Ciliopathies are inherited disorders caused by defects in motile and non-motile (primary) cilia. Ciliopathy syndromes and associated gene variants are often highly pleiotropic and represent exemplars for interrogating genotype-phenotype correlations. Towards understanding disease mechanisms in the context of ciliopathy mutations, we have employed a leading model organism for cilia and ciliopathy research, Caenorhabditis elegans, together with gene editing, to characterise two missense variants (P74S, G155S) in B9D2/mksr-2 associated with Joubert Syndrome (JBTS). B9D2 functions within the Meckel syndrome (MKS) module at the ciliary base transition zone (TZ) compartment, and regulates the cilium's molecular composition and sensory/signaling functions. Quantitative assays of cilium/TZ structure and function, together with knock-in reporters, confirm both variant alleles are pathogenic in worms. G155S causes a more severe overall phenotype and disrupts endogenous MKSR-2 organisation at the TZ. Recapitulation of the patient biallelic genotype shows that compound heterozygous worms phenocopy worms homozygous for P74S. The P74S and G155S alleles also reveal evidence of a very close functional association between the B9D2-associated B9 complex and TMEM216/MKS-2. Together, these data establish C. elegans as a paradigm for interpreting JBTS mutations, and provide further insight into MKS module organisation.

Ciliopathies are a large class of inherited disorders caused by defects in cilia. Variants in ciliopathy genes are highly pleiotropic and represent excellent case studies for interrogating genotype-phenotype correlation. However, in most cell and animal studies, ciliopathy mechanisms are not investigated using alleles as they occur in patients. We have employed Caenorhabditis elegans to model and characterise two pathogenic biallelic missense variants in B9D2/ mksr-2 associated with Joubert Syndrome (JBTS). B9D2 functions within the MKS module at the transition zone (TZ) ciliary subcompartment, and regulates the cilium's molecular composition and signaling function. Using CRISPR-Cas9 genome editing with a novel mutation detection strategy, P74S and G155S mutations were engineered in mksr-2 .Quantitative assays of cilium/TZ structure and function, as well as knock-in reporters expressed at endogenous levels, revealed that the mutations act recessively and differentially.Whilst both variants disrupt the cilium/TZ and MKS module assembly, G155S causes a more severe overall phenotype, approaching that of a null allele. In addition, G155S, but not P74S, severely disrupts endogenous MKSR-2 organisation at the TZ. Recapitulation of the JBTS patient biallelic genotype showed that compound heterozygous worms (P74S/G155S) phenocopy worms recessive for P74S. Finally, this study provides new insight into the MKS module assembly mechanism and organisation, revealing a close functional association between the B9 complex and TMEM216/MKS-2. Together, these data provide a thorough quantitative assessment of JBTS alleles, establish C. elegans as a paradigm for interpreting TZ ciliopathy mutations, and provide insight into MKS module organisation at the TZ. TZ membrane diffusion barrier revealed that an RPI-2::GFP reporter leaked into cilia of worms with the G155S, but not the P74S, mutation. Unlike the reference null, endogenous MKS module protein reporters still assemble at the TZ of the mksr-2 mutants, although the TZ level and distribution of these reporters is disrupted, especially for MKS-2. We also found that endogenous MKS module reporters do not recapitulate several previously reported TZ localisation phenotypes. Lastly, compound heterozygous worms (P74S/G155S) that reflect the biallelic genotype of the JBTS patient display a phenotype that approaches that of worms recessive for the P74S mutation. Taken together these data demonstrate the utility of C. elegans for interpreting the pathogenicity of missense alleles in JBTS. Our work also uncovers new insight into MKS module organisation at the TZ, including a close functional association between the B9 complex and TMEM216/MKS-2.Caenorhabditis elegans ciliary transition zone structure, ultrastructure, molecular composition, and function.