Skip to main content
NCBI home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1996 Jan 15;97(2):373–380. doi: 10.1172/JCI118425

A nonsense mutation in the erythrocyte band 3 gene associated with decreased mRNA accumulation in a kindred with dominant hereditary spherocytosis.

P B Jenkins 1, G K Abou-Alfa 1, D Dhermy 1, E Bursaux 1, C Féo 1, A L Scarpa 1, S E Lux 1, M Garbarz 1, B G Forget 1, P G Gallagher 1
PMCID: PMC507027  PMID: 8567957

Abstract

We studied a French kindred with typical hereditary spherocytosis (HS). Studies of erythrocytes and erythrocyte membranes from HS individuals revealed abnormal erythrocyte membrane mechanical stability as well as 15-20% deficiency of band 3, the anion transporter. Anion transport studies of red cells from two affected individuals revealed decreased sulfate flux. Nucleotide sequence of cDNA encoding the distal third of the cytoplasmic domain and the entire transmembrane domain of band 3 obtained by RT-PCR of reticulocyte RNA of an affected family member was normal. Sequence analysis of genomic DNA from an HS individual identified a nonsense mutation of the band 3 gene, Q330X, near the end of the band 3 cytoplasmic domain. This mutation was present in genomic DNA of all HS family members and absent in DNA of unaffected family members. Using an RT-PCR-based assay, a marked quantitative decrease in accumulation of the mutant band 3 RNA was detected. Thus the codon 330 nonsense mutation is responsible for the decreased accumulation of mutant band 3 RNA and the deficiency of band 3 protein in this kindred. These results have important implications for the role of band 3 defects in the membrane pathobiology of HS as well as for the techniques used in detection of HS mutations.

Full Text

The Full Text of this article is available as a PDF (268.0 KB).

Selected References

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

  1. Agre P., Asimos A., Casella J. F., McMillan C. Inheritance pattern and clinical response to splenectomy as a reflection of erythrocyte spectrin deficiency in hereditary spherocytosis. N Engl J Med. 1986 Dec 18;315(25):1579–1583. doi: 10.1056/NEJM198612183152504. [DOI] [PubMed] [Google Scholar]
  2. Agre P., Casella J. F., Zinkham W. H., McMillan C., Bennett V. Partial deficiency of erythrocyte spectrin in hereditary spherocytosis. 1985 Mar 28-Apr 3Nature. 314(6009):380–383. doi: 10.1038/314380a0. [DOI] [PubMed] [Google Scholar]
  3. Baserga S. J., Benz E. J., Jr Nonsense mutations in the human beta-globin gene affect mRNA metabolism. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2056–2060. doi: 10.1073/pnas.85.7.2056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Belgrader P., Maquat L. E. Nonsense but not missense mutations can decrease the abundance of nuclear mRNA for the mouse major urinary protein, while both types of mutations can facilitate exon skipping. Mol Cell Biol. 1994 Sep;14(9):6326–6336. doi: 10.1128/mcb.14.9.6326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boivin P., Galand C., Devaux I., Lecomte M. C., Garbarz M., Dhermy D. Spectrin alpha IIa variant in dominant and non-dominant spherocytosis. Hum Genet. 1993 Sep;92(2):153–156. doi: 10.1007/BF00219683. [DOI] [PubMed] [Google Scholar]
  6. Bursaux E., Hilly M., Bluze A., Poyart C. Organic phosphates modulate anion self-exchange across the human erythrocyte membrane. Biochim Biophys Acta. 1984 Nov 7;777(2):253–260. doi: 10.1016/0005-2736(84)90427-9. [DOI] [PubMed] [Google Scholar]
  7. Chang J. C., Kan Y. W. beta 0 thalassemia, a nonsense mutation in man. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2886–2889. doi: 10.1073/pnas.76.6.2886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cheng J., Fogel-Petrovic M., Maquat L. E. Translation to near the distal end of the penultimate exon is required for normal levels of spliced triosephosphate isomerase mRNA. Mol Cell Biol. 1990 Oct;10(10):5215–5225. doi: 10.1128/mcb.10.10.5215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Clark M. R., Mohandas N., Shohet S. B. Osmotic gradient ektacytometry: comprehensive characterization of red cell volume and surface maintenance. Blood. 1983 May;61(5):899–910. [PubMed] [Google Scholar]
  10. Cooper D. N. Human gene mutations affecting RNA processing and translation. Ann Med. 1993 Feb;25(1):11–17. doi: 10.3109/07853899309147851. [DOI] [PubMed] [Google Scholar]
  11. Costa F. F., Agre P., Watkins P. C., Winkelmann J. C., Tang T. K., John K. M., Lux S. E., Forget B. G. Linkage of dominant hereditary spherocytosis to the gene for the erythrocyte membrane-skeleton protein ankyrin. N Engl J Med. 1990 Oct 11;323(15):1046–1050. doi: 10.1056/NEJM199010113231507. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Fairbanks G., Steck T. L., Wallach D. F. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971 Jun 22;10(13):2606–2617. doi: 10.1021/bi00789a030. [DOI] [PubMed] [Google Scholar]
  14. Feo C. J., Nossal M., Jones E., Bessis M. Une nouvelle technique d'étude de la physiologie des globules rouges: la mesure de leur déformabilité en fonction de l'osmolarité. Résultats obtenus par un Ektacytomètre automatisé sur du sang normal et dans différentes anémies hémolytiques. C R Seances Acad Sci III. 1982 Dec 6;295(12):687–691. [PubMed] [Google Scholar]
  15. Foley K. P., Engel J. D. Individual stage selector element mutations lead to reciprocal changes in beta- vs. epsilon-globin gene transcription: genetic confirmation of promoter competition during globin gene switching. Genes Dev. 1992 May;6(5):730–744. doi: 10.1101/gad.6.5.730. [DOI] [PubMed] [Google Scholar]
  16. Foley K. P., Leonard M. W., Engel J. D. Quantitation of RNA using the polymerase chain reaction. Trends Genet. 1993 Nov;9(11):380–385. doi: 10.1016/0168-9525(93)90137-7. [DOI] [PubMed] [Google Scholar]
  17. Gallagher P. G., Forget B. G. Spectrin St Louis and the alpha LELY allele. Blood. 1994 Sep 1;84(5):1686–1687. [PubMed] [Google Scholar]
  18. Gallagher P. G., Lecomte M. C., Galand C., Wang Y. P., Tse W. T., Forget B. G. Location and PCR-based detection of three polymorphisms of the human erythrocyte beta-spectrin gene (SPTB). Br J Haematol. 1994 Oct;88(2):413–414. doi: 10.1111/j.1365-2141.1994.tb05043.x. [DOI] [PubMed] [Google Scholar]
  19. Gallagher P. G., Tse W. T., Forget B. G. Polymerase chain reaction analysis of an NcoI polymorphism of the human erythrocyte ankyrin gene. Blood. 1992 Oct 1;80(7):1856–1857. [PubMed] [Google Scholar]
  20. Hall G. W., Thein S. Nonsense codon mutations in the terminal exon of the beta-globin gene are not associated with a reduction in beta-mRNA accumulation: a mechanism for the phenotype of dominant beta-thalassemia. Blood. 1994 Apr 15;83(8):2031–2037. [PubMed] [Google Scholar]
  21. Hanspal M., Yoon S. H., Yu H., Hanspal J. S., Lambert S., Palek J., Prchal J. T. Molecular basis of spectrin and ankyrin deficiencies in severe hereditary spherocytosis: evidence implicating a primary defect of ankyrin. Blood. 1991 Jan 1;77(1):165–173. [PubMed] [Google Scholar]
  22. Hoffman N., Stanislovitis P., Watkins P. C., Klinger K. W., Linnenbach A. J., Forget B. G. Three RFLPs are detected by an alpha spectrin genomic clone. Nucleic Acids Res. 1987 Jun 11;15(11):4696–4696. doi: 10.1093/nar/15.11.4696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hull J., Shackleton S., Harris A. The stop mutation R553X in the CFTR gene results in exon skipping. Genomics. 1994 Jan 15;19(2):362–364. doi: 10.1006/geno.1994.1070. [DOI] [PubMed] [Google Scholar]
  24. Humphries R. K., Ley T. J., Anagnou N. P., Baur A. W., Nienhuis A. W. Beta O-39 thalassemia gene: a premature termination codon causes beta-mRNA deficiency without affecting cytoplasmic beta-mRNA stability. Blood. 1984 Jul;64(1):23–32. [PubMed] [Google Scholar]
  25. Jarolim P., Rubin H. L., Brabec V., Chrobak L., Zolotarev A. S., Alper S. L., Brugnara C., Wichterle H., Palek J. Mutations of conserved arginines in the membrane domain of erythroid band 3 lead to a decrease in membrane-associated band 3 and to the phenotype of hereditary spherocytosis. Blood. 1995 Feb 1;85(3):634–640. [PubMed] [Google Scholar]
  26. Jarolim P., Rubin H. L., Liu S. C., Cho M. R., Brabec V., Derick L. H., Yi S. J., Saad S. T., Alper S., Brugnara C. Duplication of 10 nucleotides in the erythroid band 3 (AE1) gene in a kindred with hereditary spherocytosis and band 3 protein deficiency (band 3PRAGUE). J Clin Invest. 1994 Jan;93(1):121–130. doi: 10.1172/JCI116935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jenkins P. B., Gallagher P. G., Forget B. G. Analysis of a PstI polymorphism of the human erythrocyte band 3 gene (EPB3). Br J Haematol. 1993 Dec;85(4):816–818. doi: 10.1111/j.1365-2141.1993.tb03232.x. [DOI] [PubMed] [Google Scholar]
  28. Lecomte M. C., Garbarz M., Grandchamp B., Féo C., Gautero H., Devaux I., Bournier O., Galand C., d'Auriol L., Galibert F. Sp alpha I/78: a mutation of the alpha I spectrin domain in a white kindred with HE and HPP phenotypes. Blood. 1989 Aug 15;74(3):1126–1133. [PubMed] [Google Scholar]
  29. Lux S. E., John K. M., Kopito R. R., Lodish H. F. Cloning and characterization of band 3, the human erythrocyte anion-exchange protein (AE1). Proc Natl Acad Sci U S A. 1989 Dec;86(23):9089–9093. doi: 10.1073/pnas.86.23.9089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lux S. E., Tse W. T., Menninger J. C., John K. M., Harris P., Shalev O., Chilcote R. R., Marchesi S. L., Watkins P. C., Bennett V. Hereditary spherocytosis associated with deletion of human erythrocyte ankyrin gene on chromosome 8. Nature. 1990 Jun 21;345(6277):736–739. doi: 10.1038/345736a0. [DOI] [PubMed] [Google Scholar]
  31. McIntosh I., Hamosh A., Dietz H. C. Nonsense mutations and diminished mRNA levels. Nat Genet. 1993 Jul;4(3):219–219. doi: 10.1038/ng0793-219. [DOI] [PubMed] [Google Scholar]
  32. Miraglia del Giudice E., Iolascon A., Pinto L., Nobili B., Perrotta S. Erythrocyte membrane protein alterations underlying clinical heterogeneity in hereditary spherocytosis. Br J Haematol. 1994 Sep;88(1):52–55. doi: 10.1111/j.1365-2141.1994.tb04976.x. [DOI] [PubMed] [Google Scholar]
  33. Miraglia del Giudice E., Perrotta S., Pinto L., Cappellini M. D., Fiorelli G., Cutillo S., Iolascon A. Hereditary spherocytosis characterized by increased spectrin/band 3 ratio. Br J Haematol. 1992 Jan;80(1):133–134. doi: 10.1111/j.1365-2141.1992.tb06417.x. [DOI] [PubMed] [Google Scholar]
  34. Mohandas N., Clark M. R., Health B. P., Rossi M., Wolfe L. C., Lux S. E., Shohet S. B. A technique to detect reduced mechanical stability of red cell membranes: relevance to elliptocytic disorders. Blood. 1982 Apr;59(4):768–774. [PubMed] [Google Scholar]
  35. Moriyama R., Ideguchi H., Lombardo C. R., Van Dort H. M., Low P. S. Structural and functional characterization of band 3 from Southeast Asian ovalocytes. J Biol Chem. 1992 Dec 25;267(36):25792–25797. [PubMed] [Google Scholar]
  36. Muhlrad D., Parker R. Premature translational termination triggers mRNA decapping. Nature. 1994 Aug 18;370(6490):578–581. doi: 10.1038/370578a0. [DOI] [PubMed] [Google Scholar]
  37. Palek J., Jarolim P. Clinical expression and laboratory detection of red blood cell membrane protein mutations. Semin Hematol. 1993 Oct;30(4):249–283. [PubMed] [Google Scholar]
  38. Reinhart W. H., Wyss E. J., Arnold D., Ott P. Hereditary spherocytosis associated with protein band 3 defect in a Swiss kindred. Br J Haematol. 1994 Jan;86(1):147–155. doi: 10.1111/j.1365-2141.1994.tb03266.x. [DOI] [PubMed] [Google Scholar]
  39. Saad S. T., Costa F. F., Vicentim D. L., Salles T. S., Pranke P. H. Red cell membrane protein abnormalities in hereditary spherocytosis in Brazil. Br J Haematol. 1994 Oct;88(2):295–299. doi: 10.1111/j.1365-2141.1994.tb05021.x. [DOI] [PubMed] [Google Scholar]
  40. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Savvides P., Shalev O., John K. M., Lux S. E. Combined spectrin and ankyrin deficiency is common in autosomal dominant hereditary spherocytosis. Blood. 1993 Nov 15;82(10):2953–2960. [PubMed] [Google Scholar]
  42. Schofield A. E., Martin P. G., Spillett D., Tanner M. J. The structure of the human red blood cell anion exchanger (EPB3, AE1, band 3) gene. Blood. 1994 Sep 15;84(6):2000–2012. [PubMed] [Google Scholar]
  43. Schofield A. E., Reardon D. M., Tanner M. J. Defective anion transport activity of the abnormal band 3 in hereditary ovalocytic red blood cells. Nature. 1992 Feb 27;355(6363):836–838. doi: 10.1038/355836a0. [DOI] [PubMed] [Google Scholar]
  44. Stewart E. A., Kopito R., Bowcock A. M. A PstI polymorphism for the human erythrocyte surface protein band 3 (EPB3) demonstrates close linkage of EPB3 to the nerve growth factor receptor. Genomics. 1989 Oct;5(3):633–635. doi: 10.1016/0888-7543(89)90034-7. [DOI] [PubMed] [Google Scholar]
  45. Takeshita K., Forget B. G., Scarpa A., Benz E. J., Jr Intranuclear defect in beta-globin mRNA accumulation due to a premature translation termination codon. Blood. 1984 Jul;64(1):13–22. [PubMed] [Google Scholar]
  46. Tang T. K., Tam K. B., Chien S. Two RFLPs in the human protein 4.1 gene (EL1). Nucleic Acids Res. 1991 Nov 11;19(21):6057–6057. doi: 10.1093/nar/19.21.6057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tanner M. J. Molecular and cellular biology of the erythrocyte anion exchanger (AE1). Semin Hematol. 1993 Jan;30(1):34–57. [PubMed] [Google Scholar]
  48. Trecartin R. F., Liebhaber S. A., Chang J. C., Lee K. Y., Kan Y. W., Furbetta M., Angius A., Cao A. beta zero thalassemia in Sardinia is caused by a nonsense mutation. J Clin Invest. 1981 Oct;68(4):1012–1017. doi: 10.1172/JCI110323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tse W. T., Lecomte M. C., Costa F. F., Garbarz M., Feo C., Boivin P., Dhermy D., Forget B. G. Point mutation in the beta-spectrin gene associated with alpha I/74 hereditary elliptocytosis. Implications for the mechanism of spectrin dimer self-association. J Clin Invest. 1990 Sep;86(3):909–916. doi: 10.1172/JCI114792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Urlaub G., Mitchell P. J., Ciudad C. J., Chasin L. A. Nonsense mutations in the dihydrofolate reductase gene affect RNA processing. Mol Cell Biol. 1989 Jul;9(7):2868–2880. doi: 10.1128/mcb.9.7.2868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wilmotte R., Maréchal J., Morlé L., Baklouti F., Philippe N., Kastally R., Kotula L., Delaunay J., Alloisio N. Low expression allele alpha LELY of red cell spectrin is associated with mutations in exon 40 (alpha V/41 polymorphism) and intron 45 and with partial skipping of exon 46. J Clin Invest. 1993 May;91(5):2091–2096. doi: 10.1172/JCI116432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Yannoukakos D., Vasseur C., Driancourt C., Blouquit Y., Delaunay J., Wajcman H., Bursaux E. Human erythrocyte band 3 polymorphism (band 3 Memphis): characterization of the structural modification (Lys 56----Glu) by protein chemistry methods. Blood. 1991 Aug 15;78(4):1117–1120. [PubMed] [Google Scholar]
  53. Yannoukakos D., Vasseur C., Piau J. P., Wajcman H., Bursaux E. Phosphorylation sites in human erythrocyte band 3 protein. Biochim Biophys Acta. 1991 Jan 30;1061(2):253–266. doi: 10.1016/0005-2736(91)90291-f. [DOI] [PubMed] [Google Scholar]

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

RESOURCES