Multimodal analysis of cell-free DNA identifies epigenetic biomarkers for amyotrophic lateral sclerosis diagnosis and progression
Sebastian Michels, Chaorong Chen, Wolfgang P. Ruf, M. Madhy Garcia Garcia, Frederick J. Arnold, Zhuoxing Wu, Craig L. Bennett, Daniel Shams, Leslie M. Thompson, Alyssa C. Walker, Dennis W. Dickson, Leonard Petrucelli, Johannes Dorst, Mercedes Prudencio, Wei Li, Albert R. La Spada
Sebastian Michels, Chaorong Chen, Wolfgang P. Ruf, M. Madhy Garcia Garcia, Frederick J. Arnold, Zhuoxing Wu, Craig L. Bennett, Daniel Shams, Leslie M. Thompson, Alyssa C. Walker, Dennis W. Dickson, Leonard Petrucelli, Johannes Dorst, Mercedes Prudencio, Wei Li, Albert R. La Spada
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Research Article Genetics Neuroscience

Multimodal analysis of cell-free DNA identifies epigenetic biomarkers for amyotrophic lateral sclerosis diagnosis and progression

Abstract

The role of the epigenome in age-related neurodegenerative disorders remains understudied. Here, we analyzed circulating cell-free DNA (cfDNA) from blood to detect methylation changes as a liquid biopsy for Amyotrophic Lateral Sclerosis (ALS). Our study included 20 patients with sporadic ALS, 10 patients with C9orf72-associated ALS, 10 asymptomatic carriers of the C9orf72 repeat expansion mutation, and 21 nondisease control individuals. Following targeted enzymatic methyl-sequencing (EM-seq) of approximately 4 million CpG sites, we detected numerous differentially methylated genes, including several implicated in ALS disease risk and pathogenesis. By integrating multiple epigenetic features, we delineated a distinct epigenetic signature, which achieved an average area under the curve (AUC) of 0.91 ± 0.10 upon receiver operator characteristic (ROC) analysis, which enabled detection of approximately 70% of patients with ALS with close to 100% specificity. Furthermore, we also identified a set of genes whose methylation status significantly correlated with clinical disease progression and cerebrospinal fluid (CSF) neurofilament levels. Our results reveal the potential of cfDNA-based biomarkers to accurately diagnose ALS and potentially predict disease progression.

Authors

Sebastian Michels, Chaorong Chen, Wolfgang P. Ruf, M. Madhy Garcia Garcia, Frederick J. Arnold, Zhuoxing Wu, Craig L. Bennett, Daniel Shams, Leslie M. Thompson, Alyssa C. Walker, Dennis W. Dickson, Leonard Petrucelli, Johannes Dorst, Mercedes Prudencio, Wei Li, Albert R. La Spada

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Figure 6

Expression levels of differentially methylated genes in postmortem FTD-ALS patient cortex and evaluation of effect of RIPK1 expression increase on microglia function.

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Expression levels of differentially methylated genes in postmortem FTD-A...
(A) Quantitative real-time RT-PCR analysis of RNAs isolated from the frontal cortex of patients with FTD (n = 167) or patients with FTD-ALS (n = 59) with evidence of TDP-43 histopathology on postmortem examination, as well as individuals without disease acting as controls (n = 51) to measure the expression level of NDGR2 and RIPK1. Statistical significance was calculated using the Kruskal-Wallis nonparametric test. *P < 0.05, ****P < 0.0001. (B) iTF-microglia were transduced with a RIPK1 lentivirus expression or empty vector, then left untreated or subjected to LPS treatment. We then isolated RNAs and performed qRT-PCR analysis of the indicated cytokine genes. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001; ANOVA with Tukey’s post hoc test, n = 3 biological replicates. (C) iTF-microglia were transduced with a RIPK1 lentivirus expression or empty vector, and then left untreated or subjected to LPS treatment. We then incubated iTF-microglia with pHrodo Red Zymosan BioParticles Conjugate, and measured pHrodo Red Zymosan BioParticles fluorescence as a ratio of DAPI fluorescence. *P < 0.05, **P < 0.01; ANOVA with Tukey’s post hoc test, n = 4 biological replicates. (D) iTF-microglia were transduced with a RIPK1 lentivirus expression or empty vector, then left untreated or subjected to LPS treatment. To measure migration, iTF-Microglia were plated onto PDL and ECM coated 8 μm transwells, ADP was added as the attractant, and numbers of migrating microglia were counted, *P < 0.05, ****P < 0.001; ANOVA with Tukey’s post hoc test, n = 6 biological replicates. Boxes indicate the 25th percentile to 75th percentile with respective medians marked by a line within the box, while whiskers encompass the full range of scores for each condition. Please see the supporting raw values file for exact scores for each condition. (E) iTF-microglia were transduced with a RIPK1 lentivirus expression or empty vector, and then left untreated or subjected to LPS treatment. We then stained iTF-microglia with DAPI and counted the numbers of dead cells based on amoeboid and blebbing appearance. *P < 0.05, ***P < 0.001, ****P < 0.0001; ANOVA with Tukey’s post hoc test, n = 4 biological replicates. Error bars indicate SEM.