title: 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.
authors: Gene and Cell Therapy, Vall d'Hebron Research Institute, Universitat Autònoma de Barcelona (VHIR-UAB), Barcelona, Spain.|Control of Stem Cell Potency, Institute for Bioengineering of Catalonia (IBEC), Barcelona, Spain.|Molecular Diagnosis and Therapy, Vall d'Hebron Research Institute, Universitat Autònoma de Barcelona (VHIR-UAB), Barcelona, Spain.
authors: L
authors: L
authors: Martorell
authors: Molecular Diagnosis and Therapy, Vall d'Hebron Research Institute, Universitat Autònoma de Barcelona (VHIR-UAB), Barcelona, Spain.|Congenital Coagulopathies Laboratory, Blood and Tissue Bank (BST), Barcelona, Spain.
authors: I
authors: I
authors: Corrales
authors: Molecular Diagnosis and Therapy, Vall d'Hebron Research Institute, Universitat Autònoma de Barcelona (VHIR-UAB), Barcelona, Spain.|Congenital Coagulopathies Laboratory, Blood and Tissue Bank (BST), Barcelona, Spain.
authors: L
authors: L
authors: Ramirez
authors: Congenital Coagulopathies Laboratory, Blood and Tissue Bank (BST), Barcelona, Spain.|Haemophilia Unit, Vall d'Hebron University Hospital, Barcelona, Spain.
authors: R
authors: R
authors: Parra
authors: Control of Stem Cell Potency, Institute for Bioengineering of Catalonia (IBEC), Barcelona, Spain.|Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain.|Center of Regenerative Medicine in Barcelona (CMRB), Barcelona, Spain.|Networking Center of Biomedical Research in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Barcelona, Spain.
authors: A
authors: A
authors: Raya
authors: Gene and Cell Therapy, Vall d'Hebron Research Institute, Universitat Autònoma de Barcelona (VHIR-UAB), Barcelona, Spain.
authors: J
authors: J
authors: Barquinero
authors: Molecular Diagnosis and Therapy, Vall d'Hebron Research Institute, Universitat Autònoma de Barcelona (VHIR-UAB), Barcelona, Spain.|Congenital Coagulopathies Laboratory, Blood and Tissue Bank (BST), Barcelona, Spain.
authors: F
authors: F
authors: Vidal
mesh_terms: D019295:Computational Biology / Q000379:methods; D004252:DNA Mutational Analysis; D005091:Exons; D005169:Factor VIII / Q000235:genetics*; D006467:Hemophilia A / Q000175:diagnosis* / Q000235:genetics*; D006801:Humans; D007438:Introns; D007962:Leukocytes / Q000378:metabolism*; D009154:Mutation; D010641:Phenotype; D022821:RNA Splice Sites; D012326:RNA Splicing; D012333:RNA, Messenger
chemical_list: D022821:RNA Splice Sites; D012333:RNA, Messenger; D005169:Factor VIII
keywords: RNA splicing; haemophilia A; leucocytes; splice site mutation; synonymous mutation
references: Kemball-Cook G, Tuddenham EG, Wacey AI. The factor VIII structure and mutation resource site: HAMSTeRS version 4. Nucleic Acids Res 1998; 26: 216-9.
references: Stenson PD, Ball EV, Mort M et al. Human gene mutation database (HGMD): 2003 update. Hum Mutat 2003; 21: 577-81.
references: Tavassoli K, Eigel A, Wilke K, Pollmann H, Horst J. Molecular diagnostics of 15 hemophilia A patients: characterization of eight novel mutations in the factor VIII gene, two of which result in exon skipping. Hum Mutat 1998; 12: 301-3.
references: El-Maarri O, Herbiniaux U, Graw J et al. Analysis of mRNA in hemophilia A patients with undetectable mutations reveals normal splicing in the factor VIII gene. J Thromb Haemost 2005; 3: 332-9.
references: Zimmermann MA, Gehrig A, Oldenburg J, Muller CR, Rost S. Analysis of F8 mRNA in haemophilia A patients with silent mutations or presumptive splice site mutations. Haemophilia 2013; 19: 310-7.
references: Theophilus BD, Enayat MS, Williams MD, Hill FG. Site and type of mutations in the factor VIII gene in patients and carriers of haemophilia A. Haemophilia 2001; 7: 381-91.
references: Taneri B, Asilmaz E, Gaasterland T. Biomedical impact of splicing mutations revealed through exome sequencing. Mol Med 2012; 18: 314-9.
references: McManus CJ, Graveley BR. RNA structure and the mechanisms of alternative splicing. Curr Opin Genet Dev 2011; 21: 373-9.
references: Darnell RB. Developing global insight into RNA regulation. Cold Spring Harb Symp Quant Biol 2006; 71: 321-7.
references: Liu HX, Zhang M, Krainer AR. Identification of functional exonic splicing enhancer motifs recognized by individual SR proteins. Genes Dev 1998; 12: 1998-2012.
references: Blencowe BJ. Exonic splicing enhancers: mechanism of action, diversity and role in human genetic diseases. Trends Biochem Sci 2000; 25: 106-10.
references: Cho S, Hoang A, Sinha R et al. Interaction between the RNA binding domains of Ser-Arg splicing factor 1 and U1-70K snRNP protein determines early spliceosome assembly. Proc Natl Acad Sci U S A 2011; 108: 8233-8.
references: Houdayer C. In silico prediction of splice-affecting nucleotide variants. Methods Mol Biol 2011; 760: 269-81.
references: Wang GS, Cooper TA. Splicing in disease: disruption of the splicing code and the decoding machinery. Nat Rev Genet 2007; 8: 749-61.
references: Chamary JV, Parmley JL, Hurst LD. Hearing silence: non-neutral evolution at synonymous sites in mammals. Nat Rev Genet 2006; 7: 98-108.
references: Byers PH. Killing the messenger: new insights into nonsense-mediated mRNA decay. J Clin Invest 2002; 109: 3-6.
references: Bowen DJ. Haemophilia A and haemophilia B: molecular insights. Mol Pathol 2002; 55: 127-44.
references: Vidal F, Farssac E, Altisent C, Puig L, Gallardo D. Rapid hemophilia A molecular diagnosis by a simple DNA sequencing procedure: identification of 14 novel mutations. Thromb Haemost 2001; 85: 580-3.
references: Hsu TC, Pratt KP, Thompson AR. The factor VIII C1 domain contributes to platelet binding. Blood 2008; 111: 200-8.
references: Hollestelle MJ, Thinnes T, Crain K et al. Tissue distribution of factor VIII gene expression in vivo-a closer look. Thromb Haemost 2001; 86: 855-61.
references: Marchioro TL, Hougie C, Ragde H, Epstein RB, Thomas ED. Hemophilia: role of organ homografts. Science 1969; 163: 188-90.
references: Wion KL, Kelly D, Summerfield JA, Tuddenham EG, Lawn RM. Distribution of factor VIII mRNA and antigen in human liver and other tissues. Nature 1985; 317: 726-9.
references: Cooper DN, Berg LP, Kakkar VV, Reiss J. Ectopic (illegitimate) transcription: new possibilities for the analysis and diagnosis of human genetic disease. Ann Med 1994; 26: 9-14.
references: Corrales I, Ramirez L, Altisent C, Parra R, Vidal F. The study of the effect of splicing mutations in von Willebrand factor using RNA isolated from patients’ platelets and leukocytes. J Thromb Haemost 2011; 9: 679-88.
references: Yamanaka S. Strategies and new developments in the generation of patient-specific pluripotent stem cells. Cell Stem Cell 2007; 1: 39-49.
references: Raya A, Rodriguez-Piza I, Navarro S et al. A protocol describing the genetic correction of somatic human cells and subsequent generation of iPS cells. Nat Protoc 2010; 5: 647-60.
references: Sanchez-Danes A, Richaud-Patin Y, Carballo-Carbajal I et al. Disease-specific phenotypes in dopamine neurons from human iPS-based models of genetic and sporadic Parkinson's disease. EMBO Mol Med 2012; 4: 380-95.
references: Jia B, Chen S, Zhao Z et al. Modeling of hemophilia A using patient-specific induced pluripotent stem cells derived from urine cells. Life Sci 2014; 108(1): 22-9.
references: Murphy LD, Herzog CE, Rudick JB, Fojo AT, Bates SE. Use of the polymerase chain reaction in the quantitation of mdr-1 gene expression. Biochemistry 1990; 29: 10351-6.
references: Noonan KE, Beck C, Holzmayer TA et al. Quantitative analysis of MDR1 (multidrug resistance) gene expression in human tumors by polymerase chain reaction. Proc Natl Acad Sci U S A 1990; 87: 7160-4.
references: Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 2000; 25: 169-93.
references: Schmittgen TD, Zakrajsek BA, Mills AG, Gorn V, Singer MJ, Reed MW. Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods. Anal Biochem 2000; 285: 194-204.
references: Linde L, Boelz S, Neu-Yilik G, Kulozik AE, Kerem B. The efficiency of nonsense-mediated mRNA decay is an inherent character and varies among different cells. Eur J Hum Genet 2007; 15: 1156-62.
references: Tchernitchko D, Goossens M, Wajcman H. In silico prediction of the deleterious effect of a mutation: proceed with caution in clinical genetics. Clin Chem 2004; 50: 1974-8.