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)