Human Genetics

, Volume 90, Issue 1–2, pp 41–54 | Cite as

The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: Causes and consequences

  • Michael Krawczak
  • Jochen Reiss
  • David N. Cooper
Original Investigations


A total of 101 different examples of point mutations, which lie in the vicinity of mRNA splice junctions, and which have been held to be responsible for a human genetic disease by altering the accuracy of efficiency of mRNA splicing, have been collated. These data comprise 62 mutations at 5′ splice sites, 26 at 3′ splice sites and 13 that result in the creation of novel splice sites. It is estimated that up to 15% of all point mutations causing human genetic disease result in an mRNA splicing defect. Of the 5′ splice site mutations, 60% involved the invariant GT dinucleotide; mutations were found to be non-randomly distributed with an excess over expectation at positions +1 and +2, and apparent deficiencies at positions −1 and −2. Of the 3′ splice site mutations, 87% involved the invariant AG dinucleotide; an excess of mutations over expectation was noted at position -2. This non-randomness of mutation reflects the evolutionary conservation apparent in splice site consensus sequences drawn up previously from primate genes, and is most probably attributable to detection bias resulting from the differing phenotypic severity of specific lesions. The spectrum of point mutations was also drastically skewed: purines were significantly overrepresented as substituting nucleotides, perhaps because of steric hindrance (e.g. in U1 snRNA binding at 5′ splice sites). Furthermore, splice sites affected by point mutations resulting in human genetic disease were markedly different from the splice site consensus sequences. When similarity was quantified by a ‘consensus value’, both extremely low and extremely high values were notably absent from the wild-type sequences of the mutated splice sites. Splice sites of intermediate similarity to the consensus sequence may thus be more prone to the deleterious effects of mutation. Regarding the phenotypic effects of mutations on mRNA splicing, exon skipping occurred more frequently than cryptic splice site usage. Evidence is presented that indicates that, at least for 5′ splice site mutations, cryptic splice site usage is favoured under conditions where (1) a number of such sites are present in the immediate vicinity and (2) these sites exhibit sufficient homology to the splice site consensus sequence for them to be able to compete successfully with the mutated splice site. The novel concept of a “potential for cryptic splice site usage” value was introduced in order to quantify these characteristics, and to predict the relative proportion of exon skipping vs cryptic splice site utilization consequent to the introduction of a mutation at a normal splice site.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Aebi M, Hornig H, Padgett RA, Reiser J, Weissmann C (1986) Sequence requirements for splicing of higher eukaryotic nuclear pre-mRNA. Cell 47:555–565Google Scholar
  2. Aebi M, Hornig H, Weissmann C (1987) 5′ cleavage site in eukaryotic pre-mRNA splicing is determined by the overall 5′ splice region, not by the conserved 5′ GU. Cell 50:237–246Google Scholar
  3. Atweh GF, Wong C, Reed R, Antonarakis SE, Zhu D, Ghosh PK, Maniatis T, Forget B, Kazazian HH (1987) A new mutation in IVS-1 of the human β-globin gene causing β-thalassaemia due to abnormal splicing. Blood 70:147–151Google Scholar
  4. Breathnach R, Chambon P (1981) Organization and expression of eukaryotic split genes coding for proteins. Annu Rev Biochem 50:349–383CrossRefPubMedGoogle Scholar
  5. Chen S-H, Zhang M, Thompson AR, Bray GL, Scott CR (1991) Splice junction mutations in factor IX gene resulting in severe hemophilia B. Nucleic Acids Res 19:1172Google Scholar
  6. Cooper DN, Krawczak M (1989) Cytosine methylation and the fate of CpG dinucleotides in vertebrate genomes. Hum Genet 83:181–188Google Scholar
  7. Cooper DN, Krawczak M (1990) The mutational spectrum of single base-pair substitutions causing human genetic disease: patterns and predictions. Hum Genet 85:55–74PubMedGoogle Scholar
  8. Cooper DN, Krawczak M (1991) Mechanisms of insertional mutagenesis in human genes causing genetic disease. Hum Genet 87:409–415Google Scholar
  9. Deshler JO, Rossi JJ (1991) Unexpected point mutations activate cryptic splice site by perturbing a natural secondary structure within a yeast intron. Genes Dev 5:1252–1263Google Scholar
  10. Domenjoud L, Gallinaro H, Kister L, Meyer S, Jacob M (1991) Identification of a specific exon sequence that is a major determinant in the selection between a natural and a cryptic 5′ splice site. Mol Cell Biol 11:4581–4590Google Scholar
  11. Dominski Z, Kole R (1991) Selection of splice sites in pre-mRNAs with short internal exons. Mol Cell Biol 11:6075–6083Google Scholar
  12. Dunn JM, Phillips RA, Zhu X, Becker A, Gallie BL (1989) Mutations in the RB1 gene and their effects on transcription. Mol Cell Biol 9:4596–4604Google Scholar
  13. Furdon PJ, Kole R (1986) Inhibition of splicing but not cleavage at the 5′ splice site by truncating human β-globin pre-mRNA. Proc Natl Acad Sci USA 83:927–931Google Scholar
  14. Furdon PJ, Kole R (1988) The length of the downstream exon and the substitution of specific sequences affect pre-mRNA splicing in vitro. Mol Cell Biol 8:860–866Google Scholar
  15. Garcìa-Blanco MA, Jamison SF, Sharp PA (1989) Identification and purification of a 62000-dalton protein that binds specifically to the polypyrimidine tract of introns. Genes Dev 3:1874–1886Google Scholar
  16. Gerke V, Steitz JA (1986) A protein associated with small nuclear ribonucleoprotein particles recognizes the 3′ splice site of premessenger RNA. Cell 47:973–984Google Scholar
  17. Giannelli F, Green PM, High KA, Sommer S, Lillicrap DP, Ludwig M, Olek K, Reitsma PH, Goossens M, Yoshioka A, Brownlee GG (1991) Haemophilia B: database of point mutations and short additions and deletions — second edition. Nucleic Acids Res 19:2193–2219Google Scholar
  18. Goguel V, Liao X, Rymond BC, Rosbash M (1991) U1 snRNP can influence 3′-splice site selection as well as 5′-splice site selection. Genes Dev 5:1430–1438Google Scholar
  19. Grabowski PJ, Nasim FH, Kuo HC, Burch R (1991) Combinatorial splicing of exon pairs by two-site binding of U1 small nuclear ribonucleoprotein particles. Mol Cell Biol 11:5919–5925Google Scholar
  20. Green MR (1986) Pre-mRNA splicing. Annu Rev Genet 20:671–708Google Scholar
  21. Guthrie C, Patterson B (1988) Spliceosomal snRNAs. Annu Rev Genet 22:387–419Google Scholar
  22. Konarska MM, Sharp PA (1987) Interactions between small ribonucleoprotein particles in the formation of spliceosomes. Cell 49:763–774Google Scholar
  23. Krainer AR, Maniatis T (1985) Multiple factors including the small nuclear ribonucleoproteins U1 and U2 are necessary for premRNA splicing in vitro. Cell 42:725–736Google Scholar
  24. Krainer AR, Maniatis T (1988) RNA splicing. In: Harnes BD, Glover DM (eds) Transcription and splicing. IRL Press, Oxford, pp 131–220Google Scholar
  25. Krawczak M, Cooper DN (1991) Gene deletions causing human genetic disease: mechanisms of mutagenesis and the role of the local DNA sequence environment. Hum Genet 86:425–441Google Scholar
  26. Kuivaniemi H, Kontusaari S, Tromp G, Zhao M, Sabol C, Prockop DJ (1990) Identical G+1 to A mutations in three different introns of the type III procollagen gene (COL3A1) produce different patterns of RNA splicing in three variants of EhlersDanlos syndrome IV. J Biol Chem 265:12067–12074Google Scholar
  27. Kuo H-C, Nasim FH, Grabowski PJ (1991) Control of alternative splicing by the differential binding of U1 small nuclear ribonuclearprotein particle. Science 251:1045–1050Google Scholar
  28. Lamond AI, Konarska MM, Sharp PA (1987) A mutational analysis of spliceosome assembly: evidence for splice site collaboration during spliceosome formation. Genes Dev 1:532–543Google Scholar
  29. Lührmann R, Kastner B, Bach M (1990) Structure of spliceosomal snRNPs and their role in pre-mRN A splicing. Biochim Biophys Acta 1087:265–292Google Scholar
  30. Matsuo M, Masumura T, Nishio H, Nakajima T, Kitoh Y, Takumi T, Koga J, Nakamura H (1991) Exon skipping during splicing of dystrophin mRN A precursor due to an intraexon deletion in the dystrophin gene of Duchenne muscular dystrophy Kobe. J Clin Invest 87:2127–2131Google Scholar
  31. Matsushita T, Tanimoto M, Yamamoto K, Sugiura I, Hamaguchi M, Takamatsu J, Saito H (1989) Nucleotide sequence analysis of hemophilia B with the inhibitor phenotype. Blood 74 [Suppl]:251aGoogle Scholar
  32. Mitchcll PJ, Urlaub G, Chasin L (1986) Spontaneous splicing mutations at the dihydrofolate reductase locus in Chinese hamster ovary cells. Mol Cell Biol 6:1926–1935Google Scholar
  33. Mount SM (1982) A catalogue of splice junction sequences. Nucleic Acids Res 10:459–472PubMedGoogle Scholar
  34. Mount SM, Pettersson I, Hinterberger M, Karmas A, Steitz JA (1983) The U1 small nuclear RNA-protein complex selectively binds a 5′ splice site in vitro. Cell 33:509–518Google Scholar
  35. Naylor JA, Green PM, Montandon AJ, Rizza CR, Giannelli F (1991) Detection of three novel mutations in two haemophilia A patients by rapid screening of whole essential region of factor VIII gene. Lancet 337:635–639Google Scholar
  36. Nelson KK, Green MR (1989) Mammalian U2 snRNP has a sequence-specific RNA-binding activity. Genes Dev 3:1562–1571Google Scholar
  37. Oshima Y, Gotoh Y (1987) Signals for the selection of a splice site in pre-mRNA. Computer analysis of splice junction sequences and like sequences. J Mol Biol 195:247–259Google Scholar
  38. Padgett RA, Konarska MM, Aebi M, Hornig H, Weissmann C, Sharp PA (1985) Non-consensus branch-site sequences in the in vitro splicing of transcripts of mutant rabbit β-globin genes. Proc Natl Acad Sci USA 82:8349–8353Google Scholar
  39. Padgett RA, Grabowski PJ, Konarska MM, Sciler S. Sharp PA (1986) Splicing of messenger RNA precursors. Annu Rev Biochem 55:1119–1150CrossRefPubMedGoogle Scholar
  40. Reed R (1989) The organization of 3′ splice-site sequences in mammalian introns. Genes Dev 3:2113–2123Google Scholar
  41. Reed R. Maniatis T (1985) Intron sequences involved in lariat formation during pre-mRNA splicing. Cell 41:95–105Google Scholar
  42. Reed R, Maniatis T (1986) A role for exon sequences and splicesite proximity in splice site selection. Cell 46:681–690Google Scholar
  43. Reitsma PH, Poort SR, Allaart CF, Briet E, Bertina RM (1991) The spectrum of genetic defects in a panel of 40 Dutch families with symptomatic protein C deficiency type I: heterogeneity and founder effects. Blood 78:890–894Google Scholar
  44. Robberson BL, Cote GJ, Berget SM (1990) Exon definition may facilitate splice site selection in RNAs with multiple exons. Mol Cell Biol 10:84–94Google Scholar
  45. Ruskin B, Green MR (1985) Role of the 3′ splice site consensus sequence in mammalian pre-mRNA splicing. Nature 317:732–734Google Scholar
  46. Ruskin B, Greene JM, Green MR (1985) Cryptic branch point activation allows accurate in vitro splicing of human β-globin intron mutants. Cell 41:833–844Google Scholar
  47. Ruskin B, Zamore PD, Green MR (1988) A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly. Cell 52:207–219Google Scholar
  48. Sachs L (1974) Angewandte Statistik. Springer, Berlin Heidelberg New YorkGoogle Scholar
  49. Schlösser M, Slomski R, Wagner M, Reiss J, Berg LP, Kakkar VV, Cooper DN (1990) Characterization of pathological dystrophin transcripts from the lymphocytes of a muscular dystrophy carrier. Mol Biol Med 7:519–523Google Scholar
  50. Shapiro MB, Senapathy P (1987) RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res 15:7155–7174PubMedGoogle Scholar
  51. Shelley CS, Baralle FE (1987) Deletion analyses of a unique 3′ splice site indicates that alternating guanine and thymine residues represent an efficient splicing signal. Nucleic Acids Res 15:3787–3799Google Scholar
  52. Solnick D (1985) Alternative splicing caused by RNA secondary structure. Cell 43:667–676Google Scholar
  53. Somasekhar MB, Mertz JE (1985) Exon mutations that affect the choice of splice sites used in processing the SV40 late transcripts Nucleic Acids Res 13:5591–5609Google Scholar
  54. Stolow DT, Berget SM (1991) Identification of nuclear proteins that specifically bind to RNAs containing 5′ splice sites. Proc Natl Acad Sci USA 88:320–324Google Scholar
  55. Talerico M, Berget SM (1990) Effect of 5′ splice site mutations on splicing of the preceding intron. Mol Cell Biol 10:6299–6305Google Scholar
  56. Tazi J, Alibert C, Temsamani J, Reveillaud I, Cathala G, Brunel C, Jeanteur P (1986) A protein that specifically recognizes the 3′ splice site of mammalian pre-mRNA introns is associated with a small nuclear ribonuclearprotein. Cell 47:755–766Google Scholar
  57. Vasan NS, Kuivaniemi H, Vogel BE, Minor RR, Wootton JAM, Tromp G, Weksberg R, Prockop DJ (1991) A mutation in the pro alpha2 (I) gene (COL1A2) for type I procollagen in Ehlers-Danlos syndrome type VII: evidence suggesting that skipping of exon 6 in RNA splicing may be a common cause of the phenotype. Am J Hum Genet 48:305–317Google Scholar
  58. Weil D. D'Alessio M, Ramirez F. Steinmann B, Wirtz MK, Glanville RW, Hollister DW (1990) Temperature-dependent expression of a collagen splicing defect in the fibroblasts of a patient with Ehlers-Danlos syndrome type VII. J Biol Chem 264: 16804–16809Google Scholar
  59. Wong C, Antonarakis SE, Goff SC, Orkin SH, Forget BG, Nathan DG, Giardina PJV, Kazazian HH (1989) β-thalassaemia due to two novel nucleotide substitutions in consensus acceptor splice sequences of the β-globin gene. Blood 73:914–918Google Scholar
  60. Wu J, Manley JL (1989) Mammalian pre-mRNA branch site selection by U2 snRNP involves base pairing. Genes Dev 3:1553–1561Google Scholar
  61. Zhuang Y, Weiner AM (1989) A compensatory base change in human U2 snRNA can suppress a branch site mutation. Genes Dev 3:1545–1552Google Scholar

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • Michael Krawczak
    • 1
  • Jochen Reiss
    • 2
  • David N. Cooper
    • 3
  1. 1.Abteilung Humangenetik, Medizinische HochschuleHannoverGermany
  2. 2.Institut für Humangenetik der UniversitätGöttingenGermany
  3. 3.Charter Molecular Genetics Laboratory, Thrombosis Research InstituteLondonUK

Personalised recommendations