The dopaminergic network and genetic susceptibility to schizophrenia

Background: Schizophrenia is a disabling illness with unknown pathogenesis. Estimates of heritability suggest a substantial genetic contribution; however genetic studies to date have been equivocal. Uncovering liability loci may therefore require analyses of functionally related genes. Rooted in this assumption, this dissertation describes a series of studies investigating a genetic epidemiological foundation for the commonly cited hypothesis suggesting dopaminergic dysfunction in schizophrenia pathogenesis, i.e. the 'dopamine hypothesis'. Studies: The initial study investigated DRD3 and identified novel associations across the gene. The second study considered a larger network of dopaminergic genes in two independent Caucasian samples, detecting replicated associations and epistatic interactions. This study proposed a risk model for schizophrenia centered on the dopamine transporter. Study #3 investigated a dopamine precursor, phenylalanine hydroxylase, in four independent samples, identifying a single SNP (rs1522305) that was significantly replicated in two samples. Study #4 was motivated by the hypothesis of a shared genetic etiology for schizophrenia and bipolar disorder. This study comprehensively evaluated the dopaminergic network, selecting 431 'tag' SNPs from 40 genes among large schizophrenia and bipolar cohorts contrasted with adult controls. Across all genes 60% of nominally significant schizophrenia risk factors were also associated with bipolar disorder. The results supported DRD3 variations as risk factors for both disorders, confirmed several previously reported associations, and proposed new targets for future research. Conclusion: These results suggest dopaminergic gene variations could play an etiological role in the pathogenesis of schizophrenia and possibly bipolar 1 disorder. Additional replicate studies are warranted. Public Health Significance: Schizophrenia (SZ) is devastating. When the Global Burden of Disease study calculated disability adjusted life years, weighted for the severity of disability, they determined active psychosis seen in schizophrenia produces disability equal to quadriplegia. Schizophrenia has been estimated to be among the top ten causes of disability worldwide. As schizophrenia is common (roughly 1% point prevalence worldwide), the economic burden to society is substantial. Pathogenesis is unknown and treatment is palliative. Therefore understanding the genetic etiology could facilitate development of promising therapeutics

Autism Spectrum Disorder Genetics: Diverse Genes with Diverse Clinical Outcomes

The last several years have seen unprecedented advances in deciphering the genetic etiology of autism spectrum disorders (ASDs). Heritability studies have repeatedly affirmed a contribution of genetic factors to the overall disease risk. Technical breakthroughs have enabled the search for these genetic factors via genome-wide surveys of a spectrum of potential sequence variations, from common single-nucleotide polymorphisms to essentially private chromosomal abnormalities. Studies of copy-number variation have identified significant roles for both recurrent and nonrecurrent large dosage imbalances, although they have rarely revealed the individual genes responsible. More recently, discoveries of rare point mutations and characterization of balanced chromosomal abnormalities have pinpointed individual ASD genes of relatively strong effect, including both loci with strong a priori biological relevance and those that would have otherwise been unsuspected as high-priority biological targets. Evidence has also emerged for association with many common variants, each adding a small individual contribution to ASD risk. These findings collectively provide compelling empirical data that the genetic basis of ASD is highly heterogeneous, with hundreds of genes capable of conferring varying degrees of risk, depending on their nature and the predisposing genetic alteration. Moreover, many genes that have been implicated in ASD also appear to be risk factors for related neurodevelopmental disorders, as well as for a spectrum of psychiatric phenotypes. While some ASD genes have evident functional significance, like synaptic proteins such as the SHANKs, neuroligins, and neurexins, as well as fragile x mental retardation–associated proteins, ASD genes have also been discovered that do not present a clear mechanism of specific neurodevelopmental dysfunction, such as regulators of chromatin modification and global gene expression. In their sum, the progress from genetic studies to date has been remarkable and increasingly rapid, but the interactive impact of strong-effect genetic lesions coupled with weak effect common polymorphisms has not yet led to a unified understanding of ASD pathogenesis or explained its highly variable clinical expression. With an increasingly firm genetic foundation, the coming years will hopefully see equally rapid advances in elucidating the functional consequences of ASD genes and their interactions with environmental/experiential factors, supporting the development of rational interventions

CHD8 Regulates Neurodevelopmental Pathways Associated with Autism Spectrum Disorder in Neural Progenitors

Truncating mutations of chromodomain helicase DNA-binding protein 8 (CHD8), and of many other genes with diverse functions, are strong-effect risk factors for autism spectrum disorder (ASD), suggesting multiple mechanisms of pathogenesis. We explored the transcriptional networks that CHD8 regulates in neural progenitor cells (NPCs) by reducing its expression and then integrating transcriptome sequencing (RNA sequencing) with genome-wide CHD8 binding (ChIP sequencing). Suppressing CHD8 to levels comparable with the loss of a single allele caused altered expression of 1,756 genes, 64.9% of which were up-regulated. CHD8 showed widespread binding to chromatin, with 7,324 replicated sites that marked 5,658 genes. Integration of these data suggests that a limited array of direct regulatory effects of CHD8 produced a much larger network of secondary expression changes. Genes indirectly down-regulated (i.e., without CHD8-binding sites) reflect pathways involved in brain development, including synapse formation, neuron differentiation, cell adhesion, and axon guidance, whereas CHD8-bound genes are strongly associated with chromatin modification and transcriptional regulation. Genes associated with ASD were strongly enriched among indirectly down-regulated loci (P < 10[superscript −8]) and CHD8-bound genes (P = 0.0043), which align with previously identified coexpression modules during fetal development. We also find an intriguing enrichment of cancer-related gene sets among CHD8-bound genes (P < 10[superscript −10]). In vivo suppression of chd8 in zebrafish produced macrocephaly comparable to that of humans with inactivating mutations. These data indicate that heterozygous disruption of CHD8 precipitates a network of gene-expression changes involved in neurodevelopmental pathways in which many ASD-associated genes may converge on shared mechanisms of pathogenesis.Simons FoundationNancy Lurie Marks Family FoundationNational Institutes of Health (U.S.) (Grant MH095867)National Institutes of Health (U.S.) (Grant MH095088)National Institutes of Health (U.S.) (Grant GM061354)March of Dimes Birth Defects FoundationCharles H. Hood FoundationBrain & Behavior Research FoundationAutism Genetic Resource ExchangeAutism Speaks (Organization)Pitt–Hopkins Research Foundatio

A structural variation reference for medical and population genetics

Structural variants (SVs) rearrange large segments of DNA(1) and can have profound consequences in evolution and human disease(2,3). As national biobanks, disease-association studies, and clinical genetic testing have grown increasingly reliant on genome sequencing, population references such as the Genome Aggregation Database (gnomAD)(4) have become integral in the interpretation of single-nucleotide variants (SNVs)(5). However, there are no reference maps of SVs from high-coverage genome sequencing comparable to those for SNVs. Here we present a reference of sequence-resolved SVs constructed from 14,891 genomes across diverse global populations (54% non-European) in gnomAD. We discovered a rich and complex landscape of 433,371 SVs, from which we estimate that SVs are responsible for 25-29% of all rare protein-truncating events per genome. We found strong correlations between natural selection against damaging SNVs and rare SVs that disrupt or duplicate protein-coding sequence, which suggests that genes that are highly intolerant to loss-of-function are also sensitive to increased dosage(6). We also uncovered modest selection against noncoding SVs in cis-regulatory elements, although selection against protein-truncating SVs was stronger than all noncoding effects. Finally, we identified very large (over one megabase), rare SVs in 3.9% of samples, and estimate that 0.13% of individuals may carry an SV that meets the existing criteria for clinically important incidental findings(7). This SV resource is freely distributed via the gnomAD browser(8) and will have broad utility in population genetics, disease-association studies, and diagnostic screening.Peer reviewe

CHD8 suppression impacts on histone H3 lysine 36 trimethylation and alters RNA alternative splicing

Disruptive mutations in the chromodomain helicase DNA-binding protein 8 gene (CHD8) have been recurrently associated with autism spectrum disorders (ASDs). Here we investigated how chromatin reacts to CHD8 suppression by analyzing a panel of histone modifications in induced pluripotent stem cell-derived neural progenitors. CHD8 suppression led to significant reduction (47.82%) in histone H3K36me3 peaks at gene bodies, particularly impacting on transcriptional elongation chromatin states. H3K36me3 reduction specifically affects highly expressed, CHD8-bound genes and correlates with altered alternative splicing patterns of 462 genes implicated in ‘regulation of RNA splicing’ and ‘mRNA catabolic process’. Mass spectrometry analysis uncovered a novel interaction between CHD8 and the splicing regulator heterogeneous nuclear ribonucleoprotein L (hnRNPL), providing the first mechanistic insights to explain the CHD8 suppression-derived splicing phenotype, partly implicating SETD2, a H3K36me3 methyltransferase. In summary, our results point toward broad molecular consequences of CHD8 suppression, entailing altered histone deposition/maintenance and RNA processing regulation as important regulatory processes in ASD

Complex Reorganization and Predominant Non-Homologous Repair Following Chromosomal Breakage in Karyotypically Balanced Germline Rearrangements and Transgenic Integration

We defined the genetic landscape of balanced chromosomal rearrangements at nucleotide resolution by sequencing 141 breakpoints from cytogenetically-interpreted translocations and inversions. We confirm that the recently described phenomenon of “chromothripsis” (massive chromosomal shattering and reorganization) is not unique to cancer cells but also occurs in the germline where it can resolve to a karyotypically balanced state with frequent inversions. We detected a high incidence of complex rearrangements (19.2%) and substantially less reliance on microhomology (31%) than previously observed in benign CNVs. We compared these results to experimentally-generated DNA breakage-repair by sequencing seven transgenic animals, and revealed extensive rearrangement of the transgene and host genome with similar complexity to human germline alterations. Inversion is the most common rearrangement, suggesting that a combined mechanism involving template switching and non-homologous repair mediates the formation of balanced complex rearrangements that are viable, stably replicated and transmitted unaltered to subsequent generations

Fine-mapping reveals novel alternative splicing of the dopamine transporter

Center for Human Genetic Research, Massachusetts General Hospital and Department of Neurology, Harvard Medical School, Harvard University, Boston, Massachusetts.Graduate Program in Biology and Biomedical Science, Yale University, New Haven, Connecticut.The dopamine transporter gene (, ) has been implicated in the pathogenesis of numerous psychiatric and neurodevelopmental disorders, including schizophrenia (SZ). We previously detected association between SZ and intronic variants that replicated in two independent Caucasian samples, but had no obvious function. In follow-up analyses, we sequenced the coding and intronic regions of to identify complete linkage disequilibrium patterns of common variations. We genotyped 78 polymorphisms, narrowing the potentially causal region to two correlated clusters of associated SNPs localized predominantly to introns 3 and 4. Our computational analysis of these intronic regions predicted a novel cassette exon within intron 3, designated E3b, which is conserved among primates. We confirmed alternative splicing of E3b in post-mortem human substantia nigra (SN). As E3b introduces multiple in-frame stop codons, the open reading frame is truncated and the spliced product may undergo nonsense mediated decay. Thus, factors that increase E3b splicing could reduce the amount of unspliced product available for translation. Observations consistent with this prediction were made using cellular assays and in post-mortem human SN. In mini-gene constructs, the extent of splicing is also influenced by at least two common haplotypes, so the alternative splicing was evaluated in relation to SZ risk. Meta-analyses across genome-wide association studies did not support the initial associations and further post-mortem studies did not suggest case-control differences in splicing. These studies do not provide a compelling link to schizophrenia. However, the impact of the alternative splicing on other neuropsychiatric disorders should be investigated. © 2010 Wiley-Liss, Inc

Pathogenic copy number variants and SCN1A mutations in patients with intellectual disability and childhood-onset epilepsy

Background Copy number variants (CNVs) have been linked to neurodevelopmental disorders such as intellectual disability (ID), autism, epilepsy and psychiatric disease. There are few studies of CNVs in patients with both ID and epilepsy. Methods We evaluated the range of rare CNVs found in 80 Welsh patients with ID or developmental delay (DD), and childhood-onset epilepsy. We performed molecular cytogenetic testing by single nucleotide polymorphism array or microarray-based comparative genome hybridisation. Results 8.8 % (7/80) of the patients had at least one rare CNVs that was considered to be pathogenic or likely pathogenic. The CNVs involved known disease genes (EHMT1, MBD5 and SCN1A) and imbalances in genomic regions associated with neurodevelopmental disorders (16p11.2, 16p13.11 and 2q13). Prompted by the observation of two deletions disrupting SCN1A we undertook further testing of this gene in selected patients. This led to the identification of four pathogenic SCN1A mutations in our cohort. Conclusions We identified five rare de novo deletions and confirmed the clinical utility of array analysis in patients with ID/DD and childhood-onset epilepsy. This report adds to our clinical understanding of these rare genomic disorders and highlights SCN1A mutations as a cause of ID and epilepsy, which can easily be overlooked in adults