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

Abstract

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.

Copyright © 2010 Elsevier Inc. All rights reserved.

Figures

Figure 1

A. The assay remains sensitive and in the linear range through 500 pg of recombinant frataxin protein (n=3), higher than the amount of frataxin in 10 µg of total extracted protein from buccal cells (Figure 1C). Y-error bars show standard deviation. B. Normalized to GAM signal, the assay remains sensitive and in the linear range through 4000 pg of recombinant frataxin protein (n=3), levels much higher than the amount of frataxin in 10 µg of total extracted protein from buccal cells (Figure 1C). Y-error bars show standard deviation and show that error increases with increased signal, but variance remains consistent. C. The standard curve of a representative control (n=3, 0.899 µg/µl) using 0, 1, 2, 5, 10, 20, and 50 µg of total protein extracted from a cheek swab shows the assay remains in the linear range between 0–20 µg -- levels easily obtained from a typical cheek swab. All samples were normalized to 0 µg dipsticks and corrected for the completion of the assay (GAM) of the negative control. Y-error bars show standard deviation.

Figure 1

A. The assay remains sensitive and in the linear range through 500 pg of recombinant frataxin protein (n=3), higher than the amount of frataxin in 10 µg of total extracted protein from buccal cells (Figure 1C). Y-error bars show standard deviation. B. Normalized to GAM signal, the assay remains sensitive and in the linear range through 4000 pg of recombinant frataxin protein (n=3), levels much higher than the amount of frataxin in 10 µg of total extracted protein from buccal cells (Figure 1C). Y-error bars show standard deviation and show that error increases with increased signal, but variance remains consistent. C. The standard curve of a representative control (n=3, 0.899 µg/µl) using 0, 1, 2, 5, 10, 20, and 50 µg of total protein extracted from a cheek swab shows the assay remains in the linear range between 0–20 µg -- levels easily obtained from a typical cheek swab. All samples were normalized to 0 µg dipsticks and corrected for the completion of the assay (GAM) of the negative control. Y-error bars show standard deviation.

Figure 1

A. The assay remains sensitive and in the linear range through 500 pg of recombinant frataxin protein (n=3), higher than the amount of frataxin in 10 µg of total extracted protein from buccal cells (Figure 1C). Y-error bars show standard deviation. B. Normalized to GAM signal, the assay remains sensitive and in the linear range through 4000 pg of recombinant frataxin protein (n=3), levels much higher than the amount of frataxin in 10 µg of total extracted protein from buccal cells (Figure 1C). Y-error bars show standard deviation and show that error increases with increased signal, but variance remains consistent. C. The standard curve of a representative control (n=3, 0.899 µg/µl) using 0, 1, 2, 5, 10, 20, and 50 µg of total protein extracted from a cheek swab shows the assay remains in the linear range between 0–20 µg -- levels easily obtained from a typical cheek swab. All samples were normalized to 0 µg dipsticks and corrected for the completion of the assay (GAM) of the negative control. Y-error bars show standard deviation.

Figure 2

Lateral flow immunoassays showed significant differences between controls, known carriers and patients in collected buccal cell samples. A. Frataxin protein levels in buccal cells collected from controls (n=40), known carriers (n=81), patients (n=195), and non-FA movement disorder (MD) patients (n=15). Buccal cells were collected and assayed as described in the Methods section, and data is expressed as frataxin, % of average control for that day’s experiment. The carrier group contains 3 outliers (>1.5*IQR above/below quartiles), while the patient group contains 9 outliers. The p-values (Student’s t-test) were calculated for two samples assuming equal variance. B. Average frataxin protein levels in buccal samples collected in A. (expressed as % of control). Known carriers (n=81) and patients (n=195) show significant decreases in frataxin protein (50.5% and 21.1% of control, respectively) compared to controls (n=40).

Figure 2

Lateral flow immunoassays showed significant differences between controls, known carriers and patients in collected buccal cell samples. A. Frataxin protein levels in buccal cells collected from controls (n=40), known carriers (n=81), patients (n=195), and non-FA movement disorder (MD) patients (n=15). Buccal cells were collected and assayed as described in the Methods section, and data is expressed as frataxin, % of average control for that day’s experiment. The carrier group contains 3 outliers (>1.5*IQR above/below quartiles), while the patient group contains 9 outliers. The p-values (Student’s t-test) were calculated for two samples assuming equal variance. B. Average frataxin protein levels in buccal samples collected in A. (expressed as % of control). Known carriers (n=81) and patients (n=195) show significant decreases in frataxin protein (50.5% and 21.1% of control, respectively) compared to controls (n=40).

Figure 3

Lateral flow immunoassays also showed significant differences between controls, known carriers and patients in whole blood samples. A. Frataxin protein levels in whole blood samples collected from controls (n=19), known carriers (n=18), and patients (n=52). Protein from whole blood was extracted and assayed as described in the Methods section, and data is expressed as frataxin, % of average control for that day’s experiment. FRDA patients show significant decreases in frataxin protein compared to carriers and controls. The control group contains 3 outliers (>1.5*IQR above/below quartiles) carrier and patient groups contain 1 outlier. The p-values (Student’s t-test) were calculated for two samples assuming equal variance. B. Average frataxin protein levels in whole blood collected in A. with standard error Y-error bars (expressed as % of control). Similarly to buccal cells, known carriers (n=18) and patients (n=52) show significantly less amounts of frataxin protein (81.7% and 32.2% of control, respectively) compared to controls (n=19).

Figure 3

Lateral flow immunoassays also showed significant differences between controls, known carriers and patients in whole blood samples. A. Frataxin protein levels in whole blood samples collected from controls (n=19), known carriers (n=18), and patients (n=52). Protein from whole blood was extracted and assayed as described in the Methods section, and data is expressed as frataxin, % of average control for that day’s experiment. FRDA patients show significant decreases in frataxin protein compared to carriers and controls. The control group contains 3 outliers (>1.5*IQR above/below quartiles) carrier and patient groups contain 1 outlier. The p-values (Student’s t-test) were calculated for two samples assuming equal variance. B. Average frataxin protein levels in whole blood collected in A. with standard error Y-error bars (expressed as % of control). Similarly to buccal cells, known carriers (n=18) and patients (n=52) show significantly less amounts of frataxin protein (81.7% and 32.2% of control, respectively) compared to controls (n=19).

Figure 4

There are distinct relationships between frataxin levels and severity of disease. A. We confirm the suggested inverse relationship between patient age of onset and GAA repeat length of the shorter allele (p<0.001). B. Measured frataxin levels inversely correlates with GAA repeat length, suggesting that the assay is useful for assessing severity of disease in buccal cells (p<0.001). C. Age of onset correlates directly with measured frataxin levels (p<0.001).

Figure 4

There are distinct relationships between frataxin levels and severity of disease. A. We confirm the suggested inverse relationship between patient age of onset and GAA repeat length of the shorter allele (p<0.001). B. Measured frataxin levels inversely correlates with GAA repeat length, suggesting that the assay is useful for assessing severity of disease in buccal cells (p<0.001). C. Age of onset correlates directly with measured frataxin levels (p<0.001).

Figure 4

There are distinct relationships between frataxin levels and severity of disease. A. We confirm the suggested inverse relationship between patient age of onset and GAA repeat length of the shorter allele (p<0.001). B. Measured frataxin levels inversely correlates with GAA repeat length, suggesting that the assay is useful for assessing severity of disease in buccal cells (p<0.001). C. Age of onset correlates directly with measured frataxin levels (p<0.001).

Figure 5

MLPA results for a normal control (top) and for P.A.001 (bottom) revealing a heterozygous deletion of exons 2 and 3 of the FXN gene (arrows). Mutations in APTX and SETX genes are involved in ataxia-ocular apraxia.