Molecular characterization of ten F8 splicing mutations in RNA isolated from patient's leucocytes: assessment of in silico prediction tools accuracy. (5th February 2015)

• Record Type: Journal Article
• Authors: Martorell, L.; Corrales, I.; Ramirez, L.; Parra, R.; Raya, A.; Barquinero, J.; Vidal, F.
• Is Part Of: Haemophilia. Volume 21:Number 2(2015:Mar.)
• Page Start: 249
• Page End: 257
• Publication Date: 2015-02-05
• Subjects: Hemophilia -- Periodicals; 616.1572005
• DOI: 10.1111/hae.12562
• Languages: English
• ISSNs: 1351-8216

Abstract

<abstract abstract-type="main" id="hae12562-abs-0001"> <title>Summary</title> <p>Although 8% of reported FVIII gene (<italic>F8</italic>) mutations responsible for haemophilia A (HA) affect mRNA processing, very few have been fully characterized at the mRNA level and/or systematically predicted their biological consequences by <italic>in silico</italic> analysis. This study is aimed to elucidate the effect of potential splice site mutations (PSSM) on the <italic>F8 </italic>mRNA processing, investigate its correlation with disease severity, and assess their concordance with <italic>in silico</italic> predictions. We studied the <italic>F8 </italic>mRNA from 10 HA patient's leucocytes with PSSM by RT‐PCR and compared the experimental results with those predicted <italic>in silico</italic>. The mRNA analysis could explain all the phenotypes observed and demonstrated exon skipping in six cases (c.222G>A, c.601+1delG, c.602‐11T>G, c.671‐3C>G, c.6115+9C>G and c.6116‐1G>A) and activation of cryptic splicing sites, both donor (c.1009+1G>A and c.1009+3A>C) and acceptor sites (c.266‐3delC and c.5587‐1G>A). In contrast, the <italic>in silico</italic> analysis was able to predict the score variation of most of the affected splice site, but the precise mechanism could only be correctly determined in two of the 10 mutations analysed. In addition, we have detected aberrant <italic>F8</italic> transcripts, even in healthy controls, so this must be taken into account as they could mask the actual contribution of some PSSM. We conclude that <italic>F8 </italic>mRNA analysis using leucocytes still constitutes an excellent approach to investigate the transcriptional effects of the PSSM in HA, whereas prediction <italic>in silico</italic> is not always reliable for diagnostic decision‐making.</p> </abstract>