Synergistic Interaction between Ciliary Genes Reflects the Importance of Mutational Load in Ciliopathies

The formation of renal cysts occurs in roughly 10 to 15% of the population,¹ and although simple cysts are largely asymptomatic, the formation of multiple cysts can be extremely detrimental. The two major multicystic syndromes, autosomal recessive polycystic kidney disease (PKD) and autosomal dominant PKD, are primarily linked to disruptions in the pkhd1 and pkd1/pkd2 genes, respectively²; however, several additional syndromes are also characterized by kidney cyst development, including nephronophthisis (NPHP), Joubert syndrome, Bardet-Biedl syndrome, Orofaciodigital syndrome 1, and Meckel syndrome (MKS). During the past decade, there has been a great upsurgence in identifying genes affected in these syndromes. One of the most striking discoveries is that protein products of these genes localize to the cilium and/or the associated basal body and affect ciliogenesis and cilia morphology or function when mutated,³ collectively identifying these syndromes as ciliopathies.

Why do kidney cysts form? An increasing body of evidence indicates that mutations affecting ciliogenesis or cilia function can give rise to cysts. Primary cilia extend from the apical membrane of renal epithelial cells into the lumen, where they act as mechanosensors. One model suggests that fluid flow within the kidney bends the cilia and triggers intracellular calcium signaling, which, in turn, regulates epithelial tubule morphology.² Polycystin 1 and polycystin 2, the protein products of the pkd1 and pkd2 genes, respectively, localize to renal cilia⁴ and may act as the mediators of mechanosensation in these structures.⁵

Mutations in pkd1 or pkd2 do not result in morphologic cilia defects but rather affect calcium influx, which leads to cyst formation. In addition, mutations that result in aberrant cilia growth, maintenance, or morphology give rise to cysts,⁶ suggesting that both cilia formation and function are crucial for prevention of a polycystic kidney phenotype.

NPHP and MKS belong to the growing number of ciliopathies characterized by phenotypically distinct but partially overlapping malformations. NPHP is a rare autosomal recessive renal disease with an incidence of approximately one in 100,000 live births and is the most frequent cause of ESRD in children and young adults.⁷ The hallmarks of NPHP are corticomedullary cysts, tubulointerstitial nephropathy, and disruption of the tubular basement membrane. Together, these defects lead to renal failure, typically within the first three decades of life. To date, 10 genes encoding nephrocystins have been identified as being mutated in patients with NPHP, although disruptions in these genes account for only approximately 30% of NPHP cases, suggesting the presence of additional genetic loci.

MKS is a rare autosomal recessive disorder first identified more than a century ago and has a current worldwide incidence of one in 140,000 live births.⁶ MKS is characterized by postaxial polydactyly, dysplasia of the hepatic duct, occipital meningoencephalocoele, and multicystic kidneys. In recent years, six genomic loci in humans have been linked to MKS and, with the exception of MKS2, have been mapped to individual genes. Nephrocystins and MKS proteins primarily localize to cilia or basal bodies in renal epithelial cells, placing these syndromes within the growing list of ciliopathies.

Links between NPHP and MKS have begun to emerge in recent years. The two syndromes share at least three underlying genes. rpgrip1l is mutated in MKS5/NPHP8, cep-290 in MKS4/NPHP6, and mks3 in MKS3/NPHP11. Mutations in these genes have also been found in Joubert syndrome, cerebello-oculorenal syndromes, and Senior-Løken syndrome, suggesting they form a spectrum whereby the severity of phenotype is a function of the strength of the mutation. This is reflected by the fact that ciliopathies share an overlapping set of clinical features, with MKS representing the severe end of the phenotypic spectrum comprising defects in multiple organs and perinatal lethality.

In addition to the finding that the same genes can give rise to different syndromes, one of the most exciting results from the field of ciliopathy research is that underlying mutations seem to act in a combinatorial manner to influence the phenotype.^8,9 This suggests that the mutational load within an individual can modulate the severity of the disease. Previous studies showed that MKS1 and MKS1-related proteins (MKSR1/TZA2, MKSR2/TZA1) act in the same pathway and have no ciliary defects when mutated by themselves or together in Caenorhabditis elegans^10,11; however, mks1;nphp1 double mutants display ciliary defects including shortened cilia and aberrant ciliary positioning.

In this issue, Williams et al.¹² build on these initial observations and provide data that corroborate a synergistic interaction between the ciliogenic MKS and NPHP pathways. To test the hypothesis that perturbation of either pathway alone is not sufficient to disrupt ciliogenesis but disruption of both together lead to a ciliary defect, the authors analyzed mks3 mutants, nphp4 mutants, and double mutants to determine whether they would display defects in ciliogenesis, ciliary positioning, and proper sensory function of cilia.

The authors show that in C. elegans MKS3 localizes to the membrane of the dendritic tip of ciliated sensory neurons and its cilium base and acts in the MKS1 pathway. This subcellular localization somewhat differs from MKS3 localization in mammalian cells (IMCD3, HEK293, and BEC), where it is localized mostly along the ciliary axoneme.¹³ Using a putative mks3 null mutant to analyze its function in C. elegans, the authors found no obvious ciliogenesis defect (mild elongation), which is in contrast to mammalian mks3 null models in mouse (bpck) and rat (wpk), which exhibit elongated cilia and kidney cysts.^14,15 Interestingly, although mks3 mutants showed no defect in cilia morphology, mutants showed reduced chemotaxis, suggesting impaired sensory function.

Interestingly, mks3;nphp4 double mutants exhibit defective cilia positioning and shorter or absent cilia indicative of a defect in ciliogenesis and/or maintenance of the cilium. One interpretation of the data is that MKS3 may act as a potential regulator of cargo trafficking into the cilium by tethering transition fibers to the membrane, thereby regulating vesicle trafficking at the ciliary base or defective positioning of the cargo in the cilium. A leaky transition zone could account for excess cargo import into the cilium and the elongated ciliary phenotype observed in mammals and in milder form in C. elegans. The addition of ciliary microtubule defects of nphp4 mutants to the flawed ciliary membrane and/or transition fiber regulation might be detrimental for ciliogenesis and correct positioning, resulting in the observed phenotype of the double mutants. Many key questions remain unanswered. Even though MKS1 and MKS3 physically interact, it is unclear how they act to regulate ciliogenesis, cilia length control, or cilia number. In mammals, MKS3 function regulates the number of cilia on the apical membrane in kidney tissues in vivo and in vitro, and loss of MKS3 results in an increased number of cilia. The authors of this study do not comment on cilia numbers, which may reflect a difference between a mammalian and nematode role of MKS3. Along this line, MKS3 might play additional roles in mammals versus nematodes, on the basis of its differential localization. Elucidating the function of MKS3 will also require investigating its potential genetic interaction with other ciliopathy genes, which would give important insights into the understanding of the pathogenesis of the cystic phenotype.

Several reports recently provided data indicating that phenotypic severity among MKS and NPHP is a consequence of mutational load, meaning that MKS and NPHP lie within a phenotypic continuum rather than represent multiple distinct clinical entities and that the sum of mutations in ciliary genes define the severity of the phenotype. Further studies of MKS3 in conjunction with other ciliary proteins are now required to unravel how different mutational loads can lead to the clinical variability observed in patients with MKS as well as other ciliopathies and identify further, second-site modifiers.

This study by Williams et al.¹² offers a deeper understanding of the importance of mutational load on the presentation and severity of ciliopathies and expands the understanding of the synergistic interactions between ciliopathy genes. Further analysis will hopefully allow targeted therapies to alleviate the morbidity and mortality associated with these devastating diseases.

• Copyright © 2010 by the American Society of Nephrology