Multiplex targeted sequencing identifies recurrently mutated genes in autism spectrum disorders

Abstract

Exome sequencing studies of autism spectrum disorders (ASDs) have identified many de novo mutations but few recurrently disrupted genes. We therefore developed a modified molecular inversion probe method enabling ultra-low-cost candidate gene resequencing in very large cohorts. To demonstrate the power of this approach, we captured and sequenced 44 candidate genes in 2446 ASD probands. We discovered 27 de novo events in 16 genes, 59% of which are predicted to truncate proteins or disrupt splicing. We estimate that recurrent disruptive mutations in six genes-CHD8, DYRK1A, GRIN2B, TBR1, PTEN, and TBL1XR1-may contribute to 1% of sporadic ASDs. Our data support associations between specific genes and reciprocal subphenotypes (CHD8-macrocephaly and DYRK1A-microcephaly) and replicate the importance of a β-catenin-chromatin-remodeling network to ASD etiology.

Fig. 1

Massively multiplex targeted sequencing identifies recurrently mutated genes in ASD probands. (A) Schematic showing design and general workflow of a modified MIP method enabling ultra-low-cost candidate gene resequencing in very large cohorts (figs. S1–S7 and tables S1–S9) (10). (B to E) Protein diagrams of four genes with multiple de novo mutation events. Significant protein domains for the largest protein isoform are shown (colored regions) as defined by SMART (23) with mutation locations indicated. (B) CHD8. (C) GRIN2B. (D) TBR1. (E) DYRK1A. Bold variants are nonsense, frameshifting indels or at splice-sites (intron-exon junction is indicated). Domain abbreviations: CHR-chromatin organization modifier, DEX-DEAD-like helicases superfamily, HELC-helicase superfamily c-terminal, BRK-domain in transcription and CHROMO domain helicases, GLU-ligated ion channel L-glutamate- and glycine-binding site, PBP-eukaryotic homologs of bacterial periplasmic substrate binding proteins, TM-transmembrane, STK-serine-threonine kinase catalytic, TBOX-T-box DNA binding.

Fig. 2

Locus-specific mutation probabilities and associated phenotypes. (A) Estimated p-values for the observed number of additional de novo mutations identified in the MIP screen of 44 ASD candidate genes. Probabilities shown are for observing X or more events of which at least Y belong to the severe class. The observed numbers of mutations in all 44 genes (“Total”) and CHD8 were not seen in any of 5×10⁸ simulations. Based on the simulation mean (0.0153), the Poisson probability for seven or more severe class CHD8 mutations is 3.8×10⁻¹⁷. Dashed line Bonferroni corrected significance threshold for α = 0.05. *Gene product in the 74-member PPI connected component. (B–D) Standardized head circumference (HC) Z-scores for SSC. (B) All probands screened with superimposed normal distribution (dashed). HC Z-scores for individuals with de novo truncating/splice mutations highlighted for CHD8 (red arrows), DYRK1A (blue arrows), and PTEN (black arrows). (C and D) Box and whisker plots of the HC Z-scores for the SSC. Mutations carriers are shown and linked to their respective family members. (C) All family members. (D) Only proband sex-matched family members.