DISCUSSION
CEP120 and CC2D2A both encode ciliary proteins, with a different subcellular localization and function. Mutations in these genes are associated with a spectrum of both overlapping and distinct ciliopathies illustrating the concepts of genetic heterogeneity and pleiotropy, inherent to most ciliopathy genes. To capture the genetic and clinical spectrum of CEP120 - and CC2D2A -associated disease, we reviewed literature, including previous mutation summaries (Bachmann-Gagescu et al., 2012; Lam, Albaba, Study, & Balasubramanian, 2020), and open-access tools to generate a curated, annotated and HGVS compliant database. Furthermore, we used in silico tools to identify tissue-specific basal exon skipping events. Using this database, we establish genotype-phenotype correlations, including possible insights into tissue-specific disease expression, and show that several exons in CC2D2A , but not in CEP120 , are good candidates for future exon skipping approaches. In addition to creating an updated and annotated database for CEP120 - and CC2D2A - associated disease, we provide a possible roadmap of how open-access tools can be used to identify future targets for splice-altering therapeutic approaches.
To date, only 9 patients from 9 different families have been described with biallelic genetic variants in CEP120 and 103 patients from 91 families with biallelic variants in CC2D2A . In these patient populations, 9 different genetic variants have been described inCEP120 and 75 different variants in CC2D2A . It has been previously shown that mutations in CC2D2A cluster to the C-terminal half of the protein (Bachmann-Gagescu et al., 2012) (Figure 4B). Several variants are shared between unrelated families and might follow geographical clusters. For instance, CC2D2A variant c.1762C>T was detected in 11 unrelated cases with MKS in the Finnish population but not reported outside Scandinavia (Tallila et al., 2008). In contrast, CC2D2A missense variant c.4667A>T is found in 13 unrelated cases, always in compound heterozygous state and without apparent geographical patterns. Finally, by crossing purported disease-causing variants with genomic data from the general population, we detected common variants (CC2D2A : p.Glu229del & p.Pro721Ser; CEP120 : p.Leu712Phe) that are most likely misclassified, echoing similar concerns for other ciliopathy genes (Pauli et al., 2019; Shaheen et al., 2016).
Mutations in CC2D2A is a common cause of ciliopathy accounting for 7.7% of JBTS and 10% of MKS in a previous large cohort study (Bachmann-Gagescu et al., 2012). The relative prevalence of this disease enabled more detailed analyses of genotype-phenotype correlations that are important to prioritize genetic testing (if targeted tests are performed), provide better prognostic information but can also give insights into disease mechanisms. Previous studies showed that subjects with CC2D2A -related JBTS were more likely to have ventriculomegaly and seizures than subjects without CC2D2Amutations (Bachmann-Gagescu et al., 2012). Furthermore, it has been previously noted that patients with at least one missense mutation inCC2D2A are more likely to suffer from JBTS while patients with biallelic truncating variants display more often MKS or ML, in line with a more deleterious effect of null alleles (Bachmann-Gagescu et al., 2012; Mougou-Zerelli et al., 2009). A similar correlation between biallelic truncating variants and more severe phenotypes has been suggested for the ciliopathy genes TMEM67 and RPGRIP1L(Delous et al., 2007; Iannicelli et al., 2010). In this study, we provide a systematic analysis of all reported patients withCC2D2A mutations and indeed show a strikingly more severe clinical presentation for carriers of biallelic null variants. Out of 40 patients with CC2D2A -related JBTS/JSRD, only 1 harboured biallelic truncating variants, whereas the majority ofCC2D2A -related MKS/ML was caused by biallelic null alleles. We also show that this association holds true for extra-CNS manifestations as patients with biallelic truncating variants were strikingly more likely to suffer from kidney disease (Figure 1). This observation is compatible with the notion that some of the observed genetic pleiotropy might be explained by the effects of particular mutations on total protein expression. In support of this hypothesis, Drivas et al. showed that basal exon skipping events modulate total protein expression in patients with CEP290 and CC2D2A mutations and that protein expression inversely correlated with disease severity (Drivas et al., 2015). Large-scale human sequencing projects suggest major differences in pre-mRNA splicing and basal exon skipping between different organs for most of our transcriptome. Whether these tissue-specificities contribute to genetic pleiotropy is currently unknown. Here, we provideex vivo data showing tissue-specific differences in basal exon skipping and illustrate how these effects might be exploited for a better understanding of different organ involvement in ciliopathies. While our limited data by no means prove this concept, we estimate that this is an exciting field for future studies. As alternative splicing events are conserved in human urine-derived epithelial cells (hURECs), they provide the ideal tool to investigate splicing in the kidney (Figure 3) (Molinari et al., 2018).
Using bioinformatic tools, including sequence data, domain annotations and alternative splicing predictions, we identified potentially skippable exons in CC2D2A and CEP120 and populated them with reported truncating variants to assess the applicability of therapeutic exon skipping for these two ciliopathy genes. Only exons 14 and 15 in CEP120 are skippable without inducing a frameshift or disrupting a functional domain. None of the reported genetic variants to date map into these two exons. Given the low number of mutations reported, it is currently impossible to say whether this is purely down to chance or whether this observation reflects the fact that these particular exons are functionally not important and/or skippable and therefore mutations in these exons are tolerated. In contrast, we identify 15/38 exons in CC2D2A as potentially skippable and we mapped 14 distinct truncating variants in 7 of them. Exon 30 appears to be a particularly good candidate as this exon undergoes basal exon skipping in the kidney, potentially modulating the severity of kidney disease associated with JBTS-causing truncating mutations therein. This example highlights how open-access databases for tissue-specific splicing could be used to refine targets for exon skipping therapy and suggests exon 30 skipping as a potential therapeutic option for future patients with kidney, liver or retinal involvement arising from truncating mutations in exon 30. Indeed, the CC2D2A mutations identified in a total of 26 patients are a starting point for in vitro analysis to determine if a functional rescue using antisense oligonucleotide mediated exon skipping is possible. Our group has previously applied ASO-mediated exon skipping to rescue kidney phenotypes in a mouse ciliopathy model (Ramsbottom et al., 2018). Delivery of ASO via systemic administration to the kidney appears effective in contrast to the brain and retinal tissues where blood-brain and blood-retinal barriers respectively cause reduction in delivery (Daneman & Prat, 2015; Himawan et al., 2019; Pardridge, 2002; Yu et al., 2007). In rodents, systemic administration of ASOs revealed greatest accumulation in kidney and liver (Geary, Norris, Yu, & Bennett, 2015; Zhao et al., 1998) and abundant proximal tubular uptake (Janssen et al., 2019; Oberbauer, Schreiner, & Meyer, 1995). Given the high morbidity associated with kidney disease, the potential for adequate ASO delivery, the tissue-specific splicing events that convey important information about potential target exons and the availability of relevant cell systems (hURECs) for non-invasive validation, ASO-mediated exon skipping offers exciting therapeutic perspectives for nephrology and particularly ciliopathy patients suffering from kidney disease (Molinari et al., 2018; Molinari et al., 2019). As this approach was successfully tested in pre-clinical models (Ramsbottom et al., 2018), the next big step is to bring this innovative therapy from bench to bedside, following the path set out by other diseases including Duchenne muscular dystrophy (Kole & Krieg, 2015; Komaki et al., 2018; Lee et al., 2018; Servais et al., 2015)