A Combined Nucleic Acid and Protein Analysis in Friedreich Ataxia: Implications for Diagnosis, Pathogenesis and Clinical Trial Design

Francesco Saccà,Angela Marsili,Filippo M Santorelli,Alessandra Tessa,Vincenzo Brescia Morra,Giorgia Puorro,Raffaele Piro,Pierpaolo Sorrentino,Giuseppe De Michele,Sergio Cocozza,Alessandra Denaro,Antonella Antenora,Alessandro Filla,Chiara Pane,Mel Feany

doi:10.1371/journal.pone.0017627

Abstract

BackgroundFriedreich's ataxia (FRDA) is the most common hereditary ataxia among caucasians. The molecular defect in FRDA is the trinucleotide GAA expansion in the first intron of the FXN gene, which encodes frataxin. No studies have yet reported frataxin protein and mRNA levels in a large cohort of FRDA patients, carriers and controls.Methodology/Principal FindingsWe enrolled 24 patients with classic FRDA phenotype (cFA), 6 late onset FRDA (LOFA), all homozygous for GAA expansion, 5 pFA cases who harbored the GAA expansion in compound heterozygosis with FXN point mutations (namely, p.I154F, c.482+3delA, p.R165P), 33 healthy expansion carriers, and 29 healthy controls. DNA was genotyped for GAA expansion, mRNA/FXN was quantified in real-time, and frataxin protein was measured using lateral-flow immunoassay in peripheral blood mononuclear cells (PBMCs). Mean residual levels of frataxin, compared to controls, were 35.8%, 65.6%, 33%, and 68.7% in cFA, LOFA, pFA and healthy carriers, respectively. Comparison of both cFA and pFA with controls resulted in 100% sensitivity and specificity, but there was overlap between LOFA, carriers and controls. Frataxin levels correlated inversely with GAA1 and GAA2 expansions, and directly with age at onset. Messenger RNA expression was reduced to 19.4% in cFA, 50.4% in LOFA, 52.7% in pFA, 53.0% in carriers, as compared to controls (p<0.0001). mRNA levels proved to be diagnostic when comparing cFA with controls resulting in 100% sensitivity and specificity. In cFA and LOFA patients mRNA levels correlated directly with protein levels and age at onset, and inversely with GAA1 and GAA2.Conclusion/SignificanceWe report the first explorative study on combined frataxin and mRNA levels in PBMCs from a cohort of FRDA patients, carriers and healthy individuals. Lateral-flow immunoassay differentiated cFA and pFA patients from controls, whereas determination of mRNA in q-PCR was sensitive and specific only in cFA.

Highlights

Friedreich’s ataxia (FRDA), an autosomal recessive neurodegenerative disorder, is the most common hereditary ataxia among Caucasians [1]
We report the first combined study on frataxin protein and mRNA levels in peripheral blood mononuclear cells (PBMCs) from a large cohort of FRDA patients, carriers and healthy individuals
In our study residual frataxin levels were 35.8% of mean control levels in Classic FRDA (cFA), and 68.7% in carriers. These levels are slightly dissimilar to those previously reported in FRDA patients [10,11,12], and carriers [10,11]

Summary

Introduction

Friedreich’s ataxia (FRDA), an autosomal recessive neurodegenerative disorder, is the most common hereditary ataxia among Caucasians [1]. The molecular defect in FRDA is the trinucleotide GAA expansion in the first intron of the FXN gene [4]. FXN mRNA was found to be reduced to 13–30% in FRDA patients, and to 40% in carriers, as compared to control mRNA [5]. The residual amount of frataxin protein in FRDA patients varies between 4 and 29% of the level seen in normal control, and shows an inverse correlation with the size of the GAA1 repeat [6]. Friedreich’s ataxia (FRDA) is the most common hereditary ataxia among caucasians. The molecular defect in FRDA is the trinucleotide GAA expansion in the first intron of the FXN gene, which encodes frataxin. No studies have yet reported frataxin protein and mRNA levels in a large cohort of FRDA patients, carriers and controls

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