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Summary Autism spectrum disorder (ASD) is phenotypically and genetically heterogeneous. We present a CRISPR gene editing strategy to insert a protein tag and premature termination sites creating an induced pluripotent stem cell (iPSC) knockout resource for functional studies of ten ASD-relevant genes (AFF2/FMR2, ANOS1, ASTN2, ATRX, CACNA1C, CHD8, DLGAP2, KCNQ2, SCN2A, TENM1). Neurogenin 2 (NGN2)-directed induction of iPSCs allowed production of excitatory neurons, and mutant proteins were not detectable. RNA sequencing revealed convergence of several neuronal networks. Using both patch-clamp and multi-electrode array approaches, the electrophysiological deficits measured were distinct for different mutations. However, they culminated in a consistent reduction in synaptic activity, including reduced spontaneous excitatory postsynaptic current frequencies in AFF2/FMR2-, ASTN2-, ATRX-, KCNQ2-, and SCN2A-null neurons. Despite ASD susceptibility genes belonging to different gene ontologies, isogenic stem cell resources can reveal common functional phenotypes, such as reduced functional connectivity., Highlights • iPSC knockout resource for functional studies of ten ASD-risk genes • Disruption of common transcriptional networks associated with neurons and synapses • Reduced synaptic activity commonly observed for functionally diverse ASD-risk genes, In this article, Scherer and colleagues present a human induced pluripotent stem cell (iPSC) knockout resource for functional studies of ten genes associated with autism spectrum disorder. They also show that some of these genes, pertaining to diverse functional categories, can underlie common phenotypes in CRISPR-isogenic iPSC-derived glutamatergic neurons.

Autism Spectrum Disorder (ASD) demonstrates high heritability and familial clustering, yet the genetic causes remain only partially understood as a result of extensive clinical and genomic heterogeneity. Whole-genome sequencing (WGS) shows promise as a tool for identifying ASD risk genes as well as unreported mutations in known loci, but an assessment of its full utility in an ASD group has not been performed. We used WGS to examine 32 families with ASD to detect de novo or rare inherited genetic variants predicted to be deleterious (loss-of-function and damaging missense mutations). Among ASD probands, we identified deleterious de novo mutations in six of 32 (19%) families and X-linked or autosomal inherited alterations in ten of 32 (31%) families (some had combinations of mutations). The proportion of families identified with such putative mutations was larger than has been previously reported; this yield was in part due to the comprehensive and uniform coverage afforded by WGS. Deleterious variants were found in four unrecognized, nine known, and eight candidate ASD risk genes. Examples include CAPRIN1 and AFF2 (both linked to FMR1, which is involved in fragile X syndrome), VIP (involved in social-cognitive deficits), and other genes such as SCN2A and KCNQ2 (linked to epilepsy), NRXN1, and CHD7, which causes ASD-associated CHARGE syndrome. Taken together, these results suggest that WGS and thorough bioinformatic analyses for de novo and rare inherited mutations will improve the detection of genetic variants likely to be associated with ASD or its accompanying clinical symptoms.

Objective To develop a novel clinical test using microarray technology as a high-resolution alternative to current methods for detection of known and novel microdeletions on the Y chromosome. Design Custom Agilent 8x15K array comparative genomic hybridization (aCGH) with 10,162 probes on an average probe spacing of 2.5 kb across the euchromatic region of the Y chromosome. Setting Clinical diagnostic laboratory. Patient(s) Men with infertility (n = 104) and controls with proven fertility (n = 148). Intervention(s) Microarray genotyping of DNA. Main Outcome Measure(s) Gene copy number variation determined by log ratio of probe signal intensity against a DNA reference. Result(s) Our aCGH experiments found all known AZF microdeletions as well as additional unbalanced structural alterations. In addition to complete AZF microdeletions, we found that AZFc partial deletions represent a risk factor for male infertility. In total, aCGH-based detection achieved a diagnostic yield of ∼11% and also revealed additional potentially etiologic copy number variations requiring further characterization. Conclusion(s) The aCGH approach is a reliable high-resolution alternative to multiplex polymerase chain reaction for the discovery of pathogenic chromosome Y microdeletions in male infertility.