Liquid Chromatography–Mass Spectrometry Analysis of Frataxin Proteoforms in Whole Blood as Biomarkers of the Genetic Disease Friedreich’s Ataxia

Abstract

Friedreich’s ataxia (FRDA) is caused primarily by expanded GAA repeats in intron 1 of both alleles of the FXN gene, which causes transcriptional silencing and reduced expression of frataxin mRNA and protein. FRDA is characterized by slowly progressive ataxia and cardiomyopathy. Symptoms generally appear during adolescence, and patients slowly progress to wheelchair dependency usually in the late teens or early twenties with death on average in the 4th decade. There are two known mature proteoforms of frataxin. Mitochondrial frataxin (frataxin-M) is a 130-amino acid protein with a molecular weight of 14,268 Da, and there is an alternatively spliced N-terminally acetylated 135-amino acid form (frataxin-E) with a molecular weight of 14,953 Da found in erythrocytes. There is reduced expression of frataxin in the heart and brain, but frataxin is not secreted into the systemic circulation, so it cannot be analyzed in serum or plasma. Blood is a readily accessible biofluid that contains numerous different cell types that express frataxin. We have found that pig blood can serve as an excellent surrogate matrix to validate an assay for frataxin proteoforms because pig frataxin is lost during the immunoprecipitation step used to isolate human frataxin. Frataxin-M is expressed in blood cells that contain mitochondria, whereas extra-mitochondrial frataxin-E is found in erythrocytes. This means that the analysis of frataxin in whole blood provides information on the concentration of both proteoforms without having to isolate the individual cell types. In the current study, we observed that the distributions of frataxin levels for a sample of 25 healthy controls and 50 FRDA patients were completely separated from each other, suggesting 100% specificity and 100% sensitivity for distinguishing healthy controls from FRDA cases, a very unusual finding for a biomarker assay. Additionally, frataxin levels were significantly correlated with the GAA repeat length and age of onset with higher correlations for extra-mitochondrial frataxin-E than those for mitochondrial frataxin-M. These findings auger well for using frataxin levels measured by the validated stable isotope dilution ultrahigh-performance liquid chromatography–multiple reaction monitoring/mass spectrometry assay to monitor therapeutic interventions and the natural history of FRDA. Our study also illustrates the utility of using whole blood for protein disease biomarker discovery and validation.

Calcitriol treatment is safe and increases frataxin levels in Friedreich Ataxia patients

“…These studies have measured frataxin in fibroblasts, lymphocytes, muscle biopsies (35), buccal cells, peripheral blood ( 36, 37 ) and platelets (29, 38). These studies have shown that frataxin levels in peripheral tissues ranges from 30% to 40% of control levels in FRDA patients and from 60 to 80% of control levels in FRDA carriers ( 39-42 ). As we have observed similar differences between the three groups, we can conclude that the results obtained are accurate.…”

Expression and processing of mature human frataxin after gene therapy in mice

“…While there are currently no curative treatments for the cardiac manifestations of FRDA, numerous therapeutic approaches are being tested in pre-clinical models 9 – 11 As FRDA is a genetic disease caused by an autosomal recessive mutation in the frataxin gene ( FXN ) that encodes the highly conserved frataxin protein (FXN), genetic approaches for treatment are under investigation 12 – 14 . FRDA-related mutations classically consist of a trinucleotide (GAA) repeat expansion within the first FXN intron, which results in DNA triplex formation, epigenetic silencing, and consequently reduced FXN protein production 15 – 17 . While the intracellular role of FXN has not been clearly defined 18 , 19 , FXN deficiency compromises iron-sulfur (Fe-S) cluster biosynthesis, leading to mitochondrial iron overload, mitochondrial dysfunction, and oxidative stress 20 that can culminate in the neuro- and cardio-degeneration characteristics of FRDA 21 – 24 .…”

“…Gene replacement (e.g., gene therapy) represents a promising approach to treating FRDA 10 , 11 . One of the pivotal challenges critical to the success of gene therapy is to induce sufficient FXN protein expression to achieve therapeutic efficacy while limiting the toxicity induced by its overexpression, which can result in impaired Fe-S biogenesis and cellular/mitochondrial function, and eventually lead to cell death 17 . Previous gene therapy studies conducted in mouse models have shown mature hFXN expression at levels that cause cardiotoxicity and hepatotoxicity, leading to reduced efficacy 25 , 26 .…”

Long non-coding RNA TUG1 is down-regulated in Friedreich’s ataxia

“…This is because the active form of FXN is not detectable in serum or plasma; it predominantly resides in erythrocytes 8 . A novel technique utilizing stable isotope dilution Liquid Chromatography-Mass Spectrometry to target the isoform E of FXN from whole blood has been developed 8,10 . However, questions remain regarding its cost-effectiveness, and it is mainly used to assess the efficacy of interventions that aim to increase FXN levels.…”