Confirmation of tissue-specific basal exon skipping and possible implications for organ disease manifestations
We performed RT-PCR on total RNA from whole blood, kidney and human urine-derived renal epithelial cells (hURECs) using a primer pair targeted to exon 29 and exon 31 from the canonical CC2D2Atranscript (ENST00000503292). Beside the predicted amplification product of 327bp detected in the kidney and whole blood (at lower levels), a shorter transcript is clearly seen in the kidney but not in whole blood RNA. The observed molecular weight (~150bp), in line with the expected size of an amplification product lacking exon 30 (150bp), strongly suggests basal exon 30 skipping in the kidney (Figure 3A). Furthermore, we were able to detect this basal exon skipping event in hURECs (Figure 3A, right panel), highlighting the utility of this system to study kidney-specific slicing events (Molinari et al., 2018). Given that truncating CC2D2A variants are associated with more severe disease and a generally high penetrance for kidney disease (Figure 1) as well as our observation that CC2D2A exon 30 undergoes basal exon skipping in the kidney, we wondered whether truncating variants in exon 30 are rescued and therefore tolerated in the kidney. To assess this hypothesis, we represented the relative prevalence of kidney disease associated with truncating variants in the different CC2D2A exons (Figure 3B). Unfortunately, only 2 patients harboured a truncating variant in exon 30 limiting the strength of our conclusions. However, neither of these patients showed kidney involvement as compared with kidney disease in 33.3-100% of truncating variants in the other exons (Figure 3B). Considering all patients with either monoallelic or biallelic truncating variants, 35/48 presented with kidney disease vs only 13 that did not present kidney disease (including the 2 patients with exon 30 truncating variants). Patient MTI-991 (Table 1) harboured a CEP120 biallelic intronic variant at the exon-intron boundary 3’ of exon 2 and shown to lead to intron retention (Roosing et al., 2016). However, as exon 2 appears to be skipped in the kidney (Figure 2), this variant is likely “silent” in the kidney. Indeed, this patient did not show kidney involvement (Roosing et al., 2016).