Eric C. Deutsch

A rapid, noninvasive immunoassay for frataxin: utility in assessment of Friedreich ataxia.

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by reduced amounts of the mitochondrial protein frataxin. Frataxin levels in research studies are typically measured via Western blot analysis from patient fibroblasts, lymphocytes, or muscle biopsies; none of these is ideal for rapid detection in large scale clinical studies. Recently, a rapid, noninvasive lateral flow immunoassay was developed to accurately measure picogram levels of frataxin protein and shown to distinguish lymphoblastoid cells from FRDA carriers, patients and controls. We expanded the immunoassay to measure frataxin directly in buccal cells and whole blood from a large cohort of controls, known carriers and patients typical of a clinical trial population. The assay in buccal cells shared a similar degree of variability with previous studies conducted in lymphoblastoid cells (~10% coefficient of variation in controls). Significant differences in frataxin protein quantity were seen between the mean group values of controls, carriers, and patient buccal cells (100, 50.2, and 20.9% of control, respectively) and in protein extracted from whole blood (100, 75.3, and 32.2%, respectively), although there was some overlap between the groups. In addition, frataxin levels were inversely related to GAA repeat length and correlated directly with age of onset. Subjects with one expanded GAA repeat and an identified frataxin point mutation also carried frataxin levels in the disease range. Some patients displaying an FRDA phenotype but carrying only a single identifiable mutation had frataxin levels in the FRDA patient range. One patient from this group has a novel deletion that included exons 2 and 3 of the FXN gene based on multiplex ligation-dependent probe amplification (MLPA) analysis of the FXN gene. The lateral flow immunoassay may be a useful means to noninvasively assess frataxin levels repetitively with minimal discomfort in FRDA patients in specific situations such as clinical trials, and as a complementary diagnostic tool to aid in identification and characterization of atypical patients.

A0001 in Friedreich ataxia: Biochemical characterization and effects in a clinical trial†‡§¶

This study tested the ability of A0001 (α‐tocopheryl quinone; EPI‐A0001), a potent antioxidant, to improve in vitro measures, glucose metabolism, and neurological function in Friedreich ataxia. We used an in vitro study of protection from cell toxicity followed by a double‐blind, randomized, placebo‐controlled trial of 2 doses of A0001 in 31 adults with Friedreich ataxia. The primary clinical trial outcome was the Disposition Index, a measure of diabetic tendency, from a frequently sampled intravenous glucose tolerance test, evaluated 4 weeks into therapy. Secondary neurologic measures included the Friedreich Ataxia Rating Scale. A0001 potently inhibited cell death in Friedreich ataxia models in vitro. For the clinical trial, mean guanine‐adenine‐adenine repeat length was 699, and mean age was 31 years. Four weeks after treatment initiation, differences in changes in the Disposition Index between subjects treated with A0001 and placebo were not statistically significant. In contrast, a dose‐dependent improvement in the Friedreich Ataxia Rating Scale score was observed. Patients on placebo improved 2.0 rating scale points, whereas patients on low‐dose A0001 improved by 4.9 points (P = .04) and patients on a high dose improved by 6.1 points (P < .01). Although A0001 did not alter the Disposition Index, it caused a dose‐dependent improvement in neurologic function, as measured by the Friedreich Ataxia Rating Scale. Longer studies will assess the reproducibility and persistence of neurologic benefit.

Blood cells from Friedreich ataxia patients harbor frataxin deficiency without a loss of mitochondrial function

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by GAA triplet expansions or point mutations in the FXN gene on chromosome 9q13. The gene product called frataxin, a mitochondrial protein that is severely reduced in FRDA patients, leads to mitochondrial iron accumulation, Fe-S cluster deficiency and oxidative damage. The tissue specificity of this mitochondrial disease is complex and poorly understood. While frataxin is ubiquitously expressed, the cellular phenotype is most severe in neurons and cardiomyocytes. Here, we conducted comprehensive proteomic, metabolic and functional studies to determine whether subclinical abnormalities exist in mitochondria of blood cells from FRDA patients. Frataxin protein levels were significantly decreased in platelets and peripheral blood mononuclear cells from FRDA patients. Furthermore, the most significant differences associated with frataxin deficiency in FRDA blood cell mitochondria were the decrease of two mitochondrial heat shock proteins. We did not observe profound changes in frataxin-targeted mitochondrial proteins or mitochondrial functions or an increase of apoptosis in peripheral blood cells, suggesting that functional defects in these mitochondria are not readily apparent under resting conditions in these cells.

Development of Frataxin Gene Expression Measures for the Evaluation of Experimental Treatments in Friedreich’s Ataxia

Background Friedreich ataxia is a progressive neurodegenerative disorder caused by GAA triplet repeat expansions or point mutations in the FXN gene and, ultimately, a deficiency in the levels of functional frataxin protein. Heterozygous carriers of the expansion express approximately 50% of normal frataxin levels yet manifest no clinical symptoms, suggesting that therapeutic approaches that increase frataxin may be effective even if frataxin is raised only to carrier levels. Small molecule HDAC inhibitor compounds increase frataxin mRNA and protein levels, and have beneficial effects in animal models of FRDA. Methodology/Principal Findings To gather data supporting the use of frataxin as a therapeutic biomarker of drug response we characterized the intra-individual stability of frataxin over time, determined the contribution of frataxin from different components of blood, compared frataxin measures in different cell compartments, and demonstrated that frataxin increases are achieved in peripheral blood mononuclear cells. Frataxin mRNA and protein levels were stable with repeated sampling over four and 15 weeks. In the 15-week study, the average CV was 15.6% for protein and 18% for mRNA. Highest levels of frataxin in blood were in erythrocytes. As erythrocytes are not useful for frataxin assessment in many clinical trial situations, we confirmed that PBMCs and buccal swabs have frataxin levels equivalent to those of whole blood. In addition, a dose-dependent increase in frataxin was observed when PBMCs isolated from patient blood were treated with HDACi. Finally, higher frataxin levels predicted less severe neurological dysfunction and were associated with slower rates of neurological change. Conclusions/Significance Our data support the use of frataxin as a biomarker of drug effect. Frataxin levels are stable over time and as such a 1.5 to 2-fold change would be detectable over normal biological fluctuations. Additionally, our data support buccal cells or PBMCs as sources for measuring frataxin protein in therapeutic trials.

Unanswered questions in Friedreich ataxia.

During the past 15 years, the pace of research advancement in Friedreich ataxia has been rapid. The abnormal gene has been discovered and its gene product characterized, leading to the development of new evidence-based therapies. Still, various unsettled issues remain that affect clinical trials. These include the level of frataxin deficiency needed to cause disease, the mechanism by which frataxin-deficient mitochondrial dysfunction leads to symptomatology, and the reason selected cells are most affected in Friedreich ataxia. In this review, we summarize these questions and propose testable hypotheses for their resolution.

Frataxin levels in peripheral tissue in Friedreich ataxia.

Friedreich ataxia (FRDA) is an autosomal recessive ataxia resulting from mutations in the frataxin gene (FXN). Such mutations, usually expanded guanine–adenine–adenine (GAA) repeats, give rise to decreased levels of frataxin protein in both affected and unaffected tissues. The goal was to understand the relationship of frataxin levels in peripheral tissues to disease status.

Radiosensitizing effects of the prenyltransferase inhibitor AZD3409 against RAS mutated cell lines.

Mutations at the H-, N-, and K-ras loci are among the most frequent genetic alterations in human cancers. In this study, we have investigated the effect of AZD3409, a novel, peptidomimetic prenyltransferase inhibitor (PTI), on the radiosensitivity of cells with mutated ras alleles. AZD3409, developed by AstraZeneca, inhibits both farnesyl- and geranylgeranyl transferase in cell free systems. AZD3409 inhibits the growth of a variety of human cancer cell lines, including cells that express mutant alleles of either K- or H- ras and was well tolerated when administered orally to healthy volunteers in a phase I clinical trial. We have previously shown that PTI can radiosensitize human and rodent cancer cell lines that express activated RAS. Here we assessed the ability of AZD3409 to radiosensitize human cancer cell lines in vivo and in vitro and the activation state of RAS proteins in treated cells. Once daily oral administration of AZD3409 to nude mice bearing PSN-1 and MiaPaCa-2 human pancreatic cancer xenografts expressing mutant K-ras was well tolerated and resulted in a supra-additive reduction in clonogenic cell survival after irradiation. Similarly, AZD3409 reduced clonogenic survival in cells that express either mutant K- or H- ras in vitro. We next examined the effect of AZD3409 on the processing and activation of K- and H-RAS. AZD3409-mediated radiosensitization, both in vivo and in vitro, correlates with a decrease in H-RAS processing without detectable effect on K-RAS processing. RAS activation assays show that the decreased H-RAS processing is accompanied by decreased H-RAS activation in cell lines with mutations in either K- or H-ras. However, no decrease in K-RAS activation was detected. Thus, radiosensitization of human cancer cells that express mutated K-RAS occurred under conditions where AZD3409 inhibits the activation of farnesylated H-RAS, but did not inhibit K-RAS activation.

Human platelets as a platform to monitor metabolic biomarkers using stable isotopes and LC-MS.

BACKGROUND Intracellular metabolites such as CoA thioesters are modulated in a number of clinical settings. Their accurate measurement from surrogate tissues such as platelets may provide additional information to current serum and urinary biomarkers. METHODS Freshly isolated platelets from healthy volunteers were treated with rotenone, propionate or isotopically labeled metabolic tracers. Using a recently developed LC-MS-based methodology, absolute changes in short-chain acyl-CoA thioesters were monitored, as well as relative metabolic labeling using isotopomer distribution analysis. RESULTS Consistent with in vitro experiments, isolated platelets treated with rotenone showed decreased intracellular succinyl-CoA and increased β-hydroxybutyryl-CoA, while propionate treatment resulted in increased propionyl-CoA. In addition, isotopomers of the CoAs were readily detected in platelets treated with the [(13)C]- or [(13)C(15)N]-labeled metabolic precursors. CONCLUSION Here, we show that human platelets can provide a powerful ex vivo challenge platform with potential clinical diagnostic and biomarker discovery applications.

Stable isotopes and LC–MS for monitoring metabolic disturbances in Friedreich's ataxia platelets

BACKGROUND Friedreichs ataxia (FRDA) is an autosomal recessive disease with metabolic abnormalities that have been proposed to play an important role in the resulting neurodegeneration and cardiomyopathy. The inability to access the highly affected neuronal and cardiac tissues has hampered metabolic evaluation and biomarker development. METHODS Employment of a LC-MS-based method to determine whether platelets isolated from patients with FRDA exhibit differentiable metabolism compared with healthy controls. RESULTS Isotopologue analysis showed a marked decrease in glucose incorporation with a concomitant increase in palmitate-derived acyl-CoA thioesters in FRDA platelets compared with controls. CONCLUSION Our findings demonstrate that platelets can be used as a surrogate tissue for in vivo biomarker studies to monitor new therapeutic approaches for the treatment of FRDA.

High-Throughput Immunoassay for the Biochemical Diagnosis of Friedreich Ataxia in Dried Blood Spots and Whole Blood

BACKGROUND Friedreich ataxia (FRDA) is caused by reduced frataxin (FXN) concentrations. A clinical diagnosis is typically confirmed by DNA-based assays for GAA-repeat expansions or mutations in the FXN (frataxin) gene; however, these assays are not applicable to therapeutic monitoring and population screening. To facilitate the diagnosis and monitoring of FRDA patients, we developed an immunoassay for measuring FXN. METHODS Antibody pairs were used to capture FXN and an internal control protein, ceruloplasmin (CP), in 15 μL of whole blood (WB) or one 3-mm punch of a dried blood spot (DBS). Samples were assayed on a Luminex LX200 analyzer and validated according to standard criteria. RESULTS The mean recovery of FXN from WB and DBS samples was 99%. Intraassay and interassay imprecision (CV) values were 4.9%-13% and 9.8%-16%, respectively. The FXN limit of detection was 0.07 ng/mL, and the reportable range of concentrations was 2-200 ng/mL. Reference adult and pediatric FXN concentrations ranged from 15 to 82 ng/mL (median, 33 ng/mL) for DBS and WB. The FXN concentration range was 12-22 ng/mL (median, 15 ng/mL) for FRDA carriers and 1-26 ng/mL (median 5 ng/mL) for FRDA patients. Measurement of the FXN/CP ratio increased the ability to distinguish between patients, carriers, and the reference population. CONCLUSIONS This assay is applicable to the diagnosis and therapeutic monitoring of FRDA. This assay can measure FXN and the control protein CP in both WB and DBS specimens with minimal sample requirements, creating the potential for high-throughput population screening of FRDA.