Poly(GR) and poly(GA) in cerebrospinal fluid as potential biomarkers for C9ORF72-ALS/FTD

Abstract

GGGGCC repeat expansion in C9ORF72, which can be translated in both sense and antisense directions into five dipeptide repeat (DPR) proteins, including poly(GP), poly(GR), and poly(GA), is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we developed sensitive assays that can detect poly(GA) and poly(GR) in the cerebrospinal fluid (CSF) of patients with C9ORF72 mutations. CSF poly(GA) and poly(GR) levels did not correlate with age at disease onset, disease duration, or rate of decline of ALS Functional Rating Scale, and the average levels of these DPR proteins were similar in symptomatic and pre-symptomatic patients with C9ORF72 mutations. However, in a patient with C9ORF72-ALS who was treated with antisense oligonucleotide (ASO) targeting the aberrant C9ORF72 transcript, CSF poly(GA) and poly(GR) levels decreased approximately 50% within 6 weeks, indicating they may serve as sensitive fluid-based biomarkers in studies directed against the production of GGGGCC repeat RNAs or DPR proteins.

Trial registration: ClinicalTrials.gov NCT01925196.

Fig. 1. Blinded measurement of poly(GR) levels in CSF in multiple cohorts of patients with C9ORF72 mutations using the MSD-based poly(GR) immunoassay.

a In spike-and-recovery experiments, recombinant (GR)₈ peptide was added to CSF from healthy individuals and subsequently serially diluted into a separate tube containing CSF from healthy individuals to create a 5-point series. The average recovery rate in two different samples was 81–88%. b The assay can detect purified (GR)₈ peptide at concentrations as low as 3 pg/ml. Replicates of 8-point series standard curve run on different dates are shown. A four-parameter logistic curve was used to fit the dose-response with GraphPad Prism 9.1. c Across the dynamic range of the assay, the coefficient of variation is less than 16% for all points except one concentration (100 pg/ml), below the 20% cutoff recommended by the FDA in standard variability for liquid-binding assays. d Dilution linearity was evaluated by measuring duplicates of 5 dilutions of 2 patient CSF samples spiked with 200 ng/ml (GR)₈ peptide. e Poly(GR) immunoassay specifically detects (GR)₈ peptide in control CSF and did not cross react with even up to 100 ng of (GP)₈ peptide in control CSF, n = 3 independent experiments, values are mean ± s.e.m. f CSF samples from 4 healthy and 4 patients with C9ORF72 mutations from the Mayo Clinic, Florida, values are mean ± s.e.m (p = 0.0017, Unpaired t-test with Welch’s correction). g CSF samples from 3 healthy people from the NEALS cohort and 5 CSF samples from two patients with C9ORF72 mutations from the Barrow Institute, Arizona, values are mean ± s.e.m (p = 0.0012, Unpaired t-test with Welch’s correction). h 19 healthy people, 19 patients with ALS but without C9ORF72 mutations (3 patients with duplicate samples, 16 patients with single samples), and 10 patients with C9ORF72 mutations (8 patients with duplicate samples, 2 patients with single samples) from the NEALS Consortium. Values from duplicate samples are presented as mean ± s.e.m (p = 0.9460 control vs non-C9-ALS, p < 0.0001 non-C9-ALS vs C9-ALS, p < 0.0001 control vs C9-ALS Ordinary one-way ANOVA-Tukey’s multiple comparisons test). Source data are provided as a Source Data file.

Fig. 2. Poly(GR) levels in CSF do not correlate with clinical features of patients with C9ORF72 mutations.

a–c Correlations between poly(GR) levels and rate of ALSFRS-R score decline (a), age at disease onset (b), and disease duration (c), determined by two-tailed, Spearman’s rank-correlation analysis. d Poly(GR) levels in CSF of symptomatic and asymptomatic patients with C9ORF72 mutations are similar (p = 0.114 by two-tailed unpaired t-test with Mann–Whitney test). Poly(GR) concentrations are presented as values equivalent to (GR)₈ peptide and not as an absolute poly(GR) concentration since the size of poly(GR) in CSF is unknown. 25 out of 41 symptomatic CSF samples and 10 out of 14 pre-symptomatic CSF samples had poly(GR) levels within the linear range of our standard curves and are presented here. ALS Functional Rating Scale revised (ALSFRS-R). Source data are provided as a Source Data file.

Fig. 3. Poly(GA) levels in CSF do not correlate with clinical features of patients with C9ORF72 mutations.

a–c Correlation between poly(GA) levels and the rate of ALSFRS-R score decline (a), age at disease onset (b), and disease duration (c), determined by two-tailed Spearman’s rank-correlation analysis. d Poly(GA) levels in CSF from symptomatic and asymptomatic patients with C9ORF72 mutations are similar by two-tailed unpaired t-test with Mann–Whitney test. Poly(GA) concentrations are presented as values equivalent to (GA)₆₀ and not as an absolute poly(GA) concentration since the size of poly(GA) in CSF is unknown. The samples assessed in Figs. 2, 3 are overlapping but not identical and include 17 samples for which both poly(GA) and poly(GR) were detected. Source data are provided as a Source Data file.

Fig. 4. ASO treatment in a patient with C9ORF72-ALS decreases CSF DPR levels.

a Poly(GR) levels in CSF were generally stable over time in two symptomatic and two asymptomatic patients with C9ORF72 mutations. b Longitudinal trend of poly(GR) levels in CSF from a larger cohort of patients with C9ORF72 mutations. c Longitudinal trend of poly(GA) levels in CSF from patients with C9ORF72 mutations. d After ASO treatment, CSF poly(GP), poly(GA), and poly(GR) levels in a patient with C9ORF72-ALS decreased over time. Note: colors in panels b, c do not indicate the same patients. The fifth time point value for symptomatic patient 2 in panel a and the last time point for poly(GR) value in panel b were above background but below the lower limit of detection and were not included. Source data are provided as a Source Data file.