Targeted Genetic Screen in Amyotrophic Lateral Sclerosis Reveals Novel Genetic Variants with Synergistic Effect on Clinical Phenotype

Abstract

Amyotrophic lateral sclerosis (ALS) is underpinned by an oligogenic rare variant architecture. Identified genetic variants of ALS include RNA-binding proteins containing prion-like domains (PrLDs). We hypothesized that screening genes encoding additional similar proteins will yield novel genetic causes of ALS. The most common genetic variant of ALS patients is a G4C2-repeat expansion within C9ORF72. We have shown that G4C2-repeat RNA sequesters RNA-binding proteins. A logical consequence of this is that loss-of-function mutations in G4C2-binding partners might contribute to ALS pathogenesis independently of and/or synergistically with C9ORF72 expansions. Targeted sequencing of genomic DNA encoding either RNA-binding proteins or known ALS genes (n = 274 genes) was performed in ALS patients to identify rare deleterious genetic variants and explore genotype-phenotype relationships. Genomic DNA was extracted from 103 ALS patients including 42 familial ALS patients and 61 young-onset (average age of onset 41 years) sporadic ALS patients; patients were chosen to maximize the probability of identifying genetic causes of ALS. Thirteen patients carried a G4C2-repeat expansion of C9ORF72. We identified 42 patients with rare deleterious variants; 6 patients carried more than one variant. Twelve mutations were discovered in known ALS genes which served as a validation of our strategy. Rare deleterious variants in RNA-binding proteins were significantly enriched in ALS patients compared to control frequencies (p = 5.31E-18). Nineteen patients featured at least one variant in a RNA-binding protein containing a PrLD. The number of variants per patient correlated with rate of disease progression (t-test, p = 0.033). We identified eighteen patients with a single variant in a G4C2-repeat binding protein. Patients with a G4C2-binding protein variant in combination with a C9ORF72 expansion had a significantly faster disease course (t-test, p = 0.025). Our data are consistent with an oligogenic model of ALS. We provide evidence for a number of entirely novel genetic variants of ALS caused by mutations in Cooper-Knock et al. Targeted Genetic Screen in ALS RNA-binding proteins. Moreover we show that these mutations act synergistically with each other and with C9ORF72 expansions to modify the clinical phenotype of ALS. A key finding is that this synergy is present only between functionally interacting variants. This work has significant implications for ALS therapy development.

Burden of Rare Variants in ALS and Axonal Hereditary Neuropathy Genes Influence Survival in ALS: Insights from a Next Generation Sequencing Study of an Italian ALS Cohort

“…Although an association between rare variant burden and the time to reach King stage 4 has never been explored before, our data are in accordance with previous studies, similarly showing a reduced survival in patients with variants in more than one gene [71] and a correlation with the rate of disease progression [80]. Our results did not show a relation between rare variant burden and age of onset, in line with previous reports [68].…”

High-Throughput Genetic Testing in ALS: The Challenging Path of Variant Classification Considering the ACMG Guidelines

“…It means that pathogenic variants in different genes may be needed to fully express the disease. The burden of rare variants could modify the phenotype [ 159 ], influencing the age at onset, which is anticipated in patients with multiple variants [ 160 , 161 ] and could have an impact on survival [ 162 ].…”

New Insights on the Role of N6-Methyladenosine RNA Methylation in the Physiology and Pathology of the Nervous System

“…Whole-genome sequencing analysis of Greek patients with sporadic ALS revealed a positive association between FTO gene variants and the disease ( Mitropoulos et al, 2017 ). Moreover, a targeted gene sequencing of RBPs in ALS patients identified rare deleterious polymorphisms at a significantly higher rate than control in the RBM15 gene and in its paralog RMB15B ( Cooper-Knock et al, 2017 ). In addition to being part of the m 6 A methyltransferase complex, Nito , the Drosophila homolog of human RBM15 , was reported to regulate axon outgrowth, branching and to control synaptogenesis via the activity of the bursicon-expressing neurons ( Gu et al, 2017 ).…”