The molecular genetics of Joubert syndrome and related ciliopathies: The challenges of genetic and phenotypic heterogeneity

1 Introduction

Joubert syndrome (JS; OMIM PS213300) is a predominantly autosomal recessive ciliopathy condition characterized by a distinctive cerebellar and brainstem defect on cranial MRI known as the “molar tooth sign” because of its resemblance to the cross-section of a tooth on axial imaging [64, 65]. Its three key diagnostic criteria have been established as (1) the “molar tooth sign” (MTS), (2) hypotonia in infancy with later ataxia, and (3) developmental delays/intellectual disability. There are several other more variable features, including breathing problems— often episodic tachypnea and/or apnea— especially in infancy, and abnormal eye movements typically characterized by oculomotor apraxia or the inability in initiating voluntary saccades [70]. In addition, a number of findings are sometimes found in those with JS such as retinal disease, cystic kidney disease including nephronophthisis, congenital hepatic fibrosis, and skeletal features such as polydactyly and small ribcage; many of these are also shared by other ciliopathy conditions [6, 118].

Since the first gene for JS, NPHP1, was identified in 2004 [72], over 30 genes have been implicated in its causation [70]. However, most of these genes cause less than 10% of cases each, and for several genes, the assignment is based on only one or a handful of cases. All of the genes identified thus far are localized to or play a role in the function of the sub-cellular structure, the primary cilium, particularly at the transition zone of the cilium where it joins the plasma membrane [36]. The primary cilium is the appendage on almost all cells that functions in the cell’s ability to establish a pattern of left-right symmetry during development, allows it to transduce signals from the environment, and carries out fundamental intracellular processes via signaling pathways such as Sonic Hedgehog and others [82].

This review will focus on the molecular genetics of JS, while also including genetics-based discussion of other relevant ciliary disorders with overlapping phenotypes, and will provide some examples of potential future treatments to capitalize based on what is known about gene function and ciliary biology.

2 Diagnostic criteria for Joubert syndrome

The diagnosis of JS has been established as requiring 3 key elements: the radiologic finding of the “molar tooth sign” (MTS; Fig. 1A, C) accompanied by cerebellar vermis hypoplasia, as well as hypotonia or low muscle tone and developmental delays or intellectual disability. The MTS is visualized on axial views from magnetic resonance imaging (MRI) studies through the junction of the midbrain and pons and consists of the mesencephalon (the “crown” of the tooth), typically with a deepened interpeduncular fossa, and prominent, thickened, and generally straight superior cerebellar peduncles (the “roots” of the tooth) [64, 65]. The MTS is generally associated with hypoplasia of the midline portion of the cerebellum (the cerebellar vermis), a required feature in JS. On sagittal MRI, an enlarged, misshapen and/or rostral shifting of the fastigium of the 4th ventricle is often visualized (Fig. 1B, D). Although a high-resolution MRI scan with 3 mm sections is recommended for visualizing this radiologic sign, the MTS can sometimes be viewed on good quality, high density computed tomography (CT) scans. In reality, a radiologic finding of the MTS is often considered “pathognomonic” for JS, and certainly this discovery in the prenatal or neonatal period would warrant a presumptive diagnosis of JS, as the other required features are likely to become apparent with time (see refs [27, 80] for prenatal radiologic features in JS). In addition, there is a spectrum of MTS findings, with milder versions seen in association with some causative genes [31, 72], and without examination of multiple MRI cuts for subtle findings of the MTS and vermis hypoplasia, even experienced neuroradiologists may miss this diagnostic hallmark.

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Fig.1

Molar Tooth Sign in Joubert syndrome. T2-weighted MRI images on axial view (A) and mid-sagittal view (B) through the cerebellum and brainstem of a normal individual showing intact cerebellar vermis. T2-weighted MRI image on axial view (C) and mid-sagittal view (D) through the cerebellum and brainstem of a child with JS. The Molar Tooth Sign is circled in black, with rostral shifting or elevation of the roof (fastigium) of the fourth ventricle and vermis hypoplasia (white arrow).

The hypotonia of infancy often gives way to later ataxia. Developmental delays in motor and cognitive development are universal [76, 105], although rare individuals with JS and normal IQ have been described [76]. Of note, a correlation between degree of cerebellar vermis hypoplasia and cognitive impairment has been seen [77]. In fact, a systematic evaluation of the neuropsychological profiles in 76 participants with JS showed an average Full Scale Intelligence Quotient (FSIQ) = 64±15 and relative strengths in receptive language and verbal comprehension, but challenges with processing speed most likely related to cerebellar deficits [105]. Behavioral surveys suggested more emotional and behavioral problems than in the general population, which may worsen with age, but not as severe as other populations with intellectual disability [105].

Although the 3 cardinal features are necessary for a diagnosis, additional features are often described in infants and children with JS. An abnormal respiratory pattern, particularly prominent in infancy, consists of alternating tachypnea and/or apnea; some neonates have had worrisome bouts of apnea requiring pharmacological intervention [63, 103]. In fact, for those with JS under 5 years of age who were deceased, respiratory failure was the leading cause of death [24]. Eye movements characterized as oculomotor apraxia, or the absence of or defect in controlled, voluntary, and purposeful eye movements resulting in jerky eye movements and head thrusting, as well as nystagmus, strabismus, and ptosis of the eyelids, are often identified in those with JS (Fig. 2A). The spectrum of ocular findings in JS has been prospectively ascertained in one large cohort [16] and the published literature on ocular findings in JS has also been reviewed recently by two groups [16, 119], with general findings of delayed visual development and variable visual acuity [16, 119]. Retinopathy has been reported in nearly 1/3, and ocular coloboma in nearly 30%; however, these rarely occur in the same individual. Optic nerve atrophy was reported in only 10–15% of these cohorts [16, 119].

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Fig.2

Clinical features in Joubert syndrome. A. Facial features in a girl with JS/COACH syndrome at 27 months of age showing broad forehead, arched eyebrows, strabismus, eyelid ptosis (on right eye), and open mouth configuration indicating reduced facial tone. B. Oral findings in a child with oral– facial– digital syndrome-like features of JS showing midline upper lip cleft (arrowhead), midline groove of tongue, and bumps of the lower alveolar ridge (arrow). C. Left hand of an infant with JS and postaxial polydactyly (arrow). D. Left foot of an infant with JS and preaxial polysyndactyly and fusion of the hallux. E. View from above of an infant with a small occipital encephalocele with protrusion of the occiput of the skull (arrow). (Facial photograph used with permission of the family.) (Previously published in [73] with permission).

Phenotypic variability is a key component of JS. There are pleiotropic and highly variable features described in other organ systems that can provide clues to sub-phenotyping, and in some cases, even hints at underlying genetic causes. In addition to the eye findings described above, significant morbidity can arise in those with JS and renal disease (nephronophthisis or cystic kidneys) or congenital hepatic fibrosis. Renal failure is the leading cause of death in those over the age of 5 years [24]. In addition, they can have oral or tongue hamartomas (Fig. 2B), skeletal changes (polydactyly (Fig. 2C, D), syndactyly, and/or narrow thoracic ribcage), and occipital encephalocele (Fig. 2E) [6, 118]. The polydactyly can be postaxial (Fig. 2C), preaxial (Fig. 2D), and occasionally central or mesaxial. Congenital heart defects, cleft palate, skin findings related to those of cranioectodermal dysplasia (sparse hair, small, widely spaced teeth or hypodontia, and/or abnormal nails), situs defects, and obesity are less common but occasionally described. Although ocular colobomas and occipital encephaloceles are developmental defects present at birth, many other features evolve over time and with age, so regular monitoring is necessary to ensure timely diagnosis and treatment if appropriate [70]. Many of these features are typical in other ciliopathy disorders such as Meckel syndrome (MKS) or Oral-Facial-Digital (OFD) syndrome, reinforcing the overlapping phenotypic spectrum of these conditions and confounding attempts at tidy genotype-phenotype correlations [114].

Given the broad clinical heterogeneity of Joubert syndrome, there are some useful sub-categories that can help in classifying the condition, and sometimes, in predicting which additional manifestations may develop in a given individual [14]. However, even within the same family, siblings with the same causative mutations may have variable phenotypes, illustrating that clinical heterogeneity is the norm in this condition.

4 Prevalence and epidemiology of Joubert syndrome

The prevalence of JS has been estimated to be between 1:80,000 and 1:100,000 [14, 73], but this may represent an underestimate as more causative genes and private mutations are identified, as well as a broader range of phenotypic findings. JS is a predominantly autosomal recessive disorder first described in relatively isolated groups with high rates of consanguinity, including in French-Canadian, Ashkenazi Jewish, and Arab populations [30, 100–102]. Interestingly, there are multiple founder mutations in several genes for each of these populations. The original French-Canadian family described by Joubert et al in 1969 [49] was known to have distant consanguinity related to a common founder 10 generations removed in France. However, there are at least 3 different genes known to cause JS in families from this region of Canada (CPLANE1, CC2D2A, NPHP1), and the breadth of mutations even within genes linked to an inbred population is highly variable. In fact, many affected individuals who are French-Canadian harbor heterozygous pathogenic variants [100], indicating that the concept of a single “founder” mutation in a particular consanguineous population does not apply to JS. This phenomenon also applies to families from the Middle East, where although there is a high rate of consanguinity, there is a diversity in the number of causative genes [6]. However, in many families of relatively close Arab descent, with first cousin or comparable relatedness, the pathogenic variants are likely to be homozygous, reflecting identity-by-descent. The heterogeneity of causative genes, and in some cases, mutations in an inbred population has also been reported in other ciliopathy disorders. In Newfoundland, for example, multiple different founder effects appear to be causative of Bardet-Biedl syndrome (BBS; [67]). A plausible molecular explanation has not been postulated.

5 Genes associated with Joubert syndrome

There are 35 genes implicated as causative for JS (Table 1). Although most are inherited in an autosomal recessive manner, as are the majority of ciliopathies, one, OFD1 (Oral Facial Syndrome 1) demonstrates X-linked recessive inheritance. A few genes have been suggested to have autosomal dominant inheritance, but this is highly speculative as exhaustive screening for other variants may not have been performed. Collectively, these genes account for between 62–94% of cases of JS [6, 118]. The high rate of 94% with identified genetic causes in one survey [118] may be related in part to the large proportion of subjects with hepatic involvement and COACH phenotypes, with better known and characterized genetic causes. Of note, almost all of the genes have also been implicated in other ciliopathy disorders, such as MKS, NPHP, OFD, BBS, COACH syndrome, and others (see column of “Allelic disorders” in Table 1). In general, it is difficult to identify clear-cut genotype-phenotype correlations for many of these genes, but a few distinguishing features are included in the table, as well as genes where founder effects have been identified. In some cases, a more severe or even lethal condition, such as MKS, has been associated with more severe pathogenic changes such as frameshift or nonsense mutations causing loss-of-function rather than milder missense variants often associated with a non-lethal phenotype, such as JS [14]— See Additional Challenges below.

Ciliopathy conditions are human disorders characterized by dysfunction of either the motile or immotile cilium, a subcellular organelle important for embryonic development, adult homeostasis, and in sensory and/or signaling processes [38]. JS is a ciliopathy disorder of the primary, non-motile cilium found on almost all vertebrate cells [82]. Most of the genes implicated in JS and related ciliopathies localize to either the primary cilium, the basal body and/or the mother centriole from which it arises, or the regulatory proteins and transcription factors that guide its development and function [82]. The primary cilium has been described as an antennae or appendage protruding from the plasma membrane that plays a role in sensing the environment and responding to localized signals [38]. It develops from the docking of a mother centriole via “distal appendages” or transition fibers that link the centriole to the plasma membrane; this is where the centriole extends the 9 axonemal microtubule doublets into the cilium (Fig. 3A, B). The structure at the base of the cilium and junction of plasma membrane and ciliary membrane is known as the transition zone [36]. This “transition zone,” which, along with the transition fibers and Y-fibers, comprise the “ciliary gate,” lies between the basal body and the ciliary axoneme and controls the entry and exit of proteins and lipids into and out of the cilium. Intraflagellar transport (IFT) of this cargo along the microtubules is driven by kinesin (anterograde) and dynein (retrograde) motors and associated IFT proteins and BBS proteins, with lipidated protein transport also playing a role [82].

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Fig.3

Diagram of the structure of the Primary Cilium. A. Schematic of the components of a non-motile (primary cilium) showing the axoneme (ciliary stalk), comprised of 9 pairs of microtubules. The cilium extends from a basal body, which derives from the mother centriole. Transition fibers tether the basal body to the base of the ciliary membrane. The transition zone (TZ) is adjacent to the basal body and serves as a gate for passage of proteins and lipids into and out of the cilium. Anterograde transport is accomplished by kinesin motors along with associated proteins and BBsomes propelling cargo along the microtubules; retrograde transport utilizes dynein motors rather than kinesins. Signaling proteins on the ciliary membrane represent the components of the Sonic Hedgehog (SHH) or other receptor complexes. B. Cross-section of the cilium demonstrates the typical 9+0 pattern of doublet microtubes in the axoneme (upper diagram) and the addition of Y-links that connect the doublet microtubes to the ciliary membrane and help form the barrier within the transition zone (bottom diagram). (Derived from [38, 82]).

The transition zone appears to be a hotspot for human disease genes associated with ciliopathies. In fact, there are either 2 or 3 main protein complexes, mapped by interactome studies, that form the transition zone [19, 35]: the MKS complex, which includes the Tectonic proteins, B9 domain proteins, coiled-coil proteins such as CC2D2A and CEP290, AHI1, and 5–7 transmembrane or TMEM proteins; and the NPHP complex, including the NPHP proteins, RPGRIP1 L, and others [36]. Linking these 2 domains are CEP290 and NPHP5, sometimes separated out as a distinctive protein domain [38]. These subdivisions are significant, as almost all of the proteins in the MKS complex are encoded by genes implicated in MKS and JS, while the NPHP complex is associated with nephronophthisis-causing genes predominantly [38]. The developmental function of these proteins is just now being discovered and includes roles in Sonic Hedgehog (SHH) signaling, planar cell polarity, cAMP signaling, mTOR pathway activity, and DNA Damage Response (DDR) signaling pathways [66, 99]. In spite of our ever-evolving understanding of ciliary biology, the precise pathological processes that underlie the specific manifestations of the ciliopathies remain elusive.

JS is one of several disorders of the primary cilium that have multi-systemic effects, often including involvement of the brain, eyes, kidney, liver, and skeleton. These ciliopathies also overlap in their manifestations, and in their causative genes [66]. The allelic ciliopathy disorders associated with each of the 35 JS-associated genes based on overlapping phenotypes are shown in Fig. 4. The following sub-sections will describe the most common allelic disorders to JS identified thus far:

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Fig.4

Genes that cause JS have overlapping phenotypes with other ciliopathies. The 35 genes that cause Joubert syndrome (JS) and are listed in Table 1 are distributed within the large circle in relationship to allelic ciliopathy disorders indicated within each semi-oval (or circle in the case of COACH). The name of each ciliopathy disorder is indicated within the box. Other genes that cause the ciliopathy condition but are not associated with JS are omitted in order to simplify the diagram. These relationships are not fixed, as the phenotypic spectrum associated with ciliopathy genes is constantly evolving. *Can also be associated with RP alone ∧The MKS1 gene has not been described in NPHP alone but this could not be indicated due to constraints of the diagram. Abbreviations: ACS, Acrocallosal syndrome; BBS, Bardet-Biedl syndrome; BCNS, Basal Cell Nevus Syndrome; COACH, Colobomas, cognitive impairment (“Oligophrenia”), Ataxia, Cerebellar vermis hypoplasia, and Hepatic fibrosis syndrome; HL, Hydrolethalus syndrome; JATD, Jeune asphyxiating thoracic dystrophy and related skeletal dysplasias; JS, Joubert syndrome; LCA, Leber Congenital Amaurosis; MKS, Meckel syndrome; MORM, Mental retardation, Obesity, Retinal dystrophy, Micropenis syndrome; NPHP, nephronophthisis; OFD, oral-facial-digital syndrome; RP, retinitis pigmentosa.

8 Additional challenges in genetic diagnoses of ciliopathies

There are several major conundrums in understanding the genetic basis of ciliary disorders. One is the sheer number of genes that is causative for each clinically-defined disorder. Even a seemingly simplified ciliopathy such as nephronophthisis (NPHP) can be accounted for by over 20 genes, all of which are associated with the cilia, centrosome, or mitotic spindle [99]. In fact, most surveys report that about 15–20% of individuals with nephronophthisis are reported to have additional non-renal ciliopathy symptoms, the most common being RP. The NPHP1 gene only accounts for ∼20% of individuals with NPHP, with a homozygous deletion of the entire gene being the major mutation. Even in the situation where an individual harbors this clear biallelic loss-of-function deletion, however, the heterogeneity of the phenotype can range from NPHP to Senior-Løken to JS. Each of the remaining 20+ causative genes accounts for less than 1% of cases of NPHP, suggesting that over 60% of cases do not yet have a known genetic cause, in spite of 20 years of genetic studies on this condition [99]. It is notable that many ciliopathy conditions have multiple causative genes, with few individual genes causing more than 10–20% of cases within a specific condition. This lack of predictive ability and paucity of genotype-phenotype correlations are challenging.

The second conundrum is the extreme genetic heterogeneity of molecular causes and, in particular, the overlap of many causative genes with multiple different ciliopathy conditions. As noted, many of the genes that cause JS can also cause MKS, NPHP, OFD, BBS, and/or Jeune syndrome, or even more obscure ciliopathy conditions. When looking for genotype-phenotype correlations that might explain why the same gene can cause a lethal MKS phenotype as well as a milder JS phenotype and even more focal nephronophthisis, the severity of mutation has been implicated (e.g., 2 mutations in CC2D2A or TMEM67 resulting in frameshift or stop codons cause MKS while “milder” missense mutations cause JS) [8, 68]. Similarly, mutations in TMEM231 can cause MKS, OFD, and JS, with different presentations due to the same mutation even within the same family [61, 101]. In addition, different missense mutations in the same gene can cause disparate phenotypes, such as mutations in CEP290 that can cause JS, BBS, LCA, MKS, or Senior-Løken syndrome; it is likely that alterations in this protein, which straddles the MKS and NPHP modules within the transition zone, have different effects depending on their impact on interacting protein partners or different functional domains of the protein [90, 115]. Another possible explanation for this variability in the different outcomes for pathogenic variants in the same gene is that they may impact discrete isoforms of the protein that have distinctive functions. This is certainly the case for at least one BBS gene, ARL6, where one isoform is part of the BBSome and results in the BBS phenotype, whereas a mutation in a longer isoform has a specific role in photoreceptor maintenance [78]. Finally, it is likely that modifier genes or other epistatic effects such as differences in genetic background play a role. In these situations, proteins with overlapping functions and roles can be revealed. The specific manifestations of the pleiotropic effects of BBS may be partially explained by the unique roles of proteins associated with intraflagellar transport that underlie this condition: for example, ciliary biogenesis is dependent on Hedgehog signaling and is necessary for embryonic development, particularly of the nervous system and limb bud— consequently, mutations in many IFT components are associated with intellectual disability and skeletal dysplasias such as JATD [91]. Likewise, mutations in genes that impact the tissue-specific cargo of the IFT system may result in specific manifestations of ciliopathies [82]. The condition with the most overlap with JS is MKS, and both are associated with causal mutations in overlapping genes whose protein products localize to the transition zone. There is redundancy in the system, as the MKS, NPHP, and BBSome complexes likely have partially overlapping roles in promoting the ciliary localization of proteins and forming a barrier for protein transit into and out of the cilium. For proteins within the transition zone, a non-pathogenic variant may thus modify the phenotypes caused by pathogenic variants in a gene encoding a protein with overlapping or partially redundant function [82].

The third conundrum is the finding of occasional oligogenicity (variants in 2 or more different genes cause the condition; [46]), digenic inheritance, or triallelism in JS, as well as other ciliopathies. Triallelic inheritance refers to the observation that biallelic mutations in one gene are required along with a heterozygous pathogenic variant in a different ciliary gene to cause the disease [50]. Of course, these rare reports may reflect extremes of genetic modifiers, in which variants in other genes in addition to biallelic pathogenic variants in one known causative gene, affects the spectrum and severity of the phenotypic findings [75]. Several specific examples have been described, such as a heterozygous AHI1 mutation in combination with biallelic NPHP1 mutations resulting in a more severe brain phenotype in NPHP [111]. An NPHP1 mutation or deletion may also contribute to the severity of the BBS phenotype caused by 2 BBS biallelic mutations [59]. This has also been demonstrated with a common variant in RPGRIP1 L that modifies the retinal degeneration phenotype in a number of ciliary disorders [52]. Before the advent of next-generation sequencing capabilities, identifying genetic modifiers was challenging, and even targeted gene panels might fail to provide a comprehensive evaluation of all likely contributing modifying genes. One recent effort sought to tease out the prevalence of digenic inheritance, triallelism, and genetic modifiers in a large JS cohort [75]. Although rare disease variants were identified in over 1/3 of affected individuals in addition to the causal pathogenic mutations, they did not correlate with severity of the condition, nor did they support the presence of triallelic inheritance. Importantly, the rate of discordant phenotypes (∼60%) between affected siblings with shared pathogenic variants in a known JS gene suggested that modifying genes play an important role in JS, but they may be difficult to identify [75]. Another comprehensive study of the genetic contributions to a large cohort of subjects with ciliopathy phenotypes in a mostly consanguineous population demonstrated that ciliopathies are, for the most part, Mendelian disorders, as the burden of rare variants in known ciliopathy genes in the cohort did not differ significantly from the rate in a control cohort with non-JS intellectual disability; these authors suggested that stochastic events rather than modifier alleles contributed to phenotypic variability within their population [97].

9 Future directions in Joubert syndrome biology, genetics and therapeutics

The sheer number of causative genes for ciliary disorders such as JS can seem overwhelming, and clearly, identifying the causative gene and variant for a given family can be daunting. This is compounded by the lack of clear and consistent founder effects in isolated populations with these conditions. More research to identify additional causative genes will be critical for this effort, and new methodologies such as whole exome and whole genome sequencing are clearly paving the way, although interpretation of the meaning of variants can be quite challenging given the heterogeneity of genotypes and lack of correlation with phenotypes. Certainly, these features point to some level of redundancy in the complex biology of the cilium. And the extreme pleiotropy of organ system involvement in JS and other ciliopathies suggests that a deeper understanding of the roles of ciliary proteins, their interactions, and tissue specificity is necessary. In addition, the emerging role of modifying genes, many of which are ciliary genes as well, points to the role of ciliopathies serving as a bridge between strictly mendelian disorders and more complex polygenic disorders. For the extreme and rare cases of digenic or triallelic inheritance, the concept of threshold for mutational load appears operative. And in general, many of the principles that hold for one ciliary disorder are likely to be generalizable to other ciliopathies, suggesting that there may be some concepts of ciliary genetics and biology that are applicable to several, if not most, ciliopathies. For example, although many genes and several different signaling pathways are implicated in the pathogenesis of NPHP, the different mechanistic processes converge on the final common endpoints of renal tubular damage and fibrosis leading to ESRD [99].

In spite of lack of knowledge about the functional and developmental roles of many ciliarry proteins, there remains hope for development of therapies to impact specific ciliary phenotypes. Rescuing the developmental brainstem and cerebellar malformations that underlie the MTS may not be achievable, at least in the foreseeable future, but interventions to ameliorate the co-occurring morbidities such as retinal, renal, and/or liver disease could significantly impact quality of life for those with JS. One strategy to approach the cystic renal disease of NPHP has been to treat the elevated levels of cAMP and defects in urinary concentrating ability by use of vasopressin receptor-2 antagonists such as tolvaptan; animal models paved the way for the use of this drug in clinical practice and in current trials of childhood AD-PKD [69]. Other approaches to treat NPHP have included CDK inihibitors, SHH agonists, and mTOR pathway inhibitors such as rapamycin, but many of these are in early stages of development [99]. Gene therapy holds some promise in treating cystic renal disease in JS due to CEP290 mutations; an antisense oligonucleotide (ASO) has been used to promote alternative splicing and exon skipping to rescue an intronic mutation in this gene in patient-derived renal epithelial cells and in a mouse model of JS, with reduction of cystic kidney disease burden [81]. Similarly, a comparable approach to rescuing the retinal phenotype of LCA caused by the common intronic splicing defect in the CEP290 gene using gene therapy approaches has demonstrated some success in a cell model and in mice carrying the mini-gene [29]. Finally, as other articles in this special issue point out, there are novel strategies emerging to treat these and other manifestations of ciliopathy conditions (“Retinal disease in ciliopathies: recent advances with a focus on stem cell-based therapies review” by Chen, H et al., “Novel Treatments for Polycystic Kidney Disease” by Patil, A et al., and “Using human urine-derived renal epithelial cells to model kidney disease in inherited ciliopathies” by Sayer, J and Molinari, E.).