INTRODUCTION
Human disorders arising from the dysfunction of motile and/or primary cilia are collectively referred to as ciliopathies and there are more than a dozen distinguishable ciliopathy syndromes. Within the spectrum of disease arising from defects in the primary cilium (primary ciliopathies) these include, but are not limited to, neurological diseases (e.g. Joubert syndrome (JBTS, MIM PS213300) and Meckel syndrome (MKS, MIM PS249000)), skeletal ciliopathies (e.g. Jeune asphyxiating thoracic dystrophy (JATD), MIM PS208500), kidney diseases (e.g. nephronophthisis (NPHP), MIM PS256100, autosomal dominant and recessive polycystic kidney disease (ADPKD & ARPKD), MIM PS173900) and retinal dystrophies such as Leber congenital amaurosis (LCA, MIM PS120970) (Novarino, Akizu, & Gleeson, 2011; Reiter & Leroux, 2017; Shaheen et al., 2016). Collectively, ciliopathies affect approximately 1 in every 2000 individuals with ADPKD being by far the most common (Kagan, Dufke, & Gembruch, 2017). As well as the high level of phenotypic complexity and overlap of clinical phenotypes, mutations within the same gene can give rise to distinct ciliopathy syndromes, known as genetic pleiotropy (Coppieters, Lefever, Leroy, & De Baere, 2010; Roosing et al., 2016; Shaheen et al., 2016; Shamseldin et al., 2020). In addition, mutations in different genes can cause the same ciliopathy syndrome (genetic heterogeneity) such as it is the case of JBTS, with more than 35 genes associated (Figure S1 ) (Braun & Hildebrandt, 2017; Mitchison & Valente, 2017; Parisi, 2019). In addition, we have recently shown that differences in phenotypic presentation in patients with the same mutations, is in part due to the presence of genetic modifiers (Ramsbottom et al., 2020). JBTS represents the least severe end in the spectrum of neuronal ciliopathies (Parisi, 2019; Radha Rama Devi, Naushad, & Lingappa, 2020). The cerebellar and brainstem malformation, described as the “molar tooth sign” (MTS), is the hallmark for the diagnosis (Romani, Micalizzi, & Valente, 2013). On the other extreme of the spectrum is MKS, a lethal multiorgan ciliopathy, characterised by central nervous system (CNS) malformations (frequently occipital encephalocele), cystic renal dysplasia, and hepatic abnormalities including ductal plate malformation and hepatic fibrosis (Alexiev, Lin, Sun, & Brenner, 2006; Hartill, Szymanska, Sharif, Wheway, & Johnson, 2017). As an example for genetic pleiotropy and heterogeneity, genetic variants in both CEP120 and CC2D2A have been reported to cause JBTS, with CEP120 having a tropism for skeletal ciliopathies and CC2D2A giving rise to the whole spectrum of neuronal ciliopathies (Bachmann-Gagescu et al., 2012; Mougou-Zerelli et al., 2009; Roosing et al., 2016; Shaheen et al., 2015).
Centrosomal protein of 120 kDa (encoded by CEP120 ) is expressed ubiquitously in embryonic mice tissues with a subcellular expression enriched in the daughter centriole (Mahjoub, Xie, & Stearns, 2010; Xie et al., 2007). Several studies have investigated the role of CEP120 in centriole biogenesis and ciliogenesis and revealed its requirement for centriole duplication, assembly, elongation and maturation (Table S1 & Table S2 ) (Comartin et al., 2013; Mahjoub et al., 2010). Originally, biallelic genetic variants in CEP120 have been detected in 4 families with JATD but the gene has been linked to JBTS as well in later reports (Roosing et al., 2016; Shaheen et al., 2015). Coiled-coil and C2 domain containing 2A (encoded by CC2D2A ) is a centrosome-cilia protein that is described to be expressed in multiple human adult tissues, particularly in brain, prostate, pancreas, kidney, lung, liver and retina (Noor et al., 2008). CC2D2A localises and functions at the basal body/mother centriole, in particular, at the transition zone (TZ) (Gorden et al., 2008; Veleri et al., 2014) where it has a role in ciliogenesis (Lewis et al., 2019; Tallila, Jakkula, Peltonen, Salonen, & Kestila, 2008; Veleri et al., 2014) and vesicle trafficking through the TZ (Table S1 & Table S2 ) (Bachmann-Gagescu et al., 2015; Ojeda Naharros et al., 2017; Williams et al., 2011). Mutations in CC2D2A cause a spectrum of clinical phenotypes, ranging from isolated rod-cone dystrophy (Mejecase et al., 2019) to JBTS (Bachmann-Gagescu et al., 2012; Gorden et al., 2008; Noor et al., 2008) and MKS (Mougou-Zerelli et al., 2009; Szymanska et al., 2012; Tallila et al., 2008; Tallila, Salonen, Kohlschmidt, Peltonen, & Kestila, 2009). How mutations in these two genes, encoding proteins with different ciliary localization and function, can lead to this wide spectrum of distinct clinical presentations with partially overlapping phenotypes is not fully understood.
In 2015, Drivas et al. suggested that basal levels of alternative splicing (AS) with exon skipping may be responsible for some of the genetic pleiotropy observed in CEP290- andCC2D2A -associated disease (Drivas, Wojno, Tucker, Stone, & Bennett, 2015). AS is a mechanism by which a precursor messenger RNA (pre-mRNA) is processed into multiple isoforms (Nilsen & Graveley, 2010; Tabrez, Sharma, Jain, Siddiqui, & Mukhopadhyay, 2017) and is thought to occur in around 95% of multiexon genes (Pan, Shai, Lee, Frey, & Blencowe, 2008). Basal levels of noncanonical splicing has indeed been shown to occur in patient dermal fibroblasts withCEP290 mutations but also in control samples. The authors show that deleterious mutations in CEP290 and CC2D2A falling into exons that are in-frame are associated with a higher level of residual near-full length protein, as they escape nonsense-mediated mRNA decay (NMD), and correspond with a milder clinical phenotype (Drivas et al., 2015). Nonsense-associated altered splicing (NAS), an endogenous mechanism increasing the level of alternatively spliced transcripts in response to truncating variants might contribute to this rescue, although no evidence for a selective mechanism was found in this study (Drivas et al., 2015). It is currently unclear whether tissue-specific splicing events could underlie differential organ involvement in ciliopathies.
The potential of therapies exploiting this natural mechanism and based on the specific removal of dispensable exons by exon-skipping antisense oligonucleotides (ASOs) has now been well established (Aartsma-Rus & van Ommen, 2007; Bennett & Swayze, 2010). The treatment of patients with Duchenne muscular dystrophy (DMD) by targeting specific exons within the disease-causing gene, DMD, leads to exon skipping and a subsequent restoration of reading frame and a partially functional dystrophin protein (Aartsma-Rus & van Ommen, 2007; Kole & Krieg, 2015; Komaki et al., 2018; Lee, Saito, Duddy, Takeda, & Yokota, 2018; Servais et al., 2015). The same strategy has recently been applied to nonsense mutations within CEP290 (Barny et al., 2019; Garanto et al., 2016; Molinari et al., 2019; Ramsbottom et al., 2018) building on the fundamental finding that exon skipping in CEP290 is tolerated and leads to functional transcripts (Drivas et al., 2015). ASO-mediated exon skipping rescued the ciliary phenotype and CEP290 protein levels in a humanised murine model of Leber congenital amaurosis (LCA) (Garanto et al., 2016) and intravitreal injections of ASOs improved visual acuity in LCA patients (Cideciyan et al., 2019). Systemic administration of ASOs via intravenous injections was shown to induce skipping of a gene trap in a JBTS mouse model restoring CEP290 protein levels and rescuing renal ciliary phenotype and the cystic burden in the kidneys (Ramsbottom et al., 2018). As a proof of principle, ex-vivo ASO-mediated skipping restored the ciliary phenotype in human urine-derived renal epithelial cells (hURECs) and fibroblasts derived from a JBTS patient carrying a CEP290 homozygous truncating mutation (Molinari et al., 2019; Ramsbottom et al., 2018).
In this study, we systematically review and curate genetic variants and phenotypes associated with CEP120 and CC2D2A , two genes paradigmatic for the concepts of genetic heterogeneity and pleiotropy, and investigate genotype-phenotype correlations. Extending the concept proposed by Drivas et al. (Drivas et al., 2015; Molinari, Srivastava, Sayer, & Ramsbottom, 2017; Rozet & Gerard, 2015), we detect and validate tissue-specific splicing events and, using these two genes, propose a multimodal approach to identify target exons for future exon skipping therapy approaches.