Ciliopathies : an Update

Cilia are hair-like organelles that extend from the surface of almost all human cells. Nine doublet microtubule pairs make up the core of each cilium, known as the axoneme. Cilia are classified as motile or immotile; non motile or primary cilia are involved in sensing the extracellular environment. These organelles mediate perception of chemo-, mechanoand osmosensations that are then transmitted into the cell via signaling pathways. They also play a crucial role in cellular functions including planar cell polarity, cell division, proliferation and apoptosis. Because of cilia are located on almost all polarized human cell types, cilia-related disorders, can affect many organs and systems. The ciliopathies comprise a group of genetically heterogeneous clinical entities due to the molecular complexity of the ciliary axoneme. Cilia are involved in left-right laterality in the embryo, mucociliary clearance, reproductive function, neuronal development, photoreception, olfaction, homeostasis or hormonal regulation. Ciliopathies can involve single organs or can occur as multisystem disorders with phenotypically variable and overlapping disease manifestations. However, the importance of the cilium’s role in the development of several diseases is still a matter of active research. In this review, we will define ciliary ultrastructure and function, the genetic complexity associated with ciliary dysfunction and clinical manifestations. Increasing knowledge of the role of cilia in morphogenesis pathways in conjunction with genetic studies is necessary to improve the characterization of this previously unconnected group of diseases.


Introduction
Ciliopathies are a group of overlapping disorders whose etiologies lie in defective structure and function of cilia (Davis and Katsanis, 2012).The cilia are organelles found on the apical surface of most eukaryotic cells playing essential roles during development and tissue homeostasis (Garcia-Gonzalo and Reiter, 2012).The ubiquitous presence of cilia in every cell of body tissues explains the wide range of human diseases arising from defects in cilia structure or function.Classically, the function of cilia has been attributed to motility, with propelling cell locomotion or generating flow of fluid environment (Malicki, 2012).Recently, cilia have found identified as important sensory organelles involved in signaling pathways (Yuan and Sun, 2013).Furthermore, recent discoveries have assigned novel functions to primary (nonmotile) cilia, ranging from participation in different signal transduction pathways through extracellular receptors and maintaining cellular homeostasis and cell division which direct embryonic development and organ function (Kim and Dynlacht, 2013).Because cilia, either primary or motile cilia, are present in nearly all cell types of all organs, the defects in these organelles lead to numerous and uncommon human diseases grouped collectively as ciliopathies.The production of specific disorders by ciliopathies is nowadays under research.We can speculate that mutations in ciliary gene can lead to defects in various signaling pathways.Recently, defects in proteasomal clearance of specific proteins have linked to several ciliary phenotypes (Liu et al, 2014).Thus, it is a rapidly expanding field of research involving multiple disciplines, which will improve diagnostic testing and identify new therapeutic targets.Understanding of the genetics, molecular abnormalities, and clinical manifestations in children will improve the diagnostic awareness and consequently the evidence based treatment.Our aim was integrate both clinical and molecular aspects of these genetic diseases highlighting the novel genotype-phenotype associations to facilitate their recognition by pediatricians.This review summarizes the recent advances in these interesting conditions which may explain the clinical manifestations and the understanding of these processes in childhood.

Structure and Function of the Cilium
Cilia are organelles conserved throughout evolution and are present in most cells of the human body.Emerging from the basal body, a centriole derived structure; the cilium extends from the cell surface into the extracellular space and is composed of a microtubular proteic structure known as the axoneme.The basal body contains 9 pairs of peripheral microtubule cilia anchored to the cell surface, and the orientation of these cilia determines ciliogenesis (Figure 1) (Bandano et   Cilia are classified into three principal types based on microtubule structure: motile cilia with a "9+2" axoneme pattern, motile cilia with a "9+0" axoneme pattern, and immotile cilia, also called sensory or primary cilia, with a "9+0" axoneme pattern.Recent studies have shown that both motile and immotile cilia can carry out sensory functions in the organism; we use the term "sensory cilia" to refer to non motile monocilium or primary cilium (Hildebrant et al, 2011; Fliegauf et al, 2007).
Cilia are organized in a microtubular structure, formed by helical protofilaments made up of monomers of alpha and beta tubulin.Motile cilia exhibit a whipping motion and are involved in respiratory tract mucociliary clearance, cerebrospinal flow movement and transport of ovum and sperm in the reproductive tracts.These cilia have the classical "9+2" structure: 9 pairs of peripheral microtubule surrounding a central pair all contained in the cell membrane.Each peripheral doublet microtubule contains one external and one internal dynein arm.Dynein arms contain ATPase, which drives the sliding movements between the peripheral microtubule doublets.Nexin joints limit movement in adjacent ciliary doublets maintaining intact cilium during motion.Central tubules, wrapped in a central sheath, are joined by peripheral doublet radial arms, controlling the activity of dynein arms and maintaining the structure of the cilium (Figure 2  Motile cilia with a "9+2" pattern have a synchronous motion waveform, whose direction depends on the orientation of the pair of central microtubules.The low viscosity of the periciliary liquid found on the epithelial surface allows for rapid beat frequency.The ciliary beat is coordinated by calcium signals between epithelial cells through gap junctions.
Motile cilia with a "9+0" pattern lack central microtubules and are found only in the ventral node during embryonic gastrulation.These cilia have a rotational movement and are responsible for generating the extracellular leftward flow, which establishes the left-right axis in the embryo.
Sensory primary cilia follow a pattern "9+0" pattern and lack the central microtubules and dynein arms.There are other patterns in the ciliary axoneme, as sensory cilia "9+2" present in the vestibular system and a nobel "9+4" axoneme pattern indentified by ultrastructural microscopical analysis on the notochordal of rabbit embryo.It shows that even though the cilia are evolutionarily conserved organelles, the structure of the axoneme may vary among different species of vertebrates (Feistel and Blum, 2006).

Intraflagellar Transport (IFT)
Synthesis of the structural and functional elements of the cilium takes place in the cytoplasm.Assembly of protein elements is accomplished by intraflagellar transport along the ciliary axoneme.The IFT particles are composed of two complexes; IFTA with 6 protein subunits is needed for retrograde transport and IFT B with 13 protein subunits which is responsible for anterograde transport.Protein components are assembled in the basal body and undergo anterograde transport to the upper end of the cilium driven by the kinesin II motor complex.At the upper Pediatrics Research International Journal __________________________________________________________________________________________________________________________ ______________ Alba Faus-Pérez, Amparo Sanchis-Calvo and Pilar Codoñer-Franch (2015), Pediatrics Research International Journal, DOI: 10.5171/2015.935983end of ciliary axoneme, the kinesin II motor complex is inactivated, facilitating retrograde transport to the base of the cilium through cytoplasmic dynein.Disruption of IFT complex or basal body proteins leads to abnormal cilia assembly and function disorders (Berbari et al, 2009;Bisgrove and Yost, 2006) (Figure1).

Ciliopathies
Mutations in over 100 ciliary genes has been identified as a causative of several groups of diseases due to the disfunction of cilia, collectively known as ''ciliopathies,'' that often share common phenotypic features.As a multitude of genes are required for the construction of cilia and the centrioles from which cilia originate, ciliopathies serve as a model for the complex genetic interactions seen in human genetic diseases (Pan et al, 2005;Sharma et al, 2008;Berbari et al, 2009).

Motor Ciliopathies
The motor ciliopathies include alterations in the embryonic formation of the leftright axis, primary ciliary dyskinesia, and dysregulation in cell division and oncogenesis (Table 1).

Left-Right Axis Formation in the Embryo
Embryonic gastrulation proceeds as a symmetrical process until the initial break at Hensen's node.On the ventral surface of the node, there are two types of cilia: motile "9+0" pattern cilia and immotile "9+0" pattern cilia.The motile cilia are located in the central node and move rotationally to produce a leftward flow of perinodal fluid.This flow is responsible for the onset of the signaling pathway leading to leftwards orientation formation.As axonema move in response to the perinodal flow that follows a rightleft direction, a cascade of intracellular calcium signaling on the left side of the node is triggered; immotile cilia found in the periphery of the node act as mechanoreceptors in response to this signal.The intracellular calcium signal requires the presence of the ciliary protein polycystin-2 (PKD2) (Figure 3), which is thought to be involved in mechanosensation.In this way, the nodal flow might contribute to left-right asymmetry (Afzelius, 1995;Icardo et al, 2002).

Primary Ciliary Dyskinesia
Primary ciliary dyskinesia (PCD, MIM ID#244400), or immotile ciliary syndrome, was the first clinical entity known and associated with ciliary dysfunction.It includes a group of diseases in which dyskinetic and ineffective ciliary motion, or even ciliary aplasia, render respiratory cilia are immotile (Zariwala et al, 2007).
The cilium cells present in the nasopharynx, paranasal sinuses, middle ear and respiratory tract from the trachea to the bronchioles.Each single cilium cell has approximately 200 cilia on its surface that beat in a coordinated manner to achieve mucociliary clearance.Dysfunction of respiratory ciliated cells leads to chronic respiratory tract infections and accumulation of mucous secretions present in the majority of cases since birth.As the disorder also affects the ciliary motility of the sperm flagellum and the motility of cilia in the fallopian tubes, male sterility and reduced fertility in women is rather common among the affected.Because of the inefficiency of the nodal cilia in embryonic establishment of the left-right axis, approximately 50% of these patients have a total situs inversus or situs ambiguous (called Kartagener´s syndrome, MIM ID#244400), (Afzelius, 1995;Kennedy et al, 2007).Primary ciliary dyskinesia is considered an autosomal recessive disorder, although rare cases have been described with autosomal dominant or X-linked transmission, the latter in relation to the gene RPGR (retinitis pigmentosa GTPase regulator) and OFD1 gene which has been found in a family with respiratory ciliary dyskinesia, macrocephaly and mental retardation (Coene et al, 2009).Its incidence is estimated at 1 in 15,000-30,000 live births.Theoretically, any mutation in the hundreds of protein elements that constitute the complex ciliary structure could cause PCD.The most common ultrastructural defect is the complete or partial absence of dynein arms; approximately two thirds of patients have a defect in outer dynein arm.Other less common causes of PCD include defects of radial joints in the arms of nexin, transposition of ciliary microtubules or agenesis.It should be noted that normal ultrastructure is observed in 15% of PCD cases associated with DNAH11 mutations.
PCD is a genetically heterogenous disorder and over 28 genes have been identified (table 1) DNAI1 and DNAH5 mutations are responsible of 25% PCD pacients and almost 50% with defects in outer dynein arm, making these genes a target of genetic screening (Popatia et  Commonly, these patients also suffer from recurrent headaches secondary to chronic sinusitis.However, in very rare cases the headaches are caused by hydrocephalus secondary to cerebral ependyma, which is also associated with cilia dysfunction (Davenport and Yoder, 2008;Baker and Beales, 2009).

Cilia and Cell Division: Role in Oncogenesis
Several tumor phenotypes can be adjusted to ciliary dysfunction spectrum, in which case the ciliar loss would be a necessary prerequisite for cell proliferation.However, depletion of cilia formation alone is not a sufficient event to drive tumorigenesis.The cilium is involved in multiple signaling pathways allowing coordination of different cell types.Cilia dysregulation of cells is important to oncogenesis because of the role that centrosome amplification and subsequent genomic instability play in many cancers (Basten and Giles, 2013 In the development of melanoma in situ and pancreatic ductal adenocarcinoma has been observed ciliary progressive loss at different stages of tumor (Basten and Giles, 2013).Colorectal cancer has been associated with ciliary dysfunction.The protein kinase Aurora A involved in cilia loss is frequently mutated in this cancer (Furuya and Nakatani, 2013).Murine studies demonstrate the involvement of the cilia in the development of skin tumors of the basal cell carcinoma subtype and medulloblastoma brain tumors by the "Hh signaling pathway" (Toftgård, 2009;Wong et al, 2009).

Sensory Ciliopathies
Sensory primary cilia are involved in multiple biological processes.They act as chemoreceptors or extracellular mechanoreceptors and are involved in the process of cell division, serving as a structural element for the formation of the mitotic spindle and regulating the process of cell proliferation and apoptosis (Marshall and

Renal and Liver Ciliopathies: Polycystic Kidney Disease and Congenital Hepatic Fibrosis
The autosomal dominant polycystic kidney disease (ADPKD, MIM ID#173900) was one of the first clinical entities associated with primary cilia dysfunction.ADPKD affects 1 in 500-1000 live births.The disorder is characterized by the progressive growth and development of renal cysts that destroy functional parenchyma, leading to dilation of renal collecting tubules and kidney failure.Cysts development begins in utero and progresses slowly, is often no evident until adult age.Mutations in two genes, PKD1 and PKD2, have been described in association with this entity.The PKD1 gene, which encodes the protein polycystin 1, is the most frequent alteration found in up to 85% of all cases and specifically associated with severe cases of the disease.The gene encoding the protein PKD2 polycystin 2 is also involved in establishing the left-right axis during embryonic development (Waters and Beales, 2010;Hildebrandt et al, 2011).
The primary cilium was first suspected in cystic disease after observations that most proteins implicated in the pathogenesis of the disease which are part of the molecular structure of the cilium and are involved in cystogenesis.The primary cilium can act as a flow sensor in the renal tubule.Ciliary deflection occurs in response to renal flow, triggering the entry of calcium into the cell, a process mediated by polycystin 2 (PC2), which acts as a cation channel with polycystin 1 (PC1).Intraflagellar transport is also required to regulate ciliogenesis and the levels of PC2 at the cilium.Furthermore, the absence of renal flow induces COOOHterminal proteolysis of PC1.In this way, renal cilia are essential for PC1's function as a mechanoreceptor and may modify gene transcription of PC1.The low levels of intracellular calcium lead to an increased expression of PC1 and subsequent migration of aquaporin-2 to the apical membrane increasing osmotic permeability.These data show the role of primary cilium as a mechanosesnor in the lumen of the collecting duct tubule ( Cholangiocytes, the epithelial cells lining intrahepatic bile ducts, have primary cilia detecting changes in bile flow and osmolality.Cholangiocyte cilia are sensory organelles responding to mechanical stimuli by alterations in intracellular Ca2+ signal and cAMP.These cilia can also detect changes in composition and tonicity of bile and play an important role in ductal bile formation by acting as osmosensors.The osmosensory function of primary cilia in biliary epithelia is associated with the function of transient receptor potential vanilloid 4 (TRPV4) Ca2+ ciliary channel, and is also linked to ATP release.Bile tonicity detected by the osmosensor proteinTRPV4 expressed on cholangiocyte is the main mechanism for bicarbonate secretion in bile ducts and determine the bile duct formation (Gradilona et

Nephronophthisis and other Associated Ciliopathies
Nephronophthisis (NPHP) is an autosomal recessive tubulointerstitial nephropathy being the most common genetic cause of chronic kidney disease in the first three decades of life.Initially, patients affected usually present symptoms of polyuria and polydipsia with secondary enuresis and anemia.NP can be classified clinically in infantile, juvenile and adolescent by the onset of end-stage renal failure.The presentation occurs typically during early puberty with progressive renal failure.Kidneys in NPHP have normal sized with loss of cortico-medullary differentiation.
Histologically it has been identified the presence of cortico-medullary cysts, tubulointerstitial cell infiltrates and tubular basement membrane disruption.
Mutations have been reported in 12 genes (NPHP1-11 and NPHPL1), which account approximately 30% of cases of NPHP.These genes encode nephrocystin proteins that are located in the cilium, basal bodies and centrosome or adherens junctions between cilium cells ( (Hurd and Hildebrant, 2011).

Joubert Syndrome
Joubert syndrome (JBTS, MIM ID#213300) is a rare syndrome characterized by congenital malformation of the brain stem, cerebellar vermis agenesis or hypoplasia causing hypotonia, ataxia psychomotor delay, irregular breathing patterns and oculomotor apraxia.Additional clinical features include NPHP, retinal degeneration, ocular colobomas, polydactyly or endocrine abnormalities.The defining characteristic of Joubert syndrome is the "molar tooth sign" on cranial magnetic resonance imaging a consequence of hypoplasia of the cerebellar vermis and malformations of the midbrain and hindbrain.The variable clinical manifestations associated with the molar tooth sign don´t comprise different disorders; they are part of the wide clinical range of Joubert syndrome.Genetic complexity in Joubert syndrome reveals the phenotypic variability of this syndrome, and therefore using the term" Joubert syndrome and related disorders" only leads to confusion in diagnosis (Romani et al 2013) The disease has an autosomal recessive inheritance pattern and an incidence estimated at 1 in 100,000 births.It is also a genetically heterogeneous syndrome; mutations have been identified in 23 genes all of which encode for proteins of primary cilium and are responsible of about half of cases (Table 1 (Waters, 2011).Recently, it has opened a hope in those patients with experimental gene therapy in RPE65 in dogs restoring sight in this animals models (Koenekopp, 2004).

Meckel-Gruber Syndrome
Meckel-Gruber syndrome (MKS, MIM ID#249000) is one of the most severe ciliopathies with lethality in perinatal period.It is characterized by cystic renal disease, occipital encephalocele, polydactyly and hepatic fibrosis.The incidence is 0.62 in 100,000 births (Martinez-Frías et al, 2012).
MKS is a heterogeneous genetic syndrome; mutations in MKS1, TMEM216/MKS2, TMEM67/MKS3, CEP290, RPGRIP1L, CC2DA, NPHP3, TCTN2, B9D1, B9D2, TMEM231, TMEM138, TMEM237, EVC2 and C5orf42 have been identified in this syndrome.There is an important overlap in genes involved in JBTS, NPHP and MKS even though the clinical presentation of these syndromes is different (Baker et  In both BBS and ALS, truncal obesity develops in childhood and is thought to be linked to hyperphagia (Guo et al, 2011;Minton et al, 2006;Gupta et al, 2009).In ALS, an endocrine phenotype of hyperinsulinemia, insulin resistance and type 2 diabetes are common, while diabetes is also a secondary feature of BBS suggesting that ALMS1 might have a role in β-cell function or peripheral insulin signaling pathways.In Australian studies, obese mice have a spontaneous mutation of the gene responsible for ALS in humans.Although the mice are born at a normal weight, they later exhibit hyperphagia and become obese developing insulin resistance, diabetes and features of metabolic syndrome (Girard et

Ciliopathies and Skeletal Defects
The primary cilium plays an essential role in the development of cartilage and bone growth and regulates "hedgehog signaling".
The hedgehog proteins pathway is significantly involved in the differentiation of chondrocytes and osteoblasts in limbs and skeletal axial formation.Recently, it has been shown the role of the primary cilium with mechanotransduction properties in cartilage and bone responding to dynamic fluid flow in cultures being responsible for osteogenesis and bone resorption (Nguyen and Jacobs, 2013;Serra R, 2008

Ciliopathies and Sense Organs
The primary cilia play a crucial role in vision, olfaction and hearing.Cilia act as photoreceptors in the retina and as mechanoreceptors in the olfactory epithelium and inner ear.The primary cilium dysfunction may lead to alteration of a sense organ in isolation or be involved in the pathogenesis of a complex clinical syndrome.

Retinal Ciliopathies
Photoreceptors in the retina have an inner and outer segment linked by a connecting primary cilium.Photoreceptor disc visual pigments are synthesized exclusively in the inner segment and transported by intraflagellar transport (IFT) along the connecting cilium to the outer segment.

Ciliopathies and Olfaction
The olfactory receptor cells are bipolar neurons with a "9+2" pattern.These olfactory cilia lack dynein arms and are therefore immotile.Olfactory receptors, found in the apical region of the olfactory sensory neurons, transduce odor stimuli into changes in neuronal membrane potential by a G-protein mechanism to activate adenyl cyclase type III (Jenkins et al, 2009).The depolarization initiates in response to cAMP binding and is caused by the sequence of 2 currents: an influx of cations (mainly Ca 2+ ) through cyclic nucleotide-gated channel activation and a secondary efflux of Cl − through Ca 2+ -gated Cl − channel.In this way, olfactory cilia respond to mechanical stimulus that is transduced to an electrical signal.
Reduced olfactory function is present in almost 50% of Bardet-Biedlpatients.The loss of function of BBS proteins causes defects in olfactory cilia structures and leads to olfactory impairment.Mutations in BBS1 ad BBS4 gene and also gene deletion in BBS1, BBS2 and BBS4 in mice lead to impaired olfactory function (Kulaga et al. 2004

Conclusions
The ciliopathies are a genetically heterogeneous group of clinical entities resulting from dysfunction of motile or sensory cilium.The phenotypic diversity associated with ciliary dysfunction reflects the variability of ciliary protein expression in different cells and tissues.The situation of the cilia in the cell surface facilitates its function as a sensor and transmitter of information between the cell and the extracellular space.Thus, cilia can take on different roles; for example, modulating the flow direction in the extracellular embryonic node or mediating intracellular calcium signaling in the presence of renal flow.Cilia dysfunction encompasses a large group of disorders ranging from primary ciliary dyskinesia changes in left-right laterality, retinitis pigmentosa, sensorineural deafness and oncogenesis.As a result, ciliopathies can present with different phenotypes, although they often share renal, bone, ocular and central nervous system defects.These groups of disorders are also genetically heterogeneous and it remains possible that mutation of a single gene results in different phenotype according to the time for action in the embryo.Ciliopathies types based on the participation of the motile or primary cilium not support a clear differentiation between these disorders, therefore it could be more practical to know the age of onset of symptoms, their lethality and the possibility of renal or bone involvement.Recently, cilia dysfunction has been implicated in the pathogenesis of other conditions apparently unrelated to the recognized ciliopathies including obesity, hypertension and diabetes.These findings may offer new targets for therapeutic intervention.

Figure 1 :
Figure 1: Microtubular structure of the cilium and intraflagellar transport.The ciliary axoneme is a microtubule structure anchored to the basal body and surrounded by the ciliary membrane.Molecular complexes kinesin II and cytoplasmic dynein are involved in intraflagellar transport and their function is required for proper ciliogenesis.

Figure 2 :
Figure 2: Motile cilia and non motile cilia: ciliar pattern, type of movement and system location.Motile "9+2" pattern cilia are located on the cell surface as multiple cilia and have a synchronized waveform motion.Motile "9+0" pattern cilia lack central microtubules and are responsible for establishing the left-right axis in the embryo.Immotile cilia "9+0" lack dynein arms and central microtubules and act as primary or sensory cilia in multiple organs of the body.

Figure 3 :
Figure 3: Left-right axis establishment in the embryo, Cilia flexion triggers a left-sided intracellular calcium signal.
; Jenkins et al, 2009).Olfactory dysfunction is due to a defect in localization of olfactory G proteins in primary cilia leading a nonfunctional signaling pathway, despite cilia structure in sensory neurons remains intact (Dyke et al, 2007).