Evaluation of the Function of a Rare Variant in the 3'-Untranslated Region of the β-Globin Gene

Abstract

β-Thalassemia (β-thal) is an inherited genetic disease that occurs because of the absence or reduction of β-globin chain synthesis. Genetic changes occur in different regions of the β-globin gene, but these mutations are less reported in the 3' untranslated region (3'-UTR). The objective of the present investigation was to evaluate the functional effect of a rare variant in the 3'-UTR of the β-globin gene. A variant at the first nucleotide of the 3'-UTR of the β-globin gene (HBB: c.*1G > A) was identified by DNA sequencing in an individual with low hematological indices and a normal hemoglobin (Hb) electrophoresis pattern. To evaluate the functional effect of this variant, the normal and mutated 3'-UTR of the β-globin gene was synthesized separately and sub cloned in the psiCHEK2 vector. Next, using the calcium phosphate method, the psiCHEK2 vectors containing normal and mutated 3'-UTR were transfected separately into the HEK293T cell line. Finally, the transfected cell line was analyzed by dual luciferase assay. The ratio of Renilla to firefly for the mutant sample was 1.26 ± 0.06, while for normal samples it was 1.12 ± 0.04. The results of the luciferase assay showed that there was no significant difference in the functional effect between the mutant and wild type construct. Therefore, it was concluded that this variant might not reduce the expression of the β-globin gene. Future studies by globin chain synthesis or to evaluate the expression of the gene in erythroid cells, might be necessary to understand the regulatory function of this mutation.

Keywords:

• β-Globin
• mutation
• thalassemia
• 3'-untranslated region (3'-UTR)

Acknowledgements

The authors wish to thank Mr. Amir Abbas Rahimi and Ms. Shabahang Amini (Department of Molecular Medicine, Pasteur Institute of Iran, Tehran, Iran) for their help and willingness to cooperate in this study.

Author contributions

S. Targholi performed the molecular tests and the draft manuscript; Z. Noormohammadi contributed to the data analysis and edited the manuscript; E. Tafsiri contributed in cell culture, transfection and the dual luciferase assay; M. Karimipoor designed and outlined the framework of this experiment and edited the final manuscript. All authors approved the final manuscript.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Additional information

Funding

This work was supported by the Pasteur Institute of Iran, Tehran, Iran [Grant #372].