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Short-read genome sequencing (GS) holds the promise of becoming the primary diagnostic approach for the assessment of autism spectrum disorder (ASD) and fetal structural anomalies (FSAs). However, few studies have comprehensively evaluated its performance against current standard-of-care diagnostic tests: karyotype, chromosomal microarray (CMA), and exome sequencing (ES). To assess the clinical utility of GS, we compared its diagnostic yield against these three tests in 1,612 quartet families including an individual with ASD and in 295 prenatal families. Our GS analytic framework identified a diagnostic variant in 7.8% of ASD probands, almost 2-fold more than CMA (4.3%) and 3-fold more than ES (2.7%). However, when we systematically captured copy-number variants (CNVs) from the exome data, the diagnostic yield of ES (7.4%) was brought much closer to, but did not surpass, GS. Similarly, we estimated that GS could achieve an overall diagnostic yield of 46.1% in unselected FSAs, representing a 17.2% increased yield over karyotype, 14.1% over CMA, and 4.1% over ES with CNV calling or 36.1% increase without CNV discovery. Overall, GS provided an added diagnostic yield of 0.4% and 0.8% beyond the combination of all three standard-of-care tests in ASD and FSAs, respectively. This corresponded to nine GS unique diagnostic variants, including sequence variants in exons not captured by ES, structural variants (SVs) inaccessible to existing standard-of-care tests, and SVs where the resolution of GS changed variant classification. Overall, this large-scale evaluation demonstrated that GS significantly outperforms each individual standard-of-care test while also outperforming the combination of all three tests, thus warranting consideration as the first-tier diagnostic approach for the assessment of ASD and FSAs., Competing Interests: Declaration of interests M.E.T. and H.R. receive research funding from Microsoft Inc and/or research reagents from Illumina Inc. M.E.T. also received research funding from Levo Therapeutics and research reagents from Ionis Therapeutics for unrelated research projects., (Copyright © 2023 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Point mutations and structural variants that directly disrupt the coding sequence of MEF2C have been associated with a spectrum of neurodevelopmental disorders (NDDs). However, the impact of MEF2C haploinsufficiency on neurodevelopmental pathways and synaptic processes is not well understood, nor are the complex mechanisms that govern its regulation. To explore the functional changes associated with structural variants that alter MEF2C expression and/or regulation, we generated an allelic series of 204 isogenic human induced pluripotent stem cell (hiPSC)-derived neural stem cells and glutamatergic induced neurons. These neuronal models harbored CRISPR-engineered mutations that involved direct deletion of MEF2C or deletion of the boundary points for topologically associating domains (TADs) and chromatin loops encompassing MEF2C. Systematic profiling of mutation-specific alterations, contrasted to unedited controls that were exposed to the same guide RNAs for each edit, revealed that deletion of MEF2C caused differential expression of genes associated with neurodevelopmental pathways and synaptic function. We also discovered significant reduction in synaptic activity measured by multielectrode arrays (MEAs) in neuronal cells. By contrast, we observed robust buffering against MEF2C regulatory disruption following deletion of a distal 5q14.3 TAD and loop boundary, whereas homozygous loss of a proximal loop boundary resulted in down-regulation of MEF2C expression and reduced electrophysiological activity on MEA that was comparable to direct gene disruption. Collectively, these studies highlight the considerable functional impact of MEF2C deletion in neuronal cells and systematically characterize the complex interactions that challenge a priori predictions of regulatory consequences from structural variants that disrupt three-dimensional genome organization., Competing Interests: Declaration of interests M.E.T. receives research funding and/or reagents from Levo Therapeutics, Microsoft Inc, and Illumina Inc. K.M. initiated employment with Tornado Bio during the writing of this manuscript and is at present an employee of the company. J.F.G. is a founder and member of the scientific advisory board of Triplet Therapeutics, Inc., and has been a paid consultant to Biogen, Inc., Pfizer, Inc., and Wave Biosciences, Inc., (Copyright © 2022 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Chromosome 16p11.2 reciprocal genomic disorder, resulting from recurrent copy-number variants (CNVs), involves intellectual disability, autism spectrum disorder (ASD), and schizophrenia, but the responsible mechanisms are not known. To systemically dissect molecular effects, we performed transcriptome profiling of 350 libraries from six tissues (cortex, cerebellum, striatum, liver, brown fat, and white fat) in mouse models harboring CNVs of the syntenic 7qF3 region, as well as cellular, transcriptional, and single-cell analyses in 54 isogenic neural stem cell, induced neuron, and cerebral organoid models of CRISPR-engineered 16p11.2 CNVs. Transcriptome-wide differentially expressed genes were largely tissue-, cell-type-, and dosage-specific, although more effects were shared between deletion and duplication and across tissue than expected by chance. The broadest effects were observed in the cerebellum (2,163 differentially expressed genes), and the greatest enrichments were associated with synaptic pathways in mouse cerebellum and human induced neurons. Pathway and co-expression analyses identified energy and RNA metabolism as shared processes and enrichment for ASD-associated, loss-of-function constraint, and fragile X messenger ribonucleoprotein target gene sets. Intriguingly, reciprocal 16p11.2 dosage changes resulted in consistent decrements in neurite and electrophysiological features, and single-cell profiling of organoids showed reciprocal alterations to the proportions of excitatory and inhibitory GABAergic neurons. Changes both in neuronal ratios and in gene expression in our organoid analyses point most directly to calretinin GABAergic inhibitory neurons and the excitatory/inhibitory balance as targets of disruption that might contribute to changes in neurodevelopmental and cognitive function in 16p11.2 carriers. Collectively, our data indicate the genomic disorder involves disruption of multiple contributing biological processes and that this disruption has relative impacts that are context specific., Competing Interests: Declaration of interests J.F.G. is a founder and member of the scientific advisory board of Triplet Therapeutics and has been a paid consultant to Biogen, Pfizer, and Wave Biosciences. M.E.T. receives research funding and/or reagents from Illumina, Levo Therapeutics, and Microsoft. E.M. receives research funding and reagents from PTC Therapeutics. The following co-authors are currently employed by for-profit companies or non-profit organizations: P.R. is founder and CEO of Kumuda; K.M. is currently employed by Tornado Bio; T.A. is a founder and CEO of Independent Data Lab and OmicsChart; and A.H. is currently employed by Bionano Genomics., (Copyright © 2022 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

With recent rapid advances in genomic technologies, precise delineation of structural chromosome rearrangements at the nucleotide level is becoming increasingly feasible. In this era of "next-generation cytogenetics" (i.e., an integration of traditional cytogenetic techniques and next-generation sequencing), a consensus nomenclature is essential for accurate communication and data sharing. Currently, nomenclature for describing the sequencing data of these aberrations is lacking. Herein, we present a system called Next-Gen Cytogenetic Nomenclature, which is concordant with the International System for Human Cytogenetic Nomenclature (2013). This system starts with the alignment of rearrangement sequences by BLAT or BLAST (alignment tools) and arrives at a concise and detailed description of chromosomal changes. To facilitate usage and implementation of this nomenclature, we are developing a program designated BLA(S)T Output Sequence Tool of Nomenclature (BOSToN), a demonstrative version of which is accessible online. A standardized characterization of structural chromosomal rearrangements is essential both for research analyses and for application in the clinical setting., (Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)