Decoding sporadic amyotrophic lateral sclerosis: a genomic and transcriptomic approach

<p dir="ltr">Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the progressive degeneration of upper and lower motor neurons. There is no cure for ALS, nor an effective treatment for the vast majority of cases. Gene mutations are the only established cause of ALS. To date, mutations have been identified in around two-thirds of ALS patients with a family history of disease. For patients with apparently sporadic ALS, which comprise about 90% of all ALS cases, around 10% have been shown to carry an ALS-causal mutation. Several pathological features have been described in familial and sporadic ALS, most notably intraneuronal aggregates of phosphorylated TAR DNA-binding protein (pTDP-43) which are seen in approximately 97% of all ALS patients. However, the molecular basis of most sporadic ALS remains relatively unknown, with its investigation further complicated by marked clinical, genetic and pathological heterogeneity. This heterogeneity suggests that therapeutic approaches will need to become increasingly personalised. As such, there is an urgent need to identify the molecular features that underlie the onset and progression of sporadic ALS.</p><p dir="ltr">This thesis presents a molecular-level investigation into sporadic ALS, with a focus on the application of genomic and transcriptomic techniques to inherently heterogeneous patient-derived biospecimens. The thesis commences with an overview of the current genetic landscape of ALS and an examination into the genetic contribution of short tandem repeat expansions to sporadic ALS risk. A comprehensive in silico assessment of 1,087 ALS-implicated genetic variants revealed recurrent variant- and gene-level attributes and demonstrated that genetic variants exclusively found in sporadic ALS patients were more common in the population and had lower predicted pathogenicity than those seen in familial patients (Publication 1). The less severe characteristics of variants implicated in sporadic ALS are likely a reflection of reduced penetrance and disease effect size, raising important considerations for novel variant interpretation as research seeks to understand the genetic risk factors for sporadic ALS. Short tandem repeats (STRs) are abundant, highly polymorphic tracts of repetitive DNA units that are technically difficult to genotype and consequently, are an understudied genomic feature. STR expansions are heavily implicated in neurological disorders and expansion of a repeat in C9orf72 is the most common genetic cause of ALS and the associated neurodegenerative disease, frontotemporal dementia (FTD). Bioinformatic tools, including ExpansionHunter, have recently been developed to detect repeat expansions in short-read whole-genome sequencing (WGS) data, enabling systematic genotyping of STR loci. Leveraging a large collection of WGS data and matching DNA samples from 608 Australian sporadic ALS patients, ExpansionHunter and PCR genotyping were used to assess the repeat lengths of 21 STRs associated with neurodegenerative disease (Publication 2) and two recently reported ALS-associated STRs (Publication 3). Excluding C9orf72 repeat expansions, 17.6% of sporadic ALS and FTD patients were found to carry an STR expansion across eight STR loci (ATXN1, ATXN2, ATXN8, TBP, HTT, DMPK, CNBP and FMR1) reported to be causative or intermediate for other neurodegenerative disorders. Pathogenic repeat expansions in C9orf72 and intermediate expansions in ATXN2 had a significantly higher frequency in sporadic ALS patients relative to controls (n = 4,703). Though expansions at the other STR loci were not significantly associated with disease, their occurrence in clinically-confirmed ALS and FTD patients suggests pleiotropy of neurodegenerative disease genes. Assessment of two STR loci in NEK1 and STMN2, independently reported to be associated with ALS, found no association between repeat length and ALS risk. This study highlighted the importance of replication studies for the identification of <i>bona fide</i> ALS risk variants and the need to use large population-matched control cohorts to obtain high condence allele frequency distributions.</p><p dir="ltr">Subsequently presented in this thesis is an examination of broader molecular signatures relevant to sporadic ALS, centered around the use of RNA sequencing (RNA-seq) to globally interrogate peripheral blood and post-mortem central nervous system (CNS) tissues. Though disease-relevant molecular signatures are expected to be present in pathologically-affected CNS tissues, their inaccessibility in living patients underlies a need to identify biomarkers in peripheral samples. Despite this, surprisingly few studies have reported whether peripheral blood RNA can be used to detect biomarkers for ALS patient diagnosis, prognosis or cohort stratification. To address this, a bioinformatic pipeline was developed for the comprehensive assessment of single-collection peripheral blood RNA-seq generated for 96 sporadic ALS patients and 48 controls (Publication 4). Gene-, and for the first time, transcript-level expression differences were detected between ALS patients and controls, with enrichment for immune, metabolic and stress-related pathways. A classification model distinguished ALS patients from controls with 78% accuracy however, blood gene expression was not an accurate predictor of stage of disease progression, clinical features or patient survival. Clustering analysis identified four ALS patient subgroups with distinct gene expression signatures and predicted immune cell proportions. This heterogeneity represents an important consideration for future biomarker discovery efforts.</p><p dir="ltr">The CNS is not uniformly impacted by the neurodegenerative processes in ALS, with variable occurrence of the hallmark pTDP-43 inclusion pathology seen across neuroanatomical regions. Co-examination of regions that are variably impacted by pathology in ALS could provide insight into the mechanisms that underlie selective regional vulnerability or neuroprotection. A method for RNA/DNA co-isolation from seven strategically selected post-mortem CNS regions was optimised and used to generate a pilot multi-region RNA-seq cohort of six sporadic ALS cases and four non-neurological controls (Publication 5). The RNA samples and RNA-seq data generated by this study formed the basis for a parallel investigation of SFPQ (Publication 6), an RNA-binding protein whose altered splicing and nuclear loss have been reported as molecular hallmarks of ALS. Examination of SFPQ pathology in ALS and control post-mortem CNS tissue confirmed that SFPQ intron 9 retention was significantly elevated in ALS motor cortex. SFPQ nuclear clearance was not found to be a pathological feature of ALS motor neurons however, intraneuronal SFPQ-positive protein aggregates were reported for the first time. Finally, an expanded multi-region brain RNA-seq data set was generated for 22 sporadic ALS patients and 11 controls (Publication 7). Five brain regions that are variably affected by pTDP-43 inclusion pathology (motor cortex, prefrontal cortex, hippocampus, occipital cortex, cerebellum) were examined across all individuals (n = 165 samples), enabling the direct assessment of inter-region differences. Transcriptome alterations were observed across all five brain regions, with gene expression changes demonstrating conservation between the brain regions that either always, sometimes or never present with pTDP-43 inclusions. Aberrant RNA splicing events that are associated with TDP-43 loss of function were also detected across the five brain regions. This suggests that TDP-43 dysfunction is uncoupled from pTDP-43 inclusion pathology and is widespread in the ALS brain. Despite the absence of pTDP-43 pathology in the cerebellum, sporadic ALS patients with the most extensive regional burden of pTDP-43 inclusions demonstrated a unique gene expression signature in this region, revealing a possible “C9orf72-like” subgroup of sporadic ALS patients.</p><p dir="ltr">Together, the findings presented in this thesis expand our understanding of the molecular underpinnings of sporadic ALS. The studies identified STR expansions, SFPQ dysfunction and non-motor brain regions as important areas for future research. They also raise important considerations for novel risk variant interpretation and cohort selection, particularly for biomarker discovery.</p><p dir="ltr">The following is a thesis-by-publication following Macquarie University guidelines. Seven publications (six published, one submitted and under review) to which the PhD candidate made a substantial contribution are presented in this thesis.</p>