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.
- 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
- Publikační typ
- abstrakt z konference MeSH
Přestože PCR detekce mykotických patogenů v klinických vzorcích se na stránkách odborných časopisů objevuje již více než dvacet let, je použití této metody pro rutinní diagnostiku invazivní aspergilózy stále diskutabilní. A to i přesto, že metody molekulární biologie již našly své stálé místo v mnoha odvětvích moderní mikrobiologie. Genotypizace bakteriálních kmenů rezistentních na antibiotika, molekulární epidemiologie, ale i rutinní detekce, např. virových infekcí z nejrůznějších klinických materiálů je v dnešní době zcela běžnou praxí. Ve všech těchto oblastech znamenalo zavedení metody PCR diagnostiky zrychlení, zfpřesnění a zjednodušení celého procesu. Tento přehledový článek má za cíl ukázat, v čem je detekce původců mykotických infekcí jiná a proč se ji dosud nepodařilo zavést do rutinní praxe.
PCR detection of fungal pathogens in clinical samples has been discussed in journals for more than two decades. However, its use for diagnosing invasive aspergillosis is still controversial, despite the fact that molecular methods are routinely used in various fields of modern microbiology. These are e. g. genotyping of bacterial strains resistant to antibiotics, molecular epidemiology or routine detection of viral infections in clinical material. PCR methods have made the diagnostic apphcations faster, simpler and more accurate. This review deals with issues related to molecular methods for diagnosing invasive fungal infections and the main factors limiting their use in everyday clinical practice.
