Trajectory of blood cell populations from the pre-clinical stage across disease duration in sporadic and C9orf72-associated amyotrophic lateral sclerosis

Introduction Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease defined by degeneration of upper and lower motor neurons. The hexanucleotide repeat expansion (HRE) in C9orf72 is the most common genetic cause of ALS in populations of Western ancestry. Beyond its role in neurodegeneration, C9orf72 is thought to play a role in immune regulation: murine knockout models develop neutrophilia, splenomegaly and autoimmune-like phenotypes, suggesting that C9ORF72 protein contributes to limiting inflammation. Whether this immune dysregulation is detectable in humans through analysis of blood cell count (BCC) parameters remains to be determined. Aims of the Study Our study aimed to define clinical and haematological features of ALS patients carrying the C9orf72 HRE (C9ALS), comparing them with patients without pathogenetic mutations in major ALS-associated genes (nmALS). Materials and Methods We conducted a retrospective, longitudinal, case-control study drawing on the Emilia-Romagna Region ALS Registry (ERRALS). 37 C9ALS patients were matched to 74 nmALS controls according to sex, age at symptom onset and diagnostic delay. Clinical features were compared using Student’s t-test or Mann-Whitney U test, and Chi-Square or Fisher’s exact test; survival was analysed using Kaplan-Meier curves. A total of 462 blood samples were analysed within a time window of ± 11 years. BCCs, inflammatory ratios and composite indices were computed and compared between the two groups using generalised linear mixed models (GLMMs) with Gamma distribution and log link function. Conditional logistic regression models assessed association between earliest available haematological parameters and C9orf72 status. Results C9ALS patients showed shorter median survival compared to nmALS controls (34.66 vs 46.52 months; log-rank p = 0.0022) and a higher prevalence of concomitant FTD (21.6% vs 2.7%; p = 0.002). During the pre-diagnostic period, basophil counts were significantly lower in C9ALS patients (MR = 0.505; p = 0.0005). In samples drawn after clinical diagnosis, C9ALS patients showed reduced lymphocyte (MR = 0.869; p = 0.0323), monocyte (MR = 0.873; p = 0.0458), eosinophil (MR = 0.624; p = 0.0003), and basophil (MR = 0.505; p < 0.0001) counts, alongside elevated neutrophil-to-lymphocyte ratio (NLR) (MR = 1.202; p = 0.0492) and neutrophil-to-monocyte ratio (NMR) (MR = 1.163; p = 0.037). Longitudinal analysis showed that, in C9ALS patients, neutrophil counts increased over time (MR = 1.084 per year; p = 0.0011), while lymphocyte counts declined (MR = 0.970 per year, p = 0.0076), with a progressive rise in NLR (MR = 1.108; p = 0.0132) and composite inflammatory indices. At the earliest sampling, lower eosinophil count and higher haemoglobin and platelet levels were associated with C9orf72 status, but not with survival or ALSFRS-R decline. Discussion and Conclusions Our findings revealed that C9ALS patients presented with a clinical phenotype defined by shorter survival, higher prevalence of concurrent FTD, and trends towards faster disease progression and greater autoimmune comorbidity. Regarding BCCs, C9ALS patients showed reduced basophil counts already during the pre-diagnostic period, alongside post-diagnostic reductions in eosinophils, lymphocytes and monocytes, and elevation of systemic inflammation indices, including NLR and NMR. Longitudinal trajectories revealed progressive neutrophilia and lymphopenia in both groups, with potentially more pronounced inflammatory activation over time emerging in C9ALS patients. The early haematological profile associated with C9orf72 status, but not with survival or ALSFRS-R decline, may reflect a genotype-related immune signature. Overall, these findings are consistent with C9orf72 loss-of-function driven immune dysregulation.