Pathogenic paternally inherited NLGN4X deletion in a female with autism spectrum disorder: Clinical, cytogenetic, and molecular characterization.

Neuroligin 4 X-linked (NLGN4X) is an X-linked postsynaptic scaffolding protein, with functional role in excitatory synapsis development and maintenance, that has been associated with neuropsychiatric disorders such as intellectual disability, autism spectrum disorders (ASD), anxiety, attention deficit hyperactivity disorder (ADHD), and Tourette's syndrome. Chromosomal microarray analysis identified a paternally inherited, 445 Kb deletion on Xp22.3 that includes the entire NLGN4X in a 2.5 year old female (46,XX) with congenital hypotonia, strabismus, ASD, and increased aggressive behavioral issues. Her family history is significant for a mother with learning disabilities, a father with anxiety, major depressive disorder, and substance abuse, as well as two maternal half-brothers with developmental delays. X-inactivation studies in the proband's blood showed random X-inactivation despite the presence of an abnormal X chromosome. Furthermore, trio exome sequencing did not reveal any other deleterious variant that could explain her phenotype. Our report describes the first example of a paternally inherited NLGN4X microdeletion as the genetic etiology of ASD in a female proband, and the psychiatric phenotypes in the father. It also provides further evidence that NLGN4X is sensitive to dosage changes in females, and can contribute to a variety of psychiatric features within the same family.

Functions of Gtf2i and Gtf2ird1 in the developing brain: transcription, DNA binding and long-term behavioral consequences.

Gtf2ird1 and Gtf2i are two transcription factors (TFs) among the 28 genes deleted in Williams syndrome, and prior mouse models of each TF show behavioral phenotypes. Here we identify their genomic binding sites in the developing brain and test for additive effects of their mutation on transcription and behavior. GTF2IRD1 binding targets were enriched for transcriptional and chromatin regulators and mediators of ubiquitination. GTF2I targets were enriched for signal transduction proteins, including regulators of phosphorylation and WNT. Both TFs are highly enriched at promoters, strongly overlap CTCF binding and topological associating domain boundaries and moderately overlap each other, suggesting epistatic effects. Shared TF targets are enriched for reactive oxygen species-responsive genes, synaptic proteins and transcription regulators such as chromatin modifiers, including a significant number of highly constrained genes and known ASD genes. We next used single and double mutants to test whether mutating both TFs will modify transcriptional and behavioral phenotypes of single Gtf2ird1 mutants, though with the caveat that our Gtf2ird1 mutants, like others previously reported, do produce low levels of a truncated protein product. Despite little difference in DNA binding and transcriptome-wide expression, homozygous Gtf2ird1 mutation caused balance, marble burying and conditioned fear phenotypes. However, mutating Gtf2i in addition to Gtf2ird1 did not further modify transcriptomic or most behavioral phenotypes, suggesting Gtf2ird1 mutation alone was sufficient for the observed phenotypes.

Human iPSC-Derived Neurons and Cerebral Organoids Establish Differential Effects of Germline NF1 Gene Mutations.

Neurofibromatosis type 1 (NF1) is a common neurodevelopmental disorder caused by a spectrum of distinct germline NF1 gene mutations, traditionally viewed as equivalent loss-of-function alleles. To specifically address the issue of mutational equivalency in a disease with considerable clinical heterogeneity, we engineered seven isogenic human induced pluripotent stem cell lines, each with a different NF1 patient NF1 mutation, to identify potential differential effects of NF1 mutations on human central nervous system cells and tissues. Although all mutations increased proliferation and RAS activity in 2D neural progenitor cells (NPCs) and astrocytes, we observed striking differences between NF1 mutations on 2D NPC dopamine levels, and 3D NPC proliferation, apoptosis, and neuronal differentiation in developing cerebral organoids. Together, these findings demonstrate differential effects of NF1 gene mutations at the cellular and tissue levels, suggesting that the germline NF1 gene mutation is one factor that underlies clinical variability.

Gtf2i and Gtf2ird1 mutation do not account for the full phenotypic effect of the Williams syndrome critical region in mouse models.

Williams syndrome (WS) is a neurodevelopmental disorder caused by a 1.5-1.8 Mbp deletion on chromosome 7q11.23, affecting the copy number of 26-28 genes. Phenotypes of WS include cardiovascular problems, craniofacial dysmorphology, deficits in visual-spatial cognition and a characteristic hypersocial personality. There are still no genes in the region that have been consistently linked to the cognitive and behavioral phenotypes, although human studies and mouse models have led to the current hypothesis that the general transcription factor 2 I family of genes, GTF2I and GTF2IRD1, are responsible. Here we test the hypothesis that these two transcription factors are sufficient to reproduce the phenotypes that are caused by deletion of the WS critical region (WSCR). We compare a new mouse model with loss of function mutations in both Gtf2i and Gtf2ird1 to an established mouse model lacking the complete WSCR. We show that the complete deletion (CD) model has deficits across several behavioral domains including social communication, motor functioning and conditioned fear that are not explained by loss of function mutations in Gtf2i and Gtf2ird1. Furthermore, transcriptome profiling of the hippocampus shows changes in synaptic genes in the CD model that are not seen in the double mutants. Thus, we have thoroughly defined a set of molecular and behavioral consequences of complete WSCR deletion and shown that genes or combinations of genes beyond Gtf2i and Gtf2ird1 are necessary to produce these phenotypic effects.

Exome sequencing of 85 Williams-Beuren syndrome cases rules out coding variation as a major contributor to remaining variance in social behavior.

BACKGROUND: Large, multigenic deletions at chromosome 7q11.23 result in a highly penetrant constellation of physical and behavioral symptoms known as Williams-Beuren syndrome (WS). Of particular interest is the unusual social-cognitive profile evidenced by deficits in social cognition and communication reminiscent of autism spectrum disorders (ASD) that are juxtaposed with normal or even relatively enhanced social motivation. Interestingly, duplications in the same region also result in ASD-like phenotypes as well as social phobias. Thus, the region clearly regulates human social motivation and behavior, yet the relevant gene(s) have not been definitively identified. METHOD: Here, we deeply phenotyped 85 individuals with WS and used exome sequencing to analyze common and rare variation for association with the remaining variance in social behavior as assessed by the Social Responsiveness Scale. RESULTS: We replicated the previously reported unusual juxtaposition of behavioral symptoms in this new patient collection, but we did not find any new alleles of large effect in the targeted analysis of the remaining copy of genes in the Williams syndrome critical region. However, we report on two nominally significant SNPs in two genes that have been implicated in the cognitive and social phenotypes of Williams syndrome, BAZ1B and GTF2IRD1. Secondary discovery driven explorations focusing on known ASD genes and an exome wide scan do not highlight any variants of a large effect. CONCLUSIONS: Whole exome sequencing of 85 individuals with WS did not support the hypothesis that there are variants of large effect within the remaining Williams syndrome critical region that contribute to the social phenotype. This deeply phenotyped and genotyped patient cohort with a defined mutation provides the opportunity for similar analyses focusing on noncoding variation and/or other phenotypic domains.

The anatomical distribution of genetic associations.

Deeper understanding of the anatomical intermediaries for disease and other complex genetic traits is essential to understanding mechanisms and developing new interventions. Existing ontology tools provide functional, curated annotations for many genes and can be used to develop mechanistic hypotheses; yet information about the spatial expression of genes may be equally useful in interpreting results and forming novel hypotheses for a trait. Therefore, we developed an approach for statistically testing the relationship between gene expression across the body and sets of candidate genes from across the genome. We validated this tool and tested its utility on three applications. First, we show that the expression of genes in associated loci from GWA studies implicates specific tissues for 57 out of 98 traits. Second, we tested the ability of the tool to identify novel relationships between gene expression and phenotypes. Specifically, we experimentally confirmed an underappreciated prediction highlighted by our tool: that white blood cell count--a quantitative trait of the immune system--is genetically modulated by genes expressed in the skin. Finally, using gene lists derived from exome sequencing data, we show that human genes under selective constraint are disproportionately expressed in nervous system tissues.

Moving from capstones toward cornerstones: successes and challenges in applying systems biology to identify mechanisms of autism spectrum disorders.

The substantial progress in the last few years toward uncovering genetic causes and risk factors for autism spectrum disorders (ASDs) has opened new experimental avenues for identifying the underlying neurobiological mechanism of the condition. The bounty of genetic findings has led to a variety of data-driven exploratory analyses aimed at deriving new insights about the shared features of these genes. These approaches leverage data from a variety of different sources such as co-expression in transcriptomic studies, protein-protein interaction networks, gene ontologies (GOs) annotations, or multi-level combinations of all of these. Here, we review the recurrent themes emerging from these analyses and highlight some of the challenges going forward. Themes include findings that ASD associated genes discovered by a variety of methods have been shown to contain disproportionate amounts of neurite outgrowth/cytoskeletal, synaptic, and more recently Wnt-related and chromatin modifying genes. Expression studies have highlighted a disproportionate expression of ASD gene sets during mid fetal cortical development, particularly for rare variants, with multiple analyses highlighting the striatum and cortical projection and interneurons as well. While these explorations have highlighted potentially interesting relationships among these ASD-related genes, there are challenges in how to best transition these insights into empirically testable hypotheses. Nonetheless, defining shared molecular or cellular pathology downstream of the diverse genes associated with ASDs could provide the cornerstones needed to build toward broadly applicable therapeutic approaches.