Relative and Absolute Quantification of Aberrant and Normal Splice Variants in <i>HBB<sup>IVSI−110 (G > A)</sup></i> 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 <i>HBB<sup>IVSI−110(G > A)</sup></i> β-thalassemia (HGVS name: HBB:c.93-21G > A). Its...
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MDPI AG
2020-09-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/21/18/6671 |
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| author | Petros Patsali Panayiota Papasavva Soteroulla Christou Maria Sitarou Michael N. Antoniou Carsten W. Lederer Marina Kleanthous |
| author_facet | Petros Patsali Panayiota Papasavva Soteroulla Christou Maria Sitarou Michael N. Antoniou Carsten W. Lederer Marina Kleanthous |
| author_sort | Petros Patsali |
| collection | DOAJ |
| 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 <i>HBB<sup>IVSI−110(G > A)</sup></i> β-thalassemia (HGVS name: HBB:c.93-21G > A). Its underlying mutation creates an abnormal splice acceptor site in the <i>HBB</i> gene, and while partially retaining normal splicing of <i>HBB</i>, it severely reduces HBB protein expression from the mutant locus and <i>HBB</i> loci in trans. For the assessment of the underlying mechanisms and of therapies targeting β-thalassemia, accurate quantification of aberrant and normal <i>HBB</i> 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 <i>HBB<sup>IVSI−110(G > A)</sup></i> 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. |
| first_indexed | 2024-03-10T16:24:01Z |
| format | Article |
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| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T16:24:01Z |
| publishDate | 2020-09-01 |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-efd752672bd344b0aea76ce02be0b72b2023-11-20T13:29:21ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672020-09-012118667110.3390/ijms21186671Relative and Absolute Quantification of Aberrant and Normal Splice Variants in <i>HBB<sup>IVSI−110 (G > A)</sup></i> β-ThalassemiaPetros Patsali0Panayiota Papasavva1Soteroulla Christou2Maria Sitarou3Michael N. Antoniou4Carsten W. Lederer5Marina Kleanthous6Department of Molecular Genetics Thalassaemia, The Cyprus Institute of Neurology and Genetics, Nicosia 1683, CyprusDepartment of Molecular Genetics Thalassaemia, The Cyprus Institute of Neurology and Genetics, Nicosia 1683, CyprusThalassaemia Clinic Nicosia, Ministry of Health, Nicosia 1474, CyprusThalassaemia Clinic Larnaca, Ministry of Health, Larnaca 6301, CyprusDepartment of Medical and Molecular Genetics, King’s College London, London SE1 9RT, UKDepartment of Molecular Genetics Thalassaemia, The Cyprus Institute of Neurology and Genetics, Nicosia 1683, CyprusDepartment of Molecular Genetics Thalassaemia, The Cyprus Institute of Neurology and Genetics, Nicosia 1683, CyprusThe 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 <i>HBB<sup>IVSI−110(G > A)</sup></i> β-thalassemia (HGVS name: HBB:c.93-21G > A). Its underlying mutation creates an abnormal splice acceptor site in the <i>HBB</i> gene, and while partially retaining normal splicing of <i>HBB</i>, it severely reduces HBB protein expression from the mutant locus and <i>HBB</i> loci in trans. For the assessment of the underlying mechanisms and of therapies targeting β-thalassemia, accurate quantification of aberrant and normal <i>HBB</i> 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 <i>HBB<sup>IVSI−110(G > A)</sup></i> 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.https://www.mdpi.com/1422-0067/21/18/6671β-thalassemiasplice defectduplex quantitative PCRabsolute quantificationtranscript variantssplicing |
| spellingShingle | Petros Patsali Panayiota Papasavva Soteroulla Christou Maria Sitarou Michael N. Antoniou Carsten W. Lederer Marina Kleanthous Relative and Absolute Quantification of Aberrant and Normal Splice Variants in <i>HBB<sup>IVSI−110 (G > A)</sup></i> β-Thalassemia International Journal of Molecular Sciences β-thalassemia splice defect duplex quantitative PCR absolute quantification transcript variants splicing |
| title | Relative and Absolute Quantification of Aberrant and Normal Splice Variants in <i>HBB<sup>IVSI−110 (G > A)</sup></i> β-Thalassemia |
| title_full | Relative and Absolute Quantification of Aberrant and Normal Splice Variants in <i>HBB<sup>IVSI−110 (G > A)</sup></i> β-Thalassemia |
| title_fullStr | Relative and Absolute Quantification of Aberrant and Normal Splice Variants in <i>HBB<sup>IVSI−110 (G > A)</sup></i> β-Thalassemia |
| title_full_unstemmed | Relative and Absolute Quantification of Aberrant and Normal Splice Variants in <i>HBB<sup>IVSI−110 (G > A)</sup></i> β-Thalassemia |
| title_short | Relative and Absolute Quantification of Aberrant and Normal Splice Variants in <i>HBB<sup>IVSI−110 (G > A)</sup></i> β-Thalassemia |
| title_sort | relative and absolute quantification of aberrant and normal splice variants in i hbb sup ivsi 110 g a sup i β thalassemia |
| topic | β-thalassemia splice defect duplex quantitative PCR absolute quantification transcript variants splicing |
| url | https://www.mdpi.com/1422-0067/21/18/6671 |
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