Characterisation and quantification of F8 transcripts of ten putative splice site mutations

 

 Buy Article Permissions and Reprints

Summary

Mutations affecting splice sites comprise approximately 7.5 % of the known F8 gene mutations but only a few were verified at mRNA level. In the present study, 10 putative splice site mutations were characterised by mRNA analysis using reverse transcription PCR (RT-PCR). Quantitative real-time RT-PCR (RT-qPCR) and co-amplification fluorescent PCR were used in combination to quantify the amount of each of multiple F8 transcripts. All of the mutations resulted in aberrant splicing. One of them (c.6187+1del1) generated one form of F8 transcript with exon skipping, and the remaining nine mutations (c.602-6T>C, c.1752+5_1752+6insGTTAG, c.1903+5G>A, c.5219+3A>G, c.5586+3A>T, c.969A>T, c.265+4A>G, c.601+1_601+5del5 and c.1444-8_1444del9) produced multiple F8 transcripts with exon skipping, activation of cryptic splice site and/or normal splicing. Residual wild-type F8 transcripts were produced by the first six of the nine mutations with amounts of 3.9 %>, 14.2 %>, 5.2 %>, 19.2 %>, 1.8 °% and 2.5 %> of normal levels, respectively, which were basically consistent with coagulation phenotypes in the related patients. In comparison with the mRNA findings, software Alamut v2.3 had values in the prediction of pathogenic effects on native splice sites but was not reliable in the prediction of activation of cryptic splice sites. Our quantification of F8 transcripts may provide an alternative way to evaluate the low expression levels of residue wild-type F8 transcripts and help to explain the severity of haemophilia A caused by splicing site mutations.

^* Q. Liang and M. Xiang contributed equally to this work.

• References

• 1 Mannucci PM, Tuddenham EG. The hemophilias--from royal genes to gene therapy. N Engl J Med 2001; 344: 1773-1779.
  CrossrefPubMedGoogle Scholar
• 2 Haemophilia A - Summary of Unique Mutations Reported. Available at: http://hadb.org.uk . Accessed November 30, 2012.
  PubMedGoogle Scholar
• 3 Krawczak M, Reiss J, Cooper DN. The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences. Hum Genet 1992; 90: 41-54.
  PubMedGoogle Scholar
• 4 Ars E, Kruyer H, Morell M. et al. Recurrent mutations in the NF1 gene are common among neurofibromatosis type 1 patients. J Med Genet 2003; 40: e82.
  CrossrefPubMedGoogle Scholar
• 5 Teraoka SN, Telatar M, Becker-Catania S. et al. Splicing defects in the ataxia-tel-angiectasia gene, ATM: underlying mutations and consequences. Am J Hum Genet 1999; 64: 1617-1631.
  CrossrefPubMedGoogle Scholar
• 6 De Conti L, Baralle M, Buratti E. Exon and intron definition in pre-mRNA splicing. Wiley Interdiscip Rev RNA 2013; 4: 49-60.
  CrossrefPubMedGoogle Scholar
• 7 McManus CJ, Graveley BR. RNA structure and the mechanisms of alternative splicing. Curr Opin Genet Dev 2011; 21: 373-379.
  CrossrefPubMedGoogle Scholar
• 8 Castaman G, Giacomelli SH, Mancuso ME. et al. F8 mRNA studies in haemophilia A patients with different splice site mutations. Haemophilia 2010; 16: 786-790.
  CrossrefPubMedGoogle Scholar
• 9 Parker RM, Barnes NM. mRNA: detection by in Situ and northern hybridisation. Methods Mol Biol 1999; 106: 247-283.
  PubMedGoogle Scholar
• 10 Hod Y. A simplified ribonuclease protection assay. Biotechniques 1992; 13: 852-854.
  PubMedGoogle Scholar
• 11 Gilliland G, Perrin S, Blanchard K. et al. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci USA 1990; 87: 2725-2729.
  CrossrefPubMedGoogle Scholar
• 12 Walton HS, Gebhardt FM, Innes DJ. et al. Analysis of multiple exon-skipping mRNA splice variants using SYBR Green real-time RT-PCR. J Neurosci Methods 2007; 160: 294-301.
  CrossrefPubMedGoogle Scholar
• 13 Pohl G, Shih Ie M. Principle and applications of digital PCR. Expert Rev Mol Diagn 2004; 4: 41-47.
  CrossrefPubMedGoogle Scholar
• 14 Nagaraj T, Vasanth JP, Desai A. et al. Ante mortem diagnosis of human rabies using saliva samples: comparison of real time and conventional RT-PCR techniques. J Clin Virol 2006; 36: 17-23.
  CrossrefPubMedGoogle Scholar
• 15 Attanasio C, de Moerloose P, Antonarakis SE. et al. Activation of multiple cryptic donor splice sites by the common congenital afibrinogenemia mutation, FGA IVS4 + 1 G-->T. Blood 2001; 97: 1879-1881.
  CrossrefPubMedGoogle Scholar
• 16 Spena S, Duga S, Asselta R. et al. Congenital afibrinogenemia: first identification of splicing mutations in the fibrinogen Bbeta-chain gene causing activation of cryptic splice sites. Blood 2002; 100: 4478-4484.
  CrossrefPubMedGoogle Scholar
• 17 Lu Y, Xie B, Ding Q. et al. Occurence of haemophilia A and B in a Chinese family with mosaicism of the F9 gene mutation in the HB index’s maternal grandfather. Thromb Haemost 2010; 103: 1106-1108.
  Article in Thieme ConnectPubMedGoogle Scholar
• 18 You GL, Ding QL, Lu YL. et al. Characterisation of large deletions in the F8 gene using multiple competitive amplification and the genome walking technique. J Thromb Haemost 2013; 11: 1103-1110.
  CrossrefPubMedGoogle Scholar
• 19 Vandesompele J, De Preter K, Pattyn F. et al. Accurate normalisation of realtime quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002 3. RESEARCH0034.
  PubMedGoogle Scholar
• 20 The interactive biosoftware Alamut v2.3. Available at: http://www.interactive-biosoftware.com .
  PubMedGoogle Scholar
• 21 Theophilus BD, Enayat MS, Williams MD. et al. Site and type of mutations in the factor VIII gene in patients and carriers of haemophilia A. Haemophilia 2001; 7: 381-391.
  CrossrefPubMedGoogle Scholar
• 22 Chow LT, Gelinas RE, Broker TR. et al. An amazing sequence arrangement at the 5’ ends of adenovirus 2 messenger RNA. Cell 1977; 12: 1-8.
  CrossrefPubMedGoogle Scholar
• 23 Lopez-Bigas N, Audit B, Ouzounis C. et al. Are splicing mutations the most frequent cause of hereditary disease?. FEBS Lett 2005; 579: 1900-1903.
  CrossrefPubMedGoogle Scholar
• 24 Ward AJ, Cooper TA. The pathobiology of splicing. J Pathol 2010; 220: 152-163.
  PubMedGoogle Scholar
• 25 Pagani F, Baralle FE. Genomic variants in exons and introns: identifying the splicing spoilers. Nat Rev Genet 2004; 5: 389-396.
  CrossrefPubMedGoogle Scholar
• 26 Pagani F, Buratti E, Stuani C. et al. Missense, nonsense, and neutral mutations define juxtaposed regulatory elements of splicing in cystic fibrosis transmembrane regulator exon 9. J Biol Chem 2003; 278: 26580-26588.
  CrossrefPubMedGoogle Scholar
• 27 Zimmermann MA, Gehrig A, Oldenburg J. et al. Analysis of F8 mRNA in haemophilia A patients with silent mutations or presumptive splice site mutations. Haemophilia 2013; 19: 310-317.
  CrossrefPubMedGoogle Scholar
• 28 Castaman G, Giacomelli SH, Mancuso ME. et al. Deep intronic variations may cause mild hemophilia A. J Thromb Haemost 2011; 9: 1541-1548.
  CrossrefPubMedGoogle Scholar
• 29 Ganguly A, Dunbar T, Chen P. et al. Exon skipping caused by an intronic insertion of a young Alu Yb9 element leads to severe hemophilia A. Hum Genet 2003; 113: 348-352.
  CrossrefPubMedGoogle Scholar

• Supplementary Material