Identifying Disease Modifiers of SOD1 ALS Restricted; Files Only
Assefa, Ezana (Summer 2025)
Abstract
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by a loss of motor neurons and muscular atrophy. ALS is a highly heterogeneous disease, from its clinical features to genetic landscape. There are about 40 genes that account for 75% of familial ALS and around 20-25% of sporadic ALS cases and heritability estimates suggest these genes only explain half of the genetic basis of ALS. Modifier genes, or genes that act to influence the effect of another known variant or mutation, may account for at least some of the clinical variability in disease. Discovery of modifiers may be helpful for defining patient prognosis and may provide new targets for therapeutic development. A pair of monozygotic twins carrying an aggressive ALS causing mutation (SOD1A5T) were studied because of the wide discordance in disease progression – one dying within 15 months of diagnosis and the other with very slow progression over more than 7 years. These twins served as a human model for identification of potential modifier genes of mutant SOD1 toxicity. We performed transcriptomic analyses on fibroblasts and iPSC-derived motor neurons derived from the twins and discovered 979 and 472 differentially expressed genes between the twins, respectively. Whole genome sequencing of DNA isolated from blood identified 954 variants across 410 genes that differentiated the twins, with some variants overlapping multiple datasets. We selected several of these genes to further validate in other mutant SOD1 carriers as potential modifier genes accounting for the slow disease progression in the surviving twin. GPX3 and ALS2CL were strong modifier candidates that were subsequently tested in survival model of mutant SOD1 toxicity using primary mouse cortical neurons. Though GPX3 was experimentally challenging to confirm, we tested a close family member, GPX4 along with ALS2CL in this model. Both GPX4 and ALSCL2 partially rescued SOD1A5T neurotoxicity, suggesting their potential as genetic modifiers. This multi-step approach to identification of disease modifier genes provides a template for discovering and validating disease modifiers, which can help expand the understanding of novel genetic factors in ALS and lead to better diagnostic markers and treatment options for patients.
Table of Contents
Chapter 1: An Introduction and Background of Amyotrophic Lateral Sclerosis. 1
1.1: A Brief, Defining History. 1
1.2: Amyotrophic Lateral Sclerosis: An Epidemiological Overview.. 3
1.3: Amyotrophic Lateral Sclerosis: From a Clinical Perspective/Clinical Insights. 3
1.4: The Genetic Landscape of ALS.. 6
1.4.1: Chromosome 9 open reading frame 72 (C9ORF72) 6
1.4.2: TAR DNA binding protein (TARDBP) 7
1.4.3: Fused in sarcoma RNA binding protein (FUS) 8
1.4.4: Superoxide dismutase 1 (SOD1) 8
1.4.5: Other causative mutations. 11
1.5: ALS heterogeneity, a defining characteristic. 11
1.6: Aims of this thesis. 14
Chapter 2: Whole Genome Sequencing of SOD1A5T Monozygotic Twins Discordant for ALS Disease Progression to Identify Unique Variants. 16
2.1: Background. 16
2.2: Results. 17
2.2.1: Family pedigree of SOD1A5T monozygotic twins. 17
2.2.2 Whole genome sequencing of SOD1A5T monozygotic twins. 18
2.2.3 Overlaps between transcriptomic datasets and WGS variants. 21
2.3 Conclusions and Future Directions. 25
Chapter 3: Discovery of Potential Candidate Modifiers of SOD1–ALS using transcriptomic analyses. 27
3.1 Background. 27
3.2 Results. 28
3.2.1 Characterization of SOD1 mRNA and protein expression in patient-derived fibroblasts. 28
3.2.2 Transcriptomic analysis of SOD1A5T monozygotic twins’ fibroblasts. 31
3.2.3 Pathway and disease association analyses of fibroblast DEGs. 35
3.2.4 Real-time PCR validation of potential modifiers identified in fibroblast DEGs. 36
3.2.5 Derivation of a subsequent SOD1A5T – SPT and asymptomatic SOD1A5V fibroblast lines. 39
3.2.6 Generation of stable induced pluripotent stem cells (iPSCs) from SOD1A5T monozygotic twins with a motor neuron differentiation cassette. 42
3.2.7 Differentiation and characterization of induced motor neurons (iMNs) from SOD1A5T monozygotic twins 44
3.2.8 Characterization of SOD1 mRNA and protein expression in induced motor neurons. 46
3.2.9 Transcriptomic analysis of induced motor neurons from SOD1A5T monozygotic twins. 49
3.2.10 Pathway and disease association analyses of induced motor neuron DEGs. 51
3.2.11 Real-time PCR validation of potential modifiers identified in induced motor neuron DEGs 53
3.3 Discussion and Future Directions. 54
Chapter 4: Validation of candidate modifiers in a SOD1-ALS model. 65
4.1 Background: Genetic modifiers of disease. 65
4.2 Results. 68
4.2.1 Brief overview of SOD1 model systems. 68
4.2.2 Characterization of SOD1 in patient-derived fibroblasts after proteasomal treatment from SOD1A5T monozygotic twins 69
4.2.3 Generation and overexpression of SOD1A5T–tagged constructs in vitro. 73
4.2.4 SOD1A5T–HA neurotoxicity in primary mouse cortical neurons. 78
4.2.5 GPx3 protein plasma levels in a cohort of ALS cases and asymptomatic ALS gene mutation carriers 80
4.2.6 Overexpression of GPX4 is neuroprotective in a SOD1A5T model 84
4.2.7 Overexpression of ALS2CL is neuroprotective in a SOD1A5T model and is partly mediated by unique domains 87
4.3: Discussion and Future Directions. 93
Chapter 5: Methods. 97
References. 111
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