Exome sequencing in sporadic autism spectrum disorders identifies severe de novo mutations

Abstract

Evidence for the etiology of autism spectrum disorders (ASD) has consistently pointed to a strong genetic component complicated by substantial locus heterogeneity^1,2. We sequenced the exomes of 20 sporadic cases of ASD and their parents, reasoning that these families would be enriched for de novo mutations of major effect. We identified 21 de novo mutations, of which 11 were protein-altering. Protein-altering mutations were significantly enriched for changes at highly conserved residues. We identified potentially causative de novo events in 4/20 probands, particularly among more severely affected individuals, in FOXP1, GRIN2B, SCN1A, and LAMC3. In the FOXP1 mutation carrier, we also observed a rare inherited CNTNAP2 mutation and provide functional support for a multihit model for disease risk³. Our results demonstrate that trio-based exome sequencing is a powerful approach for identifying novel candidate genes for ASD and suggest that de novo mutations may contribute substantially to the genetic risk for ASD.

ASD are characterized by pervasive impairment in language and communication, social reciprocity, and having restricted interests or stereotyped behaviors¹. Several new candidate loci for ASD have recently been identified using genome-wide approaches that discover individually rare events of major effect². A number of genetic syndromes with features of the ASD phenotype, collectively referred to as syndromic autism, have also been described⁴. Despite this progress, the genetic basis for the vast majority of ASD cases remains unknown. Several observations support the hypothesis that the genetic basis for ASD in sporadic cases may differ from that of families with multiple affected individuals, with the former more likely to result from de novo mutation events rather than inherited variants^1,5–7. In this study, we sequenced the protein-coding regions of the genome (the exome)⁸ to test the hypothesis that de novo protein-altering mutations substantially contribute to the genetic basis of sporadic ASD. In contrast with array-based analysis of large de novo copy number variants (CNVs), this approach has greater potential to implicate single genes in ASD.

We selected 20 trios with idiopathic ASD, each consistent with sporadic ASD based on clinical evaluations (Supplementary Table 1), pedigree structure, familial phenotypic evaluation, family history, and/or elevated parental age. Each family was initially screened by array comparative genomic hybridization (CGH) using a customized microarray⁹. We identified no large (>250 kbp) de novo CNVs but did identify a maternally inherited deletion (~350 kbp) at 15q11.2 in one family (Supplementary Fig. 1). This deletion has been associated with increased risk for epilepsy¹⁰ and schizophrenia^11,12 but has not been considered as causal for autism.

Similar to Vissers and colleagues¹³, who reported exome sequencing on 10 parent-child trios with sporadic cases of moderate to severe intellectual disability (ID), we performed exome sequencing on each of the 60 individuals separately, by subjecting whole-blood derived genomic DNA to in-solution hybrid capture and Illumina sequencing (Methods). We obtained sufficient coverage to call variants for ~90% of the primary target (26.4 Mb) (Table 1). Genotype concordance with SNP microarray data was high (99.7%) (Supplementary Table 2) and on average 96% of proband variant sites were also called in both parents (Supplementary Table 3). Given the expected rarity of true de novo events in the targeted exome (<1/trio) (Supplementary Table 4)¹⁴, we reasoned that most apparently de novo variants would result from undercalling in parents or systematic false positive calls in the proband. We therefore filtered variants previously observed in dbSNP, 1000 Genomes Pilot Project data¹⁵, and 1490 other exomes sequenced at the University of Washington (Supplementary Fig. 2). We performed Sanger sequencing on the remaining de novo candidates (<5/trio), validating 18 events within coding sequence and three additional events mapping to 3′ untranslated regions (Table 2). A list of predicted variant sites within these genes from the 1000 Genomes Pilot Project data¹⁵ is provided for comparison (Supplementary Table 5).

Table 1.

Summary of the exome sequencing results from of 20 sporadic ASD probands

Family SSC/SAGE ID#	Proband Sex	Fa Age months	Mo Age months	Trio Bases‡	% target +/− 2 bp	Coding SNV (− dbSNP/1kG)	Rare Disruptive SNV□	Coding Indels (− dbSNP/1kG)	Rare Indels ≠‚3n	Protein Coding De Novo Events
11048	M	358	358	23,901,726	88.19	14,095 (752)	131	74 (44)	27	0
11307	M	421	407	23,549,536	86.89	13,509 (583)	75	64 (40)	19	0
11580	M	443	305	23,823,712	87.90	13,912 (642)	89	62 (36)	24	1
11666	M	398	370	24,179,474	89.21	14,306 (622)	77	59 (40)	25	1
12325	M	363	313	24,088,772	88.88	13,866 (629)	79	65 (43)	24	1
12499	M	425	372	25,217,651	93.04	14,479 (634)	86	80 (47)	21	3
12575	M	351	317	24,259,870	89.51	14,568 (679)	78	80 (55)	26	0
12647	M	541	413	24,669,129	91.02	14,144 (830)	78	68 (42)	22	1
12680	M	502	471	24,437,989	90.16	14,124 (642)	69	70 (42)	24	2
12681	F	399	375	24,723,806	91.22	14,750 (691)	93	68 (39)	20	2
12817	M	485	430	24,520,475	90.47	14,364 (656)	83	72 (38)	24	2
12974	M	366	365	24,235,164	89.42	13,990 (555)	52	54 (37)	23	0
13095	M	337	322	24,460,239	90.25	14,605 (645)	66	89 (54)	29	0
13253	M	436	427	24,070,345	88.81	13,775 (610)	96	41 (25)	16	2
13284*	M	300	302	24,911,060	91.91	17,806 (639)	111	151 (79)	53	1
13466	M	353	385	24,676,574	91.05	14,023 (591)	72	58 (39)	23	0
13683	M	470	402	24,139,439	89.06	14,419 (725)	73	72 (49)	22	0
13708	M	397	382	23,933,169	88.30	13,997 (686)	77	78 (41)	26	2
13970	M	313	234	24,465,009	90.26	14,293 (626)	84	89 (58)	31	0
SAGE4022	F	271	283	24,130,743	89.03	14,538 (713)	141	86 (56)	29	0
AVG	18M:2F	397	362	24,319,694	89.73	14,378 (658)	86	74 (45)	25	0.9

Paternal and maternal ages at time of conception were estimated based on month-year birth information assuming a 9-month pregnancy

^‡Number of bases covered at 8x and Q30 in all three individuals

^*13284 Included Additional RefSeq Targets

^□Not observed in 1490 other exomes sequenced at the University of Washington

^#Simons Simplex Collection (SSC) or Study of Autism Genetics Exploration (SAGE) family number

Table 2.

Summary of confirmed de novo mutation events

SNV Proband	Type	Chromosome: Position	Gene Symbol	Variant	AA Change	GERP Score	Grantham Score	PolyPhen-2	CpG	Ts/TV	Mut Origin
11580.p1	missense	chr20:2239665	TGM3	R	V144I	5.15	29	probably damaging	Y	Ts	Mo
11666.p1	missense	chr9:132904111	LAMC3*	R	D339G	4.92	94	probably damaging	N	Ts	Fa
12325.p1	3′UTR	chr12:55708658	MYO1A	R		2.23			N	Ts
12325.p1	missense	chr16:19951169	GPR139	Y	S151G	1.71	56	benign	N	Ts
12499.p1	missense	chr2:166556317	SCN1A*	R	P1894L	5.55	98	probably damaging	N	Ts	Fa
12499.p1	synonymous	chr3:38033207	PLCD1	K		−8.24			Y	Tv
12499.p1	missense	chr6:152865504	SYNE1	Y	Y282C	4.48	194	probably damaging	N	Ts
12575.p1	3′UTR	chr9:32619906	TAF1L	R		−1.02			Y	Ts
12647.p1	3′UTR	chr16:23585994	DCTN5	Y		−0.989			N	Ts
12647.p1	missense	chr5:68453390	SLC30A5	S	S561R	4.6	110	possibly damaging	N	Tv
12680.p1	synonymous	chr2:101992478	IL1R2	Y		−1.53			N	Ts
12680.p1	synonymous	chr5:132251451	AFF4	Y		−11.2			Y	Ts	Fa
12681.p1	3′ splice	chr12:13614220	GRIN2B*	Y		4.17	215#		N	Ts	Fa
12681.p1	synonymous	chr7:142274902	EPHB6	Y		−3.14			Y	Ts	Fa
12817.p1	synonymous	chr2:143724639	ARHGAP15	R		3.51			N	Ts
13253.p1	missense	chr3:39204494	XIRP1	Y	V483M	2.04	21	probably damaging	N	Ts
13253.p1	synonymous	chr16:74121475	CHST5	Y		−3.22			Y	Ts
13284.p1	synonymous	chr2:179145956	TTN	Y		0.328			Y	Ts
13708.p1	missense	chr17:58033198	TLK2	Y	S595L	5.43	145	probably damaging	Y	Ts
13708.p1	missense	chr3:30004687	RBMS3	Y	T383M	5.44	81	probably damaging	Y	Ts
Indel
12817.p1	frameshift	chr3:71132860	FOXP1*	+T	A339SfsX4	5.38‡	215#		NA	NA	Fa

^*Disruptive de novo mutations that are potentially causative

^#Maximum Grantham score given for splice and frameshifting variants

^‡Average GERP score for two sites flanking the insertion

We observed subtle differences with respect to mutation rate and characteristics when compared to Vissers and colleagues¹³ (Supplementary Note). The overall protein-coding de novo rate (0.9 events/trio) was slightly higher than expected¹⁴ (0.59 events/trio), suggesting that we are identifying the majority of de novo events in these trios (Supplementary Table 4). The transition to transversion ratio was highly skewed (18:2), with eight transitions mapping to hypermutable CpG dinucleotides¹⁴. The proportion of synonymous events was higher than expected based on a neutral model and may reflect selection against embryonic lethal nonsynonymous variants. We successfully determined the parent of origin for seven events, six of which occurred on the paternal haplotype (Table 2). Notably, the eight probands with two or more validated de novo events corresponded to families with higher parental age (Mann–Whitney U, Combined Age, One-Sided P<0.004).

Eleven of the 18 coding de novo events are predicted to alter protein function. Each of these mutations occurred at a different gene, precluding a statistical assessment for any specific locus despite their deleterious nature (e.g. PolyPhen-2¹⁶). We assessed whether proband de novo mutations were enriched in the aggregate for disruptive events by considering two independent quantitative measures: the nature of the amino-acid replacement (Grantham matrix score¹⁷) and the degree of nucleotide-level evolutionary conservation (Genomic Evolutionary Rate Profiling (GERP)^18,19) (Fig. 1a,b). For comparison, we sequenced 20 exomes from unrelated ethnically matched controls (HapMap) and applied the same filters to identify coding-sequence mutations that were common or private to each of the samples. These control DNA were isolated from immortalized lymphoblasts; however, the counts of private variants in the cases and controls were highly similar suggesting that suggesting that the contribution of novel somatic events is likely minimal (Supplementary Fig. 3).

Figure 1.

Evaluation of de novo mutations by simulation, proband severity, and family 12817. a,b We compared the mean Grantham (black x-axis) and GERP scores (black y-axis) of the 10 proband de novo protein-changing substitutions to 20 HapMap control samples by building a distribution of the mean values of 10 randomly selected common or private variants over 1000 trials. Splice-site and nonsense events were given a maximum Grantham score (215) and indels were not included in the simulation. Histograms show the relative frequency (blue axes) of each distribution. Points show the proband variants, with variants from the same individual highlighted (blue=13708.p1, red=12499.p1). Proband mean values, GERP: 4.349 and Grantham: 104.3. *FOXP1 not included in proband mean values. a, Control common variants (GERP: p<0.001, Grantham: p=0.015). b, Control rare variants (GERP: p=0.026, Grantham: p=0.098). c,d We evaluated the disease severity of the mutation carriers 12817.p1-FOXP1 (brown), 12681.p1-GRIN2B (green), 12499-SCN1A (blue) and 11666.p1-LAMC3 (red). c, Box and whisker plot of Full Scale Intelligence Quotient (FSIQ) values. d, Box and whisker plot of Calibrated Severity Scores (CSS) based on the Autism Diagnostic Observation Schedule (ADOS). Data were available for 19/20 probands; CSS were estimated for two probands based on ADOS module 4 data. e, Pedigree for 12817 showing chromatogram traces surrounding FOXP1 (top) and CNTNAP2 (bottom) mutation events. Proband carries a de novo single-base (+A relative to mRNA) frameshifting mutation p.A339SfsX4 in FOXP1 and an inherited missense variant p.H275A in CNTNAP2.

We determined by simulation the expected mean GERP and Grantham distributions for 10 randomly selected common or private control single nucleotide variants (SNVs) (Methods). When we compared the observed means of the 10 de novo protein-altering ASD proband variants to the distribution of common control SNVs (Fig. 1a), they corresponded to more highly conserved (GERP: p<0.001) and disruptive amino acid mutations (Grantham: p=0.015). If we limited the analysis to the private control SNVs, which serve as a proxy for evolutionarily young mutation events (Fig. 1b), we again found the de novo events were at the right tail of these distributions. Only the mean GERP score, however, remained significant (GERP: p=0.02, Grantham: p=0.115). In total, these results suggest that these de novo mutation sites are subjected to stronger selection and likely to have functional impact.

We identified a subset of trios (4/20) with disruptive de novo mutations that are potentially causative, including genes previously associated with autism, ID, and epilepsy (Table 2 and Supplementary Note). We examined the available clinical data for each of these four families and found they were among the most severely affected individuals in our study based on intelligence quotient (IQ) measures and on calibrated severity score²⁰ (CSS), which is largely independent from IQ and focuses specifically on autistic features with a score of 10 being most severe (Fig. 1c,d). For example, in proband 12681 we identified a single-base substitution (IVS9-2A>G, CCDS8662.1) at the canonical 3′ splice site of exon 10 in Glutamate receptor, ionotropic, N-methyl D-aspartate 2B (GRIN2B) (Supplementary Fig. 4a,b). She is severely affected (CSS 9), with evidence of early onset, possible regression, and comorbid for mild ID. Expression and association studies have suggested that glutamatergic neurotransmission may play a role in ASD⁴. Recently, Endele and colleagues²¹ described GRIN2A and GRIN2B as sites of recurrent de novo mutations in individuals with mild to moderate ID and/or epilepsy suggesting variable expressivity. Our data suggest that de novo mutations in GRIN2B may also lead to an ASD presentation.

Proband 12499 has a missense variant (p.P1894L, CCDS33316.1) predicted to be functionally deleterious and at a highly conserved position in Sodium channel, voltage-gated, type I, alpha subunit (SCN1A) (Supplementary Fig. 4c). He is severely affected (CSS 8) with evidence of early onset, possible regression, language delay, a diagnosis of epilepsy and mild ID. SCN1A was previously associated with epilepsy and suggested as an ASD candidate^22,23, although limited screening has been conducted in idiopathic ASD. Hundreds of disease-associated mutations have been described in epilepsy and typically patients with de novo events show more severe phenotypes²⁴. The proband also carries the maternally inherited 15q11.2 deletion increasing the risk for epilepsy¹⁰.

Proband 11666 has a missense variant (p.D399G, CCDS6938.1) predicted to be functionally deleterious and at a highly conserved position within the second laminin-type epidermal growth factor-like domain of Laminin, gamma 3 (LAMC3) (Supplementary Fig. 4d). He is severely affected (CSS 10) with evidence of early onset and moderate ID. LAMC3 is not known to be involved in neuronal development; however, human microarray data have shown expression in many areas of the cortex and limbic system²⁵. Additional study is warranted since laminins have structural similarities to the neurexin and contactin-associated families of proteins, both of which have been associated with ASD².

The fourth example of a potentially causative mutation is a single-base insertion in Forkhead box P1 (FOXP1), introducing a frameshift and premature stop codon (p.A339SfsX4, CCDS2914.1) in proband 12817 (Fig. 1e). He is severely affected (CSS 8) with evidence for regression, language delay, and comorbidity for moderate ID and nonfebrile seizures. Recently, rare occurrences of large de novo deletions and a nonsense variant disrupting FOXP1 were reported in individuals with mild to moderate ID and language defects, with or without ASD features^26,27. FOXP1 encodes a member of the forkhead-box family of transcription factors and is closely related to FOXP2, a gene implicated in rare monogenic forms of speech and language disorder^28–31. Functional evidence of heterodimer formation and overlapping neural expression patterns suggests that FOXP1 and FOXP2 can co-regulate gene expression in the brain^32,33. We assessed relative levels of the mutant transcript in proband derived lymphoblasts finding strong evidence for nonsense-mediated decay (NMD) (Supplementary Fig. 5a). HEK293T cell-based functional assays further demonstrated that, if translated, the protein would be truncated and mislocalized from the nucleus to the cytoplasm—similar to results obtained with FOXP2 mutations³¹ (Supplementary Fig. 5b,c).

Remarkably, in addition to the FOXP1 mutation, proband 12817 also carried an inherited missense variant (p.H275A, CCDS5889.1) in Contactin associated protein-like 2 (CNTNAP2) predicted to be functionally deleterious and at a highly conserved position. This variant is likely to be extremely rare or private as it was not observed in 942 previously sequenced controls³⁴ or in 1490 other exomes. CNTNAP2 is directly downregulated by FOXP2³⁵ and has been independently associated with ASD and specific language impairment^34–37. In HEK293T cells, we found that wild-type FOXP1 significantly reduced expression of CNTNAP2 (p=0.0005), while the truncated protein was associated with a three-fold expression increase (p=0.0056) (Supplementary Note, Fig. 5d). Overall, we hypothesize that FOXP1 haploinsufficiency (due to NMD), combined with dysfunction of FOXP1 mutant proteins that escape this process, may yield overexpression of CNTNAP2 proteins, amplifying any deleterious effects of p.H275A in the proband.

Among the ~110 (85 SNVs, 25 indels) novel inherited protein-altering variants in each proband, we identified several rare inherited variants in genes overlapping the SFARI Gene³⁸, a curated database of potential ASD candidate loci, but no excessive burden in cases relative to controls (Supplementary Table 6). While the numbers from our pilot study are few, we do observe two cases with a significant de novo event and a potential inherited risk variant (12817p1:FOXP1/CNTNAP2 and 12499.p1: SCN1A/15q11.2 deletion) highlighting that in some sporadic families a multihit model may be playing a role³ (Supplementary Table 7). In the future, this hypothesis could be further explored by comparing burden in a much larger number of affected/unaffected sibling pairs.

The probands with the four potentially causative de novo events met strict criteria for a diagnosis of autistic disorder (Supplementary Note). Our finding of de novo events in genes that have also been disrupted in children with ID without ASD, ID with ASD features, and epilepsy provides further evidence that these genetic pathways may lead to a spectrum of neurodevelopmental outcomes depending on the genetic and environmental context^2,4. Recent data suggest that CNVs may also blur these lines with diverse conditions all showing association to the same loci^2,4. Distinguishing primary from secondary effects will require a better understanding of the underlying biology and identification of interacting genetic and environmental factors within the phenotypic context of the family. The identification of de novo events along with disruptive inherited mutations underlying “sporadic” ASD has the potential to fundamentally transform our understanding of the genetic basis of ASD.