Development of frataxin gene expression measures for the evaluation of experimental treatments in Friedreich\u27s 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

Integrating multiple evidence sources to predict transcription factor binding in the human genome

Information about the binding preferences of many transcription factors is known and characterized by a sequence binding motif. However, determining regions of the genome in which a transcription factor binds based on its motif is a challenging problem, particularly in species with large genomes, since there are often many sequences containing matches to the motif but are not bound. Several rules based on sequence conservation or location, relative to a transcription start site, have been proposed to help differentiate true binding sites from random ones. Other evidence sources may also be informative for this task. We developed a method for integrating multiple evidence sources using logistic regression classifiers. Our method works in two steps. First, we infer a score quantifying the general binding preferences of transcription factor binding at all locations based on a large set of evidence features, without using any motif specific information. Then, we combined this general binding preference score with motif information for specific transcription factors to improve prediction of regions bound by the factor. Using cross-validation and new experimental data we show that, surprisingly, the general binding preference can be highly predictive of true locations of transcription factor binding even when no binding motif is used. When combined with motif information our method outperforms previous methods for predicting locations of true binding

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

Frataxin protein and mRNA level is stable from day-to-day in whole blood.

<p>(A) Frataxin protein levels measured by dipstick assay in FRDA patient subjects over the course of 4 weeks (Cohort 1; n = 31). (B) Frataxin protein levels measured by dipstick assay in whole blood collected from 5 FRDA patients (red) and 5 related carriers (blue) weekly for 15 weeks (Cohort 2). (C) Frataxin mRNA levels measured by RT-qPCR in blood collected in PAXgene tubes from Cohort 2. Frataxin mRNA level is expressed relative to control donor levels and is normalized to the endogenous control gene <i>GAPDH</i>.</p

Response Rate for RG2833 in PBMC Isolated From FRDA Patients in Cohort 3 and Treated <i>Ex Vivo</i>.

<p>Protein, n = 52; mRNA, n = 81.</p

Frataxin protein and mRNA levels strongly correlated when measured in different cellular compartments (Cohort 3).

<p>(A) Frataxin mRNA in PBMCs and whole blood (R² = 0.84, p<0.0001). (B) Frataxin protein in PBMCs and whole blood (R² = 0.63, p<0.0001). (C) Frataxin mRNA and protein in whole blood (R² = 0.55, p<0.0001). (D) Frataxin mRNA and protein in PBMCs (R² = 0.58, p<0.0001). (E) Frataxin protein in PBMCs and cheek swabs (R² = 0.67, p<0.0001). (F) Frataxin protein in whole blood and cheek swabs (R² = 0.58, p<0.0001). Frataxin mRNA level in PBMCs and whole blood is expressed relative to control donor levels and is normalized to the endogenous control gene <i>GAPDH</i>.</p

Summary of Clinical Information and FRDA Disease Parameters by Frataxin Quartile.

<p>Data collected from the CCRN for FRDA patient registry.</p