Systematic analysis of splicing defects in selected primary immunodeficiencies-related genes

- 20 out of 92 in silico/55 in vitro analysed variants affected pre-mRNA splicing.
- All the four SRE-affecting variants led only to minor aberrant splicing.
- Minigene showed substantial concordance with results from patients-derived RNA.
- ESRseq score, ΔHZEI and EX-SKIP predictions showed high sensitivity on SRE affection.
- Score pattern differed between exonic and intronic activated cryptic splice sites.

Both variants affecting splice sites and those in splicing regulatory elements (SREs) can impair pre-mRNA splicing, eventually leading to severe diseases. Despite the availability of many prediction tools, prognosis of splicing affection is not trivial, especially when SREs are involved. Here, we present data on 92 in silico-/55 minigene-analysed variants detected in genes responsible for the primary immunodeficiencies development (namely BTK, CD40LG, IL2RG, SERPING1, STAT3, and WAS). Of 20 splicing-affecting variants, 16 affected splice site while 4 disrupted potential SRE. The presence or absence of splicing defects was confirmed in 30 of 32 blood-derived patients' RNAs. Testing prediction tools performance, splice site disruptions and creations were reliably predicted in contrast to SRE-affecting variants for which just ESRseq, ΔHZEI-scores and EX-SKIP predictions showed promising results. Next, we found an interesting pattern in cryptic splice site predictions. These results might help PID-diagnosticians and geneticists cope with potential splicing-affecting variants.