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Abstract

Background: Sickle Cell Anaemia (SCA) is a genetically-inherited blood disorder caused by the occurrence of a point mutation in the bases coding for the sixth amino-acid of the β-chain of haemoglobin. The presence of Fetal Haemoglobin (HbF) in blood is known to show a number of beneficial effects in improving the conditions of SCA. Hence, clinical symptoms of SCA arise only after HbF levels drop. HbF is synthesized by the HBG1 (gamma-globin) gene. From birth there is a gradual shift from fetal to adult haemoglobin triggered by the downregulation of the HBG1 gene and the upregulation of the HBB gene. Rationale: Hydroyxurea has been widely evaluated in the treatment of SCA for its ability to induce synthesis of HbF by reactivating the expression of the HBG1 gene. It functions by repressing the activity of various transcription factors involved in the downregulation of the HBG1 gene during transition to adult haemoglobin. However, hydroxyurea has proven to be potentially toxic in a number of cases and may also carry a possibility of long term carcinogenicity. Therefore it is administered under restriction and may not be used in the treatment of pregnant women and children. In this study, an attempt is made to identify and characterise the altered gene expression pattern caused by hydroxyurea treatment through Differential Display Reverse Transcriptase PCR (DDRT-PCR) and Real Time PCR (Q-PCR). Further, the phenomenon depicting the repression of the transcription factors leading to the reactivation of HBG1 gene will be reproduced using techniques of RNA interference (RNAi) and antisense. Objective: To reproduce the altered genetic conditions produced by Hydroxyurea treatment for the in-vitro induction of HbF using molecular genetic techniques. Methods: Blood is obtained from SCA patients at diagnosis and after treatment with hydroxyurea. Erythroid Progenitor cells are purified from the collected blood using the EasySep™ Human progenitor cell enrichment kit. Cells are then cultured in a suitable medium and maintained for further analysis. Treatment with hydroxyurea is performed at varying doses at different intervals of time. The evaluation of gene expression is carried out through the generation of c-DNA through a reverse transcriptase PCR cycle followed by Q-PCR for identification and quantification. Results & Conclusion: Buffy coats containing mononuclear cells were purified from peripheral blood obtained from SCA patients through density-gradient centrifugation. Further, erythroid progenitor cells were successfully isolated and maintained in culture for variable periods of time. Currently, mRNA expression analysis is ongoing through the use of reverse transcriptase PCR protocols. If proven successful, this technique holds a promising future scope; as molecular genetic techniques could be used to reproduce the effects of hydroxyurea without the actual administration of the drug. Thus, the toxicity issues of the drug are avoided while also making it available to all classes of patients including pregnant women and children.

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/content/papers/10.5339/qfarc.2014.HBPP0571
2014-11-18
2020-01-26
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