Fig 2: Region-based rare variant association analysis reveals association of the IL18RAP 3'UTR with ALS.a, Diagram of study design. A collapsed region-based rare variant (MAF = 0.01) association analysis was performed on the following: (1) 295 candidate protein-coding genes (Supplementary Table 3) encoding ALS-relevant proteins or proteins associated with miRNA biogenesis/activity; variants were included if predicted to cause frameshifting, alternative splicing, an abnormal stop codon or a deleterious non-synonymous amino acid substitution in = three of seven independent dbNSFP prediction algorithms; (2) variants in 3'UTRs of the 295 genes (Supplementary Table 3); (3) all known autosomal pre-miRNA genes in the human genome and (4) predicted networks, comprised of aggregated variants detected in a specific mature miRNA sequence and its cognate downstream 3'UTR targets; ORF, open reading frame. b, QQ (quantile-quantile) probability plot of obtained and expected P values (log scale) gained by region-based rare variant association analysis of all genomic regions described in a. Data were obtained from 3,955 individuals with ALS and 1,819 healthy individuals (Project MinE). Features positioned on the diagonal line represent results obtained under the null hypothesis; blue, open reading frames of 10 known ALS genes; red, IL18RAP 3'UTR; genomic inflation ? = 1.2. c, QQ plot of obtained and expected P values (log scale) gained by region-based rare variant association analysis for all known human 3'UTRs (RefSeq). The IL18RAP 3'UTR (red) is the most significant 3'UTR associated with ALS. P values were calculated with SKAT-O (genomic inflation ? = 0.97).

Fig 3: Evaluation of IL18RAP and IL-18 mRNA expression in motor neurons of patients with ALS.(a-b) mRNA expression of IL18RAP (a) and IL-18 (b), as reads per kilobase million (RPKM), from NGS study of laser capture microdissection–enriched surviving motor neurons from lumbar spinal cords of patients with sALS with rostral onset and caudal progression (n = 12) and non-neurodegeneration controls (n = 9112; GSE76220). Two-sided Student’s t test with Welch’s correction on log-transformed data (P = 0.0138 for panel A; P = 0.0056 for panel B). (c) IL-18 mRNA expression, as log2-normalized counts, from NGS study of induced ALS motor neurons (n = 4 different donors in duplicates) or non-neurodegeneration controls (n=3 different donors in duplicates113; DESeq analysis, P = 0.0417). For box plots, the median is indicated by the central line, upper and lower quartiles are indicated by the box, and maximum/minimum values are indicated by the whiskers. *P < 0.05; **P < 0.01.

Fig 4: IL18RAP and p-NF-?B expression is elevated in lymphoblastoid cells from patients with the C9orf72 repeat expansion.(a) IL18RAP mRNA expression (qPCR normalized to IPO8 mRNA levels) and (b) IL18RAP or (c) p-NF-?B protein expression (Western blots, normalized to Tubulin). Extracts from eight different human lymphoblastoid cell lines (listed in Supplementary Table 8): Four lines of healthy individuals (without ALS) carrying the canonical IL18RAP 3'UTR sequence (Control; Canonical IL18RAP 3'UTR, black) and four C9orf72 ALS patients carrying the canonical IL18RAP 3'UTR sequence (C9orf72; Canonical IL18RAP 3'UTR, red). (d) Representative blots processed with anti-IL18RAP, anti p-NF-?B and anti-Tubulin antibodies. Mann-Whitney test (A) or one-sided student’s t-test with Welch’s correction on log-transformed data (P = 0.056 for panel B; P = 0.0065 for panel C), was conducted based on the mean value of three independent passages for each of the eight human lymphoblastoid cell lines (Source Data Extended Data Fig. 5). Scatter dot plot with mean and SEM. **P<0.01.

Fig 5: Variant IL18RAP 3'UTR is protective in human microglia and in individuals with ALS.a, Diagram of experimental design. Coculture of human i3LMNs that express green fluorescent protein (GFP), driven by the synapsin (SYN1) promoter (healthy, non-ALS75) and human iPSC-derived isogenic IL18RAP 3'UTR microglia (on a C9orf72 repeat expansion background). Time-lapse microscopic analyses of i3LMN survival after microglia activation with a cocktail of LPS and the cytokine IL-18. b,c, i3LMN survival over 20 d in the presence of microglia harboring variant (blue) or canonical (gray) IL18RAP 3'UTR (two independent isogenic pairs based on independent human C9orf72 lines; n = 3 independent differentiation procedures from different passages per line with three to eight coculture wells per passage). b, Survival plot of i3LMNs in a representative experiment for each isogenic pair (mean and s.e.m. values are shown; two-way ANOVA; C9-ALS microglia line 1, P = 0.006; C9-ALS microglia line 2, P = 0.0065). c, Box plot depicting the percentage of i3LMN survival on day 20 of coculture of all experiments. For box plots, the median is indicated by the central line, upper and lower quartiles are indicated by the box, and maximum/minimum values are indicated by the whiskers. Two independent isogenic pairs based on independent human C9orf72 lines are shown; n = 3 independent differentiation procedures from different passages per line with three to eight coculture wells per passage (two-way ANOVA followed by Tukey’s multiple comparison test; C9-ALS microglia line 1, ****P < 0.0001; C9-ALS microglia line 2, P = 0.0006 for all repeats). d, Representative micrographs of fluorescent i3LMNs after 20 days of culture with C9-ALS microglia. e, Association of age of death (9 individuals with protective 3'UTR variants/4,263 individuals with ALS with available phenotypic data in Project MinE and NYGC cohorts). f, Association of age of diagnosis (8 individuals with protective 3'UTR variants/4,216 individuals with ALS with available phenotypic data in Project MinE and NYGC cohorts). The IL18RAP variant is associated with delayed age of death (+6.1 years; permutation P = 0.02; Cohen’s d effect size = 0.65) and age of diagnosis (+6.2 years; permutation P = 0.05, Cohen’s d effect size = 0.62) relative to the mean age of all Project MinE and NYGC individuals with ALS. For box plots, the median is indicated by the central line, upper and lower quartiles are indicated by the box, and maximum/minimum values are indicated by the whiskers; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.