The contributions of rare inherited and polygenic risk to ASD in multiplex families

Abstract

Autism spectrum disorder (ASD) has a complex genetic architecture involving contributions from both de novo and inherited variation. Few studies have been designed to address the role of rare inherited variation or its interaction with common polygenic risk in ASD. Here, we performed whole-genome sequencing of the largest cohort of multiplex families to date, consisting of 4,551 individuals in 1,004 families having two or more autistic children. Using this study design, we identify seven previously unrecognized ASD risk genes supported by a majority of rare inherited variants, finding support for a total of 74 genes in our cohort and a total of 152 genes after combined analysis with other studies. Autistic children from multiplex families demonstrate an increased burden of rare inherited protein-truncating variants in known ASD risk genes. We also find that ASD polygenic score (PGS) is overtransmitted from nonautistic parents to autistic children who also harbor rare inherited variants, consistent with combinatorial effects in the offspring, which may explain the reduced penetrance of these rare variants in parents. We also observe that in addition to social dysfunction, language delay is associated with ASD PGS overtransmission. These results are consistent with an additive complex genetic risk architecture of ASD involving rare and common variation and further suggest that language delay is a core biological feature of ASD.

Keywords: autism spectrum disorder (ASD); genetics; inherited; multiplex families; polygenic score (PGS).

Fig. 1.

Overview diagram of study analyses. WGS was performed on 1,004 AGRE ASD multiplex families. Single-nucleotide variants and small insertions/deletions were called using the Genome Analysis Toolkit. Variant, sample, and genotype quality control was performed. Rare de novo, rare inherited, and private inherited variants were included for downstream analyses. We identified ASD risk genes at FDR < 0.1 using the Transmission and De Novo Association (TADA) analysis. Risk genes were characterized using integrative genomics. We investigated known genes supported by rare inherited risk and evaluated their variants’ coinheritance and effect on phenotypic measures. We tested if ASD polygenic score was overtransmitted to autistic and nonautistic children stratified by carrier status for rare de novo and inherited variants. VCF = Variant Call Format, QC = Quality Control, VQSR = Variant Quality Score Recalibration, IBD = Identity By Descent, DP = Total Depth, GQ = Genotype Quality, AD = Allele Depth, GIAB = Genome In A Bottle.

Fig. 2.

Seventy-four ASD risk genes identified by Transmission and De novo Association (TADA) analysis at FDR < 0.1. (A) Overview of cohort subjects and additional datasets included in the TADA analysis. (B) Seventy-four ASD risk genes identified in the TADA mega-analysis (FDR < 0.1) are displayed with corresponding −log₁₀(q-value). The nine newly identified ASD risk genes from our expanded AGRE multiplex cohort in comparison to Ruzzo, et al (9) are in bold. The dashed horizontal line marks the FDR = 0.1 threshold. Bars are colored by the proportion of inherited protein-truncating variants (PTVs) for each gene (inherited PTVs/(inherited PTVs + de novo PTVs + de novo mis3 + de novo small deletions). The pie chart indicates the percentages of TADA genes with 0%, <50%, and >50% proportion of inherited PTVs. (C) The nine newly identified ASD risk genes from our expanded AGRE multiplex cohort in comparison to Ruzzo, et al (9). Bars are colored according to (B). Asterisks represent genes already identified in other recent cohort studies. Note how these genes have a very low proportion of inherited PTVs (mainly de novo support). The other seven genes are previously unrecognized risk genes for ASD. (D) Protein–protein interaction network formed by the 74 ASD risk genes using Disease Association Protein-Protein Link Evaluator (DAPPLE). Genes are colored by the proportion of inherited PTVs. The gene name for two of the seven previously unrecognized ASD risk genes are in red. (E) Gene Ontology enrichment terms for 39 significant seed genes from the protein–protein interaction network.

Fig. 3.

Characterization of TADA ASD risk genes at FDR < 0.1 during development. (A) Mean expression and percentage of cells expressing TADA genes from single-cell data of the human fetal brain cortex. Asterisks indicate cell type–specific TADA genes (Materials and Methods). (B) Cell types enriched in TADA genes tested using Expression Weighted Cell Type Enrichment (EWCE). Asterisks indicate FDR < 0.05. (C) TADA genes’ variation during development using long-term maturation human cortical spheroids (hCSs) and BrainSpan. Genes are stratified by those with a majority of inherited or de novo support. (D) Clustering of the TADA genes from hCS trajectory data identified three clusters. Days postconception, TADA class (majority of inherited or de novo), and scaled expression are shown. The average expression of each cluster and significant gene ontology biological terms are shown. vRG = ventral radial glia, PgS = cycling progenitor (S phase), PgG2M = cycling progenitor (G2M phase), Per = pericyte, oRG = outer radial glia, OPC = oligodendrocyte precursor, Mic = microglia, IP = intermediate progenitor, InMGE = interneuron medial ganglionic eminence, InCGE = interneuron caudal ganglionic eminence, ExN = excitatory neuron, ExM−U = maturing excitatory neuron upper enriched, ExM = maturing excitatory neuron, ExDp2 = excitatory neuron deep layer 2, ExDp1 = excitatory neuron deep layer 1, End = endothelial, ExMig = migrating excitatory neuron, ExDp = excitatory neuron deep.

Fig. 4.

Phenotypic effect of rare and common risk for ASD in autistic children from AGRE multiplex families. (A–F) Phenotypic distributions in autistic children, stratified by those carrying rare inherited (RI) or rare de novo (RDN) variants in known ASD risk genes (KARGs) and those not carrying such variants. (A) Verbal IQ distribution for autistic children. Standard scores from PPVT-3 (Peabody Picture Vocabulary Test, 3rd edition) were used and compared to a standard hypothesized median value of 100. Autistic noncarrier: n = 767, mu = 89; autistic RI carrier: n = 70, mu = 88; autistic RDN carrier: n = 10, mu = 84.5. (B) Nonverbal IQ distribution for autistic children. Standard scores from the Raven test were used and compared to a standard hypothesized median value of 100. Autistic noncarrier: n = 987, mu = 104; autistic RI carrier: n = 98, mu = 100; autistic RDN carrier: n = 12, mu = 108.5. (C) Age of walking distribution for autistic children whose ADI-R (Autism Diagnostic Interview-Revised) data were available. A standard hypothesized median value of 12 mo was used for comparison (SI Appendix). Autistic noncarrier: n = 1180, mu = 12; autistic RI carrier: n = 120, mu = 13; autistic RDN carrier: n = 21, mu = 13. (D) Age of first word for autistic children whose ADI-R data were available. Autistic noncarrier: n = 1223, mu = 26; autistic RI carrier: n = 126, mu = 30; autistic RDN carrier: n = 21, mu = 18; nonautistic noncarrier: n = 295; mu = 12. (E) Age of first phrase for autistic children whose ADI-R data were available. Autistic noncarrier: n = 1220, mu = 42; autistic RI carrier: n = 126, mu = 48; autistic RDN carrier: n = 21, mu = 40; nonautistic noncarrier: n = 283; mu = 18. (F) SRS (Social Responsiveness Scale) total raw score distribution for autistic and nonautistic children. Autistic noncarrier: n = 709, mu = 104; autistic RI carrier: n = 73, mu = 98; autistic RDN carrier: n = 8, mu = 113.5; nonautistic noncarrier: n = 153; mu = 19. Noncarrier distributions are depicted in black, RI carrier ones in blue, and RDN ones in red. Standard hypothesized median values used for each phenotypic comparison are reported as green vertical lines. For D–F, these median values were derived from the corresponding phenotypic distributions for nonautistic noncarriers (shown in green) for whom phenotypic data were available. Each group of autistic children was tested for differences between its phenotypic median value and the standard hypothesized one by two-sided one-sample sign test. The three tests performed for each phenotypic measure were corrected for multiple hypothesis testing using the Benjamini and Hochberg (BH) procedure as an FDR-controlling method. An asterisk is used to denote an FDR < 0.05. Median values together with their 95% CI are represented by horizontal point ranges over the distributions. (G–I) ASD polygenic score transmission stratified by TADA gene carrier type and association with language and social impairment. (G) pTDT in autistic and nonautistic children, tested for all subjects (All - autistic: n = 1231; nonautistic: n = 288) and stratified by those carrying rare inherited (TADA RI carrier - autistic: n = 70; nonautistic: n = 8) or rare de novo (TADA RDN carrier - autistic: n = 12) variants in the 74 TADA genes and those without such variants (noncarriers - autistic: n = 1149; nonautistic: n = 279). (H) pTDT in autistic noncarriers of variants in the 74 TADA genes stratified by those with delayed (age of first word: n = 407; phrase: n = 564) language development (i.e., age of first word and phrase greater than 24 and 33 mo, respectively; SI Appendix) and those with nondelayed (age of first word: n = 432; phrase: n = 275) language development. (I) pTDT in autistic noncarriers of variants in the 74 TADA genes stratified by the degree of social impairment based on SRS (Social Responsiveness Scale) T-score cut-off ranges. T-scores (based on child’s sex) equal to 59 or less were considered non-ASD (n = 122), those 60 to 75 were considered mild ASD (n = 70), and those equal to or greater than 76 were considered severe ASD (n = 275). Polygenic transmission disequilibrium is shown on the y-axis of each panel as point ranges of the SD on the mid-parent distribution mean values together with their 95% CI. The probability of each pTDT deviation distribution mean being equal to zero was tested by a two-sided one-sample t-test. An asterisk is used to denote a P-value < 0.05.