Extended Data Figure 1

Meta-analysis nASD = 11,067, nrelatives+controls = 107,965 Cohort UK-Biobank SSC SPARK DBS gnomAD ASD children - 1648 4608 4811 - Non-ASD siblings - 1209 2195 - - Parents - 3296 8288 - - Controls 42,984 - - 5214 44,779

ASD Socioeconomic Brain volume diagnosis SNP array Whole-exome sequencing profile scales

Genes from SPARK Filtering Type = Loss-of-Function Extracting Extracting Measuring Frequency < 1% Regress data available cognitive and polygenic Rare LoF variants age, sex for ASD and functioning score in SPARK genes and center non-ASD BAM validation data Quality check individuals Loftee HC-R-LoF Fluid intelligence, Frequency = 1 individual/family Brain volume ASD Townsend deprivation SCQ Filtering Protein relative position < 90% and volume of polygenic index, income, scores Exon usage in brain > 10% 13 sub-regions score HC-S-LoF qualification

Relationship LoF effect ASD Interplay between between rare on functioning diagnosis rare and common variants and in general assessment variants in ASD brain anatomy population Extended Data Figure 2

UK-Biobank admixture

1000g individuals UK-Biobank individuals

SSC admixture

HapMap individuals SSC individuals SPARK admixture

1000g individuals SPARK individuals Extended Data Figure 3 LOEUF threshold (< 0.35) 8 SCN8A

CACNA1CSCN2A KMT2A CHD8 CHD3 NF1 6 WDFY3NBEA GRIN2B NIPBL SCN1A IQSEC2TRIO SYNGAP1 CHD2 PHIP KCNB1SLC6A1 ACTB KDM3B MYT1L DDX3XNLGN2 PPP1CB DLG4 TAOK1 TRIP12 STXBP1 TLK2 CTCF UBE3A NRXN2NLGN3 CUL3 NAA15 NCKAP1 SIN3A SMARCC2 EBF3 ZBTB20 TCF4 PACS1 BCL11A CREBBPCASK SHANK3CTNNB1 TBR1 4 MED13L CSDE1 BRAF HNRNPH2 CNOT3 NRXN3 POGZ DYRK1AFOXG1 PTEN DNMT3A ASH1L RFX3 PPP2R5D NSD1 SLC9A6 ZNF462CHD7 HNRNPUPTPN11SOS1 DSCAM SETD2 MAP2K1 ATRX RORB PHF21A SHOC2 SOX5 CDKL5 MED13 CASZ1TANC2 BRSK2 FMR1 AHDC1KMT5B PCDH19 ARHGEF9 NRXN1 Top decile of missense NR4A2 RAF1 SHANK2 ANK3 ARID1BGIGYF2 TSC1 MEIS2 SOS2 ARX RIT1 KRAS FOXP1 z-score (> 2.45)

Missense z−score AUTS2 RIMS1 2 SRCAPKMT2CEP300 NRAS BCKDK HIVEP2 ANK2 ADNP RELN TSHZ3 UPF3BMAP2K2 PTCHD1 HRAS WAC RERE SON PSMD12 DEAF1 CAPRIN1 VPS13B ZNF292 KANSL1 POMGNT1 EHMT1 RAI1 AFF2 SETBP1 SETD5 BAZ2B LZTR1 KDM6B MBOAT7 ALDH5A1 ADSL MBD5 SPAST CIC NEXMIF IRF2BPL TCF20 TBCK ASXL3 PHF3 DMPK 0 TSC2 CHAMP1 GIGYF1 DHCR7 ANKRD11 MAGEL2

MECP2

0.03 0.10 0.30 1.00 LOEUF Extended Data Figure 4 a b

n=129n=123n=120n=113n=112n=105n=97 n=87 n=73 n=42 n=4 n=5 n=22 n=33 n=43 n=49 n=54 n=59 n=64 n=72 n=84 n=134 20 * ** *** **** **** ** 20 ** **** **** *** **** *** *** *** **

15 15

10 10 Relative risk Relative risk 5 5

0 0

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

= Relative position on CDS >= X Pext score <= X = Reference Reference * p<=0.05 ** p<=0.01 *** p<=0.001 **** p<=0.0001 Selected thresholds Relative risk = 1 c p=0.035

10.0

Relative risk 1.0

0.1

HC-R-LoF HC-S-LoF LoF type Extended Data Figure 5

OR=0.71 OR=0.98 p=0.03 p=0.84 OR=0.79 OR=1.1 p=0.046 p=0.17 100

80

60

40

20

Proportion of individuals carrying 0 ASD ASD Parents Parents rare synonymous mutation in SPARK gene (%) Non-ASD Non-ASD

SSC SPARK Extended Data Figure 6

GIGYF1

100,680,000 100,685,000 100,690,000 100,695,000 Position on chromosome 7 HC-S-LoF ASD child Relative Control HC-R-LoF ASD child Relative Control Pext > 0.9 N/A

NLGN2

7,405,000 7,410,000 7,415,000 7,420,000 Position on chromosome 17 Extended Data Figure 7

HC-R-LoF

1e−02

DHCR7

GIGYF1 POMGNT1 WDFY3 ANK2 VPS13B ASH1L KDM6B CHD8 ARID1B ANK3 CHD2 NF1 LZTR1 SCN2A NRXN3 ASXL3 ADNP 1e−03 KMT5B POGZ DYRK1A ALDH5A1 RIMS1 SYNGAP1 ANKRD11 TSC1 CAPRIN1 PTEN NRXN1 SHANK2 RAI1 TSC2 GRIN2B PACS1 MAGEL2 TRIO KMT2C STXBP1 SETD5 TLK2 DLG4 DSCAM RFX3 TBCK KMT2A CUL3 PPP2R5D NRXN2 MYT1L SHANK3 TBR1 EP300 RELN ZNF462 SOS1 SOX5 FOXP1

AUTS2 TRIP12 TCF20 MED13 MBD5 NSD1 ZNF292 MBOAT7 CSDE1 BAZ2B SRCAP ADSL SOS2

TAOK1 CTNNB1 CNOT3 AHDC1 HRAS SMARCC2 NLGN2 PHF3 BCKDK RERE DEAF1 KRAS 1e−04 NCKAP1 PSMD12 IRF2BPL MEIS2 NBEA CASZ1 SETBP1 SLC6A1 CHD3 CHD7 TCF4 NLGN3 CTCF SIN3A KANSL1 KCNB1 NR4A2 BRAF SETD2 DNMT3A DMPK PTCHD1 KDM3B ZBTB20 NAA15 FMR1 WAC MAP2K2 EHMT1 BRSK2

tion of ASD individuals CHAMP1 CIC TANC2 EBF3 GIGYF2 SCN8A

HIVEP2 SPAST Propo r

1e−05

1e−06

0 1e−05 1e−04 1e−03 Proportion of control individuals

HC-S-LoF

WDFY3 DYRK1A ASH1L TSC1 PACS1 1e−03 GIGYF1 SCN2A SYNGAP1 ARID1B POGZ ANK3 ANK2 GRIN2B CHD8 LZTR1 TBR1 SETD5 ASXL3 TSC2 NF1 ADNP RAI1 AUTS2 ANKRD11 SHANK2 CAPRIN1 RELN DSCAM KMT5B KMT2A SMARCC2 TLK2 CHD2EP300 ZNF292 PPP2R5D MBOAT7 VPS13B TRIP12 ZNF462 CUL3 AHDC1 RFX3 FOXP1 NRXN1 MED13 PTEN TAOK1 CTNNB1 NRXN2 MYT1L NSD1 MBD5 CHD3 CASZ1 TCF20 STXBP1 CSDE1 SETD2 PHF3 PSMD12 SHANK3 NLGN2 EBF3 RERE CNOT3 KDM3B BRAF KMT2C SRCAP DEAF1 1e−04 NAA15 GIGYF2 TANC2 SCN8A BRSK2 SPAST ADSL TBCK DNMT3A ZBTB20 PTCHD1 NCKAP1 KCNB1 CIC SOX5 HIVEP2 SOS2 RIMS1 DMPK SIN3A CTCF IRF2BPL SOS1 NBEA WAC NR4A2 EHMT1 BCKDK DLG4 MAGEL2 CHD7 SLC6A1

tion of ASD individuals CHAMP1 Propo r 1e−05

1e−06

0 1e−05 1e−04 1e−03 Proportion of control individuals Extended Data Figure 8

ANK2 0.001 ANK3 NF1 ASH1L GIGYF1 CHD8 MAGEL2 SCN2A RELN WDFY3 TSC1 POMGNT1 DYRK1A MBOAT7 ASXL3 ARID1B DLG4 GRIN2B AUTS2 NRXN3 KDM6B KMT2C PTEN SYNGAP1 TRIO ADNP PPP2R5D KMT5B POGZ CTNNB1 CSDE1 BAZ2B PSMD12 5e−04 CHD2 PACS1 TLK2 SHANK2 CAPRIN1 CUL3 SRCAPALDH5A1 TBR1 TRIP12 NRXN2DSCAM ZNF292 SETD5

le risk ANKRD11 SLC6A1 NRXN1 EP300 TCF20 BRAF MBD5 uta b STXBP1 i b RAI1 SHANK3 ZNF462 CNOT3 NSD1 SOS1 MYT1L Att r SOS2 HRAS AHDC1MED13 ADSL TAOK1 IRF2BPL 0 PHF3 SOX5 CIC NLGN2PTCHD1 NBEA NLGN3 CHD3 NR4A2 FOXP1 CASZ1 NAA15 SIN3A CTCF SCN8A SETBP1 CHAMP1 RFX3 MEIS2 SMARCC2 TANC2 TBCK KCNB1 FMR1 ZBTB20 KDM3B EHMT1BRSK2 KANSL1 DEAF1 GIGYF2 EBF3 NCKAP1 MAP2K2 WAC BCKDK CHD7 KMT2A KRAS LZTR1 −5e−04 HIVEP2 RERE SETD2 SPAST TCF4 RIMS1 DNMT3A TSC2

0.1 1 10 100 Inf. Relative risk Extended Data Figure 9 a LZTR1 b DNMT3A POMGNT1 LZTR1 DHCR7 KRAS ADSL DNMT3A ALDH5A1 HRAS ADSL TBCK BCKDK MBOAT7 1.00 PTEN 1.00 TBCK BCKDK DEAF1 SHANK2 IRF2BPL CAPRIN1 VPS13B MBOAT7 GIGYF1 VPS13B GIGYF1 NF1 MAGEL2 PPP2R5D DEAF1 PTCHD1 PTEN DMPK DMPK NF1 SHANK2 ZBTB20 DSCAM MAP2K2 NRXN1 FMR1 NAA15 BRSK2 BRSK2 ZBTB20 IRF2BPL TAOK1 MAGEL2 NRXN2 KANSL1 MEIS2 PACS1 CAPRIN1 RIMS1 PTCHD1 NRXN2 PHF3 SOS2 SOS2 AUTS2 NRXN1 AUTS2 CHAMP1 NAA15 SOX5 BAZ2B DLG4 NLGN3 RIMS1 PHF3 SETD5 0.30 SETD2 DYRK1A 0.30 BRAF SETD2 SETD5 DYRK1A CIC CIC ASXL3 FOXP1 TAOK1 CHAMP1 SPAST TBR1 DLG4 TCF4 SPAST BRAF RELN PACS1 CUL3 TBR1 SOX5 TANC2 EBF3 ASXL3 PSMD12 SLC6A1 RELN DSCAM CTCF PSMD12 CASZ1 TANC2 CTNNB1CUL3 PPP2R5D TLK2 TSC1 LOEUF score LOEUF score CSDE1 ZNF292 FOXP1 CASZ1 SOS1 CHD3 CTNNB1 MBD5 SCN2A CTCF EBF3 POGZ KDM6B SLC6A1 CHD3 MBD5 SCN2A NR4A2 SCN8A TCF20 ADNP POGZ SOS1 SHANK3 KMT2C RAI1 RERE TRIO KCNB1 ARID1B NR4A2 NLGN2 TCF20 0.10 ADNP 0.10 SHANK3 ANK2 ARID1B KMT2C TLK2 RFX3 SETBP1 EP300 TSC1 RERE SCN8A ANKRD11 STXBP1 RAI1 MYT1L MYT1L NSD1 ZNF462 KCNB1 TSC2 NLGN2 AHDC1 ANKRD11 SRCAP EHMT1 ANK2 STXBP1 ZNF292 GIGYF2 KMT5B SMARCC2 ANK3 CHD7 KMT2A ASH1L GRIN2B ASH1L SRCAP GIGYF2 ANK3 CSDE1 TSC2 CHD8 GRIN2B CHD2 KMT5B WAC RFX3 HIVEP2 CNOT3 EHMT1 HIVEP2 ZNF462 NRXN3 CHD8 KDM3B WDFY3 WDFY3 NBEA CHD7 EP300 KDM3B WAC KMT2A SMARCC2 CNOT3 SYNGAP1 SIN3A AHDC1 NSD1 SIN3A SYNGAP1 0.03 NBEA TRIP12 0.03 MED13 NCKAP1 MED13 CHD2 TRIP12 NCKAP1 0.1 1.0 10.0 100.0 Inf. 0.1 1.0 10.0 100.0 Inf. Relative risk Relative risk c p=0.00064 d p=0.00011

30.0

10.0 10.0

3.0 Relative risk Relative risk 1.0

1.0

0.3 0.1

No Yes No Yes LOEUF score < 0.35 LOEUF score < 0.35 e HC-S-LoF (n=102) pLI HC-R-LoF (n=118) p=0.011 p=0.016

LOEUF p=0.0063 p=0.01

TADA p=7.6e−06 p=3.3e−05 Deleteriousness scale

−1.0 −0.5 0.0 0.5 1.0 Pearson correlation with relative risk Extended Data Figure 10 a Developmental period

** * * * *

Cerebral Brain regions cortex 0.2 * * * * * 0.1 Subcortical 0.0 regions −0.1 −0.2 Cerebellum Kendall tau between expression and relative risk of Infancy HC-R-LoF Early fetal Late fetalChildhood AdolescenceYoung adult Early Latemid-fetal mid-fetal b c p=0.023 p=0.32 p=0.38 p=0.082 p=0.16 p=0.055 100 100

10 10 Relative risk (log10) Relative risk (log10) 1 1 No Yes No Yes No Yes No Yes No Yes No Yes Synapse Chromatin Transcription Synapse Chromatin Transcription (SynGO) organization (n=59) (SynGO) organization (n=52) (n=22) (n=31) (n=17) (n=27) Extended Data Figure 11 a Probands Probands b not carrying HC-S-LoF carrying HC-S-LoF 1.00 (n=6,091) OR=1.7, p=0.0025 (n=165)

60000 0 Yes Yes 160 0.75

0.50

19% No No 29% 0.25 40

71% Fraction of individuals 81% 120 0.00

2000 4000 Gender History of mental distress Yes Female No Male 80 HC-S-LoF Control individuals Control individuals No not carrying HC-S-LoF carrying HC-S-LoF (n=46,064) OR=1.1, p=0.65 (n=320) Yes

0 0 300

40000

45.7% 10000 44%

54.3% 56% 100

30000 200

20000 c Probands Probands d not carrying HC-R-LoF carrying HC-R-LoF 40 (n=6,049) OR=1.4, p=0.06 (n=207)

60000 200 0 30

20

1000 SCQ 5000 19% 25% 10 50 81% 75% 150 0

2000 ASD Non-ASD 4000 probands siblings

HC-R-LoF No 3000 100 Yes Control individuals Control individuals not carrying HC-R-LoF carrying HC-R-LoF e (n=45,611) OR=1, p=0.77 (n=777) 1.00 0 0 Yes Yes 40000 0.75

0.50 45.7% 10000 45.2% 600 No No 200 0.25 Fraction of individuals 54.3% 54.8% 0.00 30000 History of mental distress Yes 20000 400 No Extended Data Figure 12

a Qualification Income level 1.00 1.00 10

0.75 0.75 x 10 HC-R-LoF 5 No 0.50 0.50 Yes 0 5 wnsend ind e o

0.25 0.25 T Fratcion of individuals −5 Fluid Intelligence score 0.00 0.00 0

Income Qualification level Less than 18,000 CSEs or equivalent 18,000 to 30,999 O levels/GCSEs or equivalent 31,000 to 51,999 NVQ or HND or HNC or equivalent 52,000 to 100,000 A levels/AS levels or equivalent Greater than 100,000 College or University degree b p=0.057, n=687 Townsend index (reversed) p=0.0084, n=315 p=0.0083, n=167 p=0.739, n=527 Qualification level p=0.2851, n=231 p=0.2623, n=124 p=0.259, n=599 Income p=0.0105, n=274 p=0.0083, n=148 Relative risk p=0.259, n=667 Fluid intelligence score > 0 p=0.0480, n=305 > 1 p=0.4101, n=162 > 2

−1.5 −1.0 −0.5 0.0 0.5 1.0 Regression coefficient associated to presence of LoF variant Extended Data Figure 13

T−test p=0.9 T−test p=0.56 7.5 T−test p=0.072 7.5 T−test p=0.91

Levene test p=0.93 Levene test p=0.35 Levene test p=0.68 olume Levene test p=0.044 5.0 5.0 4 5 olume olume x v v

t e 2.5 2.5 0 ain

0 al co r 0.0

r 0.0 al white matter v otal b r r T Intracranial volume −2.5 −2.5

−4 Cereb

−5 −5.0 Cereb 8 10 T−test p=0.71 10 T−test p=0.4 10 T−test p=0.1 T−test p=0.18 Levene test p=0.46 Levene test p=0.82 Levene test p=0.24 Levene test p=0.61 olume v olume 5 v 4 olume 5 olume 5 us v allidum

0 0 P 0 0 Putamen v Thala m Caudate nucleus −5 −5 −4

7.5 T−test p=0.77 7.5 T−test p=0.71 T−test p=0.049 T−test p=0.77 Levene test p=0.6 Levene test p=0.78 Levene test p=0.66 olume 5.0 Levene test p=0.038 v 5.0 5.0 olume 5 v olume v olume

v 2.5 2.5 2.5 0 0.0

0.0 ygdala 0.0 pus callosum A m −2.5 −2.5Hippocampus −2.5 −5 Co r −5.0Nucleus accumbens No Yes No Yes T−test p=0.46 T−test p=0.46 HC-S-LoF HC-S-LoF olume

10 Levene test p=0.81 v Levene test p=0.49

olume 8 x v t e

r 5 4

0 0

Cerebellum co −5 −4 No Yes Cerebellum white matter No Yes HC-S-LoF HC-S-LoF Extended Data Figure 14 a b SOS1 (n=3) SOS2 (n=3) Regulation of small GTPase mediated signal transduction RELN (n=1) NRXN3 (n=1) p=0.002 (n=5) TRIO (n=3) HIVEP2 (n=1) DYRK1A (n=2) p=0.586 (n=7) SPAST (n=1) UBE3A (n=1) KMT5B (n=2) 1 10 100 1000 Cluster 1 MAGEL2 (n=1) IRF2BPL (n=1) ARID1B (n=1) Odds ratio CASZ1 (n=3) RAF1 (n=1) 4 EHMT1 (n=3) KRAS (n=10) SLC6A1 (n=2) Synapse organization FOXP1 (n=4) TCF4 (n=7) p=0.004 (n=6) NLGN2 (n=2) ANKRD11 (n=2) DHCR7 (n=17) p=0.029 (n=6) LZTR1 (n=23) RIMS1 (n=18) 2 VPS13B (n=35) 1 10 100 1000 ADNP (n=2) SETBP1 (n=2) DMPK (n=16) Odds ratio KMT2A (n=9) TSC2 (n=16) ALDH5A1 (n=10) ANK2 (n=2) Cluster 1 Cluster 2 BCKDK (n=2) 0 PACS1 (n=2) Other SPARK genes RFX3 (n=3) SETD2 (n=3) DEAF1 (n=7) CSDE1 (n=1) POMGNT1 (n=9) SMARCC2 (n=2) RAI1 (n=3) CHD7 (n=6) MAP2K2 (n=1)

Cluster 2 ANK3 (n=2) −2 MECP2 (n=3) PHF3 (n=1) ASXL3 (n=1) PTPN11 (n=1) TBCK (n=5) DNMT3A (n=9) Wilcoxon test estimate of volume difference

between HC-S-LoF carriers and non-carriers NF1 (n=1) KCNB1 (n=3) CACNA1C (n=8) −4 SOX5 (n=5) RERE (n=3) SHANK2 (n=2) ADSL (n=2) BRSK2 (n=1) BAZ2B (n=2) SRCAP (n=1) NSD1 (n=1) WDFY3 (n=2) PHF21A (n=1) ZNF292 (n=1) CHD2 (n=2) CREBBP (n=1) NRXN2 (n=1) POGZ (n=1) CHD3 (n=1) PTEN (n=1) x te

ral cor PutamenPallidum Total brain AmygdalaThalamus Intra-cranial Hippocampus pus callosum Cerebral white matter Caudate nucleus Cor Cerebellum cortex Nucleus accumbens Cereb c Cerebellum white matter NLGN2 p.D756Efs*35 ID1 ID2 ID3 ID4 Total brain DEAF1 p.Q402X Total brain Thalamus Thalamus Putamen Putamen Pallidum Pallidum Intra-cranial Intra-cranial Hippocampus Hippocampus Corpus callosum Corpus callosum Cerebral white matter Cerebral white matter ID1 ID2 Cerebral cortex Cerebral cortex Cerebellum white matter ID3 ID4 Cerebellum white matter 76 y.o. 64 y.o. Cerebellum cortex Cerebellum cortex Caudate nucleus 67 y.o. 63 y.o. Caudate nucleus MD MD Amygdala Amygdala Nucleus accumbens No MD No MD Nucleus accumbens (anxiety) (depression) −5.0 0.0 5.0−5.0 0.0 5.0 −5.0 0.0 5.0 −5.0 0.0 5.0

RAI1 p.Q280Hfs*82 ID5 ID6 ID7 Total brain Thalamus Putamen Pallidum Intra-cranial Hippocampus Corpus callosum ID5 ID6 ID7 Cerebral white matter Cerebral cortex 60 y.o. 51 y.o. 48 y.o. Cerebellum white matter Cerebellum cortex MD MD No MD Caudate nucleus Amygdala (anxiety, (depression) Nucleus accumbens depression) −5.0 0.0 5.0 −5.0 0.0 5.0 −5.0 0.0 5.0 ID8 ID9 DHCR7 p.W151X Total brain Thalamus Putamen Volume Pallidum Intra-cranial 5.0 Hippocampus 2.5 Corpus callosum Cerebral white matter 0.0 Cerebral cortex ID8 ID9 Cerebellum white matter −2.5 Cerebellum cortex 73 y.o. 67 y.o. Caudate nucleus −5.0 Amygdala No MD No MD Nucleus accumbens −5.0 0.0 5.0 −5.0 0.0 5.0 Extended Data Figure 15 Relative risk > 0 Relative risk > 1 Relative risk > 2 n=60 n=53 n=44 2 n=25 OR=2.4, p=4e-03 n=19 OR=3.8, p=1e-04 n=15 OR=4.4, p=1e-04 n=266 n=162 n=99 OR=1.7, p=0.4 OR=2, p=0.3 OR=3.4, p=0.1 1.5

1

0.5

0

n=129 n=91 n=71 4 n=56 OR=1, p=1 n=31 OR=2.1, p=3e-03 n=22 OR=3.1, p=8e-05 n=1005 n=418 n=232 OR=2.2, p=0.06 OR=3.2, p=0.03 OR=5.6, p=6e-03 LoF variant prevalence 3

2

1

0 <10 <33 <50 All 50< 66< 90< <10 <33 <50 All 50< 66< 90< <10 <33 <50 All 50< 66< 90< Subgroups by different percentile ASD-PGS cutoff

ASD children Non-ASD siblings UK-Biobank individuals HC-S-LoF HC-R-LoF