Molecular characterization of ten F8 splicing mutations in RNA isolated from patient's leucocytes: assessment of in silico prediction tools accuracy

Abstract

Although 8% of reported FVIII gene (F8) 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 in silico analysis. This study is aimed to elucidate the effect of potential splice site mutations (PSSM) on the F8 mRNA processing, investigate its correlation with disease severity, and assess their concordance with in silico predictions. We studied the F8 mRNA from 10 HA patient's leucocytes with PSSM by RT-PCR and compared the experimental results with those predicted in silico. 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 in silico 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 F8 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 F8 mRNA analysis using leucocytes still constitutes an excellent approach to investigate the transcriptional effects of the PSSM in HA, whereas prediction in silico is not always reliable for diagnostic decision-making.

Prediction of factor VIII inhibitor development in the SIPPET cohort by mutational analysis and factor VIII antigen measurement

“…exon skipping, activation of cryptic splice sites, and intron retention), resulting in the synthesis of a varied pattern of aberrant FVIII proteins. Ex vivo approaches, based on the analysis of illegitimate F8 transcripts and in vitro expression of the FVIII mutant proteins, are much more informative than in silico predictions, and can be used to more accurately investigate the consequences of missense or truncating variants . Moreover, breakpoint mapping and transcript analysis should better characterize large deletions involving one or more exons.…”

Splicing analysis of 26 F8 nucleotide variations using a minigene assay

“…For the remaining variant c.222G>A, no impact on exon 2 splicing was found using minigene assay. This variant has been reported in 2 studies giving discordant results: no impact on exon 2 splicing was reported by Zimmermann et al , whereas partial exon 2 skipping was described by Martorell et al Thus, the clinical significance of the c.222G>A substitution remains unclear.…”

“…5 Because of the relative inaccessibility of this tissue, several F8 mRNA studies have been performed in leucocytes as they contain an amount of ectopic F8 transcripts. 4,[6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] However, RNA sample was rarely available and only a few HA patients carrying PSSM have benefited from the study of splicing effect at the RNA level. In this context, we developed a minigene assay to characterize F8 PSSM previously identified in a large cohort of HA patients.…”

“…Liver sinusoidal endothelial cells are considered as the main site of the biosynthesis of FVIII . Because of the relative inaccessibility of this tissue, several F8 mRNA studies have been performed in leucocytes as they contain an amount of ectopic F8 transcripts . However, RNA sample was rarely available and only a few HA patients carrying PSSM have benefited from the study of splicing effect at the RNA level.…”

A Bioinformatics Toolkit: In Silico Tools and Online Resources for Investigating Genetic Variation

“…Genetic variants that occur within consensus motifs for 5′ splice acceptors, 3′ splice donors, or intronic branch points can interfere with the interaction of the spliceosome complex, influencing the splicing of intronic sequence from the mature RNA causing full/partial exon skipping [29][30][31][32] or intron retention. 30 In addition, deep intronic variants can activate cryptic splice acceptors or donors causing intron retention 33 or the formation of a pseudo-exon. 34,35 There are several in silico tools available to help predict the effect of variants on RNA splicing (►Table 2), usually based on the comparison of inputted wild-type and variant DNA sequence via specific algorithms.…”