A Screen for Modifiers of Cilia Phenotypes Reveals Novel MKS Alleles and Uncovers a Specific Genetic Interaction between osm-3 and nphp-4.

Nephronophthisis (NPHP) is a ciliopathy in which genetic modifiers may underlie the variable penetrance of clinical features. To identify modifiers, a screen was conducted on C. elegans nphp-4(tm925) mutants. Mutations in ten loci exacerbating nphp-4(tm925) ciliary defects were obtained. Four loci have been identified, three of which are established ciliopathy genes mks-1, mks-2, and mks-5. The fourth allele (yhw66) is a missense mutation (S316F) in OSM-3, a kinesin required for cilia distal segment assembly. While osm-3(yhw66) mutants alone have no overt cilia phenotype, nphp-4(tm925);osm-3(yhw66) double mutants lack distal segments and are dye-filling (Dyf) and osmotic avoidance (Osm) defective, similar to osm-3(mn357) null mutants. In osm-3(yhw66) mutants anterograde intraflagellar transport (IFT) velocity is reduced. Furthermore, expression of OSM-3(S316F)::GFP reduced IFT velocities in nphp-4(tm925) mutants, but not in wild type animals. In silico analysis indicates the S316F mutation may affect a phosphorylation site. Putative phospho-null OSM-3(S316F) and phospho-mimetic OSM-3(S316D) proteins accumulate at the cilia base and tip respectively. FRAP analysis indicates that the cilia entry rate of OSM-3(S316F) is slower than OSM-3 and that in the presence of OSM-3(S316F), OSM-3 and OSM-3(S316D) rates decrease. In the presence OSM-3::GFP or OSM-3(S316D)::GFP, OSM-3(S316F)::tdTomato redistributes along the cilium and accumulates in the cilia tip. OSM-3(S316F) and OSM-3(S316D) are functional as they restore cilia distal segment formation in osm-3(mn357) null mutants; however, only OSM-3(S316F) rescues the osm-3(mn357) null Dyf phenotype. Despite rescue of cilia length in osm-3(mn357) null mutants, neither OSM-3(S316F) nor OSM-3(S316D) restores ciliary defects in nphp-4(tm925);osm-3(yhw66) double mutants. Thus, these OSM-3 mutations cause NPHP-4 dependent and independent phenotypes. These data indicate that in addition to regulating cilia protein entry or exit, NPHP-4 influences localization and function of a distal ciliary kinesin. Moreover, data suggest human OSM-3 homolog (Kif17) could act as a modifying locus affecting disease penetrance or expressivity in NPHP patients.

Assessing the pathogenic potential of human Nephronophthisis disease-associated NPHP-4 missense mutations in C. elegans.

A spectrum of complex oligogenic disorders called the ciliopathies have been connected to dysfunction of cilia. Among the ciliopathies are Nephronophthisis (NPHP), characterized by cystic kidney disease and retinal degeneration, and Meckel-Gruber syndrome (MKS), a gestational lethal condition with skeletal abnormalities, cystic kidneys and CNS malformation. Mutations in multiple genes have been identified in NPHP and MKS patients, and an unexpected finding has been that mutations within the same gene can cause either disorder. Further, there is minimal genotype-phenotype correlation and despite recessive inheritance, numerous patients were identified as having a single heterozygous mutation. This has made it difficult to determine the significance of these mutations on disease pathogenesis and led to the hypothesis that clinical presentation in an individual will be determined by genetic interactions between mutations in multiple cilia-related genes. Here we utilize Caenorhabditis elegans and cilia-associated behavioral and morphologic assays to evaluate the pathogenic potential of eight previously reported human NPHP4 missense mutations. We assess the impact of these mutations on C. elegans NPHP-4 function, localization and evaluate potential interactions with mutations in MKS complex genes, mksr-2 and mksr-1. Six out of eight nphp-4 mutations analyzed alter ciliary function, and three of these modify the severity of the phenotypes caused by disruption of mksr-2 and mksr-1. Collectively, our studies demonstrate the utility of C. elegans as a tool to assess the pathogenicity of mutations in ciliopathy genes and provide insights into the complex genetic interactions contributing to the diversity of phenotypes associated with cilia disorders.

Normal ciliogenesis requires synergy between the cystic kidney disease genes MKS-3 and NPHP-4.

Cilia dysfunction contributes to renal cyst formation in multiple human syndromes including nephronophthisis (NPHP), Meckel-Gruber syndrome (MKS), Joubert syndrome (JBTS), and Bardet-Beidl syndrome (BBS). Although genetically heterogeneous, these diseases share several loci that affect cilia and/or basal body proteins, but the functions and interactions of these gene products are incompletely understood. Here, we report that the ciliated sensory neurons (CSNs) of C. elegans express the putative transmembrane protein MKS-3, which localized to the distal end of their dendrites and to the cilium base but not to the cilium itself. Localization of MKS-3 and other known MKS and NPHP proteins partially overlapped. By analyzing mks-3 mutants, we found that ciliogenesis did not require MKS-3; instead, cilia elongated and cilia-mediated chemoreception was abnormal. Genetic analysis indicated that mks-3 functions in a pathway with other mks genes. Furthermore, mks-1 and mks-3 genetically interacted with a separate pathway (involving nphp-1 and nphp-4) to influence proper positioning, orientation, and formation of cilia. Combined disruption of nphp and mks pathways had cell nonautonomous effects on C. elegans sensilla. Taken together, these data demonstrate the importance of mutational load on the presentation and severity of ciliopathies and expand the understanding of the interactions between ciliopathy genes.