SUPPLEMENTARY INFORMATION

Transcription

1 SUPPLEMENTARY INFORMATION doi: /nature12439 Supplementary Table 1. Coriell IDs for all exome sequenced 264 infantile spasms (IS) and Lennox- Gastaut syndrome (LGS) trios. Coriell ID Trio ID Proband Father Mother Coriell ID Trio ID Proband Father Mother aa ND27062 ND25915 ND25914 gc ND35929 ND36757 ND35930 ac ND25793 ND26080 ND26079 ge ND36610 ND36789 ND36609 ad ND26087 ND26085 ND26086 gg ND22752 ND22754 ND22753 af ND24290 ND26361 ND24285 gh ND29374 ND22981 ND22995 ag ND27949 ND26631 ND26629 gi ND23319 ND23321 ND23320 ah ND26900 ND26899 ND26895 gj ND24053 ND24055 ND24054 ai ND26970 ND26969 ND26971 gk ND24070 ND24075 ND24069 aj ND26974 ND26973 ND26972 gl ND23828 ND24283 ND23827 ak ND30086 ND27136 ND27135 gm ND24447 ND24445 ND24446 al ND27253 ND27234 ND27233 gn ND24471 ND24472 ND25665 am ND27474 ND27280 ND27281 go ND24065 ND24563 ND24064 an ND27732 ND27730 ND27731 gp ND24646 ND24639 ND24641 ao ND29711 ND27972 ND27973 gq ND24762 ND24764 ND24763 aq ND34430 ND28025 ND28024 gr ND23543 ND25364 ND25363 ar ND27935 ND28028 ND28029 gs ND25442 ND25441 ND25443 as ND30915 ND28222 ND28221 gt ND25544 ND25542 ND25543 au ND28478 ND28480 ND28479 gv ND23813 ND25797 ND25817 av ND28661 ND28663 ND28660 gw ND29554 ND25816 ND29555 ax ND28699 ND28701 ND28700 gy ND26319 ND26320 ND26321 ay ND27841 ND28748 ND27840 h ND23465 ND23489 ND23464 az ND29844 ND28816 ND28815 ha ND27753 ND26479 ND27754 b ND21451 ND21448 ND21012 hc ND25992 ND27008 ND27009 ba ND31134 ND28820 ND28821 hd ND27109 ND27152 ND27153 bc ND28982 ND28981 ND28983 he ND27155 ND27157 ND27156 be ND29057 ND29048 ND29046 hf ND28840 ND27269 ND27268 bg ND29900 ND29093 ND29094 hh ND27345 ND27346 ND27344 bi ND27521 ND29124 ND27520 hi ND27497 ND27500 ND27501 bj ND29126 ND29128 ND29127 hj ND27594 ND27593 ND27597 bk ND29199 ND29198 ND29197 hk ND27637 ND27638 ND27629 bl ND29258 ND29255 ND29259 hl ND27682 ND27683 ND27154 bm ND29267 ND29269 ND29268 hn ND27543 ND27974 ND27971 bn ND33520 ND29320 ND33521 hp ND28181 ND28180 ND28192 bo ND29319 ND29321 ND29322 hq ND28245 ND28254 ND28246 bq ND29810 ND29348 ND29349 hr ND28509 ND28365 ND28364 br ND29352 ND29353 ND29351 hs ND28866 ND28398 ND28865 bs ND29366 ND29368 ND29367 ht ND28633 ND28421 ND28422 bt ND29377 ND29375 ND29376 hu ND28432 ND28424 ND

2 RESEARCH SUPPLEMENTARY INFORMATION Coriell ID Trio ID Proband Father Mother Coriell ID Trio ID Proband Father Mother bu ND29378 ND29380 ND29379 hv ND28881 ND28880 ND28879 bv ND29383 ND29382 ND29381 hw ND28027 ND28922 ND28026 bw ND29514 ND29512 ND29513 hx ND28949 ND28948 ND28950 by ND29866 ND29865 ND29867 hz ND29055 ND29053 ND29054 ca ND29292 ND29888 ND29291 ia ND29058 ND29060 ND29059 cd ND32722 ND30001 ND30000 ic ND29146 ND29147 ND29148 ce ND32131 ND30050 ND29809 id ND31529 ND29323 ND31528 cf ND30071 ND30070 ND30069 ie ND29394 ND29396 ND29395 cg ND30610 ND30127 ND30128 if ND29446 ND29445 ND29447 ch ND30474 ND30214 ND30213 ig ND29196 ND29460 ND29459 ci ND30279 ND30274 ND30273 ih ND29528 ND29527 ND29529 cj ND30280 ND30277 ND30276 ii ND29789 ND29788 ND29786 ck ND30302 ND30304 ND30303 ij ND29838 ND29839 ND29837 cm ND30377 ND30376 ND30375 ik ND29904 ND29903 ND29901 cn ND30384 ND30382 ND30381 il ND29958 ND29957 ND29959 co ND29305 ND30418 ND29306 in ND27952 ND30019 ND27953 cp ND30431 ND30432 ND30433 ip ND28402 ND30130 ND28401 cq ND31120 ND30435 ND30434 iq ND30133 ND30134 ND30132 cr ND30441 ND30443 ND30442 ir ND30241 ND30242 ND30243 cs ND30485 ND30486 ND30487 it ND29864 ND30329 ND29863 ct ND30552 ND30551 ND30565 iu ND33014 ND30333 ND30332 cu ND30679 ND30673 ND30674 iv ND30383 ND30361 ND30362 cv ND30439 ND30686 ND30440 iw ND30631 ND30374 ND30630 cx ND30880 ND30856 ND30855 ix ND30378 ND30379 ND30380 cy ND31770 ND30989 ND30993 iy ND30216 ND30558 ND30559 cz ND30575 ND31078 ND30572 iz ND29125 ND30683 ND30682 d ND24539 ND21692 ND21691 j ND24005 ND24004 ND24003 da ND31115 ND31114 ND31113 ja ND30798 ND30799 ND30800 db ND31192 ND31191 ND31193 jb ND32224 ND30808 ND33927 dc ND31228 ND31227 ND31226 jc ND36210 ND30809 ND30807 dd ND31305 ND31301 ND31299 jd ND31159 ND30811 ND30810 de ND31308 ND31306 ND31307 je ND30052 ND30837 ND30074 df ND31364 ND31366 ND31365 jf ND30864 ND30849 ND30850 dg ND36211 ND31499 ND31474 jg ND30965 ND30962 ND30953 dh ND31702 ND31605 ND31603 jh ND31059 ND31061 ND31060 di ND31635 ND31637 ND31636 ji ND31063 ND31062 ND31076 dj ND32121 ND31698 ND31699 jj ND31153 ND31155 ND31154 dk ND30090 ND31772 ND30089 jk ND31244 ND31247 ND31246 dl ND30831 ND31785 ND30832 jl ND32890 ND31464 ND

3 SUPPLEMENTARY INFORMATION RESEARCH Coriell ID Trio ID Proband Father Mother Coriell ID Trio ID Proband Father Mother dm ND30373 ND31818 ND31817 jm ND31574 ND31576 ND31575 dn ND31821 ND31820 ND31819 jn ND31664 ND31665 ND31666 do ND31831 ND31829 ND31830 jo ND31533 ND31742 ND31534 dq ND31899 ND31909 ND31910 jp ND31961 ND31746 ND31745 dr ND31241 ND31933 ND31240 jq ND31867 ND31866 ND31864 ds ND31602 ND32000 ND31601 jr ND32497 ND31912 ND31913 dt ND34170 ND32030 ND32028 js ND30729 ND32117 ND32118 du ND32641 ND32060 ND32061 jt ND32265 ND32266 ND33882 dv ND32065 ND32063 ND32064 ju ND32289 ND32288 ND32290 dw ND32132 ND32134 ND32133 jv ND31650 ND32319 ND32317 dy ND30482 ND32385 ND30473 jw ND24191 ND32352 ND24192 dz ND32398 ND32397 ND32396 jx ND32562 ND32509 ND32500 e ND21751 ND21749 ND21750 jy ND32552 ND32555 ND32554 eb ND32464 ND32618 ND32465 jz ND31894 ND32563 ND31895 ec ND32241 ND32619 ND32240 k ND29556 ND24072 ND24071 ed ND33651 ND32640 ND32639 ka ND32630 ND32629 ND32631 ee ND32757 ND32755 ND32756 kb ND31959 ND32673 ND31958 ef ND32671 ND32843 ND32678 kc ND33323 ND32699 ND32680 eg ND33197 ND33195 ND33196 kd ND32727 ND32728 ND32726 eh ND33296 ND33298 ND33297 ke ND32763 ND32734 ND32731 ej ND33342 ND33343 ND33341 kf ND32802 ND32801 ND32803 ek ND35845 ND33454 ND33453 kg ND32239 ND32842 ND32242 el ND33322 ND33461 ND33460 kh ND33064 ND33062 ND33063 ep ND34128 ND33822 ND33820 ki ND34306 ND33169 ND32700 er ND34077 ND33883 ND33891 kj ND32879 ND33247 ND32878 es ND29429 ND33900 ND29428 kk ND32340 ND33279 ND32339 et ND35575 ND33999 ND33998 kl ND32670 ND33338 ND32647 ew ND34144 ND34143 ND34142 km ND33346 ND33348 ND33347 ex ND34274 ND34271 ND34272 kn ND33590 ND33588 ND33589 ey ND34304 ND34320 ND34302 ko ND33706 ND33704 ND33705 ez ND34338 ND34340 ND34339 kp ND35495 ND33770 ND33768 f ND22993 ND22994 ND23034 kq ND33762 ND34043 ND33761 fa ND34404 ND34402 ND34396 kr ND34131 ND34133 ND34132 fb ND34548 ND34403 ND34399 ks ND34164 ND34166 ND34165 fc ND34401 ND34407 ND34397 kt ND34593 ND34314 ND34312 fd ND36066 ND34504 ND34505 ku ND34816 ND34348 ND34346 fe ND34750 ND34753 ND34768 kv ND34424 ND34368 ND34370 ff ND34116 ND34857 ND34441 kx ND34500 ND34502 ND34501 fg ND34968 ND34946 ND34947 ky ND35136 ND34900 ND

4 RESEARCH SUPPLEMENTARY INFORMATION Coriell ID Trio ID Proband Father Mother Coriell ID Trio ID Proband Father Mother fh ND34962 ND34963 ND34969 kz ND34528 ND35053 ND34503 fi ND35054 ND35052 ND35051 l ND24104 ND24102 ND24103 fj ND34680 ND35067 ND34679 lb ND35817 ND35844 ND35818 fl ND35150 ND35148 ND35149 lc ND36163 ND36164 ND36162 fn ND35197 ND35196 ND35198 le ND36440 ND36442 ND36443 fo ND35151 ND35222 ND35223 lg ND36798 ND36799 ND36795 fp ND28895 ND35345 ND28973 n ND24188 ND24190 ND24187 fs ND35907 ND35908 ND35909 o ND24217 ND24216 ND24214 fu ND35951 ND36021 ND35980 p ND24346 ND24344 ND24345 fv ND36158 ND36156 ND36157 r ND24704 ND24703 ND24705 fw ND36206 ND36207 ND36208 s ND24782 ND24781 ND25679 fx ND36367 ND36368 ND36369 t ND24470 ND24961 ND24469 fy ND36387 ND36385 ND36386 u ND25070 ND25069 ND25073 fz ND35351 ND36497 ND35722 v ND25606 ND25607 ND25605 g ND23231 ND23233 ND23232 w ND25181 ND25622 ND25179 ga ND36561 ND36560 ND36563 x ND25582 ND25623 ND25581 gb ND30629 ND36648 ND30647 z ND25839 ND25834 ND

5 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Table 2. Comprehensive list of de novo mutations identified in 264 IS and LGS trios. Trio ID hg19 coordinates Ref/Var alleles Type Gene CCDS 1 annotation (most CCDS damaging) Polyphen-2 category (score) CCDS transcript only aa 9: C/T snv C9orf129 CCDS ' UTR - No aa 20: G/A snv KCNB1 CCDS missense probably_damaging(0.999) Yes ac 6: T/C snv GCM2 CCDS missense benign(0.007) Yes ad 17: G/A snv NFE2L1 CCDS missense benign(0.029) Yes af 19: T/C snv PRR19 CCDS missense benign(0) Yes ag 19: T/C snv MLL4 CCDS missense benign(0.325) Yes ah 6: A/C snv HSF2 CCDS missense benign(0.205) Yes ai 9: A/C snv GRIN1 CCDS missense probably_damaging(1) Yes aj 11: G/A snv CNTN5 CCDS missense probably_damaging(1) Yes ak 12: C/T snv NCOR2 CCDS missense possibly_damaging(0.877) Yes ak 8: T/C snv TEX15 CCDS synonymous - Yes an 22: T/A snv RANGAP1 CCDS missense benign(0.113) Yes an 12: G/A snv SCN8A CCDS missense probably_damaging(0.999) Yes ar 5: C/T snv GABRA1 CCDS missense probably_damaging(0.999) Yes av 10: T/C snv DIP2C CCDS missense benign(0.231) Yes av 2: C/T snv IFT172 CCDS splice donor - Yes az X: G/A snv SYTL5 CCDS synonymous - Yes b 18: C/T snv DSG2 CCDS synonymous - Yes b 11: C/T snv FAM86C1 CCDS missense probably_damaging(0.926) Yes b 6: C/T snv HBS1L CCDS missense benign(0.01) Yes b 9: C/T snv KCNT1 CCDS missense possibly_damaging(0.876) Yes ba 17: G/A snv SLC16A3 CCDS missense probably_damaging(0.995) Yes be 19: C/G snv PNMAL1 CCDS missense probably_damaging(0.922) Yes bg 2: G/A snv SCN2A CCDS missense probably_damaging(0.983) Yes bg 7: G/A snv ZSCAN21 CCDS synonymous - Yes bl 10: T/A snv PIK3AP1 CCDS missense probably_damaging(0.962) Yes bm 20: G/A snv CDC25B CCDS missense benign(0.195) Yes bn 19: A/G snv ZNF83 CCDS downstream - No bo 19: G/A snv NLRP8 CCDS missense probably_damaging(0.978) Yes bq 10: G/A snv ADD3 CCDS upstream - No bq X: G/A snv DCX CCDS stop gained - Yes br 15: G/C snv C15orf38- AP3S2 CCDS stop gained - Yes br 1: G/A snv NFASC CCDS missense possibly_damaging(0.676) Yes br 7: G/A snv TRRAP CCDS missense probably_damaging(1) Yes br 1: A/G snv ZBTB40 CCDS224.1 synonymous - Yes bs 17: G/A snv NOTUM CCDS missense probably_damaging(0.911) Yes bs 7: C/T snv SMURF1 CCDS missense probably_damaging(0.996) Yes bt 8: C/A snv SGK223 CCDS missense possibly_damaging(0.463) Yes bw 7: A/G snv FLNC CCDS missense benign(0.002) Yes by 22: C/G snv C1QTNF6* CCDS missense possibly_damaging(0.832) Yes by 22: G/A snv C1QTNF6* CCDS missense probably_damaging(0.996) Yes by 9: C/T snv RALGPS1 CCDS missense benign(0.073) Yes cg 1: C/T snv C1orf116 CCDS ' UTR - No cg X: A/G snv VAMP7 CCDS ' UTR - No cg 14: C/T snv ZNF839 CCDS missense benign(0.014) Yes ch 1: T/C snv ATP2B4 CCDS missense probably_damaging(0.992) Yes ch 19: C/T snv GPR108 CCDS missense benign(0.028) Yes ch X: G/A snv PRKX CCDS missense benign(0.295) Yes ci 1: A/T snv SLAMF1 CCDS missense probably_damaging(0.998) Yes cj 16: T/C snv GNAO1 CCDS missense probably_damaging(0.996) Yes ck 11: G/C snv OR10S1 CCDS missense probably_damaging(0.998) Yes cq 17: G/A snv NLGN2 CCDS missense probably_damaging(0.96) Yes cq 19: G/T snv NLRP5 CCDS missense possibly_damaging(0.732) Yes cq 20: C/G snv RALGAPB CCDS stop gained - Yes Main analysis DNM (CCDS, coding, multisample calling) 5

6 RESEARCH SUPPLEMENTARY INFORMATION Trio ID hg19 coordinates Ref/Var alleles Type Gene CCDS 1 annotation (most CCDS damaging) Polyphen-2 category (score) CCDS transcript only cr 3: C/T snv PLA1A CCDS missense probably_damaging(0.956) Yes cr 19: AG/A indel RASIP1 CCDS frameshift - No ct 3: G/A snv C3orf22 CCDS missense benign(0.407) Yes ct 3: AT/A indel FETUB CCDS frameshift - No ct 11: G/A snv TMPRSS5 CCDS synonymous - Yes cu 9: G/A snv STXBP1 CCDS missense probably_damaging(1) Yes cx 5: T/C snv CCDC125 CCDS missense benign(0.017) Yes cy X: C/T snv FAM50A CCDS missense probably_damaging(0.996) Yes cy 1: T/C snv FPGT- TNNI3K CCDS missense possibly_damaging(0.533) Yes cy 3: C/T snv No CCDS No cy 7: A/G snv ZNF12 CCDS ' UTR - No da 12: C/T snv ABCB9 CCDS missense benign(0.008) Yes da 10: C/G snv FRAT2 CCDS missense benign(0.022) Yes da 3: C/T snv WDR82 CCDS synonymous - Yes db 8: C/T snv VPS37A CCDS stop gained - Yes dc 14: A/G snv ACOT4 CCDS missense benign(0) Yes dc 20: G/A snv ASXL1 CCDS missense probably_damaging(0.968) Yes dc 10: A/G snv CUBN CCDS synonymous - Yes dc 16: C/T snv No CCDS No dc 2: C/T snv NTSR2 CCDS synonymous - Yes dc 4: C/T snv PALLD CCDS missense possibly_damaging(0.731) Yes de 21: C/T snv AGPAT3 CCDS missense benign(0.133) Yes de 14: C/T snv AKAP6 CCDS missense benign(0.418) Yes de 10: T/C snv DHTKD1 CCDS missense probably_damaging(0.935) Yes df 5: G/A snv PWWP2A CCDS missense benign(0.222) Yes df X: A/G snv TAF1 CCDS missense probably_damaging(0.976) Yes dg X: A/G snv ALG13 CCDS missense possibly_damaging(0.869) Yes dh X: G/A snv CDKL5 CCDS missense probably_damaging(1) Yes dj X: G/T snv SLC35A2 CCDS stop gained - Yes dk 2: C/T snv TTN CCDS missense unknown(0) Yes dl 4: T/C snv C4orf37 CCDS missense benign(0.048) Yes dl 5: C/G snv RGS14 CCDS stop gained - Yes dl 9: C/T snv STXBP1 CCDS missense probably_damaging(1) Yes dm 16: G/A snv CREBBP CCDS synonymous - Yes dn 10: T/C snv RTKN2 CCDS missense possibly_damaging(0.878) Yes do 5: G/C snv CAMK4 CCDS missense benign(0.174) Yes dr 15: T/C snv GABRB3 CCDS missense probably_damaging(0.965) Yes ds 3: G/A snv PLXNA1 CCDS missense benign(0.415) Yes dt 2: G/A snv HDAC4 CCDS synonymous - Yes dt 20: C/T snv KCNQ2 CCDS missense probably_damaging(1) Yes dt 4: C/T snv TIFA CCDS missense probably_damaging(0.939) Yes du 4: G/A snv PDCL2 CCDS synonymous - Yes dy 12: CCT/C indel B3GNT4 CCDS frameshift - No dz 3: C/T snv ALS2CL CCDS missense benign(0.094) Yes dz 2: C/A snv DTYMK CCDS missense benign(0) Yes e 10: G/T snv AKR1C4 CCDS missense probably_damaging(1) Yes e 17: G/A snv RAB5C CCDS stop gained - Yes e 11: G/C snv TRIM29 CCDS missense probably_damaging(0.999) Yes ec X: C/T snv CDKL5 CCDS stop gained - Yes ec 22: T/C snv CSNK1E CCDS missense probably_damaging(0.99) Yes ec 19: G/C snv MAST1 CCDS missense benign(0.01) Yes ed 4: G/T snv MSANTD1 CCDS missense possibly_damaging(0.451) Yes ef 10: T/C snv KIAA0913 CCDS synonymous - Yes ef 9: A/G snv RFX3 CCDS missense possibly_damaging(0.861) Yes eh 6: C/T snv PAQR8 CCDS synonymous - Yes ej 2: G/T snv ATAD2B CCDS synonymous - Yes ej 10: A/C snv CEP55 CCDS missense benign(0.158) Yes Main analysis DNM (CCDS, coding, multisample calling) 6

7 SUPPLEMENTARY INFORMATION RESEARCH Trio ID hg19 coordinates Ref/Var alleles Type Gene CCDS 1 annotation (most CCDS damaging) Polyphen-2 category (score) CCDS transcript only ej 9: C/T snv STXBP1 CCDS stop gained - Yes ek 8: C/T snv PTK2B CCDS synonymous - Yes el 15: T/A snv PAK6 CCDS synonymous - Yes er 13: G/A snv MTRF1 CCDS missense benign(0.086) Yes es 3: C/T snv KIAA2018 CCDS missense probably_damaging(0.999) Yes et 11: G/A snv GAS2 CCDS missense possibly_damaging(0.873) Yes ey 19: T/C snv No CCDS No f 15: T/G snv NIPA1 CCDS synonymous - Yes fc 10: C/T snv CAMK2G CCDS ' UTR - No fc 3: C/T snv COL7A1 CCDS missense unknown(0) Yes fc 9: A/G snv SNAPC3 CCDS ' UTR - No fc 2: T/C snv XPO1 CCDS missense benign(0.077) Yes fd 15: G/A snv MYO5A CCDS stop gained - Yes fd 8: A/C snv PPP1R3B CCDS synonymous - Yes fe 13: C/T snv DIAPH3 CCDS missense possibly_damaging(0.868) Yes fe 9: C/T snv PRDM12 CCDS missense probably_damaging(0.921) Yes ff 17: C/A snv SLC26A11 CCDS synonymous - Yes fg 14: G/A snv PACS2 CCDS missense possibly_damaging(0.775) Yes fh 9: G/A snv STXBP1 CCDS missense probably_damaging(0.999) Yes fj 2: G/A snv HDAC4 CCDS synonymous - Yes fj X: T/C snv THOC2 CCDS missense probably_damaging(0.999) Yes fl 1: A/C snv LRRC8D CCDS ' UTR - No fl 9: A/T snv SNX30 CCDS missense benign(0.019) Yes fl 13: A/G snv WDFY2 CCDS synonymous - Yes fn 6: CT/C indel MRS2 CCDS frameshift - No fn 18: C/G snv NEDD4L CCDS missense possibly_damaging(0.452) Yes fn 8: G/A snv RP1L1 CCDS synonymous - Yes fo 13: G/A snv No CCDS No fp 11: C/T snv MAPK8IP1 CCDS missense benign(0.06) Yes fs 21: CT/C indel N6AMT1 CCDS frameshift - No fs 2: C/T snv No CCDS No fs 18: G/A snv TCEB3B CCDS ' UTR - No fx 3: C/T snv EPHB1 CCDS synonymous - Yes fx 19: C/T snv SMG9 CCDS missense probably_damaging(0.962) Yes g 9: G/C snv DNM1 CCDS missense probably_damaging(0.999) Yes ga 9: A/G snv EXOSC2 CCDS missense benign(0.01) Yes ga 17: T/A snv KLHL11 CCDS missense benign(0.01) Yes gb 13: C/T snv TPTE2 CCDS missense benign(0.029) Yes gc 19: C/T snv NR1H2 CCDS missense probably_damaging(0.994) Yes ge 11: C/A snv CCND1 CCDS ' UTR - No ge 20: C/T snv SS18L1 CCDS ' UTR - No gi 20: C/A snv AHCY CCDS missense benign(0.009) Yes gi 18: G/C snv C18orf25 CCDS missense probably_damaging(0.916) Yes gi 9: A/G snv DDX58 CCDS missense possibly_damaging(0.822) Yes gj 2: G/A snv SCN1A CCDS stop gained - Yes gj 16: G/A snv ZFHX3 CCDS missense unknown(0) Yes gl 10: T/C snv CUL2 CCDS synonymous - Yes gm 1: C/T snv C1orf123 CCDS576.1 synonymous - Yes gm 6: C/T snv HFE CCDS synonymous - Yes gp 19: TC/T indel PPP6R1 CCDS downstream - No gp 2: C/T snv SCN1A CCDS missense probably_damaging(0.995) Yes gs 15: T/C snv GABRB3 CCDS missense probably_damaging(0.938) Yes gt 6: A/G snv HIST1H2BD CCDS missense benign(0.347) Yes gv 1: C/A snv LCE1A CCDS missense unknown(0) Yes gy 19: G/A snv RUVBL2 CCDS missense benign(0.007) Yes h 16: C/T snv PLCG2 CCDS synonymous - Yes ha 1: C/T snv CELA3B CCDS219.1 missense benign(0.115) Yes ha 3: T/C snv GPR128 CCDS missense possibly_damaging(0.59) Yes Main analysis DNM (CCDS, coding, multisample calling) 7

8 RESEARCH SUPPLEMENTARY INFORMATION Trio ID hg19 coordinates Ref/Var alleles Type Gene CCDS 1 annotation (most CCDS damaging) Polyphen-2 category (score) CCDS transcript only ha 5: A/G snv RANBP17 CCDS synonymous - Yes hc 10: A/G snv CRTAC1 CCDS missense probably_damaging(0.949) Yes hd 12: T/A snv SCN8A CCDS missense probably_damaging(1) Yes hf 1: G/T snv ASH1L CCDS synonymous - Yes hf 17: G/A snv ITGB4 CCDS missense possibly_damaging(0.689) Yes hf 10: TG/T indel TRIM8 CCDS frameshift - No hh 19: C/T snv CACNA1A CCDS missense probably_damaging(0.996) Yes hi 12: G/A snv CUX2 CCDS missense probably_damaging(0.997) Yes hi 1: G/A snv LDLRAD1 CCDS missense benign(0.32) Yes hi 3: A/G snv RTP1 CCDS missense benign(0.184) Yes hj 20: T/G snv SDCBP2 CCDS missense probably_damaging(0.96) Yes hk 7: G/C snv YWHAG CCDS missense probably_damaging(0.995) Yes hl 2: C/T snv ALMS1 CCDS missense probably_damaging(0.974) Yes hl 5: G/T snv GPR98 CCDS missense benign(0.001) Yes hl 20: C/T snv HCK CCDS missense benign(0.271) Yes hp 4: G/C snv WDR19 CCDS missense probably_damaging(0.964) Yes hq 4: A/T snv CYP2U1 CCDS missense benign(0.38) Yes hr 2: C/T snv COL4A4 CCDS missense unknown(0) Yes hr 2: C/G snv SCN1A CCDS missense probably_damaging(1) Yes hs 1: G/A snv CSMD2 CCDS380.1 missense possibly_damaging(0.766) Yes hs 10: G/T snv DDX50 CCDS missense possibly_damaging(0.65) Yes hs 2: A/G snv MYO7B CCDS missense benign(0.285) Yes hs 19: C/T snv No CCDS No hs 18: A/G snv RIOK3 CCDS missense probably_damaging(0.999) Yes ht 17: G/C snv MRPS23 CCDS intronic - No hu 22: T/C snv FAM116B CCDS missense probably_damaging(0.984) Yes hu 17: C/T snv SLC5A10 CCDS synonymous - Yes hv 17: G/A snv C17orf53 CCDS missense probably_damaging(0.995) Yes hv 1: CATTTG TCTTT/C indel HNRNPU CCDS splice acceptor - No hw 12: A/G snv SLCO1A2 CCDS ' UTR - No hx 5: T/C snv HNRNPH1 CCDS synonymous - Yes hz 1: C/T snv MTOR CCDS127.1 missense possibly_damaging(0.666) Yes hz 7: T/C snv No CCDS No hz 18: G/A snv TCF4 CCDS synonymous - Yes ia 15: C/G snv HCN4 CCDS synonymous - Yes ie 6: G/A snv LEMD2 CCDS synonymous - Yes if 16: A/G snv C16orf62 CCDS missense benign(0.279) Yes ig 3: C/T snv IQSEC1 CCDS missense benign(0.025) No ih 15: G/A snv CHD2 CCDS splice donor - Yes ij X: A/G snv ALG13 CCDS missense possibly_damaging(0.869) Yes il 21: T/C snv SCAF4* CCDS missense unknown(0) Yes il 21: T/C snv SCAF4* CCDS synonymous - Yes il 6: C/T snv UNC5CL CCDS missense benign(0) Yes in 1: A/C snv CR2 CCDS missense possibly_damaging(0.631) Yes in 15: G/A snv GRAMD2 CCDS missense benign(0.001) Yes in 13: C/T snv NBEA CCDS stop gained - Yes iv 9: G/C snv GLIS3 CCDS synonymous - Yes iw 20: CCTCAG TGG/C indel EMILIN3 CCDS frameshift - No iw 3: A/G snv ITPR1 CCDS synonymous - Yes ix 17: C/T snv FASN CCDS missense probably_damaging(1) Yes iy 2: C/T snv SCN1A CCDS splice donor - Yes iz 3: A/G snv HRG CCDS missense benign(0.186) Yes iz 5: A/G snv PCDHB13 CCDS missense probably_damaging(0.935) Yes j 12: G/A snv FAM19A2 CCDS ' UTR - No j X: GA/G indel GLRA2 CCDS ' UTR - No ja X: C/T snv CDKL5 CCDS missense probably_damaging(0.993) Yes Main analysis DNM (CCDS, coding, multisample calling) 8

9 SUPPLEMENTARY INFORMATION RESEARCH Trio ID hg19 coordinates Ref/Var alleles Type Gene CCDS 1 annotation (most CCDS damaging) Polyphen-2 category (score) CCDS transcript only ja 12: C/T snv PRDM4 CCDS synonymous - Yes jb 6: T/C snv COQ3 CCDS missense benign(0) Yes jb 14: T/C snv TRMT61A CCDS ' UTR - No jc 8: G/A snv KCNQ3 CCDS missense probably_damaging(0.985) Yes jc 2: C/T snv THAP4 CCDS missense benign(0.077) Yes jd 11: G/C snv GLB1L3 CCDS missense possibly_damaging(0.735) Yes jf 1: A/C snv FCGR2B CCDS missense benign(0) Yes jf 22: G/A snv MIOX CCDS synonymous - Yes jf 6: T/C snv SLC26A8 CCDS synonymous - Yes jg 19: G/A snv BEST2 CCDS missense possibly_damaging(0.769) Yes jg 5: C/T snv FLT4 CCDS ' UTR - No jg 7: C/T snv KIAA1324L CCDS missense possibly_damaging(0.653) Yes jg 5: C/T snv RARS CCDS missense probably_damaging(0.999) Yes jg 7: A/G snv SVOPL CCDS synonymous - Yes jg 5: C/T snv TRIO CCDS missense probably_damaging(0.998) Yes jg X: TGA/T indel WDR45 CCDS frameshift - No jh 12: C/A snv GRIN2B CCDS missense probably_damaging(0.999) Yes ji 10: A/T snv FAM21C CCDS synonymous - Yes ji 6: GA/G indel TAAR2 CCDS frameshift - No jj 2: A/G snv ATIC CCDS synonymous - Yes jj 12: C/T snv PTPRR CCDS missense probably_damaging(0.998) No jj 13: T/C snv SKA3 CCDS missense benign(0) Yes jk 10: T/C snv ANK3 CCDS missense probably_damaging(0.995) Yes jk 9: C/T snv STXBP1 CCDS missense probably_damaging(1) Yes jl 22: T/C snv No CCDS No jn 22: G/A snv CELSR1 CCDS stop gained - Yes jo 15: G/C snv ZSCAN2 CCDS missense possibly_damaging(0.545) Yes jq 9: T/C snv ARRDC1 CCDS synonymous - Yes jq 11: G/A snv HIPK3 CCDS missense probably_damaging(1) Yes jr 15: T/C snv GABRB3 CCDS missense probably_damaging(0.974) Yes jr 19: G/A snv SAFB2 CCDS missense benign(0.037) Yes jr 16: C/G snv USP31 CCDS ' UTR - No jt 2: C/T snv AAK1 CCDS synonymous - Yes ju 2: G/A snv SCN1A CCDS missense probably_damaging(0.999) Yes jv 16: G/C snv TSNAXIP1 CCDS missense possibly_damaging(0.467) Yes jw 15: C/T snv GABRB3 CCDS missense probably_damaging(1) Yes jw 5: G/A snv RAPGEF6 CCDS ' UTR - No jx 19: G/A snv TTYH1 CCDS synonymous - Yes jz 7: A/G snv MKLN1 CCDS missense benign(0.004) Yes ka 1: G/T snv RD3 CCDS synonymous - Yes ka 8: G/A snv WHSC1L1 CCDS missense probably_damaging(0.999) Yes kb 1: G/A snv DHDDS CCDS281.1 missense probably_damaging(1) Yes kb 7: C/T snv FAM133B CCDS synonymous - Yes kb 1: T/A snv No CCDS No kc 9: T/C snv ABCA2 CCDS synonymous - Yes kc 12: A/G snv DAO CCDS missense benign(0.067) Yes kd 2: C/T snv SCN1A CCDS splice donor - Yes kd 10: T/A snv TNKS2 CCDS synonymous - Yes ke 6: A/G snv BEND3 CCDS ' UTR - No ke 1: A/G snv CHIA CCDS832.1 missense benign(0) Yes ke 2: G/A snv PASK CCDS synonymous - Yes kf 12: G/T snv CHD4 CCDS missense possibly_damaging(0.759) Yes kf 2: G/A snv SCN2A CCDS missense probably_damaging(0.983) Yes kh 4: A/G snv MUC7 CCDS upstream - No ki 1: T/C snv IPO9 CCDS ' UTR - No ki 7: G/A snv MEOX2 CCDS missense probably_damaging(0.977) Yes ki 16: CT/C indel STX1B CCDS frameshift - No kj 6: C/T snv CENPW CCDS upstream - No Main analysis DNM (CCDS, coding, multisample calling) 9

10 RESEARCH SUPPLEMENTARY INFORMATION Trio ID hg19 coordinates Ref/Var alleles Type Gene CCDS 1 annotation (most CCDS damaging) Polyphen-2 category (score) CCDS transcript only kj 9: G/C snv DNM1 CCDS missense probably_damaging(1) Yes km 1: G/A snv ABL2 CCDS downstream - No km 14: G/A snv MYH6 CCDS missense probably_damaging(1) Yes km 6: T/C snv QRSL1 CCDS synonymous - Yes ko X: G/A snv IQSEC2 CCDS stop gained - Yes kq 1: G/GA indel DNAJC6 CCDS frameshift - No kr 2: C/G snv DNAH7 CCDS missense possibly_damaging(0.603) Yes kr 9: C/T snv DPP7 CCDS missense probably_damaging(0.999) Yes kr 4: G/T snv RXFP1 CCDS missense possibly_damaging(0.893) Yes ks 1: A/G snv FLG CCDS synonymous - Yes ks 7: G/A snv GET4 CCDS ' UTR - No kt 15: G/A snv FAM63B CCDS stop gained - Yes kt 9: CAGA/C indel NRARP CCDS ' UTR - No kt 16: T/C snv SPG7 CCDS missense probably_damaging(0.999) Yes ku 1: C/T snv RYR2 CCDS stop gained - Yes ku 2: A/AT indel SCN1A CCDS frameshift - No kx X: A/G snv FLNA CCDS missense probably_damaging(0.943) Yes kx 17: C/T snv KRTAP1-3 CCDS missense benign(0.332) Yes kx 11: A/G snv MMP27 CCDS splice donor - No kx 10: A/G snv MYO3A CCDS missense probably_damaging(0.944) Yes kx 2: T/C snv SULT1C4 CCDS ' UTR - No kz 1: G/A snv CTTNBP2NL CCDS845.1 missense benign(0.315) Yes kz 11: C/T snv MLL CCDS missense probably_damaging(0.995) Yes kz 1: C/T snv MYOM3 CCDS missense possibly_damaging(0.886) Yes kz 11: C/A snv SLC5A12 CCDS ' UTR - No l 7: T/C snv ZC3HAV1 CCDS intronic - No lb 9: C/A snv FAM102A CCDS missense possibly_damaging(0.584) Yes lb 7: GT/G indel USP42 CCDS ' UTR - No lc 11: T/C snv RHOG CCDS synonymous - Yes lg 2: G/T snv SCN1A CCDS missense probably_damaging(0.999) Yes lg 9: CAGT/C indel TRIM32 CCDS inframe deletion - No n 11: C/T snv OSBPL5 CCDS missense benign(0.068) Yes n 11: C/T snv SLC1A2 CCDS missense probably_damaging(1) Yes n 2: T/A snv STK36 CCDS missense benign(0.001) Yes o 17: G/A snv KRT34 CCDS missense benign(0.402) Yes o 2: CTTTG/C indel TTN CCDS frameshift - No t 12: TC/T indel WNK1 CCDS intronic - No u 10: C/T snv PTEN CCDS missense benign(0.113) Yes u 12: A/G snv SLCO1B7 CCDS missense benign(0.169) Yes w 17: G/A snv CANT1 CCDS synonymous - Yes x 18: G/A snv ANKRD12 CCDS missense probably_damaging(0.999) Yes x 20: A/G snv CDS2 CCDS missense benign(0.076) Yes x 21: G/T snv RRP1B CCDS missense probably_damaging(1) Yes z 4: T/C snv GABRB1 CCDS missense probably_damaging(0.997) Yes z 1: A/G snv PDIK1L CCDS274.1 missense possibly_damaging(0.752) Yes z 7: T/A snv ZNF467 CCDS ' UTR - No *two confirmed de novo mutations within close proximity of each other in the same individual Main analysis DNM (CCDS, coding, multisample calling) 10

11 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Table 3. Summary of de novo mutations identified in 264 IS and LGS trios. De novo mutation count Initially identified/putative 439 Confirmed 283 False positive 32 False positive (due to B-cell immortalization process) 67 Inherited 3 Target sequence could not be successfully amplified 54 Confirmed (identified by alternate de novo variant calling methods*) 46 TOTAL CONFIRMED 329 *See Additional Methods. The overall confirmation rates for the de novo mutations discovered by sequencing primary DNA were 89% for single nucleotide variants (SNVs) and 93% for small insertion-deletion mutations (indels). 100" 90" Number"of"EE"probands" 80" 70" 60" 50" 40" 30" 20" 10" 0" 0"""""1""""2"""""3""""4"""""5""""6" Number'of'de#novo## muta1ons'per'ee'proband' Supplementary Figure 1. Distribution of de novo mutations detected in 264 IS/LGS probands. Histogram is based on all Sanger sequencing confirmed SNV de novo mutations (n=309) from the 264 IS/LGS trios. 11

12 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Table 4. Estimating probability of multiple de novo events amongst control exomes. Genes with greater than one de novo SNV mutation in the combined cohorts of 610 control trios 2-4, and their probabilities of getting greater than or equal observed de novo mutation tally by chance. Average effectively Weighted De novo Gene Chromosome p-value a captured length (bp) mutation rate mutation No. ADAMTS x x10-3 AGBL x x10-3 AHNAK x x10-2 BYSL x x10-4 EIF4G x x10-3 GLIS x x10-4 KIF x x10-3 KIF4A b X x x x10-4 MUC x x10-1 RGS x x10-4 SNRNP x x10-3 TDRD x x10-3 TTN x x10-1 UGT2B x x10-4 USP x x10-2 a Adjusted α is equivalent to (0.05 / 18,091) = 2.76x10-6. b KIF4A is on the X chromosome. Because the gender information of these control samples is not available, we calculated two p-values assuming all samples are male and female, respectively. The true p- value should be between these two numbers. 12

13 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Methods: Investigating instances of multiple hits at the same site across distinct probands. To find whether there are homologs with high sequencing similarity to the sequences around the de novo mutations detected in gene ALG13 and SCN2A, we retrieved 500bp from each side of the mutations and performed BLAST comparison with the human whole genome sequence. We turn the F parameter off so that it can also detect low complexity sequences. Except for themselves, no homologs with >= 90% similarity were detected. Sequencing coverage at the do novo mutations also look, with enough uniquely mapped high quality reads (Supplementary Figure 2). Taken together, the same de novo mutations of ALG13 and SCN2A detected in different samples are unlikely artifacts caused by sequencing or mapping errors. Supplementary Figure 2. The sequencing coverage of four de novo mutations. The black curves represent the coverage of the total mapped reads; the blue curves represent uniquely mapped reads; the red curves represent high quality (>=30) mapped reads. Vertical lines represent the de novo mutation position, where cumulative distribution functions (CDF) were calculated. Sample size: 10,

14 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Methods: Genetic Architecture Likelihood model formulation. We extend the approach of Neale et al. and develop a likelihood model that expresses the distribution of de novo coding variants among affected individuals by relating it to the distribution of coding de novos in the general population through a parametric model characterizing the genetic architecture of disease. Specifically, if X denotes the number of de novo SNVs in an individual and indicates that the individual is affected, we wish to characterize (1) where denotes the distribution of coding de novos in the general population. Let H be an indicator of whether an individual has least one bad hit, i.e., contains a mutation that is deleterious with respect to the disease phenotype. Thus we can write where we have assumed that once it is known that an individual has a bad hit or not, the number of de novo variants that person has is irrelevant to disease risk. We define the relative risk as allowing us to write, [ ] Let the probability that a de novo mutation is deleterious with respect to the disease phenotype be decomposed into the product of two components: C, the probability that a de novo coding variant is deleterious with respect to the protein in which it is found, and, the probability that a de novo is found in a gene related to the disease phenotype. With this, we have that.since, we can write [ ] Plugging this back into equation (1) gives [ ] [ ] which can be further simplified as [ ] where is the probability generating function of (i.e., distribution of de novo coding variants in the general population). When, as in Neale et al., we assume the distribution of de novos in the general population is Poisson with rate parameter λ, the distribution among affecteds becomes [ ] As in Neale et al., we assume that C and are known constants (we discuss values for C and below), therefore, the likelihood function for and can be written as 14

15 SUPPLEMENTARY INFORMATION RESEARCH { [ ] } (2) where denotes the number of trios with x coding de novo variants. This model allows us to preform likelihood-based inference on the key parameters (γ and η) characterizing genetic architecture of IS/LGS. Note that though, under the null, η takes on a value on the boundary of the parameter space, we found very close agreement between a standard chi-square approximation of the likelihood ratio statistic and a Monte Carlo based approximation based on sampling directly from. Constants. As in Neale et al. we estimate C and from external data and assume they are fixed with respect to the likelihood analysis. The constant C is the probability that a de novo mutation deleteriously affects the protein it is found in. To characterize this probability, we consider the frequency of different categories of de novo mutations and their deleteriousness. From control samples, we estimate that 69.4% of de novo coding variants are nonsynonymous, 3.5% are nonsense, and 0.7% are splice site mutations. As in Neale et al., we assume that 30% of non-synonymous mutations are deleterious 5 while both nonsense and splice site mutations are always deleterious. Thus. We used the same trinucleotide-based mutation rate estimation procedure detailed above to compute de novo mutation rate in the general population ( to be 0.88 per exome. We note that this is in close agreement with Neale et al. estimate of 0.87 per exome. The same procedure, applied to intolerant genes gave a value of. Application to autism spectrum disorder. We applied the likelihood model to analyze autism spectrum disorder de novo coding variant data presented by Neale et al. (Supplementary Table 5). To be consistent with the values used by Neale et al., we used and (note that they considered different categories of mutations). A likelihood ratio test based on equation (2) above, confirmed their assertion that the data were not significantly different from that expected from a random sample of coding de novos from the general population. Supplementary Figure 3, shows the likelihood surface for and implied by these data. The blue Xs in this figure represent the 6 sets of parameter values simulated by Neale et al. Note that the likelihood analysis yields the same conclusions with respect to these 6 points as those made by Neale et al., i.e., while the bottom 3 points are consistent with the observed data, the top three points are not. However, the likelihood analysis makes it clear that this does not imply, as concluded in Neale et al., that large relative risks are inconsistent with the observed data. In fact, the likelihood analysis shows that for any relative risk,, there is always a value of, such that is within the 95% confidence region (illustrated by red lines in figure) given by the data. Supplementary Table 5. Observed number of de novo coding mutations observed in autism spectral disorder trios as reported by Neale et al. Number of de novo mutations per trio (x) Number of trios ( ) Application to IS/LGS trios. We applied the likelihood model to the IS/LGS trio de novo coding variant data given in Supplementary Table 6. Similar to that observed in autism, a likelihood ratio test based of equation (2) above showed that the data, when considering the exome as a whole, were not significantly different from that expected from a random sample of de novo coding mutations from the general population. A plot of the likelihood surface for these data is presented in Supplementary Figure 4. Though this analysis can exclude a large portion of all combinations of and, marginally, it cannot limit the range of values possible, i.e., for each possible value of, there is always a value of, such that is within the 95% confidence region. The likelihood ratio test based on equation (2) is a two degree of 15

16 RESEARCH SUPPLEMENTARY INFORMATION freedom test. We also considered a one degree of freedom negative-binomial-based test that tests whether the rate of de novo mutations among IS/LGS trios differs from that predicted by the trinucleotide-based mutation rate estimation procedure detailed above. To construct this test we fit a negative binomial regression model with an offset equal to the log of the null rate. In this model a test of whether the observed rate differs significantly from the null corresponds to a test of the intercept parameter being zero. Using this test, we found evidence of an increase from the null rate of 0.88 (p=0.045). When we subset to that portion of the exome harboring intolerant genes (Supplementary Table 7), we find that the likelihood ratio test is significant (p=2.9x10-3 ). A plot of the likelihood surface given in Supplementary Figure 5 shows that a 95% confidence region for is distinct from the null. Maximum likelihood point estimates suggest that a limited number of intolerant genes (2.1%) contain highly penetrant mutations ( ) affecting IS/LGS risk. However, caution must be taken when interpreting these results as, though distinct from the null, the likelihood is quite flat in the neighborhood of the mle, resulting in a large number of combinations that are consistent with the data. Supplementary Table 6. Observed number of de novo coding mutations observed in IS/LGS trio exomes. Number of de novo mutations per trio (x) Number of trios ( ) Supplementary Table 7. Observed number of de novo coding mutations observed in intolerant genes among IS/LGS trios. Number of de novo mutations per trio (x) Number of trios ( )

17 SUPPLEMENTARY INFORMATION RESEARCH log(grr) (0.018,log(15.6)) % exome related to autism Supplementary Figure 3. Likelihood surface for autism spectrum data given in Neale et al. (Supplementary Table 5). Gray lines represent contours of the likelihood surface. Red lines represent 95% likelihood ratio confidence regions for and. Maximum likelihood estimate given by black X ( ). Blue Xs represent 6 sets of parameter values for and simulated by Neale et al. 17

18 RESEARCH SUPPLEMENTARY INFORMATION log(grr) (0.007,log(86)) % exome related to IS/LGS Supplementary Figure 4. Likelihood surface for IS/LGS exome data (Supplementary Table 6). Gray lines represent contours of the likelihood surface. Red lines represent 95% likelihood ratio confidence regions for and. Maximum likelihood estimate given by black X ( ). 18

19 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 5. Likelihood surface for IS/LGS intolerant gene data (Supplementary Table 7). Gray lines represent contours of the likelihood surface. Red lines represent 95% likelihood ratio confidence regions for and. Maximum likelihood estimate given by black X ( ). 19

20 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Table 8. Summary statistics of the opportunity space to call a de novo coding variant in the known early epileptic encephalopathy MIM genes [last accessed OMIM December 2012]. Phenotype MIM number OMIM Phenotype Gene Average % of CCDS transcript with de novo mutation call opportunity Intolerance score and (percentile) Epileptic encephalopathy, early infantile, 9 PCDH % ( ) (10.4%tile) Epileptic encephalopathy, early infantile, 8 ARHGEF % ( ) (44.9%tile) Epileptic encephalopathy, early infantile, 2 CDKL % ( ) (15.9%tile) Epileptic encephalopathy, early infantile, 1 ARX 20.57% (0 853) Unassessed Epileptic encephalopathy, Lennox-Gastaut type MAPK % ( ) (24.0%tile) Dravet syndrome SCN1A 98.86% ( ) (4.0%tile) Epileptic encephalopathy, early infantile, 3 SLC25A % ( ) 0.13 (63.2%tile) Epileptic encephalopathy, early infantile, 4 STXBP % ( ) (15.0%tile) Epileptic encephalopathy, early infantile, 10 PNKP 86.67% ( ) (20.9%tile) Epileptic encephalopathy, early infantile, 5 SPTAN % ( ) (0.3%tile) Epileptic encephalopathy, early infantile, 7 KCNQ % ( ) (15.9%tile) Epileptic encephalopathy, early infantile, 11 SCN2A 93.78% ( ) (1.8%tile) Epileptic encephalopathy, early infantile, 12 PLCB % ( ) (11.5%tile) Epileptic encephalopathy, early infantile, 13 SCN8A 96.15% ( ) (2.3%tile) Epileptic encephalopathy, early infantile, 14 KCNT % ( ) (1.6%tile) 20

21 SUPPLEMENTARY INFORMATION RESEARCH Reproduced from Petrovski et al (manuscript submitted). *OMIM genes are based on qualifying genes within MIM database (October 2012). Supplementary Figure 6. Classification of known EE genes as intolerant. 21

22 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Table 9. List of intolerant CCDS-defined genes harboring a de novo mutation in an IS or LGS trio (n=109). Previously reported EE genes are highlighted in red. SCN1A [indel] + [SG] + [2xESS] + [4xNS] TRIM32 [indel] C16orf62 [NS] FASN [NS] MAPK8IP1 [NS] OSBPL5 [NS] THOC2 [NS] EPHB1 [SYN] STXBP1 [SG] + [4xNS] TRIM8 [indel] CACNA1A [NS] FLNA [NS] MAST1 [NS] PACS2 [NS] TRIO [NS] ITPR1 [SYN] CDKL5 [SG] + [2xNS] IFT172 [ESS] CDC25B [NS] FLNC [NS] MKLN1 [NS] PLXNA1 [NS] TRRAP [NS] KIAA0913 [SYN] TSNAXIP1 C15orf38-AP3S2 [SG] CHD2 [ESS] CHD4 [NS] GABRA1 [NS] MLL [NS] PRKX [NS] [NS] PAQR8 [SYN] CELSR1 [SG] GABRB3 [4xNS] COL7A1 [NS] GLB1L3 [NS] MLL4 [NS] RFX3 [NS] WHSC1L1 [NS] PLCG2 [SYN] IQSEC2 [SG] SCN8A [2xNS] CRTAC1 [NS] GNAO1 [NS] MTOR [NS] RUVBL2 [NS] XPO1 [NS] PRDM4 [SYN] MYO5A [SG] SCN2A [2xNS] CSMD2 [NS] GRIN1 [NS] MYH6 [NS] SAFB2 [NS] ZFHX3 [NS] PTK2B [SYN] NBEA [SG] ALG13 [2xNS] CUX2 [NS] GRIN2B [NS] NCOR2 [NS] SCAF4 [NS] ZSCAN2 [NS] SLC5A10 [SYN] HDAC4 RALGAPB [SG] DNM1 [2xNS] DAO [NS] HIPK3 [NS] NEDD4L [NS] SLC16A3 [NS] [2xSYN] TCF4 [SYN] RYR2 [SG] ABCB9 [NS] DDX50 [NS] HSF2 [NS] NFASC [NS] SLC1A2 [NS] AAK1 [SYN] TNKS2 [SYN] VPS37A [SG] AGPAT3 [NS] DIP2C [NS] ITGB4 [NS] NFE2L1 [NS] SMG9 [NS] ABCA2 [SYN] ZBTB40 [SYN] DNAJC6 [indel] ALS2CL [NS] DTYMK [NS] KCNQ2 [NS] NLGN2 [NS] SMURF1 [NS] ASH1L [SYN] EMILIN3 [indel] ANK3 [NS] FAM102A [NS] KCNT1 [NS] NOTUM [NS] SNX30 [NS] CREBBP [SYN] KIAA2018 HNRNPU [indel] ATP2B4 [NS] FAM116B [NS] [NS] NR1H2 [NS] SPG7 [NS] CUL2 [SYN] [SG] = Stop gained; [ESS] = splice acceptor/donor site; [indel] = Frameshift or inframe deletion; [NS] = Non-Synonymous; [SYN] = Synonymous. 22

23 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Methods: Assessment of mutation consequences in intolerant genes To assess whether the more damaging classes of de novo mutations in our cohort are enriched for the intolerant genes, we stratify our de novo mutations into four variant effect categories: Loss-Of-Function (LoF, which includes indels, splice acceptor splice donor, and nonsense mutations), Missense (PolyPhen2 probably/possibly damaging), Missense (PolyPhen2 benign), and Synonymous. We assess whether there is an observed enrichment for intolerant genes amongst the more damaging mutation categories. We do not include the six missense de novo mutations without a currently predicted PolyPhen2 score, or the eight de novo mutations that occurred in genes that did not have an intolerance score available. We calculate the expected proportion of the sequenced exome that contains the intolerant genes to be 37.77% of the overall CCDS opportunity space (Additional Methods). We find that LoF de novo variants occur in intolerant genes 61.8% (21/34) of observations (p=6.94x10-3, an excess of approximately 8 LoF de novo mutations). Missense mutations with a PolyPhen2 probably/possibly damaging prediction occur in intolerant genes 56.4% (66/117) of the observations (p=5.16x10-5, an excess of approximately 21 PolyPhen2 damaging missense de novo mutations). While, missense mutations with a PolyPhen2 benign prediction (22/65), and synonymous mutations (18/51), occur in intolerant genes 33.85% and 35.29% of the observations, respectively (both p>0.6). Moreover, the two resulting groups of de novo mutation types: benign (synonymous and missense PolyPhen2 benign predictions) and damaging (LoF and missense de novos with a damaging PolyPhen2 prediction), occur in intolerant genes 34.48% of the time (p=0.50) and 57.62% (p=8.48x10-7 ), respectively. Comparing the raw intolerance scores from the benign group of de novo mutations (n=116 de novo, median intolerance score = percentile) and likely damaging (n=151 de novo, median intolerance score = percentile) we observe that the damaging group of de novo mutations have a higher propensity to occur in genes more intolerant to standing functional variation (p=1.34x10-4, 2-tail Mann Whitney U test). Moreover, the signal remains significant after excluding the 22 de novo mutations occurring in known genes (p=6.37x10-3 ). 23