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.

中文翻译：

---

解释秀丽隐杆线虫Joubert综合征错义变异的致病性

纤毛病是一类由纤毛缺陷引起的遗传性疾病。纤毛病基因的变异具有高度的多效性，代表了研究基因型与表型相关性的优秀案例研究。但是，在大多数细胞和动物研究中，并未使用等位基因来研究睫状体病的发病机理，因为这些等位基因会出现在患者体内。我们已经使用秀丽隐杆线虫来建模和表征与乔伯特综合征（JBTS）相关的B9D2 / mksr-2中的两个致病性双等位基因错义变体。B9D2在过渡区（TZ）睫状小室的MKS模块中起作用，并调节纤毛的分子组成和信号传导功能。使用具有新颖突变检测策略的CRISPR-Cas9基因组编辑，在mksr-2中工程化了P74S和G155S突变。纤毛/ TZ结构和功能的定量测定，以及内源水平表达的敲入报告基因，均表明该突变具有隐性和差异性作用。两种变体都破坏纤毛/ TZ和MKS模块的装配，而G155S导致更严重的总体表型，接近无效等位基因的表型。此外，G155S而非P74S严重破坏了TZ的内源性MKSR-2组织。对JBTS患者双等位基因型的概述表明，复合杂合蠕虫（P74S / G155S）表型蠕虫对P74S具有隐性。最后，这项研究提供了对MKS模块组装机制和组织的新见解，揭示了B9复合体与TMEM216 / MKS-2之间的紧密功能联系。这些数据一起提供了对JBTS等位基因的全面定量评估，建立了C。