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Exon Skipping Mutations in Neurofibromatosis

  • Emanuele Buratti
  • Diana BaralleEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 867)

Abstract

Defects at the level of pre-mRNA splicing represent a common source of disease mutations in almost all known diseases with a genetic aetiology. In general, it is commonly accepted that 15% of all pathogenic mutations are caused by splicing defects. However, this is probably a conservative estimate since clinical practice has only recently begun to routinely assess for this types of abnormalities. Therefore, it is expected that many currently unclassified or apparently harmless genetic variants will really turn out to be splicing-affecting defects. It is also well known that some genes are more susceptible than others to alterations in their splicing processes. Among these genes, one of the most representative is the NF-1 gene. In this gene, almost 50% of all reported disease-causing mutations can be directly attributed to alterations of the pre-mRNA process. In this chapter, we review the splicing process of the NF-1 gene and the most commonly used methods to identify splicing alterations. In particular, we provide practical notes on how to perform this analysis to maximize the chance of correctly identifying aberrant pre-mRNA splicing events in this gene.

Key words

NF-1 gene Neurofibromatosis Neurofibromin Pre-mRNA splicing Minigene systems Exon skipping 

Notes

Acknowledgments

EB is supported by a European community grant (EURASNET). DB is supported by Action Medical Research grant no. SP4175, EURASNET and Cancer Research UK.

References

  1. 1.
    Baralle D, Lucassen A, Buratti E (2009) Missed threads. The impact of pre-mRNA splicing defects on clinical practice. EMBO Rep 10:810–816PubMedCrossRefGoogle Scholar
  2. 2.
    Raponi M, Baralle D (2010) Alternative splicing: good and bad effects of translationally silent substitutions. FEBS J 277:836–840PubMedCrossRefGoogle Scholar
  3. 3.
    Buratti E, Baralle M, Baralle FE (2006) Defective splicing, disease and therapy: searching for master checkpoints in exon definition. Nucleic Acids Res 34:3494–3510PubMedCrossRefGoogle Scholar
  4. 4.
    Krawczak M, Thomas NS, Hundrieser B, Mort M, Wittig M, Hampe J, Cooper DN (2007) Single base-pair substitutions in exon-intron junctions of human genes: nature, distribution, and consequences for mRNA splicing. Hum Mutat 28:150–158PubMedCrossRefGoogle Scholar
  5. 5.
    Vorechovsky I (2006) Aberrant 3′ splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization. Nucleic Acids Res 34:4630–4641PubMedCrossRefGoogle Scholar
  6. 6.
    Buratti E, Chivers M, Kralovicova J, Romano M, Baralle M, Krainer AR, Vorechovsky I (2007) Aberrant 5′ splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization. Nucleic Acids Res 35:4250–4263PubMedCrossRefGoogle Scholar
  7. 7.
    Roca X, Sachidanandam R, Krainer AR (2003) Intrinsic differences between authentic and cryptic 5′ splice sites. Nucleic Acids Res 31:6321–6333PubMedCrossRefGoogle Scholar
  8. 8.
    Galante PA, Sakabe NJ, Kirschbaum-Slager N, de Souza SJ (2004) Detection and evaluation of intron retention events in the human transcriptome. RNA 10:757–765PubMedCrossRefGoogle Scholar
  9. 9.
    Dhir A, Buratti E (2010) Alternative splicing: role of pseudoexons in human disease and potential therapeutic strategies. FEBS J 277:841–855PubMedCrossRefGoogle Scholar
  10. 10.
    Ars E, Serra E, Garcia J, Kruyer H, Gaona A, Lazaro C, Estivill X (2000) Mutations affecting mRNA splicing are the most common molecular defects in patients with neurofibromatosis type 1. Hum Mol Genet 9:237–247PubMedCrossRefGoogle Scholar
  11. 11.
    Teraoka SN, Telatar M, Becker-Catania S, Liang T, Onengut S, Tolun A, Chessa L, Sanal O, Bernatowska E, Gatti RA, Concannon P (1999) Splicing defects in the ataxia-telangiectasia gene, ATM: underlying mutations and consequences. Am J Hum Genet 64:1617–1631PubMedCrossRefGoogle Scholar
  12. 12.
    Stenson PD, Ball EV, Howells K, Phillips AD, Mort M, Cooper DN (2009) The Human Gene Mutation Database: providing a comprehensive central mutation database for molecular diagnostics and personalized genomics. Hum Genomics 4:69–72PubMedGoogle Scholar
  13. 13.
    Pros E, Gomez C, Martin T, Fabregas P, Serra E, Lazaro C (2008) Nature and mRNA effect of 282 different NF1 point mutations: focus on splicing alterations. Hum Mutat 29:E173–E193PubMedCrossRefGoogle Scholar
  14. 14.
    Wimmer K, Roca X, Beiglbock H, Callens T, Etzler J, Rao AR, Krainer AR, Fonatsch C, Messiaen L (2007) Extensive in silico analysis of NF1 splicing defects uncovers determinants for splicing outcome upon 5′ splice-site disruption. Hum Mutat 28:599–612PubMedCrossRefGoogle Scholar
  15. 15.
    Ars E, Kruyer H, Morell M, Pros E, Serra E, Ravella A, Estivill X, Lazaro C (2003) Recurrent mutations in the NF1 gene are common among neurofibromatosis type 1 patients. J Med Genet 40:e82PubMedCrossRefGoogle Scholar
  16. 16.
    Bernards A (1995) Neurofibromatosis type 1 and Ras-mediated signaling: filling in the GAPs. Biochim Biophys Acta 1242:43–59PubMedGoogle Scholar
  17. 17.
    Williams VC, Lucas J, Babcock MA, Gutmann DH, Korf B, Maria BL (2009) Neurofibromatosis type 1 revisited. Pediatrics 123:124–133PubMedCrossRefGoogle Scholar
  18. 18.
    Trovo-Marqui AB, Tajara EH (2006) Neurofib-romin: a general outlook. Clin Genet 70:1–13PubMedCrossRefGoogle Scholar
  19. 19.
    Cichowski K, Jacks T (2001) NF1 tumor suppressor gene function: narrowing the GAP. Cell 104:593–604PubMedCrossRefGoogle Scholar
  20. 20.
    Costa RM, Silva AJ (2002) Molecular and cellular mechanisms underlying the cognitive deficits associated with neurofibromatosis 1. J Child Neurol 17:622–626, discussion 627-629, 646-651PubMedCrossRefGoogle Scholar
  21. 21.
    Viskochil D, Buchberg AM, Xu G, Cawthon RM, Stevens J, Wolff RK, Culver M, Carey JC, Copeland NG, Jenkins NA et al (1990) Deletions and a translocation interrupt a cloned gene at the neurofibromatosis type 1 locus. Cell 62:187–192PubMedCrossRefGoogle Scholar
  22. 22.
    Gutmann DH, Zhang Y, Hirbe A (1999) Develop-mental regulation of a neuron-specific neurofibromatosis 1 isoform. Ann Neurol 46:777–782PubMedCrossRefGoogle Scholar
  23. 23.
    Gutmann DH, Geist RT, Rose K, Wright DE (1995) Expression of two new protein isoforms of the neurofibromatosis type 1 gene product, neurofibromin, in muscle tissues. Dev Dyn 202:302–311PubMedCrossRefGoogle Scholar
  24. 24.
    Danglot G, Regnier V, Fauvet D, Vassal G, Kujas M, Bernheim A (1995) Neurofibromatosis 1 (NF1) mRNAs expressed in the central nervous system are differentially spliced in the 5′ part of the gene. Hum Mol Genet 4:915–920PubMedCrossRefGoogle Scholar
  25. 25.
    Gutmann DH, Andersen LB, Cole JL, Swaroop M, Collins FS (1993) An alternatively-spliced mRNA in the carboxy terminus of the neurofibromatosis type 1 (NF1) gene is expressed in muscle. Hum Mol Genet 2:989–992CrossRefGoogle Scholar
  26. 26.
    Andersen LB, Ballester R, Marchuk DA, Chang E, Gutmann DH, Saulino AM, Camonis J, Wigler M, Collins FS (1993) A conserved alternative splice in the von Recklinghausen neurofibromatosis (NF1) gene produces two neurofibromin isoforms, both of which have GTPase-activating protein activity. Mol Cell Biol 13:487–495PubMedGoogle Scholar
  27. 27.
    Zhu H, Hinman MN, Hasman RA, Mehta P, Lou H (2008) Regulation of neuron-specific alternative splicing of neurofibromatosis type 1 pre-mRNA. Mol Cell Biol 28:1240–1251PubMedCrossRefGoogle Scholar
  28. 28.
    Barron VA, Zhu H, Hinman MN, Ladd AN, Lou H (2010) The neurofibromatosis type I pre-mRNA is a novel target of CELF protein-mediated splicing regulation. Nucleic Acids Res 38:253–264PubMedCrossRefGoogle Scholar
  29. 29.
    Park VM, Kenwright KA, Sturtevant DB, Pivnick EK (1998) Alternative splicing of exons 29 and 30 in the neurofibromatosis type 1 gene. Hum Genet 103:382–385PubMedCrossRefGoogle Scholar
  30. 30.
    Vandenbroucke I, Callens T, De Paepe A, Messiaen L (2002) Complex splicing pattern generates great diversity in human NF1 transcripts. BMC Genomics 3:13PubMedCrossRefGoogle Scholar
  31. 31.
    Vandenbroucke I, Vandesompele J, De Paepe A, Messiaen L (2002) Quantification of NF1 transcripts reveals novel highly expressed splice variants. FEBS Lett 522:71–76PubMedCrossRefGoogle Scholar
  32. 32.
    Thomson SA, Wallace MR (2002) RT-PCR splicing analysis of the NF1 open reading frame. Hum Genet 110:495–502PubMedCrossRefGoogle Scholar
  33. 33.
    Buratti E, Baralle M, De Conti L, Baralle D, Romano M, Ayala YM, Baralle FE (2004) hnRNP H binding at the 5′ splice site correlates with the pathological effect of two intronic mutations in the NF-1 and TSHbeta genes. Nucleic Acids Res 32:4224–4236PubMedCrossRefGoogle Scholar
  34. 34.
    Baralle M, Skoko N, Knezevich A, De Conti L, Motti D, Bhuvanagiri M, Baralle D, Buratti E, Baralle FE (2006) NF1 mRNA biogenesis: effect of the genomic milieu in splicing regulation of the NF1 exon 37 region. FEBS Lett 580:4449–4456PubMedCrossRefGoogle Scholar
  35. 35.
    Raponi M, Buratti E, Dassie E, Upadhyaya M, Baralle D (2009) Low U1 snRNP dependence at the NF1 exon 29 donor splice site. FEBS J 276:2060–2073PubMedCrossRefGoogle Scholar
  36. 36.
    Raponi M, Upadhyaya M, Baralle D (2006) Functional splicing assay shows a pathogenic intronic mutation in neurofibromatosis type 1 (NF1) due to intronic sequence exonization. Hum Mutat 27:294–295PubMedCrossRefGoogle Scholar
  37. 37.
    Raponi M, Buratti E, Llorian M, Stuani C, Smith CW, Baralle D (2008) Polypyrimidine tract binding protein regulates alternative splicing of an aberrant pseudoexon in NF1. FEBS J 275:6101–6108PubMedCrossRefGoogle Scholar
  38. 38.
    Aartsma-Rus A, van Ommen GJ (2007) Antisense-mediated exon skipping: a versatile tool with therapeutic and research applications. RNA 13:1609–1624PubMedCrossRefGoogle Scholar
  39. 39.
    Garcia-Blanco MA (2005) Making antisense of splicing. Curr Opin Mol Ther 7:476–482PubMedGoogle Scholar
  40. 40.
    Tazi J, Bakkour N, Stamm S (2009) Alternative splicing and disease. Biochim Biophys Acta 1792:14–26PubMedGoogle Scholar
  41. 41.
    Pros E, Fernandez-Rodriguez J, Canet B, Benito L, Sanchez A, Benavides A, Ramos FJ, Lopez-Ariztegui MA, Capella G, Blanco I, Serra E, Lazaro C (2009) Antisense therapeutics for neurofibromatosis type 1 caused by deep intronic mutations. Hum Mutat 30:454–462PubMedCrossRefGoogle Scholar
  42. 42.
    Pros E, Fernandez-Rodriguez J, Benito L, Ravella A, Capella G, Blanco I, Serra E, Lazaro C (2010) Modulation of aberrant NF1 pre-mRNA splicing by kinetin treatment. Eur J Hum Genet 18:614–617PubMedCrossRefGoogle Scholar
  43. 43.
    Vibe-Pedersen K, Kornblihtt AR, Baralle FE (1984) Expression of a human alpha-globin/fibronectin gene hybrid generates two mRNAs by alternative splicing. EMBO J 3:2511–2516PubMedGoogle Scholar
  44. 44.
    Baralle D, Baralle M (2005) Splicing in action: assessing disease causing sequence changes. J Med Genet 42:737–748PubMedCrossRefGoogle Scholar
  45. 45.
    Cooper TA (2005) Use of minigene systems to dissect alternative splicing elements. Methods 37:331–340PubMedCrossRefGoogle Scholar
  46. 46.
    Singh G, Cooper TA (2006) Minigene reporter for identification and analysis of cis elements and trans factors affecting pre-mRNA splicing. Biotechniques 41:177–181PubMedCrossRefGoogle Scholar
  47. 47.
    Orengo JP, Bundman D, Cooper TA (2006) A bichromatic fluorescent reporter for cell-based screens of alternative splicing. Nucleic Acids Res 34:e148PubMedCrossRefGoogle Scholar
  48. 48.
    Bonano VI, Oltean S, Brazas RM, Garcia-Blanco MA (2006) Imaging the alternative silencing of FGFR2 exon IIIb in vivo. RNA 12:2073–2079PubMedCrossRefGoogle Scholar
  49. 49.
    Bonano VI, Oltean S, Garcia-Blanco MA (2007) A protocol for imaging alternative splicing regulation in vivo using fluorescence reporters in transgenic mice. Nat Protoc 2:2166–2181PubMedCrossRefGoogle Scholar
  50. 50.
    Newman EA, Muh SJ, Hovhannisyan RH, Warzecha CC, Jones RB, McKeehan WL, Carstens RP (2006) Identification of RNA-binding proteins that regulate FGFR2 splicing through the use of sensitive and specific dual color fluorescence minigene assays. RNA 12:1129–1141PubMedCrossRefGoogle Scholar
  51. 51.
    Nasim MT, Chowdhury HM, Eperon IC (2002) A double reporter assay for detecting changes in the ratio of spliced and unspliced mRNA in mammalian cells. Nucleic Acids Res 30:e109PubMedCrossRefGoogle Scholar
  52. 52.
    Kishore S, Khanna A, Stamm S (2008) Rapid generation of splicing reporters with pSpliceExpress. Gene 427:104–110PubMedCrossRefGoogle Scholar
  53. 53.
    Pros E, Larriba S, Lopez E, Ravella A, Gili ML, Kruyer H, Valls J, Serra E, Lazaro C (2006) NF1 mutation rather than individual genetic variability is the main determinant of the NF1-transcriptional profile of mutations affecting splicing. Hum Mutat 27:1104–1114PubMedCrossRefGoogle Scholar
  54. 54.
    Skoko N, Baralle M, Buratti E, Baralle FE (2008) The pathological splicing mutation c.6792C  >  G in NF1 exon 37 causes a change of tenancy between antagonistic splicing factors. FEBS Lett 582:2231–2236PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Molecular PathologyICGEBTriesteItaly
  2. 2.Human Genetics DivisionUniversity of Southampton, Southampton General HospitalSouthamptonUK

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