In silico analysis of gene expression and tissue-specific
basal exon skipping
Based on these results suggesting that truncating variants are
associated with a more severe clinical picture, we were interested to
assess the applicability of exon skipping therapies to rescue truncating
variants (Ramsbottom et al., 2018). Basal exon skipping events are
particularly informative as they indicate that skipping of these
particular exons is likely well tolerated.
CEP120 and CC2D2A are ubiquitously expressed in human
tissues, with highest expression levels in the female reproductive
system and cerebellum for the former and smooth muscle and female
reproductive system for the latter (Figure S3). Expression of both genes
has also been reported in the human kidney (Figure S3 &
http://www.proteinatlas.org, (Uhlén et al., 2015)). Cerebellum and
kidney phenotypes are classically encountered in ciliopathies
(Bachmann-Gagescu et al., 2012; Vilboux et al., 2017). Using human RNA
sequencing data available through the Genotype-Tissue Expression (GTEx)
project (https://www.gtexportal.org/home/), we investigated the
tissue-specific expression and splicing of CEP120 andCC2D2A in kidney and cerebellum. ENST00000306481 (“transcript
1”) is the main CEP120 transcript detected in the kidney medulla
and the cerebellar hemisphere. Abundant expression of ENST00000328236
(“transcript 2”) and ENST00000306467 (“transcript 3”) were also
detected in the cerebellum but nearly absent in the kidney (Figure 2Ai).
These transcript isoforms are generated through alternative splicing
events at the pre-mRNA 5’-end, with exon 2 (ref.: ENST00000328236 or
NM_153223) predicted to be skipped in the kidney (Figure 2Aii). These
changes are reflected by a predicted protein product lacking the first
26 amino acids for transcript 1 (Figure 2Aiii).
For CC2D2A , the main protein coding transcripts in kidney medulla
are ENST00000515124 (“transcript 1”) and ENST00000503292 (“transcript
2”) and in the cerebellar hemisphere are “transcript 2” and
ENST00000389652 (“transcript 3”) (Figure 2Bi). Transcript 1 is short
(1474bp), lacking functional CC2D2A domains and generated by splicing in
an additional exon after exon 5 (ref.: ENST00000503292), leading to a
premature stop codon. This transcript is supported by the detection in
GTEx of the specific junction in nearly all tissues but enriched in the
kidney (Figure 2Bii). Transcript 3 is detected in cerebellum but not
kidney and has an incomplete open reading frame with the 5’-end not
fully annotated. However, based on GTEx junction expression data, exon 2
appears to be spliced in the kidney but not the cerebellum, while an
exon predicted in the cerebellum (between exons 30 and 31) is skipped in
the kidney. Of interest, a splice junction
leading to skipping of exon 30 is
detected at low frequency and almost exclusively in the kidney medulla
(Figure 2Bii). On the protein level, transcript 1 encodes a 111 amino
acid product, sharing the first 82 amino acids with canonical transcript
2 (Figure 2Biii). In summary, human RNAseq data suggest the presence of
tissue-specific transcripts for CEP120 and CC2D2A . Exons
that are predicted to undergo organ-specific splicing events, such as
exon 30 of CC2D2A , represent optimal candidates to apply exon
skipping strategies. Isoform expression predicted from RNA sequencing
data must however be interpreted with caution and specific isoforms
should be confirmed by dedicated RT-PCR (Molinari et al., 2018).