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.