associated organ anomalies, age at onset, severity and prognosis (138). While ADPKD is characterized by large cysts originating from the distal nephrons and collecting ducts, which grow in volume and number with age, and by the presence of cysts in the liver, pancreas or other epithelial organs, intracranial aneurysms and mitral valve prolapse (139), the cysts in ARPKD are small, originate from the distal tubules and collecting ducts and display a salt-and-pepper pattern, and the liver is always affected by fibrosis (138). In contrast to ADPKD, which starts in late adulthood and slowly progresses to end-stage renal disease (ESRD), ARPKD is more severe, with antenatal onset and diagnosis during late pregnancy or at birth, leading to increased perinatal death rate (30-50%). Death occurs as a consequence of respiratory insufficiency due to pulmonary hypoplasia and thoracic compression by the extremely expanded kidneys (75). NPHP, which is characterized by corticomedullary cysts, atrophy and interstitial fibrosis resulting in nephron disintegration, is the main cause of ESRD in children (140). The severity and, subsequently, the progression to ESRD, depend on the clinical variant, namely the infantile, juvenile or adolescent variant. The infantile variant is the most severe, with prenatal manifestations consisting of oligohydramnios and bilateral enlarged cystic kidneys. Thus, ESRD develops in the first year of life. The first symptoms of the classical juvenile form, which is characterized by renal interstitial fibrosis and inflammation, with progression to tubular atrophy and small cyst formation, develop during the first decade of life and ESRD occurs at the mean age of 13 years (74). NPHP may be limited to the kidneys or may be part of other ciliopathic conditions, such as Joubert/COACH syndrome, SLSN, BBS, Meckel-Gruber syndrome (MKS) or skeletal disorders (141). BBS (MIM, 209900) is the most extensively investigated ciliopathy, and it has provided valuable data for the entire spectrum of human cilia-related disorders due to its overlapping characteristics at the level of phenotype, genotype, protein-protein interactions and participation in signaling pathways (142). BBS is a multisystem disorder, but renal impairment is its most prominent cause of morbidity and mortality. The major clinical characteristics, including retinal dystrophy, obesity, post-axial polydactyly, renal anomalies, cognitive impairment and hypogonadism, are suggestive of the diagnosis. The presence of four of those characteristics, or association of three primary characteristics with two secondary features is considered as sufficient for clinical diagnosis (143). Secondary features include speech delay, developmental delay, diabetes and congenital heart disease. BBS is characterized by marked clinical variability, which cannot be fully attributed to the 24 genes identified to date (144). MKS (MIM, 249000), which displays renal (cystic kidney dysplasia) as well as neurological [occipital encephalocele (OE)] manifestations, may be considered as either a renal or a CNS-related ciliopathy. Hepatic fibrosis completes the specific clinical triad of this condition, although polydactyly is often considered as the 4th pathognomonic feature (145). MKS has a heterogeneous, severe phenotype, which is not compatible with life, with death occurring in utero or shortly after birth. Renal dysfunction may often lead to oligohydramnios or anhydramnios. Apart from OE, which is the most frequent finding, additional CNS malformations found in MKS include olfactory bulb dysgenesis, optic nerve hypoplasia, agenesis of the corpus callosum, holoprosencephaly, cerebellar hypoplasia or total anencephaly. Cleft lip and palate, shortening of the long bones, congenital heart defects and pulmonary hypoplasia may further complicate the clinical picture (45,101).

CNS-related ciliopathies

CNS-related ciliopathies comprise a group of conditions, the hallmark of which is the MTS, which is required for diagnosis. Impairment of the Wnt pathway, which is a major signaling pathway involved in cerebellar development, may be responsible for defective cerebellar vermis hypoplasia, one of the components of MTS. In addition to this pathway, other neuronal primary cilium-specific pathways are required for normal brain development, regulating neuronal fate, proliferation, migration and differentiation. Dysregulation of these pathways, including Shh, PDGFRα and GRCR, may manifest with malformations during cortical development or midline defects, which are often found in JS or CNS-related ciliopathies (71). Depending on the additional clinical characteristics, JS (MIM, 213300) has been classified into several groups as follows: i) Pure or classic JS, characterized by hypotonia, developmental delay, abnormal eye movements, breathing abnormalities, ataxia and ID; ii) JS with ocular defects, including retinal dystrophy or LCA; iii) JS with renal defects (NPHP); iv) JS with oculorenal defects, also named cerebello-oculorenal syndrome, comprising SLSN (retinal dystrophy, LCA and NPHP) associated with MTS, and Dekaban-Arima syndrome (cerebrooculohepatorenal syndrome) characterized by chorioretinal coloboma or retinal dystrophy, PKD, MTS and hepatic fibrosis in some cases; v) JS with congenital hepatic fibrosis; vi) JS with congenital hepatic fibrosis and associated chorioretinal coloboma, also known as COACH syndrome; and vii) JS with orofaciodigital defects, including a lobulated or bifid tongue, hamartomas, cleft lip and/or palate and polydactyly, also known as orofaciodigital syndrome type VI (83,87). To date, ~40 causative genes covering >90% of clinical subjects have been identified (146-152).

Ciliopathies with skeletal involvement

This group of disorders is characterized by variable severity, ranging from mild to severe or even lethal phenotypes. Two subgroups have been distinguished: Those with major skeletal system involvement, including craniofacial, thoracic cage and long bone involvement, known as short-rib thoracic dysplasias (SRTDs), with or without polydactyly or ciliary condrodysplasias, and OFDS, with milder involvement of the skeletal system (153).

Development of the cartilage and bones is a complex process that is modulated mainly by the IFT and Hh pathways. Disruption of Ihh signaling in chondral primary cilia affects chondrocyte maturation during the ossification process. Consequently, various skeletal abnormalities, including polydactyly, shortening of the ribs or long bones and craniofacial abnormalities, may occur (154,155). Dysregulation of IFT, which is involved in the trafficking of the transmembrane SMO receptor, a signal transducer in Hh signaling, may lead to premature differentiation and decreased proliferation of chondrocytes, manifesting as specific SRTDs and defects of long bone growth plates (156).

There are >19 types of SRTDs, classified based on phenotype severity, radiological findings and confirmation of genetic defects (100,157). Chondroectodermal dysplasia or Ellis-van-Creveld syndrome (EVC; MIM, 2255000), Weyers acrodental dysostosis (WAD; MIM, 193530) and Sensenbrenner syndrome or CED (MIM, 218330) are the milder disorders in this group. EVC is characterized by disproportionate short limb dwarfism, short ribs, polydactyly, cardiac malformations and ectodermal defects affecting the hair, teeth and nails (158-160). WAD is an allelic disorder to EVC, but displays a milder phenotype, consisting of moderate short stature, postaxial polydactyly, and nail and dental anomalies, and is inherited in an autosomal dominant manner (161). CED is characterized by craniofacial abnormalities, such as sagittal craniosynostosis, leading to dolichocephaly, frontal bossing and dental defects, in conjunction with skeletal abnormalities (short stature, rhizomelic limbs, brachydactyly and narrow thorax) and ectodermal anomalies (thin/sparse hair, hypoplastic nails and skin laxity) (162,163). Kidney involvement (NPHP progressing to renal failure) and liver involvement (ranging from asymptomatic hepatomegaly to acute cholangitis, liver cirrhosis and severe cholestasis) are common findings in CED (164).

The second group with more severe phenotypes is comprised of Jeune asphyxiating thoracic dystrophy (JATD; MIM, 208500) and conorenal syndrome or Mainzer-Saldino syndrome (MZSDS; MIM, 266920). The specific presentation of JATD includes a constrictive thoracic cage and secondary respiratory distress due to restrictive pulmonary hypoplasia. Respiratory distress is the main cause of mortality in ~60% of the patients (100). Additional skeletal findings may include a short stature, short limbs with irregular metaphyses, cone-shaped epiphyses in the hands, foot polydactyly, a shortened ilium and a trident-shaped acetabulum. Retinal degeneration, NPHP-like or cystic renal disease, pancreatic and liver involvement or brain malformations are occasionally found in patients with JATD (100,165). MZSDS is characterized by the triad of retinal dystrophy, renal disease (typically NPHP) and phalangeal cone-shaped epiphyses (166). The thorax is less narrow compared with that in patients with JATD. Short stature, hepatic fibrosis and cerebellar ataxia are variable traits that may be observed in MZSDS (166,167).

The last subtype is the perinatally lethal SRPS, the core features of which include a constrictive thoracic cage, significantly shortened long bones, polydactyly, brahydactyly and pelvic abnormalities (100). Different types have been characterized, based mainly on radiological findings: SRPS types I (Saldino-Noonan syndrome) and III (Verma-Naumoff syndrome) (MIM, 613091); SRP type II or Majewski syndrome (MIM, 263520); SRPS type IV or Beemer-Langer syndrome (MIM, 269860); and SRPS type V (MIM, 614091) (98). In addition to skeletal abnormalities, involvement of the brain, heart, kidneys, liver, pancreas and genitalia have often been recorded in SRPS. In some cases, facial dysmorphism may also be observed (100,165).

Apart from the typical manifestations, some 'unusual' features may be observed in each group, further expanding and complicating the phenotype; these include atlantoaxial instability and spinal cord compression (168,169), short irregularly bent ribs, hypoplastic and bent mesomelic bones, short campomelic long bones, undermineralized bones (170,171), OE or MTS (172).

OFDS (MIM, 311200) describes a heterogeneous group of diseases caused by defects in ~18 genes (173-175). Clinical manifestations include anomalies of the face (micrognathia, hypertelorism, telecanthus, cleft lips and low-set ears), the oral cavity (gingival frenulae, lingual hamartomas, cleft/lobulated tongue and cleft palate) and the digits (polydactyly, brachydactyly, oligodactyly and bifid digits), associated with an extensive spectrum of additional features affecting the CNS, the kidneys, the heart or the eyes, outlining the 13 forms described to date (173,176). In addition to renal involvement, which is commonly found in OFDS, a series of features overlapping with other ciliopathies (JS, SRPS and EVC) have been reported, such as MTS identified in OFD types 4, 6 and 14, and tibial abnormalities observed in OFD types 4, 8 and 12 (177,178).

Other unclassified subtypes have also been described, which are characterized, in addition to the typical features, by fused kidneys (179), tetralogy of Fallot (179,180), coarctation of the aorta (181), corpus callosum agenesis (179), cerebellar vermis hypoplasia, DW malformation, ID, 12th rib hypoplasia (174) and short mesoaxial phalanges (182).

4. Conclusions

Increasing use of whole exome sequencing has enabled the discovery of new causal genes in ciliopathies. Combined efforts have been made in the fields of proteomics, cell biology and model organisms to link the genes with their phenotypic effect. Taken together, all these studies have improved our knowledge on recognized ciliopathies, confirmed the proposed cilia-related disorders or identified new ciliary diseases.

Since Baker and Beales (183) proposed 72 conditions as candidates for ciliopathic disorders in 2009, several have been included in the group of known ciliopathies (Table II), increasing their number to 35 (184-202).

Table II

Newly defined ciliopathies.

MIM ID	Disease name	Gene name	Protein localization	(Refs.)
616287	Lethal congenital contracturesyndrome; hypomyelination neuropathy-arthrogryposis syndrome	ADCY6	Axoneme	(184)
243605	Stromme syndrome; lethal fetal brain malformation-duodenal atresia-bilateral renal hypoplasia syndrome; microcephaly	CENPF	Basal body	(185,186)
135150	Birt-Hogg-Dubé syndrome	FLCN	Basal body; axoneme	(187)
201000	Carpenter syndrome	RAB23	Axoneme	(200,201)
616897	Complex lethal osteochondrodysplasia	TAPT1	Basal body	(189)
NO MIM ID	A novel syndrome with multiple congenital malformations and developmental delay	USP9X	Axoneme	(190)
601707	Curry-Jones syndrome	SMO	Axoneme	(191)
607131	Al-Gazali-Bakalinova syndrome	KIF7	Axoneme	(192)
236680 614120	Hydrolethalus	HYLS1; KIF7;	Basal body; axoneme; basal body	(193-195)
175700	Greig cephalopolysyndactyly syndrome	GLI3	Axoneme (tip)	(196)
612651	Lethal endocrine-cerebro-osteodysplasia syndrome	ICK	IFT	(197)
NO MIM ID	Pituitary stalk interruption syndrome	GPR161	Axoneme	(198)
300707	Syndactyly-telecanthus-anogenital and renal malformations syndrome	FAM58A	Probably cytosolic	(202)

MIM, Mendelian Inheritance in Man; IFT, intraflagellar transport.

By contrast, other conditions were excluded from the list of possible or likely ciliopathies following the determination of their genetic background, including Kabuki syndrome (type 1 MIM, 147920; type 2 MIM, 300867) following identification of its causal genes, MLL2 (MIM, 602113) and KDM6A (MIM, 300128) (203,204), or Neu-Laxova syndrome (type 1: MIM, 256520; type 2: MIM, 616038) due to the discovery of its causal genes, PHGDG (MIM, 606879) and PSAT1 (MIM, 610936) (205,206).

The delineation of the ciliary proteome and its interaction with extraciliary molecules opens new perspectives in reclassifying cilia-related disorders. Thus, the disorders characterized by ciliopathy-overlapping phenotypes, the causal genes of which are not expressed in the ciliary assembly, but interfere with ciliogenesis or the cilia signaling network, have been termed ciliopathy-like disorders. A representative example is Cohen syndrome (MIM, 216550), which is defined by obesity, developmental delay, retinal degeneration and intermittent neutropenia (207), and is caused by mutations in VPS13B (MIM, 607817) (208). The expression product, which is localized to the Golgi apparatus, may impair processing of ciliary components (207). Townes-Brocks syndrome (MIM, 107480), which is characterized by hearing impairment, PKD, ESRD, imperforate anus and digit malformations (209), is caused by mutations in the SALL1 (MIM, 602218) gene, which encodes a zinc-finger transcription factor; its interaction with two ciliogenesis suppressors, CEP97 (MIM, 615864) and CCP110 (MIM, 609544), leads to cilia formation and function impairment (210). For these ciliopathy-like conditions, Reiter and Leroux (110) proposed the term second-order ciliopathies, whereas first-order ciliopathies are defined as disorders in which disease-associated proteins are expressed in the primary ciliary compartment. Although applying this classification is seemingly straightforward, unexpected evidence has uncovered the possibility of a condition being either first- or second-order, thus complicating the picture. One such example is MKS, a well-known ciliopathy caused by mutations in genes encoding proteins that are localized in the transition zone (101). A recent study identified a new causal gene for MKS, TXNDC15 (MIM, 617778), a non-ciliary gene, the bi-allelic mutations of which lead to abnormal cilia biogenesis (211).

Numerous conditions remain to be elucidated, either due to the fact that the genetic cause has not been uncovered or since the pathophysiological mechanism underlying the phenotype remains elusive. Predicting organ involvement and, consequently, phenotype severity based on genetic defects also represents a major challenge.

Future research will hopefully provide new insights that may help reorganize and further elucidate the striking field of ciliopathies.