Artículos de revistas
Determination Of Neutral Polymorphisms (frameworks) Of The Human Beta Globin Gene In Beta Thalassaemias By Pcr/dgge
Registro en:
Sangre. , v. 42, n. 1, p. 21 - 24, 1997.
364355
2-s2.0-0031063763
Autor
Moreira H.W.
De Oliveira C.M.
Martins C.S.B.
De Sales T.S.
Costa F.F.
Institución
Resumen
Purpose: Considering the importance of type beta thalassaemias as hereditary syndromes of high significance in different populations of Mediterranean origin and, by extension, in the Brazilian population, the objective of the present study was to determine by PCR/DGGE the gene structures responsible for neutral polymorphisms (frameworks) observed in the human beta globin gene associated with the mutations responsible for type beta thalassaemias in a sample of the Brazilian population and, more specifically, of the population of the State of São Paulo. Patients and methods: Thirty individuals with beta thalassaemic mutations were analyzed: 22 mutations were in codon 39 (C->T), 5 in IVS1-110 (G->A), 2 in IVS1-6 (T->C) and 1 in IVS1-1 (G->A). DNA was extracted and selective amplification was performed by PCR extending from position IVS1 nt 46 to IVS2 nt 126 (474 pb). The product was then analyzed by polyacrylamide gel electrophoresis on a denaturing 10-60% urea/formamide gradient. Results: The results demonstrated that, as expected, the mutations responsible for type beta thalassaemia observed in this population are of Mediterranean origin, with 73% distribution represented by codon 39,17% by IVS1-110, 7% by IVS1-6 and 3% by IVS1-1. In turn, framework distribution seems to indicate a higher frequency of Fr 1-1 in codon 39 and IVS1-110, of Fr 1-3 in IVS1-6 and of Fr 1-2 in IVS1-1. Conclusions: These results permit us to conclude that gene amplification by PCR followed by DGGE is an appropriate method for the separation of DNA molecules that differ even by a single base change and therefore can be utilized to detect the alterations observed in the human beta globin gene. This methodology shows that, using only a pair of primers, it is possible to define the frameworks that are observed in the beta globin gene. 42 1 21 24 Permutt, M.A., Use of DNA polymorphisms for genetic analysis of non-insulin depedent diabetes mellitus (1991) Baillière's Clin Endocrinol Metab, 5, pp. 495-526 Orkin, S.H., Kazazian, H.H., Antonarakis, S.F., Goff, S.C., Bohem, C.D., Sexton, J.P., Linkage of beta-thalassemia mutations and beta-globin gene polymorphisms with DNA polymorphisms in the human gene cluster (1982) Nature, 296, pp. 627-631 Myers, R.M., Sheffield, V.C., Cox, D.R., Mutation detection by PCR, GC-clamps. and denaturing gradient gel electrophoresis (1989) PCR Technology: Principles and Applications for DNA Amplification Stochton Press, pp. 71-88. , Erlich HA. New York Losekoot, M., Van Heeren, H., Schipper, J.J., Giordano, P.C., Bernint, L.F., Fodde, R., Rapid detection of highly polymorphic beta globin framework by denaturing gradient gel electrophoresis (1992) J Med Genet, 29, pp. 574-577 Cao, A., Goosens, M., Pirastu, M., Beta-thalassaemia mutations in Mediterranean populations (1989) Br J Haematol, 71, pp. 309-312 Martins, C.S.B., (1992) Caractcrização Molecular de Heterozigotos da Talassemia Beta, , Doctor's thesis. UNICAMP. Campinas Goosens, M., Kan, Y.W., DNA analysis in the diagnostic of hemoglobin disorders (1981) Methods Enzymol, 76, pp. 805-817 Kaplan, J.C., Delpech, M., (1990) Biologic Moléculaire et Médicine 2nd Ed., , Flammarion Médicine-Sciencies. Paris Lerman, L.S., Silverstein, K., Computational stimulation of DNA melting and its application to denaturing gradient gel electrophoresis (1987) Methods Enzymol, 155, pp. 482-501 Myers, R.M., Maniatis, T., Lerman, L.S., Detection and localization of single base changes by denaturing gel electrophoresis (1987) Methods Enzymol, 155, pp. 501-527 Myers, R.M., Fischer, S.G., Maniatis, T., Lerman, L.S., Modification of the melting properties of duplex DNA by attachment of a GC-rich DNA sequence as determined by denaturing gradient gel electrophoresis (1985) Nucl Acids Res, 13, pp. 3111-3129 Sheffield, V.C., Cox, D.R., Lerman, L.S., Myers, R.M., Attachment of a 40-base-pair G+C- rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reation results in improved detection of single-base changes (1989) Proc Natl Acad Sci USA, 86, pp. 232-236 Diegues Jr., M., (1980) Etnias e Culturas no Brasil, , Biblioteca do Exército Editoria. Rio de Janeiro Sonati, M.F., Kimura, E.M., Grotto, H.Z.W., Costa, F.F., Hemoglobinopatias hereditárias em uma população hospitalar (1991) XII Congresso Nacional do Colégio Brasileiro de Hematologia, p. 111 Costa, F.F., Tavella, M.A., Zago, M.A., Molecular bases of beta thalassemia in Brazil (1990) Blood, 76, pp. 58a Goossens, M., Personal communication, 1991