Pipkiia and Beta-Globin: Transcripts Differentially Expressed in Reticulocytes and Associated with High Levels of Hb H in Two Siblings with Hb H Disease.

Heterogeneous amounts of Hb H have been detected in peripheral blood of alpha-thalassemic patients even when they carry the same mutation association. This suggests the involvement of other modulating factors besides the thalassemia determinants. To address this issue, we investigated differential gene expression in reticulocytes from two Brazilian siblings of mixed ethnical origin (Chinese and African) in whom Hb H disease is caused by the -a^3.7/--^SEA genotype. One is a 21-year-old male, and the other a 19-year-old female. Their hemoglobin H levels are 18.7 and 5.0%, respectively. By using the mRNA differential-display reverse-transcription polymerase chain reaction (DDRT-PCR) and suppression subtractive hybridization (SSH) techniques, we identified two main transcripts in both procedures, one corresponding to the phosphatidylinositol-4-phosphate-5-kinase type II-alpha (PIP5KIIA) gene and the other corresponding to the beta-globin gene, both overexpressed in the patient with the higher percentage of Hb H. This result was validated by quantitative RT-PCR using two endogenous genes (GAPDH and BAC). PIP5KIIA expression, in arbitrary units (AU), was 0.108701 in the patient with the higher Hb H, 0.031646 in the other and 0.01561 in the normal control, while beta-globin gene expression was 1.13882, 0.71080 and 0.37631 AU, respectively. Reticulocyte RNA samples obtained from three beta-thalassemia intermedia patients showed a very low level of PIP5KIIA expression. Type II PIP kinases produce phosphatidylinositol 4.5 biphosphate (PI4.5P2) by phosphorylating phosphatidylinositol-5-phosphate. PI4.5P2 is a pleiotropic regulatory molecule involved in diverse cellular activities, including modulation of actin cytoskeleton, protein localization and gene expression. Our results suggest that PIP5KIIA may be one of the factors related to the beta-globin gene expression and to the different levels of Hb H in alpha-thalassemic patients. Its exact mechanism of action, however, remains to be determined.