Exploiting the role of CSF NfL, CHIT1, and miR-181b as potential diagnostic and prognostic biomarkers for ALS

Abstract

Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disorder characterized by relentless and progressive loss of motor neurons. A molecular diagnosis, supported by the identification of specific biomarkers, might promote the definition of multiple biological subtypes of ALS, improving patient stratification and providing prognostic information. Here, we investigated the levels of neurofilament light chain (NfL), chitotriosidase (CHIT1) and microRNA-181b (miR-181b) in the cerebrospinal fluid (CSF) of ALS subjects (N = 210) as well as neurologically healthy and neurological disease controls (N = 218, including N = 74 with other neurodegenerative diseases) from a large European multicentric cohort, evaluating their specific or combined utility as diagnostic and prognostic biomarkers. NfL, CHIT1 and miR-181b all showed significantly higher levels in ALS subjects compared to controls, with NfL showing the most effective diagnostic performance. Importantly, all three biomarkers were increased compared to neurodegenerative disease controls and, specifically, to patients with Alzheimer's disease (AD; N = 44), with NfL and CHIT1 being also higher in ALS than in alpha-synucleinopathies (N = 22). Notably, ALS patients displayed increased CHIT1 levels despite having, compared to controls, a higher prevalence of a polymorphism lowering CHIT1 expression. While no relationship was found between CSF miR-181b and clinical measures in ALS (disease duration, functional disability, and disease progression rate), CSF NfL was the best independent predictor of disease progression and survival. This study deepens our knowledge of ALS biomarkers, highlighting the relative specificity of CHIT1 for ALS among neurodegenerative diseases and appraising the potential diagnostic utility of CSF miR-181b.

Keywords: ALS; Biomarker; CHIT1; CSF; MiR-181b; NfL.

Fig. 1

CSF NfL, CHIT1 and miR-181b distribution in ALS and controls. A–C CSF NfL, CHIT1 and miR-181b levels in ALS compared to all control individuals. D–F CSF NfL, CHIT1 and miR-181b levels in ALS compared to patients with AD and synucleinopathies (CTRL-3 group). Scatter dot plot values represent median and interquartile range. Symbols of statistically significant differences: *p < 0.05; **p < 0.01; ****p < 0.0001 (Kruskal–Wallis test). G–I ROC curves of CSF NfL, CHIT1, miR-181b in ALS patients vs. all control individuals

Fig. 2

Analysis of CHIT1 polymorphism. A distribution of wild-type and mutated alleles in individuals with CHIT1 levels equal to 390 pg/mL vs. above 390 pg/mL (****p < 0.0001). B distribution of wild-type and mutated alleles in ALS patients and controls (*p = 0.021). C comparison of CSF CHIT1 levels in wild-type homozygotes vs. heterozygous/homozygous polymorphism carriers in ALS cohort (*p = 0.014)

Fig. 3

Correlation between biomarkers and clinical variables. Correlation analyses of CSF NfL, CHIT1 and miR-181b with age at evaluation (A-C), disease duration (D-F), ALSFRS-R (G-I) and FVC (L-N) (r = Spearman’s coefficient). ALSFRS-R Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised. FVC forced vital capacity (expressed as % of predicted value)

Fig. 4

Correlation between biomarkers and disease progression rate. A-C correlation of CSF NfL, CHIT1 and miR-181b with disease progression rate (DPR) (r = Spearman’s coefficient). D-F CSF NfL, CHIT1 and miR-181b levels in fast progressing ALS cases compared to slow progressing patients (*p < 0.05; ****p < 0.0001; Mann–Whitney test). Scatter dot plot values represent median and interquartile range

Fig. 5

Survival analysis. A-C Kaplan–Meier curves according to CSF NfL, CHIT1, and miR-181b levels. D survival estimates according to CHIT1 levels in patients with CSF NfL levels below the median. Survival time was calculated from disease onset and median values of these biomarkers were used as cut-offs