Sequencing chromosomal abnormalities reveals neurodevelopmental loci that confer risk across diagnostic boundaries.

Balanced chromosomal abnormalities (BCAs) represent a relatively untapped reservoir of single-gene disruptions in neurodevelopmental disorders (NDDs). We sequenced BCAs in patients with autism or related NDDs, revealing disruption of 33 loci in four general categories: (1) genes previously associated with abnormal neurodevelopment (e.g., AUTS2, FOXP1, and CDKL5), (2) single-gene contributors to microdeletion syndromes (MBD5, SATB2, EHMT1, and SNURF-SNRPN), (3) novel risk loci (e.g., CHD8, KIRREL3, and ZNF507), and (4) genes associated with later-onset psychiatric disorders (e.g., TCF4, ZNF804A, PDE10A, GRIN2B, and ANK3). We also discovered among neurodevelopmental cases a profoundly increased burden of copy-number variants from these 33 loci and a significant enrichment of polygenic risk alleles from genome-wide association studies of autism and schizophrenia. Our findings suggest a polygenic risk model of autism and reveal that some neurodevelopmental genes are sensitive to perturbation by multiple mutational mechanisms, leading to variable phenotypic outcomes that manifest at different life stages.

FOXG1 targets BMP repressors and cell cycle inhibitors in human neural progenitor cells.

FOXG1 is a critical transcription factor in human brain where loss-of-function mutations cause a severe neurodevelopmental disorder, while increased FOXG1 expression is frequently observed in glioblastoma. FOXG1 is an inhibitor of cell patterning and an activator of cell proliferation in chordate model organisms but different mechanisms have been proposed as to how this occurs. To identify genomic targets of FOXG1 in human neural progenitor cells (NPCs), we engineered a cleavable reporter construct in endogenous FOXG1 and performed chromatin immunoprecipitation (ChIP) sequencing. We also performed deep RNA sequencing of NPCs from two females with loss-of-function mutations in FOXG1 and their healthy biological mothers. Integrative analyses of RNA and ChIP sequencing data showed that cell cycle regulation and Bone Morphogenic Protein (BMP) repression gene ontology categories were over-represented as FOXG1 targets. Using engineered brain cell lines, we show that FOXG1 specifically activates SMAD7 and represses CDKN1B. Activation of SMAD7 which inhibits BMP signaling may be one way that FOXG1 patterns the forebrain, while repression of cell cycle regulators such as CDKN1B may be one way that FOXG1 expands the NPC pool to ensure proper brain size. Our data reveal novel mechanisms on how FOXG1 may control forebrain patterning and cell proliferation in human brain development.

Kabuki syndrome stem cell models reveal locus specificity of histone methyltransferase 2D (KMT2D/MLL4).

Kabuki syndrome is frequently caused by loss-of-function mutations in one allele of histone 3 lysine 4 (H3K4) methyltransferase KMT2D and is associated with problems in neurological, immunological and skeletal system development. We generated heterozygous KMT2D knockout and Kabuki patient-derived cell models to investigate the role of reduced dosage of KMT2D in stem cells. We discovered chromosomal locus-specific alterations in gene expression, specifically a 110 Kb region containing Synaptotagmin 3 (SYT3), C-Type Lectin Domain Containing 11A (CLEC11A), Chromosome 19 Open Reading Frame 81 (C19ORF81) and SH3 And Multiple Ankyrin Repeat Domains 1 (SHANK1), suggesting locus-specific targeting of KMT2D. Using whole genome histone methylation mapping, we confirmed locus-specific changes in H3K4 methylation patterning coincident with regional decreases in gene expression in Kabuki cell models. Significantly reduced H3K4 peaks aligned with regions of stem cell maps of H3K27 and H3K4 methylation suggesting KMT2D haploinsufficiency impact bivalent enhancers in stem cells. Preparing the genome for subsequent differentiation cues may be of significant importance for Kabuki-related genes. This work provides a new insight into the mechanism of action of an important gene in bone and brain development and may increase our understanding of a specific function of a human disease-relevant H3K4 methyltransferase family member.

Characterization of Stress Granule Protein Turnover in Neuronal Progenitor Cells Using Correlative STED and NanoSIMS Imaging.

Stress granules (SGs) are stress-induced biomolecular condensates which originate primarily from inactivated RNA translation machinery and translation initiation factors. SG formation is an important defensive mechanism for cell survival, while its dysfunction has been linked to neurodegenerative diseases. However, the molecular mechanisms of SG assembly and disassembly, as well as their impacts on cellular recovery, are not fully understood. More thorough investigations into the molecular dynamics of SG pathways are required to understand the pathophysiological roles of SGs in cellular systems. Here, we characterize the SG and cytoplasmic protein turnover in neuronal progenitor cells (NPCs) under stressed and non-stressed conditions using correlative STED and NanoSIMS imaging. We incubate NPCs with isotopically labelled (15N) leucine and stress them with the ER stressor thapsigargin (TG). A correlation of STED and NanoSIMS allows the localization of individual SGs (using STED), and their protein turnover can then be extracted based on the 15N/14N ratio (using NanoSIMS). We found that TG-induced SGs, which are highly dynamic domains, recruit their constituents predominantly from the cytoplasm. Moreover, ER stress impairs the total cellular protein turnover regimen, and this impairment is not restored after the commonly proceeded stress recovery period.

Human iPSC-derived Down syndrome astrocytes display genome-wide perturbations in gene expression, an altered adhesion profile, and increased cellular dynamics.

Down syndrome (DS), caused by the triplication of human chromosome 21, leads to significant alterations in brain development and is a major genetic cause of intellectual disability. While much is known about changes to neurons in DS, the effects of trisomy 21 on non-neuronal cells such as astrocytes are poorly understood. Astrocytes are critical for brain development and function, and their alteration may contribute to DS pathophysiology. To better understand the impact of trisomy 21 on astrocytes, we performed RNA-sequencing on astrocytes from newly produced DS human induced pluripotent stem cells (hiPSCs). While chromosome 21 genes were upregulated in DS astrocytes, we found consistent up- and down-regulation of genes across the genome with a strong dysregulation of neurodevelopmental, cell adhesion and extracellular matrix molecules. ATAC (assay for transposase-accessible chromatin)-seq also revealed a global alteration in chromatin state in DS astrocytes, showing modified chromatin accessibility at promoters of cell adhesion and extracellular matrix genes. Along with these transcriptomic and epigenomic changes, DS astrocytes displayed perturbations in cell size and cell spreading as well as modifications to cell-cell and cell-substrate recognition/adhesion, and increases in cellular motility and dynamics. Thus, triplication of chromosome 21 is associated with genome-wide transcriptional, epigenomic and functional alterations in astrocytes that may contribute to altered brain development and function in DS.

Cocaine-related DNA methylation in caudate neurons alters 3D chromatin structure of the IRXA gene cluster.

Epigenetic mechanisms, like those involving DNA methylation, are thought to mediate the relationship between chronic cocaine dependence and molecular changes in addiction-related neurocircuitry, but have been understudied in human brain. We initially used reduced representation bisulfite sequencing (RRBS) to generate a methylome-wide profile of cocaine dependence in human post-mortem caudate tissue. We focused on the Iroquois Homeobox A (IRXA) gene cluster, where hypomethylation in exon 3 of IRX2 in neuronal nuclei was associated with cocaine dependence. We replicated this finding in an independent cohort and found similar results in the dorsal striatum from cocaine self-administering mice. Using epigenome editing and 3C assays, we demonstrated a causal relationship between methylation within the IRX2 gene body, CTCF protein binding, three-dimensional (3D) chromatin interaction, and gene expression. Together, these findings suggest that cocaine-related hypomethylation of IRX2 contributes to the development and maintenance of cocaine dependence through alterations in 3D chromatin structure in the caudate nucleus.

Umap and Bismap: quantifying genome and methylome mappability.

Short-read sequencing enables assessment of genetic and biochemical traits of individual genomic regions, such as the location of genetic variation, protein binding and chemical modifications. Every region in a genome assembly has a property called 'mappability', which measures the extent to which it can be uniquely mapped by sequence reads. In regions of lower mappability, estimates of genomic and epigenomic characteristics from sequencing assays are less reliable. These regions have increased susceptibility to spurious mapping from reads from other regions of the genome with sequencing errors or unexpected genetic variation. Bisulfite sequencing approaches used to identify DNA methylation exacerbate these problems by introducing large numbers of reads that map to multiple regions. Both to correct assumptions of uniformity in downstream analysis and to identify regions where the analysis is less reliable, it is necessary to know the mappability of both ordinary and bisulfite-converted genomes. We introduce the Umap software for identifying uniquely mappable regions of any genome. Its Bismap extension identifies mappability of the bisulfite-converted genome. A Umap and Bismap track hub for human genome assemblies GRCh37/hg19 and GRCh38/hg38, and mouse assemblies GRCm37/mm9 and GRCm38/mm10 is available at https://bismap.hoffmanlab.org for use with genome browsers.

A Dual Noradrenergic Mechanism for the Relief of Neuropathic Allodynia by the Antidepressant Drugs Duloxetine and Amitriptyline.

In addition to treating depression, antidepressant drugs are also a first-line treatment for neuropathic pain, which is pain secondary to lesion or pathology of the nervous system. Despite the widespread use of these drugs, the mechanism underlying their therapeutic action in this pain context remains partly elusive. The present study combined data collected in male and female mice from a model of neuropathic pain and data from the clinical setting to understand how antidepressant drugs act. We show two distinct mechanisms by which the selective inhibitor of serotonin and noradrenaline reuptake duloxetine and the tricyclic antidepressant amitriptyline relieve neuropathic allodynia. One of these mechanisms is acute, central, and requires descending noradrenergic inhibitory controls and α2A adrenoceptors, as well as the mu and delta opioid receptors. The second mechanism is delayed, peripheral, and requires noradrenaline from peripheral sympathetic endings and ß2 adrenoceptors, as well as the delta opioid receptors. We then conducted a transcriptomic analysis in dorsal root ganglia, which suggested that the peripheral component of duloxetine action involves the inhibition of neuroimmune mechanisms accompanying nerve injury, including the downregulation of the TNF-α-NF-κB signaling pathway. Accordingly, immunotherapies against either TNF-α or Toll-like receptor 2 (TLR2) provided allodynia relief. We also compared duloxetine plasma levels in the animal model and in patients and we observed that patients' drug concentrations were compatible with those measured in animals under chronic treatment involving the peripheral mechanism. Our study highlights a peripheral neuroimmune component of antidepressant drugs that is relevant to their delayed therapeutic action against neuropathic pain.SIGNIFICANCE STATEMENT In addition to treating depression, antidepressant drugs are also a first-line treatment for neuropathic pain, which is pain secondary to lesion or pathology of the nervous system. However, the mechanism by which antidepressant drugs can relieve neuropathic pain remained in part elusive. Indeed, preclinical studies led to contradictions concerning the anatomical and molecular substrates of this action. In the present work, we overcame these apparent contradictions by highlighting the existence of two independent mechanisms. One is rapid and centrally mediated by descending controls from the brain to the spinal cord and the other is delayed, peripheral, and relies on the anti-neuroimmune action of chronic antidepressant treatment.

A new microdeletion syndrome involving TBC1D24, ATP6V0C, and PDPK1 causes epilepsy, microcephaly, and developmental delay.

PURPOSE: Contiguous gene deletions are known to cause several neurodevelopmental syndromes, many of which are caused by recurrent events on chromosome 16. However, chromosomal microarray studies (CMA) still yield copy-number variants (CNVs) of unknown clinical significance. We sought to characterize eight individuals with overlapping 205-kb to 504-kb 16p13.3 microdeletions that are distinct from previously published deletion syndromes. METHODS: Clinical information on the patients and bioinformatic scores for the deleted genes were analyzed. RESULTS: All individuals in our cohort displayed developmental delay, intellectual disability, and various forms of seizures. Six individuals were microcephalic and two had strabismus. The deletion was absent in all 13 parents who were available for testing. The area of overlap encompasses seven genes including TBC1D24, ATP6V0C, and PDPK1 (also known as PDK1). Bi-allelic TBC1D24 pathogenic variants are known to cause nonsyndromic deafness, epileptic disorders, or DOORS syndrome (deafness, onychodystrophy, osteodystrophy, mental retardation, seizures). Sanger sequencing of the nondeleted TBC1D24 allele did not yield any additional pathogenic variants. CONCLUSIONS: We propose that 16p13.3 microdeletions resulting in simultaneous haploinsufficiencies of TBC1D24, ATP6V0C, and PDPK1 cause a novel rare contiguous gene deletion syndrome of microcephaly, developmental delay, intellectual disability, and epilepsy.

Correction: A new microdeletion syndrome involving TBC1D24, ATP6V0C, and PDPK1 causes epilepsy, microcephaly, and developmental delay.

The original version of this Article contained an error in the spelling of the author Siddharth Banka, which was incorrectly given as Siddhart Banka. This has now been corrected in both the PDF and HTML versions of the Article.

Strategies to Advance Drug Discovery in Rare Monogenic Intellectual Disability Syndromes.

Some intellectual disability syndromes are caused by a mutation in a single gene and have been the focus of therapeutic intervention attempts, such as Fragile X and Rett Syndrome, albeit with limited success. The rate at which new drugs are discovered and tested in humans for intellectual disability is progressing at a relatively slow pace. This is particularly true for rare diseases where so few patients make high-quality clinical trials challenging. We discuss how new advances in human stem cell reprogramming and gene editing can facilitate preclinical study design and we propose new workflows for how the preclinical to clinical trajectory might proceed given the small number of subjects available in rare monogenic intellectual disability syndromes.

Medium throughput bisulfite sequencing for accurate detection of 5-methylcytosine and 5-hydroxymethylcytosine.

BACKGROUND: Epigenetic modifications of DNA, such as 5-methylcytosine and 5-hydroxymethycytosine, play important roles in development and disease. Here, we present a cost-effective and versatile methodology for the analysis of DNA methylation in targeted genomic regions, which comprises multiplexed, PCR-based preparation of bisulfite DNA libraries followed by customized MiSeq sequencing. RESULTS: Using bisulfite and oxidative bisulfite conversion of DNA, we have performed multiplexed targeted sequencing to analyse several kilobases of genomic DNA in up to 478 samples, and achieved high coverage data of 5-methylcytosine and 5-hydroxymethycytosine at single-base resolution. Our results demonstrate the ability of this methodology to detect all levels of cytosine modifications at greater than 100× coverage in large sample sets at low cost compared to other targeted methods. CONCLUSIONS: This approach can be applied to multiple settings, from candidate gene to clinical studies, and is especially useful for validation of differentially methylated or hydroxymethylated regions following whole-genome analyses.

Molecular convergence of neurodevelopmental disorders.

Neurodevelopmental disorders (NDDs) are caused by mutations in diverse genes involved in different cellular functions, although there can be crosstalk, or convergence, between molecular pathways affected by different NDDs. To assess molecular convergence, we generated human neural progenitor cell models of 9q34 deletion syndrome, caused by haploinsufficiency of EHMT1, and 18q21 deletion syndrome, caused by haploinsufficiency of TCF4. Using next-generation RNA sequencing, methylation sequencing, chromatin immunoprecipitation sequencing, and whole-genome miRNA analysis, we identified several levels of convergence. We found mRNA and miRNA expression patterns that were more characteristic of differentiating cells than of proliferating cells, and we identified CpG clusters that had similar methylation states in both models of reduced gene dosage. There was significant overlap of gene targets of TCF4 and EHMT1, whereby 8.3% of TCF4 gene targets and 4.2% of EHMT1 gene targets were identical. These data suggest that 18q21 and 9q34 deletion syndromes show significant molecular convergence but distinct expression and methylation profiles. Common intersection points might highlight the most salient features of disease and provide avenues for similar treatments for NDDs caused by different genetic mutations.

Transcriptional consequences of 16p11.2 deletion and duplication in mouse cortex and multiplex autism families.

Reciprocal copy-number variation (CNV) of a 593 kb region of 16p11.2 is a common genetic cause of autism spectrum disorder (ASD), yet it is not completely penetrant and can manifest in a wide array of phenotypes. To explore its molecular consequences, we performed RNA sequencing of cerebral cortex from mouse models with CNV of the syntenic 7qF3 region and lymphoblast lines from 34 members of 7 multiplex ASD-affected families harboring the 16p11.2 CNV. Expression of all genes in the CNV region correlated well with their DNA copy number, with no evidence of dosage compensation. We observed effects on gene expression outside the CNV region, including apparent positional effects in cis and in trans at genomic segments with evidence of physical interaction in Hi-C chromosome conformation data. One of the most significant positional effects was telomeric to the 16p11.2 CNV and includes the previously described "distal" 16p11.2 microdeletion. Overall, 16p11.2 CNV was associated with altered expression of genes and networks that converge on multiple hypotheses of ASD pathogenesis, including synaptic function (e.g., NRXN1, NRXN3), chromatin modification (e.g., CHD8, EHMT1, MECP2), transcriptional regulation (e.g., TCF4, SATB2), and intellectual disability (e.g., FMR1, CEP290). However, there were differences between tissues and species, with the strongest effects being consistently within the CNV region itself. Our analyses suggest that through a combination of indirect regulatory effects and direct effects on nuclear architecture, alteration of 16p11.2 genes disrupts expression networks that involve other genes and pathways known to contribute to ASD, suggesting an overlap in mechanisms of pathogenesis.

Implication of LRRC4C and DPP6 in neurodevelopmental disorders.

We performed whole-genome sequencing on an individual from a family with variable psychiatric phenotypes that had a sensory processing disorder, apraxia, and autism. The proband harbored a maternally inherited balanced translocation (46,XY,t(11;14)(p12;p12)mat) that disrupted LRRC4C, a member of the highly specialized netrin G family of axon guidance molecules. The proband also inherited a paternally derived chromosomal inversion that disrupted DPP6, a potassium channel interacting protein. Copy Number (CN) analysis in 14,077 cases with neurodevelopmental disorders and 8,960 control subjects revealed that 60% of cases with exonic deletions in LRRC4C had a second clinically recognizable syndrome associated with variable clinical phenotypes, including 16p11.2, 1q44, and 2q33.1 CN syndromes, suggesting LRRC4C deletion variants may be modifiers of neurodevelopmental disorders. In vitro, functional assessments modeling patient deletions in LRRC4C suggest a negative regulatory role of these exons found in the untranslated region of LRRC4C, which has a single, terminal coding exon. These data suggest that the proband's autism may be due to the inheritance of disruptions in both DPP6 and LRRC4C, and may highlight the importance of the netrin G family and potassium channel interacting molecules in neurodevelopmental disorders. © 2016 Wiley Periodicals, Inc.

A de novo frameshift mutation in chromodomain helicase DNA-binding domain 8 (CHD8): A case report and literature review.

Mutations in chromodomain helicase DNA-binding domain 8 (CHD8) have been identified in independent genotyping studies of autism spectrum disorder. To better understand the phenotype associated with CHD8 mutations, we genotyped all CHD8 exons in carefully assessed cohorts of autism (n = 142), schizophrenia (SCZ; n = 143), and intellectual disability (ID; n = 94). We identified one frameshift mutation, seven non-synonymous variants, and six synonymous variants. The frameshift mutation, p.Asn2092Lysfs*2, which creates a premature stop codon leading to the loss of 212 amino acids of the protein, was from an autism case on whom we present multiple clinical assessments and pharmacological treatments spanning more than 10 years. RNA and protein analysis support a model where the transcript generated from the mutant allele results in haploinsufficiency of CHD8. This case report supports the association of CHD8 mutations with classical autism, macrocephaly, infantile hypotonia, speech delay, lack of major ID, and psychopathology in late adolescence caused by insufficient dosage of CHD8. Review of 16 other CHD8 mutation cases suggests that clinical features and their severity vary considerably across individuals; however, these data support a CHD8 mutation syndrome, further highlighting the importance of genomic medicine to guide clinical assessment and treatment.

Characterizing 5-hydroxymethylcytosine in human prefrontal cortex at single base resolution.

BACKGROUND: The recent discovery that methylated cytosines are converted to 5-hydroxymethylated cytosines (5hmC) by the family of ten-eleven translocation enzymes has sparked significant interest on the genomic location, the abundance in different tissues, the putative functions, and the stability of this epigenetic mark. 5hmC plays a key role in the brain, where it is particularly abundant and dynamic during development. RESULTS: Here, we comprehensively characterize 5hmC in the prefrontal cortices of 24 subjects. We show that, although there is inter-individual variability in 5hmC content among unrelated individuals, approximately 8 % of all CpGs on autosomal chromosomes contain 5hmC, while sex chromosomes contain far less. Our data also provide evidence suggesting that 5hmC has transcriptional regulatory properties, as the density of 5hmC was highest in enhancer regions and within exons. Furthermore, we link increased 5hmC density to histone modification binding sites, to the gene bodies of actively transcribed genes, and to exon-intron boundaries. Finally, we provide several genomic regions of interest that contain gender-specific 5hmC. CONCLUSIONS: Collectively, these results present an important reference for the growing number of studies that are interested in the investigation of the role of 5hmC in brain and mental disorders.

Investigation of genes important in neurodevelopment disorders in adult human brain.

Several neurodevelopmental disorders (NDDs) are caused by mutations in genes expressed in fetal brain, but little is known about these same genes in adult human brain. Here, we test the hypothesis that genes associated with NDDs continue to have a role in adult human brain to explore the idea that NDD symptoms may be partially a result of their adult function rather than just their neurodevelopmental function. To demonstrate adult brain function, we performed expression analyses and ChIPseq in human neural stem cell(NSC) lines at different developmental stages and adult human brain, targeting two genes associated with NDDs, SATB2 and EHMT1, and the WNT signaling gene TCF7L2, which has not been associated with NDDs. Analysis of DNA interaction sites in neural stem cells reveals high (40-50 %) overlap between proliferating and differentiating cells for each gene in temporal space. Studies in adult brain demonstrate that consensus sites are similar to NSCs but occur at different genomic locations. We also performed expression analyses using BrainSpan data for NDD-associated genes SATB2, EHMT1, FMR1, MECP2, MBD5, CTNND2, RAI1, CHD8, GRIN2A, GRIN2B, TCF4, SCN2A, and DYRK1A and find high expression of these genes in adult brain, at least comparable to developing human brain, confirming that genes associated with NDDs likely have a role in adult tissue. Adult function of genes associated with NDDs might be important in clinical disease presentation and may be suitable targets for therapeutic intervention.

Disruption of a large intergenic noncoding RNA in subjects with neurodevelopmental disabilities.

Large intergenic noncoding (linc) RNAs represent a newly described class of ribonucleic acid whose importance in human disease remains undefined. We identified a severely developmentally delayed 16-year-old female with karyotype 46,XX,t(2;11)(p25.1;p15.1)dn in the absence of clinically significant copy number variants (CNVs). DNA capture followed by next-generation sequencing of the translocation breakpoints revealed disruption of a single noncoding gene on chromosome 2, LINC00299, whose RNA product is expressed in all tissues measured, but most abundantly in brain. Among a series of additional, unrelated subjects referred for clinical diagnostic testing who showed CNV affecting this locus, we identified four with exon-crossing deletions in association with neurodevelopmental abnormalities. No disruption of the LINC00299 coding sequence was seen in almost 14,000 control subjects. Together, these subjects with disruption of LINC00299 implicate this particular noncoding RNA in brain development and raise the possibility that, as a class, abnormalities of lincRNAs may play a significant role in human developmental disorders.

BisQC: an operational pipeline for multiplexed bisulfite sequencing.

BACKGROUND: Bisulfite sequencing is the most efficient single nucleotide resolution method for analysis of methylation status at whole genome scale, but improved quality control metrics are needed to better standardize experiments. RESULTS: We describe BisQC, a step-by-step method for multiplexed bisulfite-converted DNA library construction, pooling, spike-in content, and bioinformatics. We demonstrate technical improvements for library preparation and bioinformatic analyses that can be done in standard laboratories. We find that decoupling amplification of bisulfite converted (bis) DNA from the indexing reaction is an advantage, specifically in reducing total PCR cycle number and pre-selecting high quality bis-libraries. We also introduce a progressive PCR method for optimal library amplification and size-selection. At the sequencing stage, we thoroughly test the benefits of pooling non-bis DNA library with bis-libraries and find that BisSeq libraries can be pooled with a high proportion of non-bis DNA libraries with minimal impact on BisSeq output. For informatics analysis, we propose a series of optimization steps including the utilization of the mitochondrial genome as a QC standard, and we assess the validity of using duplicate reads for coverage statistics. CONCLUSION: We demonstrate several quality control checkpoints at the library preparation, pre-sequencing, post-sequencing, and post-alignment stages, which should prove useful in determining sample and processing quality. We also determine that including a significant portion of non-bisulfite converted DNA with bisulfite converted DNA has a minimal impact on usable bisulfite read output.