Transient expression assay of Agamma-588 (A/G) mutations in the K562 cell line.

Mohammad Hamid (1) , Frouzandeh Mahjoubi (2) , Mohammad Taghi Akbari (3) , Hossein Khanahmad (4) , Fatemeh Jamshidi (1) , Sirous Zeinali (1) , Morteza Karimipoor (1)

1 Biotechnology Research Center
2 NIGEB - Department of Clinical Genetics
3 Department of Medical Genetics
4 BCG department

BACKGROUND: In the previous study, we have shown that the presence of A allele at position -588 in Agamma-globin gene was highly frequent and closely associated with fetal hemoglobin elevation among beta-thalassemia intermedia patients. Therefore, we decided to investigate whether this allele (A allele at -588) could result in an increase in Agamma-globin gene expression to ameliorate the severity of the disease in thalassemia patients. METHODS: Three constructs containing mu locus control region, Agamma-globin and beta-globin genes were designed and employed in the transient expression assay. The difference among constructs was in the promoter region of Agamma-globin gene (A and G alleles at -588). A construct with T to C base substitution at -175 of Agamma-globin, created by site-directed mutagenesis, was selected as positive control. The K562 cell line was transfected with the above constructs. Subsequently, the expression of Agamma-globin gene was determined by quantitative real-time reverse transcription-PCR. RESULTS: There was not a significant increase in the expression of Agamma-globin gene in the construct containing A allele comparing the one with G allele at -588. CONCLUSIONS: -588 (A>G) mutation does not play a major role in regulation of Agamma-globin gene, suggesting that other factors may be involved.

Mots clés: Β-Thalassemia; globin; k652 cells

Domaines: Génétique humaine

Mohammad Hamid, Frouzandeh Mahjoubi, Mohammad Taghi Akbari, Hossein Khanahmad, Fatemeh Jamshidi, et al.. Transient expression assay of Agamma-588 (A/G) mutations in the K562 cell line.. Iranian Biomedical Journal, 2011, 15 (1-2), pp.15-21. ⟨pasteur-00746421⟩