Supplementary Information to

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1 Supplementary Information to Exome sequencing to identify de novo mutations in sporadic ALS trios Alessandra Chesi 1,11, Brett T. Staahl 2, Ana Jovicic 1, Julien Couthouis 1, Maria Fasolino 3, Alya R. Raphael 1, Tomohiro Yamazaki 4, Laura Elias 2, Meraida Polak 5, Crystal Kelly 5, Kelly L. Williams 6,7,8, Jennifer A. Fifita 6,8, Nicholas J. Maragakis 9, Garth A. Nicholson 6,7, Oliver D. King 10, Robin Reed 4, Gerald R. Crabtree 2, Ian P. Blair 6,7,8, Jonathan D. Glass 5, and Aaron D. Gitler 1,11 Table S1. Clinical and demographic characteristics of ALS patients Trio # Sex Race Site of onset Age onset Disease Duration (YRS) Familial? 1 Male Caucasian bulbar sporadic 2 Female Caucasian bulbar sporadic 3 Male Black UE sporadic 4 Male Caucasian UE sporadic 6 Male Caucasian LE sporadic 7 Female Black/Latino LE 48.7 ALIVE sporadic 8 Male Caucasian UE sporadic 10 Male Caucasian bulbar sporadic 11 Male Caucasian UE 38.0 ALIVE sporadic 13 Male Caucasian UE sporadic 16 Male Caucasian UE 31.1 ALIVE sporadic * 17 Female Caucasian UE sporadic 18 Male Caucasian UE sporadic 19 Male Caucasian LE 37.3 ALIVE sporadic 20 Female Caucasian UE 29.9 ALIVE sporadic 1

2 21 Male Caucasian UE 50.6 ALIVE sporadic 22 Male Caucasian UE 42.1 ALIVE sporadic 23 Male Caucasian UE sporadic ** 24 Female Caucasian LE 20.6 ALIVE sporadic 25 Male Caucasian UE sporadic 26 Female Caucasian UE sporadic 27 Male Caucasian bulbar sporadic 28 Female Caucasian LE 38.1 ALIVE sporadic 29 Male Caucasian UE 38.4 ALIVE sporadic 30 Male Caucasian UE sporadic 31 Male Caucasian bulbar 38.4 ALIVE sporadic 33 Male Caucasian UE sporadic 34 Male Caucasian LE sporadic 35 Female Caucasian UE sporadic 37 Male Caucasian UE 43.1 ALIVE sporadic 41 Female Caucasian UE 33.3 ALIVE sporadic 43 Female Caucasian UE 34.9 ALIVE sporadic 46 Female Caucasian UE 49.3 ALIVE sporadic 47 Male Caucasian UE 45.7 ALIVE sporadic 48 Male Caucasian UE 48.5 ALIVE sporadic 49 Male Caucasian UE 40.4 ALIVE sporadic 50 Male Caucasian LE 37.7 ALIVE sporadic 51 Male Caucasian bulbar 51.4 ALIVE sporadic *patient also has a diagnosis of autism; **father with benign fasciculations. UE, upper extremity; LE, lower extremity. 2

3 Table S2. Mapping and coverage overview of exome sequencing data in all ALS trio samples (n=141). Measure Mean (±95% CI) Total reads (millions) 61.6 (±2.6) % reads aligned 89.5% (±0.2%) % duplicate reads 18.1% (±1.7%) Average coverage 55.7X (±2.4X) % on target bases 63.8% (±0.5%) % target at 2X 95.2% (±0.2%) % target at 10X 86.8% (±0.8%) % target at 20X 76.5% (±1.5%) Table S3. Comparison of rates of de novo amino acid-altering events (non-synonymous, NS) in this study (ALS) and those reported in 4 recently published studies of autism spectrum disorders (ASD). Study # NS events # probands # NS events per proband Sanders et al. (ASD) 125 (SNPs only) (SNPs only) O'Roak et al. (ASD) Neale et al. (ASD) Iossifov et al. (ASD) Chesi et al. (ALS) 30*

4 * 25 out of 30 de novo variants are novel (not present in dbsnpv135, the NHLBI Exome Sequencing Project (ESP 5400) and the 1000 Genomes Project (Feb 22), see Table 1) Number of subjects NS de novo events per subject Fig. S1. Frequency distribution of non-synonymous de novo novel events in probands. The distribution is well fitted with the Poisson distribution (red line, P=0.5), suggesting that multiple de novo mutations within a single individual do not contribute to ALS risk. 4

5 Figure. S2. Functional enrichment analysis of genes with de novo mutations in 47 ALS trios and 50 control trios (from O Roak et al. (Nature , ). After multiple comparison correction, the ALS trios are enriched in chromatin regulator genes, while the control genes do not show significant enrichment in any category. A. List of genes containing amino acid-altering de novo mutations in 47 ALS trios and 50 control trios used for functional enrichment analysis. ALS trios AIM1L CEP70 CHRM1 CNOT1 COL19A1 CSNK1G3 EHMT1 ELL FOXA1 FOXK1 GPR132 HDAC10 HS3ST2 KIF13A NTM PLEKHO2 RP1L1 SRCAP SS18L1 STARD13 UTP6 VCL WDR1 ZNF410 ZNF778 Control trios AK1 AMIGO2 BYSL CBLN3 CCDC15 CPM CRIP3 DCX DHCR7 DHX37 ENOX2 EP300 FAM196A GYLTL1B HIPK1 ITGB3 MACC1 MAML3 MAPK8 MKNK2 MOBKL2C NAA40 PCSK7 PIK3CG SEMA3F SERPINC1 SIN3A SRRM5 TMEM218 TRIM9 ZFYVE26 ZNF133 ZNF780A ZYG11A B. Functional enrichment in ALS trios. Chromatin regulator genes are enriched after multiple comparison correction. Term Count % Genes Fold Enrichment PValue Bonferroni 5

6 chromatin regulator EHMT1, FOXA1, HDAC10, SRCAP, SS18L1 transcription regulation ZNF778, FOXK1, ELL, FOXA1, HDAC10, CNOT1, SRCAP, SS18L1, ZNF410 Transcription ZNF778, FOXK1, ELL, FOXA1, HDAC10, CNOT1, SRCAP, SS18L1, ZNF410 activator FOXK1, FOXA1, SRCAP, SS18L1, ZNF E E E E E E E E- C. Functional enrichment in control trios. No functional categories are enriched after multiple comparison correction. Term Co unt regulation of cell size transferase regulation of cellular 4 11 component size.8 zinc % Genes Fold Enrichme nt PVa lue ENOX2, EP300, SEMA3F, DCX E -03 PIK3CG, GYLTL1B, EP300, HIPK1, AK1, E NAA40, MKNK2, MAPK8-03 ENOX2, EP300, SEMA3F, DCX E -02 CPM, EP300, CRIP3, ZFYVE26, ZNF133, E TRIM9, MKNK2, MOBKL2C, ZNF780A -02 Bonfe rroni 8.4E- 5.4E- 9.8E- 9.3E- 6

7 Figure S3. Predicted prion-like regions in SS18L1/CREST and SS18. Parses of proteins using a hidden Markov model for prion-domain (PrD)-like (red) vs. background (black) amino acid composition are indicated by the tracks 'MAP' (Maximum a Posteriori parse) and 'Vit' (Viterbi parse), with per-residue marginal probabilities indicated by the curves above the parses. Following King et al. (Brain Res , 61-80), the curves beneath the parses show rescaled versions of the log-likelihood ratio scores (PrD LLR) from the Alberti et al. algorithm in red (Cell , ), the predicted prion propensity (PPP) log-odds ratio scores from the Toombs et al. algorithm in green (Mol Cell Biol , ) and FoldIndex scores for predicted disorder in gray (Bioinformatics , ), each averaged over sliding windows of 41 residues. Negative scores are suggestive of both disorder and prion propensity, with the rescaled cutoff corresponding to PPP > 0.05 indicated by the dashed green line. 7