Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1996 Oct 15;98(8):1745–1754. doi: 10.1172/JCI118973

Glanzmann thrombasthenia. Cooperation between sequence variants in cis during splice site selection.

Y Jin 1, H C Dietz 1, R A Montgomery 1, W R Bell 1, I McIntosh 1, B Coller 1, P F Bray 1
PMCID: PMC507612  PMID: 8878424

Abstract

Glanzmann thrombasthenia (GT), an autosomal recessive bleeding disorder, results from abnormalities in the platelet fibrinogen receptor, GP(IIb)-IIIa (integrin alpha(IIb)beta3). A patient with GT was identified as homozygous for a G-->A mutation 6 bp upstream of the GP(IIIa) exon 9 splice donor site. Patient platelet GP(IIIa) transcripts lacked exon 9 despite normal DNA sequence in all of the cis-acting sequences known to regulate splice site selection. In vitro analysis of transcripts generated from mini-gene constructs demonstrated that exon skipping occurred only when the G-->A mutation was cis to a polymorphism 116 bp upstream, providing precedence that two sequence variations in the same exon which do not alter consensus splice sites and do not generate missense or nonsense mutations, can affect splice site selection. The mutant transcript resulted from utilization of a cryptic splice acceptor site and returned the open reading frame. These data support the hypothesis that pre-mRNA secondary structure and allelic sequence variants can influence splicing and provide new insight into the regulated control of RNA processing. In addition, haplotype analysis suggested that the patient has two identical copies of chromosome 17. Markers studied on three other chromosomes suggested this finding was not due to consanguinity. The restricted phenotype in this patient may provide information regarding the expression of potentially imprinted genes on chromosome 17.

Full Text

The Full Text of this article is available as a PDF (377.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bajt M. L., Ginsberg M. H., Frelinger A. L., 3rd, Berndt M. C., Loftus J. C. A spontaneous mutation of integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) helps define a ligand binding site. J Biol Chem. 1992 Feb 25;267(6):3789–3794. [PubMed] [Google Scholar]
  2. Barlow D. P. Imprinting: a gamete's point of view. Trends Genet. 1994 Jun;10(6):194–199. doi: 10.1016/0168-9525(94)90255-0. [DOI] [PubMed] [Google Scholar]
  3. Bennett J. S., Hoxie J. A., Leitman S. F., Vilaire G., Cines D. B. Inhibition of fibrinogen binding to stimulated human platelets by a monoclonal antibody. Proc Natl Acad Sci U S A. 1983 May;80(9):2417–2421. doi: 10.1073/pnas.80.9.2417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bennett J. S., Vilaire G., Cines D. B. Identification of the fibrinogen receptor on human platelets by photoaffinity labeling. J Biol Chem. 1982 Jul 25;257(14):8049–8054. [PubMed] [Google Scholar]
  5. Bennett J. S., Vilaire G. Exposure of platelet fibrinogen receptors by ADP and epinephrine. J Clin Invest. 1979 Nov;64(5):1393–1401. doi: 10.1172/JCI109597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bray P. F., Barsh G., Rosa J. P., Luo X. Y., Magenis E., Shuman M. A. Physical linkage of the genes for platelet membrane glycoproteins IIb and IIIa. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8683–8687. doi: 10.1073/pnas.85.22.8683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bray P. F. Inherited diseases of platelet glycoproteins: considerations for rapid molecular characterization. Thromb Haemost. 1994 Oct;72(4):492–502. [PubMed] [Google Scholar]
  8. Bray P. F., Rosa J. P., Johnston G. I., Shiu D. T., Cook R. G., Lau C., Kan Y. W., McEver R. P., Shuman M. A. Platelet glycoprotein IIb. Chromosomal localization and tissue expression. J Clin Invest. 1987 Dec;80(6):1812–1817. doi: 10.1172/JCI113277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bray P. F., Shuman M. A. Identification of an abnormal gene for the GPIIIa subunit of the platelet fibrinogen receptor resulting in Glanzmann's thrombasthenia. Blood. 1990 Feb 15;75(4):881–888. [PubMed] [Google Scholar]
  10. Calvete J. J., Henschen A., González-Rodríguez J. Assignment of disulphide bonds in human platelet GPIIIa. A disulphide pattern for the beta-subunits of the integrin family. Biochem J. 1991 Feb 15;274(Pt 1):63–71. doi: 10.1042/bj2740063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Calvete J. J., Mann K., Alvarez M. V., López M. M., González-Rodríguez J. Proteolytic dissection of the isolated platelet fibrinogen receptor, integrin GPIIb/IIIa. Localization of GPIIb and GPIIIa sequences putatively involved in the subunit interface and in intrasubunit and intrachain contacts. Biochem J. 1992 Mar 1;282(Pt 2):523–532. doi: 10.1042/bj2820523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Carothers A. M., Urlaub G., Grunberger D., Chasin L. A. Splicing mutants and their second-site suppressors at the dihydrofolate reductase locus in Chinese hamster ovary cells. Mol Cell Biol. 1993 Aug;13(8):5085–5098. doi: 10.1128/mcb.13.8.5085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chen I. T., Chasin L. A. Direct selection for mutations affecting specific splice sites in a hamster dihydrofolate reductase minigene. Mol Cell Biol. 1993 Jan;13(1):289–300. doi: 10.1128/mcb.13.1.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chen Y. P., Djaffar I., Pidard D., Steiner B., Cieutat A. M., Caen J. P., Rosa J. P. Ser-752-->Pro mutation in the cytoplasmic domain of integrin beta 3 subunit and defective activation of platelet integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) in a variant of Glanzmann thrombasthenia. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10169–10173. doi: 10.1073/pnas.89.21.10169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Chen Y. P., O'Toole T. E., Ylänne J., Rosa J. P., Ginsberg M. H. A point mutation in the integrin beta 3 cytoplasmic domain (S752-->P) impairs bidirectional signaling through alpha IIb beta 3 (platelet glycoprotein IIb-IIIa). Blood. 1994 Sep 15;84(6):1857–1865. [PubMed] [Google Scholar]
  16. Coller B. S., Cheresh D. A., Asch E., Seligsohn U. Platelet vitronectin receptor expression differentiates Iraqi-Jewish from Arab patients with Glanzmann thrombasthenia in Israel. Blood. 1991 Jan 1;77(1):75–83. [PubMed] [Google Scholar]
  17. Coller B. S., Peerschke E. I., Scudder L. E., Sullivan C. A. A murine monoclonal antibody that completely blocks the binding of fibrinogen to platelets produces a thrombasthenic-like state in normal platelets and binds to glycoproteins IIb and/or IIIa. J Clin Invest. 1983 Jul;72(1):325–338. doi: 10.1172/JCI110973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Cuticchia A. J., Fasman K. H., Kingsbury D. T., Robbins R. J., Pearson P. L. The GDB human genome data base anno 1993. Nucleic Acids Res. 1993 Jul 1;21(13):3003–3006. doi: 10.1093/nar/21.13.3003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Dietz H. C., Kendzior R. J., Jr Maintenance of an open reading frame as an additional level of scrutiny during splice site selection. Nat Genet. 1994 Oct;8(2):183–188. doi: 10.1038/ng1094-183. [DOI] [PubMed] [Google Scholar]
  20. Dietz H. C., Valle D., Francomano C. A., Kendzior R. J., Jr, Pyeritz R. E., Cutting G. R. The skipping of constitutive exons in vivo induced by nonsense mutations. Science. 1993 Jan 29;259(5095):680–683. doi: 10.1126/science.8430317. [DOI] [PubMed] [Google Scholar]
  21. Djaffar I., Rosa J. P. A second case of variant of Glanzmann's thrombasthenia due to substitution of platelet GPIIIa (integrin beta 3) Arg214 by Trp. Hum Mol Genet. 1993 Dec;2(12):2179–2180. doi: 10.1093/hmg/2.12.2179. [DOI] [PubMed] [Google Scholar]
  22. Djaffar I., Vilette D., Bray P. F., Rosa J. P. Quantitative isolation of RNA from human platelets. Thromb Res. 1991 May 1;62(3):127–135. doi: 10.1016/0049-3848(91)90187-2. [DOI] [PubMed] [Google Scholar]
  23. Eldadah Z. A., Brenn T., Furthmayr H., Dietz H. C. Expression of a mutant human fibrillin allele upon a normal human or murine genetic background recapitulates a Marfan cellular phenotype. J Clin Invest. 1995 Feb;95(2):874–880. doi: 10.1172/JCI117737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Faraday N., Goldschmidt-Clermont P., Dise K., Bray P. F. Quantitation of soluble fibrinogen binding to platelets by fluorescence-activated flow cytometry. J Lab Clin Med. 1994 May;123(5):728–740. [PubMed] [Google Scholar]
  25. Fasman K. H., Cuticchia A. J., Kingsbury D. T. The GDB Human Genome Data Base anno 1994. Nucleic Acids Res. 1994 Sep;22(17):3462–3469. doi: 10.1093/nar/22.17.3462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Fitzgerald L. A., Steiner B., Rall S. C., Jr, Lo S. S., Phillips D. R. Protein sequence of endothelial glycoprotein IIIa derived from a cDNA clone. Identity with platelet glycoprotein IIIa and similarity to "integrin". J Biol Chem. 1987 Mar 25;262(9):3936–3939. [PubMed] [Google Scholar]
  27. George J. N., Caen J. P., Nurden A. T. Glanzmann's thrombasthenia: the spectrum of clinical disease. Blood. 1990 Apr 1;75(7):1383–1395. [PubMed] [Google Scholar]
  28. Gogstad G. O., Brosstad F., Krutnes M. B., Hagen I., Solum N. O. Fibrinogen-binding properties of the human platelet glycoprotein IIb-=IIIa complex: a study using crossed-radioimmunoelectrophoresis. Blood. 1982 Sep;60(3):663–671. [PubMed] [Google Scholar]
  29. Gyapay G., Morissette J., Vignal A., Dib C., Fizames C., Millasseau P., Marc S., Bernardi G., Lathrop M., Weissenbach J. The 1993-94 Généthon human genetic linkage map. Nat Genet. 1994 Jun;7(2 Spec No):246–339. doi: 10.1038/ng0694supp-246. [DOI] [PubMed] [Google Scholar]
  30. Heidenreich R., Eisman R., Surrey S., Delgrosso K., Bennett J. S., Schwartz E., Poncz M. Organization of the gene for platelet glycoprotein IIb. Biochemistry. 1990 Feb 6;29(5):1232–1244. doi: 10.1021/bi00457a020. [DOI] [PubMed] [Google Scholar]
  31. Hynes R. O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992 Apr 3;69(1):11–25. doi: 10.1016/0092-8674(92)90115-s. [DOI] [PubMed] [Google Scholar]
  32. Iwamoto S., Nishiumi E., Kajii E., Ikemoto S. An exon 28 mutation resulting in alternative splicing of the glycoprotein IIb transcript and Glanzmann's thrombasthenia. Blood. 1994 Feb 15;83(4):1017–1023. [PubMed] [Google Scholar]
  33. Jin Y., Dietz H. C., Nurden A., Bray P. F. Single-strand conformation polymorphism analysis is a rapid and effective method for the identification of mutations and polymorphisms in the gene for glycoprotein IIIa. Blood. 1993 Oct 15;82(8):2281–2288. [PubMed] [Google Scholar]
  34. Kato A., Yamamoto K., Miyazaki S., Jung S. M., Moroi M., Aoki N. Molecular basis for Glanzmann's thrombasthenia (GT) in a compound heterozygote with glycoprotein IIb gene: a proposal for the classification of GT based on the biosynthetic pathway of glycoprotein IIb-IIIa complex. Blood. 1992 Jun 15;79(12):3212–3218. [PubMed] [Google Scholar]
  35. Lanza F., Stierlé A., Fournier D., Morales M., André G., Nurden A. T., Cazenave J. P. A new variant of Glanzmann's thrombasthenia (Strasbourg I). Platelets with functionally defective glycoprotein IIb-IIIa complexes and a glycoprotein IIIa 214Arg----214Trp mutation. J Clin Invest. 1992 Jun;89(6):1995–2004. doi: 10.1172/JCI115808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lapalme G., Cedergren R. J., Sankoff D. An algorithm for the display of nucleic acid secondary structure. Nucleic Acids Res. 1982 Dec 20;10(24):8351–8356. doi: 10.1093/nar/10.24.8351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Loftus J. C., O'Toole T. E., Plow E. F., Glass A., Frelinger A. L., 3rd, Ginsberg M. H. A beta 3 integrin mutation abolishes ligand binding and alters divalent cation-dependent conformation. Science. 1990 Aug 24;249(4971):915–918. doi: 10.1126/science.2392682. [DOI] [PubMed] [Google Scholar]
  38. Marguerie G. A., Plow E. F., Edgington T. S. Human platelets possess an inducible and saturable receptor specific for fibrinogen. J Biol Chem. 1979 Jun 25;254(12):5357–5363. [PubMed] [Google Scholar]
  39. Matsuo M., Nishio H., Kitoh Y., Francke U., Nakamura H. Partial deletion of a dystrophin gene leads to exon skipping and to loss of an intra-exon hairpin structure from the predicted mRNA precursor. Biochem Biophys Res Commun. 1992 Jan 31;182(2):495–500. doi: 10.1016/0006-291x(92)91759-j. [DOI] [PubMed] [Google Scholar]
  40. McEver R. P., Baenziger N. L., Majerus P. W. Isolation and quantitation of the platelet membrane glycoprotein deficient in thrombasthenia using a monoclonal hybridoma antibody. J Clin Invest. 1980 Dec;66(6):1311–1318. doi: 10.1172/JCI109983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Menichini P., Inga A., Fronza G., Iannone R., Degan P., Campomenosi P., Abbondandolo A. Defective splicing induced by 4NQO in the hamster hprt gene. Mutat Res. 1994 Apr;323(4):159–165. doi: 10.1016/0165-7992(94)90028-0. [DOI] [PubMed] [Google Scholar]
  42. Mitchell G. A., Looney J. E., Brody L. C., Steel G., Suchanek M., Engelhardt J. F., Willard H. F., Valle D. Human ornithine-delta-aminotransferase. cDNA cloning and analysis of the structural gene. J Biol Chem. 1988 Oct 5;263(28):14288–14295. [PubMed] [Google Scholar]
  43. Nachman R. L., Leung L. L. Complex formation of platelet membrane glycoproteins IIb and IIIa with fibrinogen. J Clin Invest. 1982 Feb;69(2):263–269. doi: 10.1172/JCI110448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Nakai K., Sakamoto H. Construction of a novel database containing aberrant splicing mutations of mammalian genes. Gene. 1994 Apr 20;141(2):171–177. doi: 10.1016/0378-1119(94)90567-3. [DOI] [PubMed] [Google Scholar]
  45. Nicholls R. D. New insights reveal complex mechanisms involved in genomic imprinting. Am J Hum Genet. 1994 May;54(5):733–740. [PMC free article] [PubMed] [Google Scholar]
  46. Nurden A. T., Caen J. P. An abnormal platelet glycoprotein pattern in three cases of Glanzmann's thrombasthenia. Br J Haematol. 1974 Oct;28(2):253–260. doi: 10.1111/j.1365-2141.1974.tb06660.x. [DOI] [PubMed] [Google Scholar]
  47. O'Toole T. E., Loftus J. C., Plow E. F., Glass A. A., Harper J. R., Ginsberg M. H. Efficient surface expression of platelet GPIIb-IIIa requires both subunits. Blood. 1989 Jul;74(1):14–18. [PubMed] [Google Scholar]
  48. Padgett R. A., Grabowski P. J., Konarska M. M., Seiler S., Sharp P. A. Splicing of messenger RNA precursors. Annu Rev Biochem. 1986;55:1119–1150. doi: 10.1146/annurev.bi.55.070186.005351. [DOI] [PubMed] [Google Scholar]
  49. Peretz H., Rosenberg N., Usher S., Graff E., Newman P. J., Coller B. S., Seligsohn U. Glanzmann's thrombasthenia associated with deletion-insertion and alternative splicing in the glycoprotein IIb gene. Blood. 1995 Jan 15;85(2):414–420. [PubMed] [Google Scholar]
  50. Plow E. F., Ginsberg M. H. Cellular adhesion: GPIIb-IIIa as a prototypic adhesion receptor. Prog Hemost Thromb. 1989;9:117–156. [PubMed] [Google Scholar]
  51. Poncz M., Eisman R., Heidenreich R., Silver S. M., Vilaire G., Surrey S., Schwartz E., Bennett J. S. Structure of the platelet membrane glycoprotein IIb. Homology to the alpha subunits of the vitronectin and fibronectin membrane receptors. J Biol Chem. 1987 Jun 25;262(18):8476–8482. [PubMed] [Google Scholar]
  52. Poncz M., Rifat S., Coller B. S., Newman P. J., Shattil S. J., Parrella T., Fortina P., Bennett J. S. Glanzmann thrombasthenia secondary to a Gly273-->Asp mutation adjacent to the first calcium-binding domain of platelet glycoprotein IIb. J Clin Invest. 1994 Jan;93(1):172–179. doi: 10.1172/JCI116942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Rainier S., Johnson L. A., Dobry C. J., Ping A. J., Grundy P. E., Feinberg A. P. Relaxation of imprinted genes in human cancer. Nature. 1993 Apr 22;362(6422):747–749. doi: 10.1038/362747a0. [DOI] [PubMed] [Google Scholar]
  54. Ramírez-Dueñas M. L., Medina C., Ocampo-Campos R., Rivera H. Severe Silver-Russell syndrome and translocation (17;20) (q25;q13) Clin Genet. 1992 Jan;41(1):51–53. doi: 10.1111/j.1399-0004.1992.tb03630.x. [DOI] [PubMed] [Google Scholar]
  55. Rosa J. P., Bray P. F., Gayet O., Johnston G. I., Cook R. G., Jackson K. W., Shuman M. A., McEver R. P. Cloning of glycoprotein IIIa cDNA from human erythroleukemia cells and localization of the gene to chromosome 17. Blood. 1988 Aug;72(2):593–600. [PubMed] [Google Scholar]
  56. Schlegel N., Gayet O., Morel-Kopp M. C., Wyler B., Hurtaud-Roux M. F., Kaplan C., Mc Gregor J. The molecular genetic basis of Glanzmann's thrombasthenia in a gypsy population in France: identification of a new mutation on the alpha IIb gene. Blood. 1995 Aug 1;86(3):977–982. [PubMed] [Google Scholar]
  57. Sharp P. A. Splicing of messenger RNA precursors. Science. 1987 Feb 13;235(4790):766–771. doi: 10.1126/science.3544217. [DOI] [PubMed] [Google Scholar]
  58. Simsek S., Heyboer H., de Bruijne-Admiraal L. G., Goldschmeding R., Cuijpers H. T., von dem Borne A. E. Glanzmann's thrombasthenia caused by homozygosity for a splice defect that leads to deletion of the first coding exon of the glycoprotein IIIa mRNA. Blood. 1993 Apr 15;81(8):2044–2049. [PubMed] [Google Scholar]
  59. Smith C. W., Chu T. T., Nadal-Ginard B. Scanning and competition between AGs are involved in 3' splice site selection in mammalian introns. Mol Cell Biol. 1993 Aug;13(8):4939–4952. doi: 10.1128/mcb.13.8.4939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Smith C. W., Porro E. B., Patton J. G., Nadal-Ginard B. Scanning from an independently specified branch point defines the 3' splice site of mammalian introns. Nature. 1989 Nov 16;342(6247):243–247. doi: 10.1038/342243a0. [DOI] [PubMed] [Google Scholar]
  61. Steingrimsdottir H., Rowley G., Dorado G., Cole J., Lehmann A. R. Mutations which alter splicing in the human hypoxanthine-guanine phosphoribosyltransferase gene. Nucleic Acids Res. 1992 Mar 25;20(6):1201–1208. doi: 10.1093/nar/20.6.1201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Talerico M., Berget S. M. Effect of 5' splice site mutations on splicing of the preceding intron. Mol Cell Biol. 1990 Dec;10(12):6299–6305. doi: 10.1128/mcb.10.12.6299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Tomiyama Y., Kashiwagi H., Kosugi S., Shiraga M., Kanayama Y., Kurata Y., Matsuzawa Y. Abnormal processing of the glycoprotein IIb transcript due to a nonsense mutation in exon 17 associated with Glanzmann's thrombasthenia. Thromb Haemost. 1995 May;73(5):756–762. [PubMed] [Google Scholar]
  64. Villa-Garcia M., Li L., Riely G., Bray P. F. Isolation and characterization of a TATA-less promoter for the human beta 3 integrin gene. Blood. 1994 Feb 1;83(3):668–676. [PubMed] [Google Scholar]
  65. Weissenbach J., Gyapay G., Dib C., Vignal A., Morissette J., Millasseau P., Vaysseix G., Lathrop M. A second-generation linkage map of the human genome. Nature. 1992 Oct 29;359(6398):794–801. doi: 10.1038/359794a0. [DOI] [PubMed] [Google Scholar]
  66. Wilcox D. A., Paddock C. M., Lyman S., Gill J. C., Newman P. J. Glanzmann thrombasthenia resulting from a single amino acid substitution between the second and third calcium-binding domains of GPIIb. Role of the GPIIb amino terminus in integrin subunit association. J Clin Invest. 1995 Apr;95(4):1553–1560. doi: 10.1172/JCI117828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Wilcox D. A., Wautier J. L., Pidard D., Newman P. J. A single amino acid substitution flanking the fourth calcium binding domain of alpha IIb prevents maturation of the alpha IIb beta 3 integrin complex. J Biol Chem. 1994 Feb 11;269(6):4450–4457. [PubMed] [Google Scholar]
  68. Wu J. Y., Maniatis T. Specific interactions between proteins implicated in splice site selection and regulated alternative splicing. Cell. 1993 Dec 17;75(6):1061–1070. doi: 10.1016/0092-8674(93)90316-i. [DOI] [PubMed] [Google Scholar]
  69. Zimrin A. B., Gidwitz S., Lord S., Schwartz E., Bennett J. S., White G. C., 2nd, Poncz M. The genomic organization of platelet glycoprotein IIIa. J Biol Chem. 1990 May 25;265(15):8590–8595. [PubMed] [Google Scholar]
  70. Zuker M., Stiegler P. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res. 1981 Jan 10;9(1):133–148. doi: 10.1093/nar/9.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES