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.