Targeted DNA Sequencing from Autism Spectrum Disorder Brains Implicates Multiple Genetic Mechanisms

Abstract

Single nucleotide variants (SNVs), particularly loss-of-function mutations, are significant contributors to autism spectrum disorder (ASD) risk. Here we report the first systematic deep sequencing study of 55 postmortem ASD brains for SNVs in 78 known ASD candidate genes. Remarkably, even without parental samples, we find more ASD brains with mutations that are protein-altering (26/55 cases versus 12/50 controls, p = 0.015), deleterious (16/55 versus 5/50, p = 0.016), or loss-of-function (6/55 versus 0/50, p = 0.028) compared to controls, with recurrent deleterious mutations in ARID1B, SCN1A, SCN2A, and SETD2, suggesting these mutations contribute to ASD risk. In several cases, the identified mutations and medical records suggest syndromic ASD diagnoses. Two ASD and one Fragile X premutation case showed deleterious somatic mutations, providing evidence that somatic mutations occur in ASD cases, and supporting a model in which a combination of germline and/or somatic mutations may contribute to ASD risk on a case-by-case basis.

Figure 1. Targeted deep sequencing identifies germline and somatic mutations in ASD candidate genes in postmortem ASD brain

A. Study schematic. DNA extracted from ASD and control brain samples was sequenced at high coverage using a targeted panel of candidate ASD genes. After variant calling and filtering, protein-altering variants were validated using Sanger sequencing and subcloning, and somatic variants were traced across the brain and non-brain tissues, when available. B. Fraction of cases and controls harboring synonymous, protein-altering, deleterious (subset of protein-altering), or loss-of-function (subset of deleterious) variants. p values were calculated using a two-tailed Fisher’s exact test. C. Validation of germline and somatic variants according to total depth of sequence coverage. Essentially all germline variants validated; somatic variants were more likely to validate at higher total depth (≥100X). D. Validation of somatic variants according to alternate allele depth of coverage. Somatic variants were more likely to validate at alternate allele depth ≥25X. Each data point represents the validation fraction for all variants with b. total or c. alternate allele depth from the previous data point to that data point. See also Tables S1, S3-S5.

Figure 2. Regional distribution of somatic mutations and transcriptomic analysis of ASD brains

A. Protein-altering somatic mutations in ASD (5278, UK20244), suspected ASD (967), Social Anxiety Disorder (5378) or Fragile X premutation (5006) brain. Blue indicates brain region tested where the mutation was detected, red indicates brain region tested where the mutation was not detected, and grey indicates regions not tested. Arrows show approximate location of the sample tested. B-E. Detection and validation of protein-altering somatic mutations in ASD brains. Each panel depicts representative NGS reads, Sanger sequencing trace from bulk brain tissue, and Sanger sequencing traces from individual clones following subcloning for Case UK20244, mutation in SETD2, B. PFC and C. CER; Case 5278, mutation in SCN1A, D. PFC and E. CER. In addition, D depicts single nuclei Sanger sequencing traces for Case 5278, mutation in SCN1A, PFC. F. PCA plot of samples analyzed for RNA-sequencing. G-L. Detection of mutations in RNA-seq data. Each panel depicts representative RNA seq reads and read counts for Case 5278, mutation in SCN1A, G. PFC and H. CER; Case 5278, mutation in CACNA1H, I.PFC and J. CER; Case 4849, mutation in SCN2A, J. PFC and L. CER. CER: cerebellum; PFC: prefrontal cortex. See also Tables S6-7.