Characterization of alpha thalassemic genotypes by multiplex ligation-dependent probe amplification in the Brazilian population

Alpha-thalassemia is the most common inherited disorder of hemoglobin synthesis. Genomic deletions involving the alpha-globin gene cluster on chromosome 16p13.3 are the most frequent molecular causes of the disease. Although common deletions can be detected by a single multiplex gap-PCR, the rare and novel deletions depend on more laborious techniques for their identification. The multiplex ligation-dependent probe amplification (MLPA) technique has recently been used for this purpose and was successfully used in the present study to detect the molecular alterations responsible for the alpha-thalassemic phenotypes in 8 unrelated individuals (3 males and 5 females; age, 4 months to 30 years) in whom the molecular basis of the disease could not be determined by conventional methods. A total of 44 probe pairs were used for MLPA, covering approximately 800 kb from the telomere to the MSLN gene in the 16p13.3 region. Eight deletions were detected. Four of these varied in size from 240 to 720 kb and affected a large region including the entire alpha-globin gene cluster and its upstream regulatory element (alpha-MRE), while the other four varied in size from 0.4 to 100 kb and were limited to a region containing this element. This study is the first in Brazil to use the MLPA method to determine the molecular basis of alpha-thalassemia. The variety of rearrangements identified highlights the need to investigate all cases presenting microcytosis and hypochromia, but without iron deficiency or elevated hemoglobin A2 levels and suggests that these rearrangements may be more frequent in our population than previously estimated.162

Characterization of alpha thalassemic genotypes by multiplex ligation-dependent probe amplification in the Brazilian population

Alpha-thalassemia is the most common inherited disorder of hemoglobin synthesis. Genomic deletions involving the alpha-globin gene cluster on chromosome 16p13.3 are the most frequent molecular causes of the disease. Although common deletions can be detected by a single multiplex gap-PCR, the rare and novel deletions depend on more laborious techniques for their identification. The multiplex ligation-dependent probe amplification (MLPA) technique has recently been used for this purpose and was successfully used in the present study to detect the molecular alterations responsible for the alpha-thalassemic phenotypes in 8 unrelated individuals (3 males and 5 females; age, 4 months to 30 years) in whom the molecular basis of the disease could not be determined by conventional methods. A total of 44 probe pairs were used for MLPA, covering approximately 800 kb from the telomere to the MSLN gene in the 16p13.3 region. Eight deletions were detected. Four of these varied in size from 240 to 720 kb and affected a large region including the entire alpha-globin gene cluster and its upstream regulatory element (alpha-MRE), while the other four varied in size from 0.4 to 100 kb and were limited to a region containing this element. This study is the first in Brazil to use the MLPA method to determine the molecular basis of alpha-thalassemia. The variety of rearrangements identified highlights the need to investigate all cases presenting microcytosis and hypochromia, but without iron deficiency or elevated hemoglobin A2 levels and suggests that these rearrangements may be more frequent in our population than previously estimated

Hb S-são Paulo: A New Sickling Hemoglobin With Stable Polymers And Decreased Oxygen Affinity

Hb S-São Paulo (SP) [HBB:c.20A > T p.Glu6Val; c.196A > G p.Lys65Glu] is a new double-mutant hemoglobin that was found in heterozygosis in an 18-month-old Brazilian male with moderate anemia. It behaves like Hb S in acid electrophoresis, isoelectric focusing and solubility testing but shows different behavior in alkaline electrophoresis, cation-exchange HPLC and RP-HPLC. The variant is slightly unstable, showed reduced oxygen affinity and also appeared to form polymers more stable than the Hb S. Molecular dynamics simulation suggests that the polymerization is favored by interfacial electrostatic interactions. This provides a plausible explanation for some of the reported experimental observations. © 2012 Elsevier Inc. All rights reserved.51912331Steinberg, M.H., (1999) New Engl. J. Med., 340, pp. 1021-1030Stuart, M.J., Nagel, R.L., (2004) Lancet, 364, pp. 1343-1360Barabino, G.A., Platt, M.O., Kaul, D.K., (2010) Annu. Rev. Biomed. Eng., 12, pp. 345-367Goossens, M., Garel, M.C., Auvinet, J., Basset, O., Ferreira Gomes, P., Rosa, J., Arous, N., (1975) FEBS Lett., 58, pp. 149-154Bookchin, R.M., Nagel, R.L., Ranney, H.M., (1967) J. Biol. Chem., 242, pp. 248-255Langdown, J.V., Williamson, D., Knight, C.B., Rubenstein, D., Carrell, R.W., (1989) Br. J. Haematol., 71, pp. 443-444Moo-Penn, W.F., Schmidt, R.M., Jue, D.L., Bechtel, K.C., Wright, J.M., Horne III, M.K., Haycraft, G.L., Nagel, R.L., (1977) Eur. J. Biochem., 77, pp. 561-566Monplaisir, N., Merault, G., Poyart, C., Rhoda, M.D., Craescu, C., Vidaud, M., Galacteros, F., Rosa, J., (1986) Proc. Natl. Acad. Sci. USA, 83, pp. 9363-9367Geva, A., Clark, J.J., Zhang, Y., Popowicz, A., Manning, J.M., Neufeld, E.J., (2004) New Engl. J. Med., 351, pp. 1532-1538Karplus, M., McCammon, J.A., (2002) Nat. Struct. Biol., 9, pp. 646-652Dacie, J.V., Lewis, S.M., (1995) Practical Haematology, , Churchill Livingstone EdinburghAdachi, K., Asakura, T., (1979) J. Biol. Chem., 254, pp. 7765-7771Miranda, S.R.P., Fonseca, S.F., Figueiredo, M.S., Yamamoto, M., Grotto, H.Z.W., Saad, S.T.O., Costa, F.F., (1997) Braz. J. Genet., 20, pp. 745-748Chong, S.S., Boehm, C.D., Higgs, D.R., Cutting, G.R., (2000) Blood, 95, pp. 360-362Kattamis, A.C., Camaschella, C., Sivera, P., Surrey, S., Fortina, P., (1996) Am. J. Hematol., 53, pp. 81-91Rossi-Fanelli, A., Antonini, E., (1958) Arch. Biochem. Biophys., 77, pp. 478-492Benesch, R.E., Benesch, R., Yu, C.I., (1969) Biochemistry-Us, 8, pp. 2567-2571Martinez, L., Andrade, R., Birgin, E.G., Martinez, J.M., (2009) J. Comput. Chem., 30, pp. 2157-2164MacKerell, A.D., Bashford, D., Bellott, M., Dunbrack, R.L., Evanseck, J.D., Field, M.J., Fischer, S., Karplus, M., (1998) J. Phys. Chem. B, 102, pp. 3586-3616Ryckaert, J.-P., Ciccotti, G., Berendsen, H.J.C., (1977) J. Comp. Phys., pp. 327-341Park, S.-Y., (2006) J. Mol. Biol., 360, pp. 690-701Humphrey, W., Dalke, A., Schulten, K., (1996) J. Mol. Graph., 14, pp. 33-38Harrington, D.J., Adachi, K., Royer, Jr.W.W., (1997) J. Mol. Biol., 272, pp. 398-407Adachi, K., Konitzer, P., Pang, J., Reddy, K.S., Surrey, S., (1997) Blood, 90, pp. 2916-2920Benesch, R., Benesch, R.E., Yu, C.I., (1968) Proc. Natl. Acad. Sci. USA, 59, pp. 526-532Edalji, R., Benesch, R.E., Benesch, R., (1976) J. Biol. Chem., 251, pp. 7720-7721Antonini, E., Brunoni, M., (1971) Hemoglobin and Myoglobin in Their Reactions with Ligands, , North-Holland Publishing Company AmsterdanCapece, L., Marti, M.A., Crespo, A., Doctorovich, F., Estrin, D.A., (2006) J. Am. Chem. Soc., 128, pp. 12455-12461Marti, M.A., Crespo, A., Capece, L., Boechi, L., Bikiel, D.E., Scherlis, D.A., Estrin, D.A., (2006) J. Inorg. Biochem., 100, pp. 761-770Young, R.C., Rachal, R.E., Del Pilar Aguinaga, M., Nelson, B.L., Kim, B.C., Winter, W.P., Castro, O., (2000) J. Natl. Med. Assoc., 92, pp. 430-435Eaton, W.A., Hofrichter, J., (1987) Blood, 70, pp. 1245-1266Kuczera, K., Gao, J., Tidor, B., Karplus, M., (1990) Proc. Natl. Acad. Sci. USA, 87, pp. 8481-8485Bihoreau, M.T., Baudin, V., Marden, M., Lacaze, N., Bohn, B., Kister, J., Schaad, O., Pagnier, J., (1992) Protein Sci., 1, pp. 145-150Salzano, F.M., Bortolini, M.C., (2002) The Evolution and Genetics of Latin American Population, , Cambridge University Press CambridgeMosca, A., Paleari, R., Ivaldi, G., Galanello, R., Giordano, P.C.P.C., (2009) J. Clin. Pathol., 62, pp. 13-1