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Simultaneous Quantification of Mitochondrial Mature Frataxin and Extra-Mitochondrial Frataxin Isoform E in Friedreich’s Ataxia Blood
Friedreich’s ataxia (FRDA) is an autosomal recessive disease caused by an intronic guanine-adenine-adenine (GAA) triplet expansion in the frataxin (FXN) gene, which leads to reduced expression of full-length frataxin (1–210) also known as isoform 1. Full-length frataxin has a mitochondrial targeting...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9098139/ https://www.ncbi.nlm.nih.gov/pubmed/35573317 http://dx.doi.org/10.3389/fnins.2022.874768 |
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author | Wang, Qingqing Laboureur, Laurent Weng, Liwei Eskenazi, Nicolas M. Hauser, Lauren A. Mesaros, Clementina Lynch, David R. Blair, Ian A. |
author_facet | Wang, Qingqing Laboureur, Laurent Weng, Liwei Eskenazi, Nicolas M. Hauser, Lauren A. Mesaros, Clementina Lynch, David R. Blair, Ian A. |
author_sort | Wang, Qingqing |
collection | PubMed |
description | Friedreich’s ataxia (FRDA) is an autosomal recessive disease caused by an intronic guanine-adenine-adenine (GAA) triplet expansion in the frataxin (FXN) gene, which leads to reduced expression of full-length frataxin (1–210) also known as isoform 1. Full-length frataxin has a mitochondrial targeting sequence, which facilitates its translocation into mitochondria where it is processed through cleavage at G(41)-L(42) and K(80)-S(81) by mitochondrial processing (MPP) to release mitochondrial mature frataxin (81–210). Alternative splicing of FXN also leads to expression of N-terminally acetylated extra-mitochondrial frataxin (76–210) named isoform E because it was discovered in erythrocytes. Frataxin isoforms are undetectable in serum or plasma, and originally whole blood could not be used as a biomarker in brief therapeutic trials because it is present in erythrocytes, which have a half-life of 115-days and so frataxin levels would remain unaltered. Therefore, an assay was developed for analyzing frataxin in platelets, which have a half-life of only 10-days. However, our discovery that isoform E is only present in erythrocytes, whereas, mature frataxin is present primarily in short-lived peripheral blood mononuclear cells (PBMCs), granulocytes, and platelets, meant that both proteins could be quantified in whole blood samples. We now report a quantitative assay for frataxin proteoforms in whole blood from healthy controls and FRDA patients. The assay is based on stable isotope dilution coupled with immunoprecipitation (IP) and two-dimensional-nano-ultrahigh performance liquid chromatography/parallel reaction monitoring/high resolution mass spectrometry (2D-nano-UHPLC-PRM/HRMS). The lower limit of quantification was 0.5 ng/mL for each proteoform and the assays had 100% sensitivity and specificity for discriminating between healthy controls (n = 11) and FRDA cases (N = 100 in year-1, N = 22 in year-2,3). The mean levels of mature frataxin in whole blood from healthy controls and homozygous FRDA patients were significantly different (p < 0.0001) at 7.5 ± 1.5 ng/mL and 2.1 ± 1.2 ng/mL, respectively. The mean levels of isoform E in whole blood from healthy controls and homozygous FRDA patients were significantly different (p < 0.0001) at 26.8 ± 4.1 ng/mL and 4.7 ± 3.3 ng/mL, respectively. The mean levels of total frataxin in whole blood from healthy controls and homozygous FRDA patients were significantly different (p < 0.0001) at 34.2 ± 4.3 ng/mL and 6.8 ± 4.0 ng/mL, respectively. The assay will make it possible to rigorously monitor the natural history of the disease and explore the potential role of isoform E in etiology of the disease. It will also facilitate the assessment of therapeutic interventions (including gene therapy approaches) that attempt to increase frataxin protein expression as a treatment for this devastating disease. |
format | Online Article Text |
id | pubmed-9098139 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-90981392022-05-13 Simultaneous Quantification of Mitochondrial Mature Frataxin and Extra-Mitochondrial Frataxin Isoform E in Friedreich’s Ataxia Blood Wang, Qingqing Laboureur, Laurent Weng, Liwei Eskenazi, Nicolas M. Hauser, Lauren A. Mesaros, Clementina Lynch, David R. Blair, Ian A. Front Neurosci Neuroscience Friedreich’s ataxia (FRDA) is an autosomal recessive disease caused by an intronic guanine-adenine-adenine (GAA) triplet expansion in the frataxin (FXN) gene, which leads to reduced expression of full-length frataxin (1–210) also known as isoform 1. Full-length frataxin has a mitochondrial targeting sequence, which facilitates its translocation into mitochondria where it is processed through cleavage at G(41)-L(42) and K(80)-S(81) by mitochondrial processing (MPP) to release mitochondrial mature frataxin (81–210). Alternative splicing of FXN also leads to expression of N-terminally acetylated extra-mitochondrial frataxin (76–210) named isoform E because it was discovered in erythrocytes. Frataxin isoforms are undetectable in serum or plasma, and originally whole blood could not be used as a biomarker in brief therapeutic trials because it is present in erythrocytes, which have a half-life of 115-days and so frataxin levels would remain unaltered. Therefore, an assay was developed for analyzing frataxin in platelets, which have a half-life of only 10-days. However, our discovery that isoform E is only present in erythrocytes, whereas, mature frataxin is present primarily in short-lived peripheral blood mononuclear cells (PBMCs), granulocytes, and platelets, meant that both proteins could be quantified in whole blood samples. We now report a quantitative assay for frataxin proteoforms in whole blood from healthy controls and FRDA patients. The assay is based on stable isotope dilution coupled with immunoprecipitation (IP) and two-dimensional-nano-ultrahigh performance liquid chromatography/parallel reaction monitoring/high resolution mass spectrometry (2D-nano-UHPLC-PRM/HRMS). The lower limit of quantification was 0.5 ng/mL for each proteoform and the assays had 100% sensitivity and specificity for discriminating between healthy controls (n = 11) and FRDA cases (N = 100 in year-1, N = 22 in year-2,3). The mean levels of mature frataxin in whole blood from healthy controls and homozygous FRDA patients were significantly different (p < 0.0001) at 7.5 ± 1.5 ng/mL and 2.1 ± 1.2 ng/mL, respectively. The mean levels of isoform E in whole blood from healthy controls and homozygous FRDA patients were significantly different (p < 0.0001) at 26.8 ± 4.1 ng/mL and 4.7 ± 3.3 ng/mL, respectively. The mean levels of total frataxin in whole blood from healthy controls and homozygous FRDA patients were significantly different (p < 0.0001) at 34.2 ± 4.3 ng/mL and 6.8 ± 4.0 ng/mL, respectively. The assay will make it possible to rigorously monitor the natural history of the disease and explore the potential role of isoform E in etiology of the disease. It will also facilitate the assessment of therapeutic interventions (including gene therapy approaches) that attempt to increase frataxin protein expression as a treatment for this devastating disease. Frontiers Media S.A. 2022-04-28 /pmc/articles/PMC9098139/ /pubmed/35573317 http://dx.doi.org/10.3389/fnins.2022.874768 Text en Copyright © 2022 Wang, Laboureur, Weng, Eskenazi, Hauser, Mesaros, Lynch and Blair. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Neuroscience Wang, Qingqing Laboureur, Laurent Weng, Liwei Eskenazi, Nicolas M. Hauser, Lauren A. Mesaros, Clementina Lynch, David R. Blair, Ian A. Simultaneous Quantification of Mitochondrial Mature Frataxin and Extra-Mitochondrial Frataxin Isoform E in Friedreich’s Ataxia Blood |
title | Simultaneous Quantification of Mitochondrial Mature Frataxin and Extra-Mitochondrial Frataxin Isoform E in Friedreich’s Ataxia Blood |
title_full | Simultaneous Quantification of Mitochondrial Mature Frataxin and Extra-Mitochondrial Frataxin Isoform E in Friedreich’s Ataxia Blood |
title_fullStr | Simultaneous Quantification of Mitochondrial Mature Frataxin and Extra-Mitochondrial Frataxin Isoform E in Friedreich’s Ataxia Blood |
title_full_unstemmed | Simultaneous Quantification of Mitochondrial Mature Frataxin and Extra-Mitochondrial Frataxin Isoform E in Friedreich’s Ataxia Blood |
title_short | Simultaneous Quantification of Mitochondrial Mature Frataxin and Extra-Mitochondrial Frataxin Isoform E in Friedreich’s Ataxia Blood |
title_sort | simultaneous quantification of mitochondrial mature frataxin and extra-mitochondrial frataxin isoform e in friedreich’s ataxia blood |
topic | Neuroscience |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9098139/ https://www.ncbi.nlm.nih.gov/pubmed/35573317 http://dx.doi.org/10.3389/fnins.2022.874768 |
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