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Open AccessArticle

Relative and Absolute Quantification of Aberrant and Normal Splice Variants in HBBIVSI−110 (G > A) β-Thalassemia

1
Department of Molecular Genetics Thalassaemia, The Cyprus Institute of Neurology and Genetics, Nicosia 1683, Cyprus
2
Cyprus School of Molecular Medicine, Nicosia 1683, Cyprus
3
Thalassaemia Clinic Nicosia, Ministry of Health, Nicosia 1474, Cyprus
4
Thalassaemia Clinic Larnaca, Ministry of Health, Larnaca 6301, Cyprus
5
Department of Medical and Molecular Genetics, King’s College London, London SE1 9RT, UK
*
Author to whom correspondence should be addressed.
These authors have contributed equally to this study.
Int. J. Mol. Sci. 2020, 21(18), 6671; https://doi.org/10.3390/ijms21186671
Received: 18 July 2020 / Revised: 8 September 2020 / Accepted: 8 September 2020 / Published: 11 September 2020
(This article belongs to the Section Molecular Biology)
The β-thalassemias are an increasing challenge to health systems worldwide, caused by absent or reduced β-globin (HBB) production. Of particular frequency in many Western countries is HBBIVSI−110(G > A) β-thalassemia (HGVS name: HBB:c.93-21G > A). Its underlying mutation creates an abnormal splice acceptor site in the HBB gene, and while partially retaining normal splicing of HBB, it severely reduces HBB protein expression from the mutant locus and HBB loci in trans. For the assessment of the underlying mechanisms and of therapies targeting β-thalassemia, accurate quantification of aberrant and normal HBB mRNA is essential, but to date, has only been performed by approximate methods. To address this shortcoming, we have developed an accurate, duplex reverse-transcription quantitative PCR assay for the assessment of the ratio and absolute quantities of normal and aberrant mRNA species as a tool for basic and translational research of HBBIVSI−110(G > A) β-thalassemia. The method was employed here to determine mRNA ratios and quantities in blood and primary cell culture samples and correlate them with HBB protein levels. Moreover, with its immediate utility for β-thalassemia and the mutation in hand, the approach can readily be adopted for analysis of alternative splicing or for quantitative assays of any disease-causing mutation that interferes with normal splicing. View Full-Text
Keywords: β-thalassemia; splice defect; duplex quantitative PCR; absolute quantification; transcript variants; splicing β-thalassemia; splice defect; duplex quantitative PCR; absolute quantification; transcript variants; splicing
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Patsali, P.; Papasavva, P.; Christou, S.; Sitarou, M.; Antoniou, M.N.; Lederer, C.W.; Kleanthous, M. Relative and Absolute Quantification of Aberrant and Normal Splice Variants in HBBIVSI−110 (G > A) β-Thalassemia. Int. J. Mol. Sci. 2020, 21, 6671.

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