mRNA analysis in reticulocytes of subjects with Hb D, Hb Porto Alegre, Hb E, and different types of unstable hemoglobin variants

Abstract

Using e reverse transcription-polymerase chain reaction (RT-PCR) technique we determined the α2/α1, α/β, and γ/β mRNA ratios in reticulocytes of 11 patients with seven different unstable β chain variants, of 4 patients with two unstable α chain variants, in hemoglobin (Hb) D, Hb Porto Alegre, and Hb E heterozygotes, and in 8 patients with Hb X-β(o)-thalassemia (thai) (three D-β(o)-thal, one Porto Alegre-β(o)- thal, one Lulu Island-β(o)-thal, and three E-β(o)-thal). In addition, we determined the β(X)/β(A) mRNA ratios (X = unstable) in some Hb D heterozygotes and in 6 subjects with an unstable β chain variant. Normal α/β and β(X)β(A) mRNA ratios were found in all heterozygotes tested, indicating that the respective mutations did not alter the stability of the mRNAs. The α/β mRNA ratio in four Hb E heterozygotes averaged 4.21 (normal, 4.47), and that in 2 patients with Hb E-β(o)-thal and four α- globin genes (αα/αα) averaged a high 22.4. The γ mRNA level in the Hb E heterozygotee was <1% but varied greatly in patients with Hb E-β(o)-thal; the α/(γ + β) mRNA ratios in the 2 patients were 15.5 and 16.7, respectively. The large differences in α/β and α/(γ + β) mRNA ratios in reticulocytes of subjects with AE and with E-β(o)-thal may be due to differences in the levels of normally-spliced β(E) and abnormally-spliced α(E) mRNAs. Only the latter is unstable and is preferentially produced in bone marrow and reticulocytes of Hb E-β(o)-thal patients, where it is rapidly degraded.