Ectopic mRNA analysis and molecular modelling substantiate severe haemophilia in a patient with a FVIII gene splice mutation

Bicocchi, M. P.; Pasino, M; Lanza, Tiziana; Bottini, F.; Molinari, Angelo Claudio; Rosano, Camillo; Acquila, Maura

doi:10.1055/s-0037-1616251

Cited by 2 publications

(4 citation statements)

References 12 publications

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“…Further mRNA analyses of different mutations resulting in exon 19 skipping are described in literature: Tavassoli et al [24] and Bicocchi et al [27] published the splice site mutations c.6115+5G>A and c.6115+4A>G and Theophilus et al [19] described the mutations c.5999-18_6003del23 and c.6046C>T (p.Arg1997Trp). Moreover, Ganguly et al [26] referred to an Alu insertion at c.5999-19 leading to exon 19 skipping.…”

Section: Discussionmentioning

confidence: 99%

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Analysis of F8 mRNA in haemophilia A patients with silent mutations or presumptive splice site mutations

et al. 2012

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Mutation screenings in haemophilia A (HA) patients identified a great variety of mutations in the factor VIII gene (F8): intron 22 or intron 1 inversions, missense mutations, nonsense mutations, small or large deletions, insertions, duplications and splice site mutations. Mutations which do not result in amino acid substitutions (silent mutations) and intronic variants located outside the splice site consensus sequences cannot be easily classified as causative for HA. In these cases, special prediction software algorithms are applied to estimate their impact on splicing. Here, we present mRNA analysis of novel F8 mutations with possible impact on splicing in four HA patients with silent mutations and seven patients with intronic variants close to or within splice site consensus sequences. Seven of eleven mutations examined in vitro could be shown to have an effect on F8 mRNA splicing and the results were compared to in silico predictions. In addition, to validate the splice site prediction software Alamut v2.0 (Interactive Biosoftware), we compared published F8 mRNA analyses with the results of the in silico prediction. In general, the results of the splice site prediction tools of Alamut were in good accordance with the experimental F8 mRNA analyses, but a fundamental discrepancy between in silico and in vitro analyses was obtained in some cases. In conclusion, this study shows that the functional classification of potential splicing mutations should not only rely on prediction software, but be rather based on mRNA analysis experiments.

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Section: Discussionmentioning

confidence: 99%

“… and Bicocchi et al . published the splice site mutations c.6115+5G>A and c.6115+4A>G and Theophilus et al . described the mutations c.5999‐18_6003del23 and c.6046C>T (p.Arg1997Trp).…”

Section: Discussionmentioning

confidence: 99%

Analysis of F8 mRNA in haemophilia A patients with silent mutations or presumptive splice site mutations

et al. 2012

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“…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.…”

Section: Introductionmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

Splicing analysis of 26 F8 nucleotide variations using a minigene assay

et al. 2019

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Background Classically, the study of splicing impact of variation located near the splice site is performed by both in silico and mRNA analysis. However, RNA sample was rarely available. Objective To characterize a panel of putative haemophilia A splicing variations. Materials and methods Twenty‐six F8 variations identified from a cohort of 2075 haemophilia A families were studied using both bioinformatic tools and in vitro minigene assays in HeLa and Huh7 cells. Results An aberrant splicing was demonstrated for 21/26 tested sequence variations. A good correlation between in silico and in vitro analysis was obtained for variations affecting donor splice site (12/14) and for the synonymous variations located inside an exon (6/6). Conversely, no concordant results were observed for the six variations affecting acceptor splice sites. The variations resulted more frequently in exon skipping (n = 13) than in activation of nearby cryptic splice sites (n = 5), in use of a de novo splice site (n = 2) or in insertion of large intronic sequences (n = 1). This study allowed to reclassify 5 synonymous substitutions c.1167A>G (p.Gln389Gln), c.1569G>T (p.Leu523Leu), c.1752G>A (p.Gln584Gln), c.5586G>A (p.Leu1862Leu) and c.6066C>T (p.Gly2022Gly) as splicing variations. The pathological significance of five variations remained unclear (c.222G>A [p.Thr74Thr], c.237C>T [p.Asn79Asn], c.240C>T [p.Ile80Ile], c.2113+5_2113+8del and c.2113+5G>A). Discussion The minigene assay herein gave additional evidences for the clinical significance of 21/26 F8 putative splice site mutations. Such investigation should be performed for each F8 putative splice site variation for which no mRNA sample is available, notably to greatly improve the genetic counselling given to female carriers.

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Ectopic mRNA analysis and molecular modelling substantiate severe haemophilia in a patient with a FVIII gene splice mutation

Cited by 2 publications

References 12 publications

Analysis of F8 mRNA in haemophilia A patients with silent mutations or presumptive splice site mutations

Analysis of F8 mRNA in haemophilia A patients with silent mutations or presumptive splice site mutations

Splicing analysis of 26 F8 nucleotide variations using a minigene assay

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