Figure 7. Distinct functional subgroups of autism mutations correlate with differing clinical profiles of language development.

(A) Unbiased hierarchical clustering of phenotypic data from six multiplex assays reveals two major functional groups of autism mutations from the MIX30 library. Class 1 mutations fall into cluster A, while Class 2 mutations fall into cluster B. (B) Strategy for clinical phenotype analysis. Probands from the Simons Simplex Collection (SSC) were segregated into cohorts based on the presence of a de novo mutation in genes from Cluster A (17 patients) or Cluster B (55 patients). Control groups were generated by defining IQ-matched patient cohorts with any other de novo mutation not in the MIX30 library (de novo control, 263 patients) or patients without any known de novo mutation (idiopathic control, 482 patients). First, the ADI-R was used to screen for differences in core autism behavioral domains (communication, social behavior, restricted and repetitive behaviors). Specific behavioral phenotypes were further investigated based on the initial screening. (C), Cluster B patients exhibit increased severity in ADI-R non-verbal communication scores (Kruskal-Wallis p = 0.0072, corrected for multiple comparison of each behavioral domain using Holm-Sidak method; post-hoc Dunn’s multiple comparison test, B vs. DN p = 0.0025; B vs. Idiopathic corrected p = 0.0008). (D) Cluster B patients are on average reduced in the communication domain of the Vineland adaptive behavior scale (ANOVA p = 0.0004; Tukey’s multiple comparison’s test, A vs. B p = 0.037, B vs. DN p = 0.0027, B vs. Idiopathic p = 0.0002). (E) Average trajectories of language development. Control cohorts speak single words at ~24 months (DN=24.4 mo; idiopathic=24.4 mo) and speak their first phrases at ~39 months (other=38.8 mo; idiopathic=39.3 mo). Cluster B patients speak words at 28.02 months and first phrases at 46.1 months. Cluster A patients speak single words at 17.4 months and first phrases at 28.7 months. Typical language development is depicted in gray. (F) Cluster B cohort contains an increased fraction of patients with severe language deficit when compared to control cohorts (chi-squared p < 0.0001; Fisher’s exact tests with Holm-Sidak correction for multiple comparison B vs. DN p = 0.0018, B vs. Idiopathic p = 0.0018). (G) No significant difference in word delay across groups (chi-squared p = 0.114). (H) Cluster A contains a decreased fraction of patients with phrase delay compared to other cohorts (Chi-squared p = 0.0002; Fisher’s exact tests with Holm-Sidak correction for multiple comparison A vs. B p = 0.0018, A vs. DN p = 0.0018, A vs. idiopathic p = 0.0018). (I) Class 0 exhibits an intermediate phenotype of average language development, between Class 1 and 2. (J) Correlation of language development with in vitro PFC neurogenesis. PFC neurogenesis values (Fig. 2b, DCX/SOX2 ratio) were assigned to probands using proband genotypes. Both first word (R² = 0.1334, p = 0.0024) and first phrases milestone (R² = 0.09551, p = 0.0182) showed significant positive correlations with the extent of PFC neurogenesis. Each dot represents one proband. (K) Summary of phenotypic segregation of autism patients defined using hPSC-based multiplex analysis platform. ASD, autism spectrum disorder. ADOS, Autism Diagnostic Observation Schedule. ADI-R, Autism Diagnostic Interview-Revised. See also Figure S7.