ABSTRACT
Due to genetic heterogeneity of beta-thalassemia (beta-thal) patients, several efforts have been undertaken to determine the efficacy of hydroxyurea treatment. The aim of this work is to determine the responder and nonresponder for hydroxyurea treatment in beta-thal intermedia based on gamma-globin mRNA and fetal hemoglobin (HbF) induction in human erythroid progenitor cells purified from a patient's peripheral blood. Eighteen beta-thal/hemoglobin E patients [13 beta(E)/codon41/42(-TCTT), 4 beta(E)/codon17, and 1 beta(E)/IVS-654], requiring blood transfusion occasionally, with Hb levels of 5.20-8.50 g/dl were studied. The relative levels of gamma-globin mRNA was measured by real-time reverse-transcription polymerase chain reaction and HbF by high-performance liquid chromatography. The results indicated that erythroid progenitor cells treated with 30 mumol/l hydroxyurea for 96 h preferentially enhanced (G)gamma-and (A)gamma-globin mRNA. The mean values of (G)gamma-globin mRNA fold induction were higher than (A)gamma-globin mRNA (12+/-4 vs 4+/-0.30), the Pearson's correlation of (G)gamma-and (A)gamma-globin mRNA was r=0.80. Induction of (G)gamma/(A)gamma globin mRNA is up to ninefold. A 30% increase in the proportion of HbF out of the total Hb was found in cultures derived from four patients, 20-30% in cultures from nine patients, and less than 20% in cultures from five patients. In cultures from only two patients, increase in the proportion of HbF was less than 3%, and (G)gamma/(A)gamma globin mRNA is less than 0.50.
Subject(s)
Enzyme Inhibitors/pharmacology , Erythroid Precursor Cells/metabolism , Erythropoiesis/drug effects , Fetal Hemoglobin/biosynthesis , Hydroxyurea/pharmacology , beta-Thalassemia/metabolism , Blood Transfusion , Cells, Cultured , Chromatography, High Pressure Liquid/methods , Codon/genetics , Dose-Response Relationship, Drug , Erythroid Precursor Cells/drug effects , Female , Fetal Hemoglobin/analysis , Fetal Hemoglobin/genetics , Humans , Male , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Up-Regulation/drug effects , beta-Thalassemia/genetics , beta-Thalassemia/therapyABSTRACT
OBJECTIVE: Some, but not all, beta-thalassemia/hemoglobin E (beta-thal/HbE) patients respond to hydroxyurea treatment. It would be helpful if patient responses to hydroxyurea could be screened in vitro to identify responders and nonresponders before beginning in vivo treatment. MATERIALS AND METHODS: Thirteen beta-Thal/HbE patients were treated with hydroxyurea orally for 2 years at a starting dose of 5 mg/kg/day for 5 days/week with escalation to a maximum of 10 mg/kg/day. For comparison, erythroid cells obtained from peripheral blood of the same patients 1 year after they had stopped hydroxyurea treatment were treated with hydroxyurea in vitro. The gamma-globin mRNA was measured by real-time reverse-transcription polymerase chain reaction, fetal hemoglobin (HbF) by high-performance liquid chromatography, (G)gamma- and (A)gamma-globin chains by Triton X-100 acid urea polyacrylamide gel electrophoresis. RESULTS: Treatment of cells in primary culture with 30 microM hydroxyurea for 96 hours significantly increased the fractional HbF content in beta-Thal/HbE patients. The (G)gamma:(A)gamma-globin mRNA was induced 0.30- to 8-fold in vitro and 0.30- to 6-fold in vivo (r(2) = 0.51, p = 0.16 by paired t-test); the fractional HbF content was induced 0.50- to 19-fold in vitro and 0.30- to 12-fold in vivo (r(2) = 0.61, p = 0.20) and the (G)gamma:(A)gamma-globin chain ratio was increased 0.80- to 1.40-fold in vitro and 1- to 1.20-fold in vivo (r(2) = 0.62, p = 0.13). CONCLUSION: The correlation of in vivo and in vitro results of HbF synthesis and globin mRNA suggest that in vitro testing may predict the in vivo response.
