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Relative and Absolute Quantification of Aberrant and Normal Splice Variants in HBB(IVSI−110 (G > A)) β-Thalassemia
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 HBB(IVSI−110(G > A)) β-thalassemia (HGVS name: HBB:c.93-21G > A). Its underlying mutation creates an abnormal...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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MDPI
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7555009/ https://www.ncbi.nlm.nih.gov/pubmed/32933098 http://dx.doi.org/10.3390/ijms21186671 |
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author | Patsali, Petros Papasavva, Panayiota Christou, Soteroulla Sitarou, Maria Antoniou, Michael N. Lederer, Carsten W. Kleanthous, Marina |
author_facet | Patsali, Petros Papasavva, Panayiota Christou, Soteroulla Sitarou, Maria Antoniou, Michael N. Lederer, Carsten W. Kleanthous, Marina |
author_sort | Patsali, Petros |
collection | PubMed |
description | 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 HBB(IVSI−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 HBB(IVSI−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. |
format | Online Article Text |
id | pubmed-7555009 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-75550092020-10-14 Relative and Absolute Quantification of Aberrant and Normal Splice Variants in HBB(IVSI−110 (G > A)) β-Thalassemia Patsali, Petros Papasavva, Panayiota Christou, Soteroulla Sitarou, Maria Antoniou, Michael N. Lederer, Carsten W. Kleanthous, Marina Int J Mol Sci Article 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 HBB(IVSI−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 HBB(IVSI−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. MDPI 2020-09-11 /pmc/articles/PMC7555009/ /pubmed/32933098 http://dx.doi.org/10.3390/ijms21186671 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Patsali, Petros Papasavva, Panayiota Christou, Soteroulla Sitarou, Maria Antoniou, Michael N. Lederer, Carsten W. Kleanthous, Marina Relative and Absolute Quantification of Aberrant and Normal Splice Variants in HBB(IVSI−110 (G > A)) β-Thalassemia |
title | Relative and Absolute Quantification of Aberrant and Normal Splice Variants in HBB(IVSI−110 (G > A)) β-Thalassemia |
title_full | Relative and Absolute Quantification of Aberrant and Normal Splice Variants in HBB(IVSI−110 (G > A)) β-Thalassemia |
title_fullStr | Relative and Absolute Quantification of Aberrant and Normal Splice Variants in HBB(IVSI−110 (G > A)) β-Thalassemia |
title_full_unstemmed | Relative and Absolute Quantification of Aberrant and Normal Splice Variants in HBB(IVSI−110 (G > A)) β-Thalassemia |
title_short | Relative and Absolute Quantification of Aberrant and Normal Splice Variants in HBB(IVSI−110 (G > A)) β-Thalassemia |
title_sort | relative and absolute quantification of aberrant and normal splice variants in hbb(ivsi−110 (g > a)) β-thalassemia |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7555009/ https://www.ncbi.nlm.nih.gov/pubmed/32933098 http://dx.doi.org/10.3390/ijms21186671 |
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