PURPOSE: Hereditary angioedema (HAE) is a rare autosomal dominant life-threatening disease characterized by low levels of C1 inhibitor (type I HAE) or normal levels of ineffective C1 inhibitor (type II HAE), typically occurring as a consequence of a SERPING1 mutation. In some cases, a causal mutation remains undetected after using a standard molecular genetic analysis. RESULTS: Here we show a long methodological way to the final discovery of c.1029 + 384A > G, a novel deep intronic mutation in intron 6 which is responsible for HAE type I in a large family and has not been identified by a conventional diagnostic approach. This mutation results in de novo donor splice site creation and subsequent pseudoexon inclusion, the mechanism firstly described to occur in SERPING1 in this study. We additionally discovered that the proximal part of intron 6 is a region potentially prone to pseudoexon-activating mutations, since natural alternative exons and additional cryptic sites occur therein. Indeed, we confirmed the existence of at least two different alternative exons in this region not described previously. CONCLUSIONS: In conclusion, our results suggest that detecting aberrant transcripts, which are often low abundant because of nonsense-mediated decay, requires a modified methodological approach. We suggest SERPING1 intron 6 sequencing and/or tailored mRNA analysis to be routinely used in HAE patients with no mutation identified in the coding sequence.

• Klíčová slova
• Hereditary angioedema, SERPING1, donor splice site, pre-mRNA splicing, pseudoexon activation,
• MeSH
• dítě MeSH
• dospělí MeSH
• exony genetika   MeSH
• hereditární angioedém, typy I a II genetika   MeSH
• inhibiční protein komplementu C1 genetika   MeSH
• introny genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• místa sestřihu RNA genetika   MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mutace genetika   MeSH
• mutační analýza DNA metody   MeSH
• rodokmen MeSH
• senioři MeSH
• splicing proteinů genetika   MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• inhibiční protein komplementu C1 MeSH
• místa sestřihu RNA MeSH

UNLABELLED: Mutations affecting splicing underlie the development of many human genetic diseases, but rather rarely through mechanisms of pseudoexon activation. Here, we describe a novel c.1092T>A mutation in the iduronate-2-sulfatase (IDS) gene detected in a patient with significantly decreased IDS activity and a clinical diagnosis of mild mucopolysaccharidosis II form. The mutation created an exonic de novo acceptor splice site and resulted in a complex splicing pattern with multiple pseudoexon activation in the patient's fibroblasts. Using an extensive series of minigene splicing experiments, we showed that the competition itself between the de novo and authentic splice site led to the bypass of the authentic one. This event then resulted in activation of several cryptic acceptor and donor sites in the upstream intron. As this was an unexpected and previously unreported mechanism of aberrant pseudoexon inclusion, we systematically analysed and disproved that the patient's mutation induced any relevant change in surrounding splicing regulatory elements. Interestingly, all pseudoexons included in the mature transcripts overlapped with the IDS alternative terminal exon 7b suggesting that this sequence represents a key element in the IDS pre-mRNA architecture. These findings extend the spectrum of mechanisms enabling pseudoexon activation and underscore the complexity of mutation-induced splicing aberrations. KEY MESSAGE: Novel exonic IDS gene mutation leads to a complex splicing pattern. Mutation activates multiple pseudoexons through a previously unreported mechanism. Multiple cryptic splice site (ss) activation results from a bypass of authentic ss. Authentic ss bypass is due to a competition between de novo and authentic ss.

• Klíčová slova
• Complex splicing aberration, De novo splice site, IDS, Pseudoexon, Splice site competition,
• MeSH
• bodová mutace MeSH
• exony MeSH
• glykoproteiny genetika   MeSH
• introny MeSH
• lidé MeSH
• messenger RNA genetika   MeSH
• místa sestřihu RNA MeSH
• mladiství MeSH
• mukopolysacharidóza II genetika   MeSH
• mutace MeSH
• sestřih RNA MeSH
• Check Tag
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glykoproteiny MeSH
• IDS protein, human MeSH Prohlížeč
• messenger RNA MeSH
• místa sestřihu RNA MeSH

The prevalent c.903+469T>C mutation in MTRR causes the cblE type of homocystinuria by strengthening an SRSF1 binding site in an ESE leading to activation of a pseudoexon. We hypothesized that other splicing regulatory elements (SREs) are also critical for MTRR pseudoexon inclusion. We demonstrate that the MTRR pseudoexon is on the verge of being recognized and is therefore vulnerable to several point mutations that disrupt a fine-tuned balance between the different SREs. Normally, pseudoexon inclusion is suppressed by a hnRNP A1 binding exonic splicing silencer (ESS). When the c.903+469T>C mutation is present two ESEs abrogate the activity of the ESS and promote pseudoexon inclusion. Blocking the 3'splice site or the ESEs by SSOs is effective in restoring normal splicing of minigenes and endogenous MTRR transcripts in patient cells. By employing an SSO complementary to both ESEs, we were able to rescue MTRR enzymatic activity in patient cells to approximately 50% of that in controls. We show that several point mutations, individually, can activate a pseudoexon, illustrating that this mechanism can occur more frequently than previously expected. Moreover, we demonstrate that SSO blocking of critical ESEs is a promising strategy to treat the increasing number of activated pseudoexons.

• MeSH
• buněčné linie MeSH
• exony * MeSH
• ferredoxin-NADP-reduktasa genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• homocystinurie enzymologie   genetika   MeSH
• kultivované buňky MeSH
• lidé MeSH
• megaloblastová anemie enzymologie   genetika   MeSH
• místa sestřihu RNA MeSH
• mutace * MeSH
• oligonukleotidy * MeSH
• regulační sekvence ribonukleových kyselin * MeSH
• sestřih RNA * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ferredoxin-NADP-reduktasa MeSH
• methionine synthase reductase MeSH Prohlížeč
• místa sestřihu RNA MeSH
• oligonukleotidy * MeSH
• regulační sekvence ribonukleových kyselin * MeSH