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...
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| Language: | English |
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Frontiers Media S.A.
2022-04-01
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| Series: | Frontiers in Neuroscience |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fnins.2022.874768/full |
| _version_ | 1811303194251231232 |
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| author | Qingqing Wang Qingqing Wang Laurent Laboureur Laurent Laboureur Liwei Weng Liwei Weng Nicolas M. Eskenazi Nicolas M. Eskenazi Lauren A. Hauser Lauren A. Hauser Lauren A. Hauser Clementina Mesaros Clementina Mesaros David R. Lynch David R. Lynch David R. Lynch Ian A. Blair Ian A. Blair |
| author_facet | Qingqing Wang Qingqing Wang Laurent Laboureur Laurent Laboureur Liwei Weng Liwei Weng Nicolas M. Eskenazi Nicolas M. Eskenazi Lauren A. Hauser Lauren A. Hauser Lauren A. Hauser Clementina Mesaros Clementina Mesaros David R. Lynch David R. Lynch David R. Lynch Ian A. Blair Ian A. Blair |
| author_sort | Qingqing Wang |
| collection | DOAJ |
| 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 G41-L42 and K80-S81 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. |
| first_indexed | 2024-04-13T07:42:23Z |
| format | Article |
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| institution | Directory Open Access Journal |
| issn | 1662-453X |
| language | English |
| last_indexed | 2024-04-13T07:42:23Z |
| publishDate | 2022-04-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Neuroscience |
| spelling | doaj.art-5d26bbbea7fa4e5189e4f73f8a4d0f4c2022-12-22T02:55:50ZengFrontiers Media S.A.Frontiers in Neuroscience1662-453X2022-04-011610.3389/fnins.2022.874768874768Simultaneous Quantification of Mitochondrial Mature Frataxin and Extra-Mitochondrial Frataxin Isoform E in Friedreich’s Ataxia BloodQingqing Wang0Qingqing Wang1Laurent Laboureur2Laurent Laboureur3Liwei Weng4Liwei Weng5Nicolas M. Eskenazi6Nicolas M. Eskenazi7Lauren A. Hauser8Lauren A. Hauser9Lauren A. Hauser10Clementina Mesaros11Clementina Mesaros12David R. Lynch13David R. Lynch14David R. Lynch15Ian A. Blair16Ian A. Blair17Center of Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United StatesPenn/CHOP Center of Excellence in Friedreich’s Ataxia, Philadelphia, PA, United StatesCenter of Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United StatesPenn/CHOP Center of Excellence in Friedreich’s Ataxia, Philadelphia, PA, United StatesCenter of Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United StatesPenn/CHOP Center of Excellence in Friedreich’s Ataxia, Philadelphia, PA, United StatesCenter of Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United StatesPenn/CHOP Center of Excellence in Friedreich’s Ataxia, Philadelphia, PA, United StatesPenn/CHOP Center of Excellence in Friedreich’s Ataxia, Philadelphia, PA, United StatesDepartments of Pediatrics and Neurology, Children’s Hospital of Philadelphia, Philadelphia, PA, United StatesDepartments of Pediatrics and Neurology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United StatesCenter of Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United StatesPenn/CHOP Center of Excellence in Friedreich’s Ataxia, Philadelphia, PA, United StatesPenn/CHOP Center of Excellence in Friedreich’s Ataxia, Philadelphia, PA, United StatesDepartments of Pediatrics and Neurology, Children’s Hospital of Philadelphia, Philadelphia, PA, United StatesDepartments of Pediatrics and Neurology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United StatesCenter of Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United StatesPenn/CHOP Center of Excellence in Friedreich’s Ataxia, Philadelphia, PA, United StatesFriedreich’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 G41-L42 and K80-S81 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.https://www.frontiersin.org/articles/10.3389/fnins.2022.874768/fullassay validationsmass spectrometrystable isotope dilutionneurodegenerative diseaseliquid chromatographyhomozygous Friedreich’s ataxia |
| spellingShingle | Qingqing Wang Qingqing Wang Laurent Laboureur Laurent Laboureur Liwei Weng Liwei Weng Nicolas M. Eskenazi Nicolas M. Eskenazi Lauren A. Hauser Lauren A. Hauser Lauren A. Hauser Clementina Mesaros Clementina Mesaros David R. Lynch David R. Lynch David R. Lynch Ian A. Blair Ian A. Blair Simultaneous Quantification of Mitochondrial Mature Frataxin and Extra-Mitochondrial Frataxin Isoform E in Friedreich’s Ataxia Blood Frontiers in Neuroscience assay validations mass spectrometry stable isotope dilution neurodegenerative disease liquid chromatography homozygous Friedreich’s ataxia |
| 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 | assay validations mass spectrometry stable isotope dilution neurodegenerative disease liquid chromatography homozygous Friedreich’s ataxia |
| url | https://www.frontiersin.org/articles/10.3389/fnins.2022.874768/full |
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