Introduction

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder characterized by persistent deficits in social communication and social interaction across multiple contexts, and restricted, repetitive patterns of behavior, interests, or activities, with onset during the first 3 years of life. The prevalence of ASD is estimated to be 1 in 54–100 children, and the male-to-female ratio has been reported as 4.2–4.3:1 [1, 2].

Both genetic and environmental factors have long been investigated as contributors to ASD. A genetic component of ASD was first suspected in twin studies; the concordance of ASD in identical twins (60–96% concordance) is much higher than that in dizygotic twins (0–36%) [3, 4]. Furthermore, the heritability of ASD is estimated to be 60–90% (reviewed by Ruzzo et al. [5]). In the past few decades, many genetic studies have revealed that a proportion of ASD cases can be explained by rare inherited or de novo single-nucleotide variants (SNVs), including small insertions and deletions (indels) and copy number variants (CNVs) [5,6,7,8,9], as well as by commonly inherited SNVs/indels and CNVs [4, 10]. It is important to distinguish between individual and population risks for ASD; common variants can explain a genetic contribution at the population level but not at the individual level, whereas rare and/or de novo variants can explain ASD at the individual level but not at the population level [11]. Indeed, both rare and common variants are thought to contribute to the pathomechanism of ASD, but it remains unclear how this actually occurs.

The diagnostic yield of ASD using chromosomal microarray analyses has been reported as 3.0–9.3% [12,13,14], and that using exome sequencing (ES) as 6.1–8.4% [12, 14]. There are relatively few reports of the molecular diagnosis of ASD using ES to detect both SNVs/indels and CNVs. Feliciano et al. [15] reported that the diagnostic yield of ES for detecting SNVs/indels and CNVs in ASD was 10.4% in 457 families. Although the diagnostic rate of ES depends on the cohort and target disease that is being assessed, its rate in ASD is lower than that in other genetic conditions, which have usually been reported as 25%–44% [16, 17]. However, many aberrant disease-causing genes have been identified over the years, and 10–15% of unresolved cases may obtain a molecular diagnosis by reanalysis (reviewed by Lee and Nelson [18]). Here, we investigated monogenic causes that may be clinically relevant for the molecular diagnosis of ASD, and simulated how the re-analysis of ASD cohorts might improve diagnostic rates (i.e., the identification of genetic causes in ASD patients).

Materials and methods

Study cohort

A total of 405 affected individuals (283 males and 122 females) from 377 families clinically diagnosed with ASD based on the Diagnostic and Statistical Manual of Mental Disorders-V (Table 1) and their unaffected parents were included in this multi-center cohort. Our ASD cohort consisted of 351 trios, 24 quads (two affected siblings in each), and two quintets (three affected siblings in each). Samples were collected after obtaining written informed consent. Part of this cohort (N = 261) has been previously analyzed [19]. However, the analytical approach and aim of the present study are very different to those of the previous investigation; this study evaluated the pathogenicity of the respective variants to reach a molecular diagnosis at the individual level considering the American College of Medical Genetics (ACMG)/Association for Molecular Pathology (AMP) guidelines [20], whereas the previous study was more explorative and sought to understand de novo variants in ASD in a population-based manner. The present study was approved by the Institutional Review Boards of Yokohama City University School of Medicine and the other collaborating hospitals.

Table 1 Cohort overview and molecular diagnostic rate in this cohort.

Genetic analysis

DNA was extracted from human peripheral leukocytes or saliva. ES was performed as previously reported [21]. In brief, genomic DNA was sheared using the Covaris S2 system (Covaris) and genome partitioning was performed using SureSelect Human All Exon kits (Agilent Technology) according to the manufacturers’ instructions. Prepared samples were run on a HiSeq 2000/2500 instrument (Illumina) with 101-bp paired-end reads. The reads were mapped to the human reference genome hg19 using Novoalign (Novocraft). The SNVs/indels were called using the Genome Analysis Toolkit 3 (UnifiedGenotyper) and annotated using ANNOVAR. We selected candidate SNVs/indels in all possible inheritance modes: autosomal dominant (de novo), autosomal recessive, X-linked dominant, and X-linked recessive (for male individuals only). Each filtering setting is described in the Supplementary text. The candidate variants were validated by Sanger sequencing. The pathogenicity of candidate variants was classified in accordance with the ACMG/AMP guidelines [20]. We used SIFT, Polyphen-2, MutationTaster, and CADD for the in silico prediction of missense variants. All variant descriptions were confirmed by Mutalyzer (2.0.35).

We also examined candidate CNVs in ES data using the eXome Hidden Markov Model (XHMM) [22]. We picked up the candidate CNV calls with a QSOME score of 60 or more. CNVs were excluded from the candidates when their regions overlapped with polymorphic structural variations (Database of Genomic Variants), overlapped with regions frequently observed in our in-house control data, or did not contain protein-coding genes. The rest of the candidate regions and any CNVs of more than 200 kb in length were manually evaluated in terms of whether they overlapped with dose-sensitive regions or genes with a score of 3 (Sufficient Evidence) or 2 (Emerging Evidence) for haploinsufficiency or triplosensitivity in ClinGen, were in regions known to be associated with CNV syndromes [DatabasE of genomiC varIation and Phenotype in Humans using Ensembl Resources (DECIPHER) syndromes, n = 66], or were dose-sensitive genes as determined using DECIPHER. The pathogenicity of each CNV was classified in accordance with ACMG/AMP guidelines [23]. The candidate CNVs were validated by quantitative polymerase chain reaction (PCR) in two different regions within the CNV-called regions, with normalization using two independent control regions (FBN1 and STXBP1). Primer sequences and PCR conditions are available on request.

Simulation of diagnostic yield by year

By referring to the Human Genome Mutation Database Professional and the Online Mendelian Inheritance in Man (OMIM) database, we noted the years in which many aberrant genes were first identified in neurodevelopmental disorders (including ASD). We then calculated the number of cases in which a molecular diagnosis would have been obtained (together with the diagnostic rate) if exome analysis were to have been performed each year.

Gene ontology (GO) analysis

We performed GO analysis using DAVID Bioinformatics Resources version 6.8 to determine the enrichment of genes in this ASD cohort. We input 52 genes (Supplementary Table 1) harboring disease-causing variants or associated with disease-causing CNVs in our cohort, and visualized the results using the GOseq package [24] of the R program. We also performed GO analysis with the enrichGO function from the ClusterProfiler package [25] of R program with Benjamini–Hochberg adjustment of p values. GO terms with the 25 lowest p values were visualized.

Results

SNV/indel detection

Among the 405 affected individuals, 55 SNVs that can explain the clinical phenotype were detected in 53 individuals (13.1%), including 45 SNVs and 10 indels (Fig. 1A, Table 1, and Supplementary Table 2). All variants were confirmed by Sanger sequencing. Among them, 21 variants had not previously been reported (Supplementary Table 2). On the basis of the ACMG/AMP guidelines [20], 28 and 27 variants were classified as “Pathogenic” and “Likely pathogenic,” respectively (Supplementary Table 2). The inheritance modes of the variants were classified as autosomal dominant (de novo, n = 39), autosomal recessive (n = 2, one individual with compound heterozygous variants, each inherited either from the father or the mother), X-linked dominant (de novo, n = 9), and X-linked recessive (de novo, n = 3; maternal inheritance, n = 2) (Fig. 1B, Supplementary Table 2).

Fig. 1: Genetic architecture in our autism spectrum disorder (ASD) cohort.
figure 1

A Identified monogenic single-nucleotide variants (SNVs) including small insertions and deletions and copy number variations (CNVs) in our ASD cohort. B The number of individuals with pathogenic SNVs and CNVs leading to a molecular diagnosis under the respective genetic inheritance modes. C Size distribution of 25 candidate CNVs. D The diagnostic yields in male and female individuals with either SNVs or CNVs.

One male patient (Individual ID: 8553) had two disease-causing variants: a de novo BRAF missense variant [NM_004333.6, c.722 C > T, p.(Thr241Met)] and a de novo CAMK2B missense variant [NM_001220.5, c.1991C > T, p.(Pro664Leu)]. He was diagnosed with BRAF-associated cardio-facio-cutaneous syndrome with acute encephalopathy [26]. This BRAF variant is classified as “Pathogenic” based on ACMG/AMP guidelines, and it was previously reported as pathogenic in multiple patients [27]. The CAMK2B variant is classified as “Likely pathogenic” based on ACMG/AMP guidelines, and its pathogenicity may contribute to some of the patient’s clinical features (developmental delay, speech delay, and no reacquisition of language skills after acute encephalopathy) because pathogenic CAMK2B variants are found in mental retardation, autosomal dominant 54 (MIM#617799).

Another male patient (Individual ID: 22716) had a de novo splicing variant at an intronic region of FOXP1 (NM_032682.6, c.1428 + 5 G > A). This variant is not registered in gnomAD, Exome Variant Server, or the Human Genetic Variation Database. It is not located at a canonical splicing site, but three different splice site prediction software tools gave a decreased score at the canonical splice donor site of intron 16 for the altered allele compared with the wild-type one (ESE finder, 10.49 to 7.05; NetGene2, 0.86 to 0.67; BDGP splice site predictor, 1.0 to 0.97). In addition, SpliceAI Lookup [28] predicted the splice acceptor loss 84 bp upstream from the variant at the pre-mRNA level (delta score 0.79) and the splice donor loss 5 bp upstream from the variant at the pre-mRNA level (delta score 0.52) in the variant allele; these predictions indicate the impaired splicing of exon 16. In addition, this base is highly conserved with a Genomic Evolutionary Rate Profiling score of 6.17. This patient’s clinical features could be explained by the FOXP1 variant, which causes mental retardation with language impairment with or without autistic features (MIM#613670). On the basis of ACMG/AMP guidelines, this variant can be classified as “Likely pathogenic.”

We also found five variants of unknown significance (VUSs) in five genes that may potentially explain the patients’ phenotypes (Supplementary Table 3). They were all missense variants in X-linked genes inherited from unaffected mothers. Among the five variants, three (NLGN4X, TMLHE, and IL1RAPL1) are thought to be X-linked recessive. These three variants match PM2 (extremely low frequency if recessive) and PP3 (multiple in silico prediction) and are classified as VUSs in accordance with ACMG/AMP guidelines [20].

The other two genes—SRPK3 and PCDH11X—have not been conclusively shown to be related to diseases in OMIM. A missense SRPK3 variant [NM_014370.4, c.475 C > G, p.(His159Asp)] was previously reported in intellectual disability [29]. As for PCDH11X, at least three truncating variants, four gross deletions, and eight gross insertions have been reported previously in individuals with neurodevelopmental disorders [30], including autism and developmental dyslexia, according to the Human Gene Mutation Database (as of October 17, 2022). Meanwhile, at least 21 loss-of-function PCDH11X variants are registered in gnomAD SVs v2.1, but only six loss-of-function hemizygous variants in 10 individuals and no homozygous loss-of-function variants were observed in control populations. The most common hemizygous loss-of-function variant [chrX: 91873457 C > T, p.(Arg1188*)] was observed in 5 of 183,192 alleles (minor allele frequency = 0.000027), implying that PCDH11X null variants produce variable neurodevelopmental symptoms including ASD. Our case (Individual ID 19010) had a novel missense variant, which remains a VUS in line with ACMG/AMP guidelines [20].

CNV detection

Among 405 affected individuals, 27 possible candidate CNVs were detected in 25 patients. Two of these 27 CNVs were unable to be confirmed by quantitative PCR (likely false positive). Another 4 of the remaining 25 CNVs in two affected individuals were unable to be validated because of the limited residual DNA samples (Supplementary Table 4). Finally, the remaining 21 CNVs were validated in 21 patients (5.2% of our cohort), including 10 deletions and 11 duplications. The sizes of the CNVs ranged from 289 to 21,972 kb, with XHMM QSOME scores of 92–99 (Fig. 1C, Supplementary Table 4).

Among the 21 CNVs, 13 led to a molecular diagnosis in 13 patients (3.2% of our cohort; five male and eight female patients) (Fig. 1A, Tables 1 and 2). Eight pathogenic CNVs occurred de novo, three were paternally inherited, and two were maternally inherited. Twelve disease-causing CNVs were classified as having autosomal dominant inheritance (including seven occurring de novo) and one as having X-linked dominant inheritance (Fig. 1B, Table 2). Seven CNVs were associated with microdeletion/duplication syndromes and the other six can be explained by the haploinsufficiency of single genes: BCL11A deletion related to Dias–Logan syndrome (MIM# 617101), NBEA deletion related to neurodevelopmental disorder with or without early-onset generalized epilepsy (MIM# 619157), SHANK3 deletion related to Phelan–McDermid syndrome (MIM#606232), NRXN1 deletion related to complex neurodevelopmental disorder [31, 32] (no MIM# is given), KDM6A deletion related to Kabuki syndrome 2 (MIM#300867), and EBF3 deletion related to hypotonia, ataxia, and delayed development syndrome (MIM#617330) (Table 2, Supplementary Table 4).

Table 2 Disease-causing CNVs for ASD.

The most common pathogenic CNVs in our ASD cohort were copy number gains of chromosome 15q11–q13 (n = 4), known as maternal 15q duplication syndrome associated with either maternal isodicentric 15q11.2–q13.1 supernumerary chromosome (tetrasomy for 15q11.2–q13.1) or maternal interstitial 15q11.2–q13.1 duplication (trisomy for 15q11.2–q13.1). Deletion of this region causes Prader–Willi syndrome or Angelman syndrome, depending on whether the deletion occurs in a paternally or maternally inherited chromosome. Regarding the copy numbers, two patients had trisomy and the other two possessed tetrasomy of this region (Supplementary Fig. 1). Three 15q11–q13 copy number gains occurred de novo (Individual IDs 15441, 17649, and 22728) and one was maternally inherited (Individual ID 15664 with trisomy).

Affected sibling analysis

In 24 quad and 2 quintet families, we examined two (in quad families) or three (in quintet families) affected siblings by ES to determine whether they shared an identical genetic cause. Surprisingly, only one quad family shared an indel variant [IRF2BPL, NM_024496.4:c.1484_1486delinsCGT, p.(Leu495_Pro496delinsProSer)] in affected siblings (Individual IDs 16771 and 16774) (Supplementary Table 5). In this family, germline mosaicism in either of their parents was suspected based on the observation that there were no mutant alleles in >50 reads of ES in both parental samples.

Interestingly, in 2 of the 26 familial cases, one of the affected siblings had disease-causing variants but the other(s) did not (Supplementary table 5). The molecular diagnostic rate in familial cases was calculated to be 7.4% (4/54), which was lower than that in simplex cases (17.7%, 62/351) in this cohort, implying a possible difference in genetic architecture between simplex and multiplex families.

Autosomal versus sex chromosomal genes

We identified disease-causing SNVs in 32 (11.3%) and CNVs in 5 (1.8%) of 283 male patients, and disease-causing SNVs in 21 (17.2%) and CNVs in 8 (6.6%) of 122 female patients. Interestingly, the diagnostic rate was nearly twice as high in female patients than it was in male patients in our cohort (13.1% in male and 23.8% in female patients; p = 0.0075, chi-squared test) (Fig. 1D, Table 1). Of all of the disease-causing SNVs/indels and CNVs in our cohort, 31 and 8 disease-causing variants in male patients were autosomal and X-linked, respectively (ratio of X-linked/all variants = 8/39, 20.5%), while 22 and 7 disease-causing variants in female patients were autosomal and X-linked, respectively (ratio of X-linked/all variants = 7/29, 24.1%). The molecular diagnostic rate of X-linked variants in female individuals was almost twice that in male individuals [5.7% (7/122) in females and 2.8% (8/283) in males].

Diagnostic yield improvement over the years

Recent technological developments in comprehensive genomic sequencing and the identification of many aberrant genes, susceptibility genes, and chromosomal abnormalities associated with ASD and its associated disorders should have led to improvements in the diagnostic yield over the years. We therefore simulated the diagnostic yield of the exome data using known disease-related aberrant genes for each year since 1992 (Supplementary Fig. 2). Among 65 patients, only 10 could have been diagnosed before 2000. However, 19 patients could have been newly diagnosed from 2000 to 2010, and 38 patients from 2011 onwards. It has previously been reported that the number of known disease-causing variants has rapidly increased [18, 33], especially since 2012. A rapid increase in diagnostic yield in 2012 was also expected in our cohort (Supplementary Fig. 2). Thus, the periodic reanalysis of ASD patients for whom disease causation remains unresolved is highly recommended. The average percentage of newly resolved cases among unresolved cases was 0.63% (range 0%–2.5%) per year in our ASD cohort.

GO analysis of genes with disease-causing SNVs and CNVs in this cohort

GO analysis was performed using the 52 genes in which we identified SNVs or CNVs in this cohort using DAVID 6.8 with the default settings (Supplementary Table 1). The cluster that was most enriched by functional annotation clustering was that containing genes related to transcription and DNA binding (Enrichment score: 2.23, Supplementary Table 6). The second and fourth clusters were also related to transcription; namely, helicase and chromatin regulation (Enrichment score: 1.87) and repression of transcription (Enrichment score: 1.76), respectively. The enriched terms were “Mental retardation,” “Disease mutation,” “Phosphorylation,” “Methylation,” “Epilepsy,” “Visual learning,” “Chromosomal rearrangement,” “Nucleus,” and “Autism spectrum disorder” with false discovery rates of <0.01 (Fig. 2A, Supplementary Table 7). Another GO enrichment analysis with enrichGO also showed that the ontology terms were mainly associated with nervous system development, synapses, behavior, and memory (Fig. 2B, Supplementary Table 8).

Fig. 2: Gene ontology (GO) enrichment analysis for our autism spectrum disorder (ASD) cohort.
figure 2

GO analysis was performed for 52 genes with pathogenic variants. A Visualization of GO enrichment analyzed with DAVID 6.8. Top 9 categories with a false discovery rate of <0.01 are shown. B Top 25 enriched GO terms analyzed with enrichGO are shown. Count: the number of hit genes within each category. Hits (%): proportion of the 52 input genes included in each GO term.

Discussion

We analyzed 405 individuals with ASD and identified pathogenic variants in 66 affected individuals (SNVs in 53 individuals, 13.1%, and CNVs in 13 individuals, 3.2%). We achieved a molecular diagnostic yield of 16.3% (66/405) in ASD patients by ES (Fig. 1A). The diagnostic yield in our cohort was much higher than those of previous reports (6.0–10.4%) [12, 14, 15], while the pathogenic CNV detection rate was similar to those of previous reports (3.0–9.3%) [12,13,14]. In families with affected individuals who received a molecular diagnosis, recurrent risk may be partially predicted. A molecular diagnosis may also provide a better understanding of the natural history of each patient and lead to appropriate medical care.

Some of our identified genes (PBX1 and ABCC8) have not yet been established as ASD-related genes. In OMIM, “autistic behavior” is not listed as a phenotype of PBX1 or ABCC8 aberrations; however, pathogenic PBX1 variants lead to speech and developmental delays, and pathogenic ABCC8 variants can result in neurological phenotypes because of repeated episodes of hypoglycemia. ASD-related features can therefore be observed under these genetic conditions. Nonetheless, it remains possible that other substantial genetic factors may coexist for ASD.

We analyzed 26 multiplex families (24 quads and 2 quintets) and identified a shared pathogenic variant in only one quad (Supplementary Table 5). In two other quads, we identified disease-causing variants in only one of the two affected siblings (i.e., not in both siblings). We initially expected that some of the familial cases would be explained by recessive variants shared by affected siblings; however, all identified variants were de novo. It has been reported that recessive variants contribute to a small proportion of ASD cases (Lim et al. estimated autosomal recessive contribution as 3% [34]), or up to 30% of cases in consanguineous families [35]. However, our cohort did not include any consanguineous families.

Notably, the diagnostic yield of simplex families (17.7%, 62/351) was much higher than that of multiplex families (7.4%, 4/54) in the present study. Similarly, it has been reported that rare de novo protein truncating events are more frequently observed in simplex families than in multiplex families [5, 36, 37]. Together, these findings suggest different genetic architecture in simplex and multiplex families.

Monogenic causative variants were more commonly identified in female patients than in male patients in this study (23.8% vs. 13.1%, respectively). Similarly, a two-fold enrichment of de novo protein-truncating variants in highly constrained genes in ASD females versus males has been reported [9]. These observations are consistent with the “female protective effect” model in which fewer women are diagnosed despite having the same risk as men for developing ASD [9]. A gender bias also tends to be more prominent in groups with higher intelligence quotient scores (male bias is as high as 9:1 among cases with normal-to-high-range intelligence quotient scores [high-functioning] but as low as 1.6:1 among cases with intellectual disability), and it has been suggested that ASD may be masked by higher language skills, especially in high-functioning females (reviewed by Werling [38]). Fewer high-functioning females may therefore be diagnosed with ASD, resulting in a higher proportion of females with intellectual disabilities in the diagnosed population; consequently, more monogenic causes might be identified in female ASD patients.

The molecular diagnostic rate of X-linked variants in female individuals was approximately twice that of male individuals (5.7% vs. 2.8%, respectively) in the present study. In a neurodevelopmental disorder cohort, it was reported that de novo variants are enriched in some X-linked genes in female patients [39]. The most likely explanation for this phenomenon involves hemizygous male lethality [39]. Another possibility involves difficulties in defining the pathogenicity of variants in X-linked recessive traits—especially for maternally inherited missense variants—when previously unreported as disease-causing. Thus, even if the variants are truly disease-causing, they might be classified as VUSs under the current ACMG/AMP guidelines. Further functional and/or genetic evidence may be needed for confirming pathogenicity.

In our GO analysis using DAVID 6.8, the most enriched cluster contained genes related to transcription and DNA binding. In previous studies [9, 40], gene expression regulation including chromatin regulation and transcription factors, neuronal communication including synaptic function, cytoskeleton, and others were enriched, also supporting the GO enrichment of “transcription” in our ASD cohort focusing on the monogenic causes of ASD.

Interestingly, another GO analysis using enrichGO showed that “Face development” was also enriched (p.adjust: 0.00082). This might reflect that some ASD patients are affected by syndromic ASD presenting with a characteristic face; in our cohort, ANKRD11 aberration is known for KBG syndrome (MIM#148050), CHD7 for CHARGE syndrome (MIM#214800), EP300 for Menke–Hennekam syndrome 2 (MIM#618333) or Rubinstein–Taybi syndrome 2 (MIM#613684), and PTPN11 for Noonan syndrome 1 (MIM#163950) or LEOPARD syndrome 1 (MIM#151100). In addition, the enrichment of “GABA signaling pathway” (p.adjust: 0.0036) was also detected (Fig. 2B, Supplementary Table 8). The association between ASD and GABA has been previously reported and considered as a potential treatment target [41,42,43].

When analyzing the potential phenotypic expression of monogenic disorders including ASD, genomic imprinting must be considered. 15q11–q13 duplication is expected to be present in approximately 1 in 5000 individuals in the general population and its penetrance has been calculated as 54% [44]. Interestingly, penetrance differs depending on the parent from whom the abnormal copy originated and the type of chromosomal abnormality. It is 100% in maternal isodicentric 15q11.2–q13.1 supernumerary chromosome and almost 100% in maternal duplication, but is less than 50% in paternal abnormalities, with some cases even expressing a normal phenotype [45,46,47]. Thus, knowledge of the parental origin of such duplications (if inherited by the offspring) may be useful for predicting phenotype in genetic counseling.

Our analytical flow had some limitations. One is that we were likely to miss pathogenic SNVs/indels with incomplete penetrance in dominantly inherited traits because we selected candidate variants by picking up rare variants with a minor allele frequency of <0.1%; we filtered out all inherited variants for autosomal and X-linked dominant models. Furthermore, we focused on CNVs involving known disease genes or >200 kb CNVs that overlapped with known pathogenic regions. As a result, all of the detected CNVs were larger than 200 kb in size. As we have previously reported, XHMM is less powerful for detecting CNVs of less than 200 kb [48]. We might therefore have missed pathogenic CNVs smaller than 200 kb. In addition, we selected the pathogenic regions for which solid evidence was available based on the most recently updated public databases, including ClinGen and DECIPHER. Although these databases are very useful, not all genes have been curated in them yet. For example, we found an interesting case (Individual ID 8397) with a de novo 3.9 Mb duplication at 22q13, which partially overlaps with the critical region (chr22:51045516–51187844 based on hg19) for 22q13 deletion syndrome (Phelan–McDermid syndrome, MIM#606232) (Supplementary Table 4). Although there were 49 genes in the 3.9 Mb duplicated region of the patient, only 7 had been completely curated in ClinGen (Supplementary table 9). At present, there is no evidence that this region contains any triplosensitive genes based on ClinGen. Recently, however, the dosage sensitivity of all protein-coding genes has been reported [49]. Based on the recommended threshold (pHaplo score ≥0.86 and pTriplo score ≥0.94), candidate genes were identified in 8 of 12 CNVs unrelated to known diseases/syndromes (Supplementary Table 10). If more genetic evidence is accumulated, the diagnostic rate may thus be improved. In addition, variants in noncoding regions, mitochondrial dysfunction, and mosaic variants have been suggested to be involved in ASD [50,51,52,53]. Because the current ES analysis in the present cohort may have missed any of these, genome sequencing and/or deep sequencing may detect further disease-causing variants.

In conclusion, we performed a comprehensive analysis to detect SNVs/indels and CNVs using ES data, and achieved a molecular diagnosis in 66 of 405 affected individuals (16.3%). In addition, we demonstrated the effectiveness of reanalyzing ES data for unresolved cases with ASD. The higher diagnostic rates in simplex cases than in multiplex families in the present study support two genetic components, of monogenic and polygenic factors, in ASD genomic architecture. Because ASD is genetically and phenotypically heterogeneous, one medication is unlikely to successfully treat all patients. Disease-causing variants of monogenic diseases may have strong effects on phenotypes and thus signal potential treatment targets. We believe that a comprehensive analysis of the monogenic causes of ASD, to understand its pathomechanism, may be important for new drug discoveries.