Poly-GP in cerebrospinal fluid links C9orf72-associated dipeptide repeat expression to the asymptomatic phase of ALS/FTD

Abstract

The C9orf72 GGGGCC repeat expansion is a major cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). Non-conventional repeat translation results in five dipeptide repeat proteins (DPRs), but their clinical utility, overall significance, and temporal course in the pathogenesis of c9ALS/FTD are unclear, although animal models support a gain-of-function mechanism. Here, we established a poly-GP immunoassay from cerebrospinal fluid (CSF) to identify and characterize C9orf72 patients. Significant poly-GP levels were already detectable in asymptomatic C9orf72 mutation carriers compared to healthy controls and patients with other neurodegenerative diseases. The poly-GP levels in asymptomatic carriers were similar to symptomatic c9ALS/FTD cases. Poly-GP levels were not correlated with disease onset, clinical scores, and CSF levels of neurofilaments as a marker for axonal damage. Poly-GP determination in CSF revealed a C9orf72 mutation carrier in our cohort and may thus be used as a diagnostic marker in addition to genetic testing to screen patients. Presymptomatic expression of poly-GP and likely other DPR species may contribute to disease onset and thus represents an alluring therapeutic target.

Keywords: C9orf72; amyotrophic lateral sclerosis; biomarker; cerebrospinal fluid; frontotemporal dementia.

Figure 1. Validation of a novel poly‐GP‐specific immunoassay

1. A

   Immunohistochemistry of frontal cortex from ALS/FTD cases with or without C9orf72 repeat expansion using poly‐GP antibodies 18H8 and 3F9. Both antibodies detect neuronal cytoplasmic inclusions specifically in the C9orf72 case (arrows). Hybridoma supernatants were used at 1:250 dilution as described previously (Schludi et al, 2015). Scale bar 20 μm.

2. B, C

   Poly‐GP sandwich immunoassay with anti‐GP antibodies 18H8 and 3F9 detects purified GST‐GP₁₅ below 0.03 ng/ml (B), but no other 15‐mer DPRs fused to GST at 1 μg/ml. Data are shown as mean ± SD (n = 2) (C). A four‐parameter logistic curve was used to fit the dose–response using Prism 7.01 software.

Figure EV1. The poly‐GP immunoassay is reproducible

1. A–D

   Poly‐GP sandwich immunoassay with anti‐GP antibodies 18H8 and 3F9 was used to analyze the GST‐GP₁₅ standard at four concentrations. Background‐corrected absolute values, mean, and standard deviation (SD) for n = 4 GST‐GP₁₅ intra‐plate replicates (A), n = 3 inter‐plate replicates (B), and n = 3 day‐to‐day replicates (C). Mean, SD, and the coefficient of variance (CV) for all conditions are listed in (D).

Figure 2. Poly‐GP expression is increased in CSF of asymptomatic and symptomatic C9orf72 mutation carriers

1. A

   Poly‐GP was measured using immunoassay in an age‐matched control population without signs of a neurodegenerative disease (ND‐CON, n = 18–20), C9orf72‐negative offspring of C9orf72 mutation carriers (NonC9‐F1, n = 8) in patients with other neurodegenerative diseases, that is, Alzheimer's (AD, n = 24) and Parkinson's disease (PD, n = 14), sporadic ALS (sALS, n = 18) and FTD (sFTD, n = 11) patients, and asymptomatic (C9‐F1, n = 10) and symptomatic C9orf72 mutation carriers with ALS (c9ALS, n = 9) and FTD (c9FTD, n = 11). The c9FTD patient indicated by the filled, red circle was initially seen under the differential diagnosis of AD, but after poly‐GP measurement followed by C9orf72 genotyping reclassified as c9FTD.

2. B

   Receiver operating characteristic (ROC) curve analysis of poly‐GP levels for the discrimination of C9orf72 mutation carriers vs. non‐carriers. The cutoff (43.5) was calculated using the Youden index and is shown as a dotted line in (A). AUC, area under the curve; Sens, sensitivity; Spec, specificity.

3. C, D

   (C) Phosphorylated neurofilament heavy chain (pNfH) and (D) neurofilament light chain (NfL) were measured using an established ELISA.

Data information: Groups were compared by Kruskal–Wallis test and Dunn's post hoc test. Bars and whiskers are median and interquartile range, and circles are individual values. Exact P‐values poly‐GP (A): ND‐CON vs. c9FTD: P = 0.0477; PD vs. AD: P = 0.0053; ND‐CON vs. c9ALS: P = 0.0483; ND‐CON vs. C9‐F1: P = 0.0236; NonC9‐F1 vs. c9FTD: P = 0.0365; NonC9‐F1 vs. c9ALS: P = 0.0334; NonC9‐F1 vs. C9‐F1: P = 0.0194; sALS vs. c9FTD: P = 0.0006; sALS vs. c9ALS: P = 0.0007; sALS vs. C9‐F1: P = 0.0003; sFTD vs. c9FTD, sFTD vs. c9ALS, sFTD vs. C9‐F1, PD vs. c9FTD, PD vs. c9ALS, and PD vs. C9‐F1: P < 0.0001. Exact P‐values pNfH (C): PD vs. C9‐F1: P = 0.0121; PD vs. NonC9‐F1: P = 0.0261; sALS vs. ND‐CON: P = 0.0103; C9‐F1 vs. AD: P = 0.0334; ND‐CON vs. c9ALS: P = 0.0142; NonC9‐F1 vs. c9ALS, C9‐F1 vs. c9ALS, sALS vs. C9‐F1, and sALS vs. NonC9‐F1: P < 0.0001. Exact P‐values NfL (D): sFTD vs. C9‐F1: P = 0.0013; sFTD vs. NonC9‐F1: P = 0.0038; PD vs. C9‐F1: P = 0.0122; PD vs. NonC9‐F1: P = 0.0245; c9ALS vs. AD: P = 0.0107; sALS vs. ND‐CON: P = 0.0017; sALS vs. NonC9‐F1: P = 0.0001; sALS vs. C9‐F1, ND‐CON vs. c9ALS, NonC9‐F1 vs. c9ALS, and C9‐F1 vs. c9ALS: P < 0.0001.

Figure 3. Poly‐GP expression in CSF correlates neither with markers of neurodegeneration nor with clinical disease severity

1. A–F

   Correlation analysis of poly‐GP levels in CSF of c9ALS (A, C, E) and c9FTD cases (B, D, F). Correlation with phosphorylated neurofilament heavy chain (pNfH) and neurofilament light chain (NfL) (A, B), with disease duration at lumbar puncture (LP) and the ALSFRS‐R or FTLD‐CDR score (C, D) and with age at disease onset (E, F).

2. G

   Correlation of poly‐GP levels in CSF with the largest repeat length estimated by Southern blotting.

3. H

   Association of poly‐GP levels in CSF with disease duration at LP in c9ALS/FTD patients and with time to expected disease onset in C9‐F1 cases. Time to expected disease onset was calculated using parental age at disease onset.

Data information: Correlation analysis was performed using Spearman’s rank correlation coefficient.