RNA sequencing of peripheral blood in amyotrophic lateral sclerosis reveals distinct molecular subtypes: Considerations for biomarker discovery.

AIM: Amyotrophic lateral sclerosis (ALS) is a heterogeneous neurodegenerative disease with limited therapeutic options. A key factor limiting the development of effective therapeutics is the lack of disease biomarkers. We sought to assess whether biomarkers for diagnosis, prognosis or cohort stratification could be identified by RNA sequencing (RNA-seq) of ALS patient peripheral blood. METHODS: Whole blood RNA-seq data were generated for 96 Australian sporadic ALS (sALS) cases and 48 healthy controls (NCBI GEO accession GSE234297). Differences in sALS-control gene expression, transcript usage and predicted leukocyte proportions were assessed, with pathway analysis used to predict the activity state of biological processes. Weighted Gene Co-expression Network Analysis (WGCNA) and machine learning algorithms were applied to search for diagnostic and prognostic gene expression patterns. Unsupervised clustering analysis was employed to determine whether sALS patient subgroups could be detected. RESULTS: Two hundred and forty-five differentially expressed genes were identified in sALS patients relative to controls, with enrichment of immune, metabolic and stress-related pathways. sALS patients also demonstrated switches in transcript usage across a small set of genes. We established a classification model that distinguished sALS patients from controls with an accuracy of 78% (sensitivity: 79%, specificity: 75%) using the expression of 20 genes. Clustering analysis identified four patient subgroups with gene expression signatures and immune cell proportions reflective of distinct peripheral effects. CONCLUSIONS: Our findings suggest that peripheral blood RNA-seq can identify diagnostic biomarkers and distinguish molecular subtypes of sALS patients however, its prognostic value requires further investigation.

Short tandem repeat expansions in sporadic amyotrophic lateral sclerosis and frontotemporal dementia.

Pathogenic short tandem repeat (STR) expansions cause over 20 neurodegenerative diseases. To determine the contribution of STRs in sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), we used ExpansionHunter, REviewer, and polymerase chain reaction validation to assess 21 neurodegenerative disease-associated STRs in whole-genome sequencing data from 608 patients with sporadic ALS, 68 patients with sporadic FTD, and 4703 matched controls. We also propose a data-derived outlier detection method for defining allele thresholds in rare STRs. Excluding C9orf72 repeat expansions, 17.6% of clinically diagnosed ALS and FTD cases had at least one expanded STR allele reported to be pathogenic or intermediate for another neurodegenerative disease. We identified and validated 162 disease-relevant STR expansions in C9orf72 (ALS/FTD), ATXN1 [spinal cerebellar ataxia type 1 (SCA1)], ATXN2 (SCA2), ATXN8 (SCA8), TBP (SCA17), HTT (Huntington's disease), DMPK [myotonic dystrophy type 1 (DM1)], CNBP (DM2), and FMR1 (fragile-X disorders). Our findings suggest clinical and pathological pleiotropy of neurodegenerative disease genes and highlight their importance in ALS and FTD.

NEK1 and STMN2 short tandem repeat lengths are not associated with Australian amyotrophic lateral sclerosis risk.

Sporadic amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a complex genetic architecture. The lengths of two short tandem repeats (STRs), at the NEK1 and STMN2 loci, were recently associated with ALS risk in cohorts of European descent. The STMN2 STR was proposed to be predictive of clinical features including the age of onset and disease duration in bulbar onset cases. We sought to investigate NEK1 and STMN2 STR lengths in a cohort of Australian sporadic ALS cases (n = 608) and neurologically healthy controls (n = 4689) of European ancestry. ExpansionHunter was used to determine NEK1 and STMN2 STR length genotypes from whole-genome sequencing data followed by PCR validation of predicted lengths. No significant association was identified between sporadic ALS risk and the length of either STR. Further, neither NEK1 nor STMN2 STR lengths were indicative of the age of onset or disease duration. We report that the NEK1 and STMN2 STRs were not associated with ALS risk or clinical features in this Australian sporadic ALS cohort.