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

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.

Atypical Friedreich ataxia in patients with FXN p.R165P point mutation or comorbid hemochromatosis

“…Such spatial specificity may cause more damage to neurons involved in lower limb functioning and vision, but relatively milder impairment of the neuronal substrates of speech and upper limb function. In classical FRDA, frataxin levels are very low [1,9]. p.R165P compound heterozygotes had higher mean frataxin levels than GAA-TRE homozygotes in both the present study and previous analyses of two Italian siblings [9].…”

“…In classical FRDA, frataxin levels are very low [1,9]. p.R165P compound heterozygotes had higher mean frataxin levels than GAA-TRE homozygotes in both the present study and previous analyses of two Italian siblings [9]. Their atypical disease phenotype may be related to altered function of mutated frataxin in these patients, combined with lower total frataxin expression.…”

“…We compared our findings with the published data on the only two patients with FXN p.R165P mutation previously reported in the literature [4,9].…”

Friedreich ataxia today—preparing for the final battle

“…The expansion of the intronic GAA repeat beyond the pathologic threshold inhibits FXN expression, resulting in decreased levels of FXN protein. 3,4 The EFACTS cohort study found that the number of GAA repeats on the patients' shorter allele inversely correlated with age at onset, with a mean of 745 repeats in the earlyonset group, 500 repeats in the intermediateonset group and 234 repeats in the lateonset group. The EFACTS paper 2 confirms previous reports 5, 6 that disease severity is associated with the size of the GAA expansion.…”

Gene Expression Profile in Peripheral Blood Cells of Friedreich Ataxia Patients

“…A desired theoretical drug would not only increase mRNA and protein levels of frataxin [13] but also modulate the downstream changes associated with it.…”