A Blood Transcriptomic Resource for ALS Highlights Disease-Associated Signatures and Alternative Splicing Events

Amyotrophic lateral sclerosis (ALS) is a neurogenerative disease resulting from progressive degeneration of motor neurons leading to systemic consequences. Despite being the most common motor neuron disease, with increasing global prevalence, limited treatment options exist. Emerging evidence from genetic studies and pathology analyses implicates RNA dysregulation in ALS pathogenesis, however, deep, comprehensive RNA sequencing studies have not been carried out.

Here, we analysed >240 ALS and control whole blood transcriptome samples. Cross-sectional (N_cases=121, N_controls=53) and longitudinal (N_observations=103) cohorts supported complementary expression analyses of disease mechanisms across disease stages. Both short (N=241) and long-read (N=16) technologies were utilised to discover splicing changes. Total RNA was extracted from PAXgene whole blood RNA tubes before libraries (Illumina Stranded Total RNA RiboZero Plus) were prepared and sequenced (∼50M PE reads per sample). Long-read sequencing was performed using the Mas-Seq protocol with Kinnex full-length RNA prep kit and sequenced (PacBio Revio platform, 10M reads per sample) for full-length transcripts.

Case-control cohort analyses identified 50 significantly differentially expressed genes, with pathway analyses implicating RNA processing and immune system regulation. Findings were corroborated using existing ALS RNAseq datasets from blood (correlation >0.4), iPSC-MN and post-mortem tissues. Alternative splicing (AS) analyses (LeafCutter) identified 62 clusters. Within-case analyses involved ALS cases with multiple (2-4) visits, detected 144 genes associated with disability progression over time. The long-read sequencing (N_cases=8, N_controls=8) provided novel discovery insights, in particular in the HLA region.

This comprehensive blood-based transcriptomic dataset reveals both known and novel disease mechanisms in ALS, offering valuable insights that could inform future research and therapeutic development. The results of this study may inform and refine the prioritization of candidate genes and loci in future ALS research.