Evaluating the regulatory function of non-coding autism-associated single nucleotide polymorphisms on gene expression in human brain tissue.

Common variants account for most of the estimated heritability associated with autism spectrum disorder (autism). Although several replicable single nucleotide polymorphisms (SNPs) for the condition have been detected using genome-wide association study (GWAS) methodologies, their pathophysiological relevance remains elusive. Examining this is complicated, however, as all detected loci are situated within non-coding regions of the genome. It is therefore likely that they possess roles of regulatory function as opposed to directly affecting gene coding sequences. To bridge the gap between SNP discovery and mechanistic insight, we applied a comprehensive bioinformatic pipeline to functionally annotate autism-associated polymorphisms and their non-coding linkage disequilibrium (i.e., non-randomly associated) partners. We identified 82 DNA variants of probable regulatory function that may contribute to autism pathogenesis. To validate these predictions, we measured the impact of 11 high-confidence candidates and their GWAS linkage disequilibrium partners on gene expression in human brain tissue from Autistic and non-Autistic donors. Although a small number of the surveyed variants exhibited measurable influence on gene expression as determined via quantitative polymerase chain reaction, these did not survive correction for multiple comparisons. Additionally, no significant genotype-by-diagnosis effects were observed for any of the SNP-gene associations. We contend that this may reflect an inability to effectively capture the modest, neurodevelopmental-specific impact of individual variants on biological dysregulation in available post-mortem tissue samples, as well as limitations in the existing autism GWAS data.

Integration of xeno-free single-cell cloning in CRISPR-mediated DNA editing of human iPSCs improves homogeneity and methodological efficiency of cellular disease modeling.

The capability to generate induced pluripotent stem cell (iPSC) lines, in tandem with CRISPR-Cas9 DNA editing, offers great promise to understand the underlying genetic mechanisms of human disease. The low efficiency of available methods for homogeneous expansion of singularized CRISPR-transfected iPSCs necessitates the coculture of transfected cells in mixed populations and/or on feeder layers. Consequently, edited cells must be purified using labor-intensive screening and selection, culminating in inefficient editing. Here, we provide a xeno-free method for single-cell cloning of CRISPRed iPSCs achieving a clonal survival of up to 70% within 7-10 days. This is accomplished through improved viability of the transfected cells, paralleled with provision of an enriched environment for the robust establishment and proliferation of singularized iPSC clones. Enhanced cell survival was accompanied by a high transfection efficiency exceeding 97%, and editing efficiencies of 50%-65% for NHEJ and 10% for HDR, indicative of the method's utility in stem cell disease modeling.

Generation of induced pluripotent stem cell lines from three individuals with autism spectrum disorder.

Uncovering the molecular mechanisms of autism spectrum disorder (autism) necessitates development of relevant experimental models that are capable of recapitulating features of the clinical phenotype. Using non-integrative episomal vectors, peripheral blood mononuclear cells derived from three unrelated individuals diagnosed with autism were reprogrammed to induced pluripotent stem cells (iPSCs). The resultant lines exhibited the expected cellular morphology, karyotype, and evidence of pluripotency. These iPSCs constitute a valuable resource to support investigations of the underlying aetiology of autism.

Trans-ancestry meta-analysis of genome wide association studies of inhibitory control.

Deficits in effective executive function, including inhibitory control are associated with risk for a number of psychiatric disorders and significantly impact everyday functioning. These complex traits have been proposed to serve as endophenotypes, however, their genetic architecture is not yet well understood. To identify the common genetic variation associated with inhibitory control in the general population we performed the first trans-ancestry genome wide association study (GWAS) combining data across 8 sites and four ancestries (N = 14,877) using cognitive traits derived from the stop-signal task, namely - go reaction time (GoRT), go reaction time variability (GoRT SD) and stop signal reaction time (SSRT). Although we did not identify genome wide significant associations for any of the three traits, GoRT SD and SSRT demonstrated significant and similar SNP heritability of 8.2%, indicative of an influence of genetic factors. Power analyses demonstrated that the number of common causal variants contributing to the heritability of these phenotypes is relatively high and larger sample sizes are necessary to robustly identify associations. In Europeans, the polygenic risk for ADHD was significantly associated with GoRT SD and the polygenic risk for schizophrenia was associated with GoRT, while in East Asians polygenic risk for schizophrenia was associated with SSRT. These results support the potential of executive function measures as endophenotypes of neuropsychiatric disorders. Together these findings provide the first evidence indicating the influence of common genetic variation in the genetic architecture of inhibitory control quantified using objective behavioural traits derived from the stop-signal task.

Dissecting Schizotypy and Its Association With Cognition and Polygenic Risk for Schizophrenia in a Nonclinical Sample.

Schizotypy is a multidimensional construct that captures a continuum of risk for developing schizophrenia-spectrum psychopathology. Existing 3-factor models of schizotypy, consisting of positive, negative, and disorganized dimensions have yielded mixed evidence of genetic continuity with schizophrenia using polygenic risk scores. Here, we propose an approach that involves splitting positive and negative schizotypy into more specific subdimensions that are phenotypically continuous with distinct positive symptoms and negative symptoms recognized in clinical schizophrenia. We used item response theory to derive high-precision estimates of psychometric schizotypy using 251 self-report items obtained from a non-clinical sample of 727 (424 females) adults. These subdimensions were organized hierarchically using structural equation modeling into 3 empirically independent higher-order dimensions enabling associations with polygenic risk for schizophrenia to be examined at different levels of phenotypic generality and specificity. Results revealed that polygenic risk for schizophrenia was associated with variance specific to delusional experiences (γ = 0.093, P = .001) and reduced social interest and engagement (γ = 0.076, P = .020), and these effects were not mediated via the higher-order general, positive, or negative schizotypy factors. We further fractionated general intellectual functioning into fluid and crystallized intelligence in 446 (246 females) participants that underwent onsite cognitive assessment. Polygenic risk scores explained 3.6% of the variance in crystallized intelligence. Our precision phenotyping approach could be used to enhance the etiologic signal in future genetic association studies and improve the detection and prevention of schizophrenia-spectrum psychopathology.

Potential role for immune-related genes in autism spectrum disorders: Evidence from genome-wide association meta-analysis of autistic traits.

LAY ABSTRACT: Autism spectrum disorders are complex, with a strong genetic basis. Genetic research in autism spectrum disorders is limited by the fact that these disorders are largely heterogeneous so that patients are variable in their clinical presentations. To address this limitation, we investigated the genetics of individual dimensions of the autism spectrum disorder phenotypes, or autistic-like traits. These autistic-like traits are continuous variations in autistic behaviours that occur in the general population. Therefore, we meta-analysed data from four different population cohorts in which autistic-like traits were measured. We performed a set of genetic analyses to identify common variants for autistic-like traits, understand how these variants related to autism spectrum disorders, and how they contribute to neurobiological processes. Our results showed genetic associations with specific autistic-like traits and a link to the immune system. We offer an example of the potential to use a dimensional approach when dealing with heterogeneous, complex disorder like autism spectrum disorder. Decomposing the complex autism spectrum disorder phenotype in its core features can inform on the specific biology of these features which is likely to account to clinical variability in patients.

Environmental exposures associated with elevated risk for autism spectrum disorder may augment the burden of deleterious de novo mutations among probands.

Although the full aetiology of autism spectrum disorder (ASD) is unknown, familial and twin studies demonstrate high heritability of 60-90%, indicating a predominant role of genetics in the development of the disorder. The genetic architecture of ASD consists of a complex array of rare and common variants of all classes of genetic variation usually acting additively to augment individual risk. The relative contribution of heredity in ASD persists despite selective pressures against the classic autistic phenotype; a phenomenon thought to be explained, in part, by the incidence of spontaneous (or de novo) mutations. Notably, environmental exposures attributed as salient risk factors for ASD may play a causal role in the emergence of deleterious de novo variations, with several ASD-associated agents having significant mutagenic potential. To explore this hypothesis, this review article assesses published epidemiological data with evidence derived from assays of mutagenicity, both in vivo and in vitro, to determine the likely role such agents may play in augmenting the genetic liability in ASD. Broadly, these exposures were observed to elicit genomic alterations through one or a combination of: (1) direct interaction with genetic material; (2) impaired DNA repair; or (3) oxidative DNA damage. However, the direct contribution of these factors to the ASD phenotype cannot be determined without further analysis. The development of comprehensive prospective birth cohorts in combination with genome sequencing is essential to forming a causal, mechanistic account of de novo mutations in ASD that links exposure, genotypic alterations, and phenotypic consequences.

The Monash Autism-ADHD genetics and neurodevelopment (MAGNET) project design and methodologies: a dimensional approach to understanding neurobiological and genetic aetiology.

BACKGROUND: ASD and ADHD are prevalent neurodevelopmental disorders that frequently co-occur and have strong evidence for a degree of shared genetic aetiology. Behavioural and neurocognitive heterogeneity in ASD and ADHD has hampered attempts to map the underlying genetics and neurobiology, predict intervention response, and improve diagnostic accuracy. Moving away from categorical conceptualisations of psychopathology to a dimensional approach is anticipated to facilitate discovery of data-driven clusters and enhance our understanding of the neurobiological and genetic aetiology of these conditions. The Monash Autism-ADHD genetics and neurodevelopment (MAGNET) project is one of the first large-scale, family-based studies to take a truly transdiagnostic approach to ASD and ADHD. Using a comprehensive phenotyping protocol capturing dimensional traits central to ASD and ADHD, the MAGNET project aims to identify data-driven clusters across ADHD-ASD spectra using deep phenotyping of symptoms and behaviours; investigate the degree of familiality for different dimensional ASD-ADHD phenotypes and clusters; and map the neurocognitive, brain imaging, and genetic correlates of these data-driven symptom-based clusters. METHODS: The MAGNET project will recruit 1,200 families with children who are either typically developing, or who display elevated ASD, ADHD, or ASD-ADHD traits, in addition to affected and unaffected biological siblings of probands, and parents. All children will be comprehensively phenotyped for behavioural symptoms, comorbidities, neurocognitive and neuroimaging traits and genetics. CONCLUSION: The MAGNET project will be the first large-scale family study to take a transdiagnostic approach to ASD-ADHD, utilising deep phenotyping across behavioural, neurocognitive, brain imaging and genetic measures.

Genetic influences on hub connectivity of the human connectome.

Brain network hubs are both highly connected and highly inter-connected, forming a critical communication backbone for coherent neural dynamics. The mechanisms driving this organization are poorly understood. Using diffusion-weighted magnetic resonance imaging in twins, we identify a major role for genes, showing that they preferentially influence connectivity strength between network hubs of the human connectome. Using transcriptomic atlas data, we show that connected hubs demonstrate tight coupling of transcriptional activity related to metabolic and cytoarchitectonic similarity. Finally, comparing over thirteen generative models of network growth, we show that purely stochastic processes cannot explain the precise wiring patterns of hubs, and that model performance can be improved by incorporating genetic constraints. Our findings indicate that genes play a strong and preferential role in shaping the functionally valuable, metabolically costly connections between connectome hubs.

A rare missense variant in the ATP2C2 gene is associated with language impairment and related measures.

At least 5% of children present unexpected difficulties in expressing and understanding spoken language. This condition is highly heritable and often co-occurs with other neurodevelopmental disorders such as dyslexia and ADHD. Through an exome sequencing analysis, we identified a rare missense variant (chr16:84405221, GRCh38.p12) in the ATP2C2 gene. ATP2C2 was implicated in language disorders by linkage and association studies, and exactly the same variant was reported previously in a different exome sequencing study for language impairment (LI). We followed up this finding by genotyping the mutation in cohorts selected for LI and comorbid disorders. We found that the variant had a higher frequency in LI cases (1.8%, N = 360) compared with cohorts selected for dyslexia (0.8%, N = 520) and ADHD (0.7%, N = 150), which presented frequencies comparable to reference databases (0.9%, N = 24 046 gnomAD controls). Additionally, we observed that carriers of the rare variant identified from a general population cohort (N = 42, ALSPAC cohort) presented, as a group, lower scores on a range of reading and language-related measures compared to controls (N = 1825; minimum P = 0.002 for non-word reading). ATP2C2 encodes for an ATPase (SPCA2) that transports calcium and manganese ions into the Golgi lumen. Our functional characterization suggested that the rare variant influences the ATPase activity of SPCA2. Thus, our results further support the role of ATP2C2 locus in language-related phenotypes and pinpoint the possible effects of a specific rare variant at molecular level.

Evidence against benefits from cognitive training and transcranial direct current stimulation in healthy older adults.

Cognitive training and brain stimulation show promise for ameliorating age-related neurocognitive decline. However, evidence for this is controversial. In a Registered Report, we investigated the effects of these interventions, where 133 older adults were allocated to four groups (left prefrontal cortex anodal transcranial direct current stimulation (tDCS) with decision-making training, and three control groups) and trained over 5 days. They completed a task/questionnaire battery pre- and post-training, and at 1- and 3-month follow-ups. COMT and BDNF Val/Met polymorphisms were also assessed. Contrary to work in younger adults, there was evidence against tDCS-induced training enhancement on the decision-making task. Moreover, there was evidence against transfer of training gains to untrained tasks or everyday function measures at any post-intervention time points. As indicated by exploratory work, individual differences may have influenced outcomes. But, overall, the current decision-making training and tDCS protocol appears unlikely to lead to benefits for older adults.

Functional validation of CHMP7 as an ADHD risk gene.

Attention deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder of childhood with a strong genetic component. Despite the success of mapping ADHD risk loci, little work has been done to experimentally verify the contribution of these loci to ADHD phenotypes. Meta-analysis of four genome-wide association studies in ADHD suggested CHMP7 as a predisposing gene for ADHD. A DNA variant (rs2294123) mapped to CHMP7 has been shown (via bioinformatic analysis) to have a high likelihood for functionality and correlate with reduced transcript levels. We used CRISPR-Cas9 genome editing to generate a chmp7 zebrafish model for ADHD. chmp7+/- fish showed comparable reductions in mRNA levels to individuals homozygous for the CHMP7 ADHD risk allele. These fish displayed significant hyperactivity over a 24-h period at 6 days post-fertilisation compared to chmp7+/+, but this effect did not persist into juvenile and adulthood stages. In addition, chmp7+/- fish had significantly smaller total brain volumes than chmp7+/+ fish. Finally, the hyperactivity at 6 days post-fertilisation was significantly reduced through the application of methylphenidate, a mainstay pharmacological treatment for ADHD. Overall, this study highlights an important role for CHMP7 in the neurodevelopment of ADHD, and demonstrates the utility of zebrafish for modelling the functional effects of genes conferring risk to ADHD.

Impact of CYP2C19 genotype-predicted enzyme activity on hippocampal volume, anxiety, and depression.

Cytochrome P450 C19 (CYP2C19) metabolizes exogenous and endogenous compounds. Although CYP2C19 is highly expressed in the liver, it is also expressed in the brain during early life. Previous human and animal studies have linked CYP2C19 genotype-predicted enzyme activity to hippocampal volumes, depressive symptoms, and anxiety-like behaviors. We examined these promising associations in a general community sample comprising 386 Caucasian adults with no history of psychiatric or neurological illnesses. Contrary to previous findings, CYP2C19 genotype-predicted enzyme activity was not associated with hippocampal volumes, nor depressive and anxiety symptoms. Interstudy differences in CYP2C19 frequencies and/or study methodology may explain this discrepancy.

Intensity Matters: High-intensity Interval Exercise Enhances Motor Cortex Plasticity More Than Moderate Exercise.

A single bout of cardiovascular exercise can enhance plasticity in human cortex; however, the intensity required for optimal enhancement is debated. We investigated the effect of exercise intensity on motor cortex synaptic plasticity, using transcranial magnetic stimulation. Twenty healthy adults (Mage = 35.10 ± 13.25 years) completed three sessions. Measures of cortico-motor excitability (CME) and inhibition were obtained before and after a 20-min bout of either high-intensity interval exercise, moderate-intensity continuous exercise, or rest, and again after intermittent theta burst stimulation (iTBS). Results showed that high-intensity interval exercise enhanced iTBS plasticity more than rest, evidenced by increased CME and intracortical facilitation, and reduced intracortical inhibition. In comparison, the effect of moderate-intensity exercise was intermediate between high-intensity exercise and rest. Importantly, analysis of each participant's plasticity response profile indicated that high-intensity exercise increased the likelihood of a facilitatory response to iTBS. We also established that the brain-derived neurotrophic factor Val66Met polymorphism attenuated plasticity responses following high-intensity exercise. These findings suggest that high-intensity interval exercise should be considered not only when planning exercise interventions designed to enhance neuroplasticity, but also to maximize the therapeutic potential of non-invasive brain stimulation. Additionally, genetic profiling may enhance efficacy of exercise interventions for brain health.

Generation of four iPSC lines from peripheral blood mononuclear cells (PBMCs) of an attention deficit hyperactivity disorder (ADHD) individual and a healthy sibling in an Australia-Caucasian family.

Peripheral blood mononuclear cells were donated by a male teenager with clinically diagnosed attention deficit hyperactivity disorder (ADHD) under the Diagnostic and Statistical Manual of Mental Disorders IV criteria and his unaffected male sibling. Induced pluripotent stem cells were developed using integration-free Sendai Reprogramming factors containing OCT4, SOX2, KLF4, and c-MYC. All four iPSC lines displayed pluripotent cell morphology, pluripotency-associated factors at the DNA and protein level, alkaline phosphatase enzymatic activity and a male karyotype of 46, XY. All lines had capacity for in vitro differentiation into all the three germ layers. All were negative for Mycoplasma.

A case-control genome-wide association study of ADHD discovers a novel association with the tenascin R (TNR) gene.

It is well-established that there is a strong genetic contribution to the aetiology of attention deficit hyperactivity disorder (ADHD). Here, we employed a hypothesis-free genome-wide association study (GWAS) design in a sample of 480 clinical childhood ADHD cases and 1208 controls to search for novel genetic risk loci for ADHD. DNA was genotyped using Illumina's Human Infinium PsychArray-24v1.2., and the data were subsequently imputed to the 1000 Genomes reference panel. Rigorous quality control and pruning of genotypes at both individual subject and single nucleotide polymorphism (SNP) levels was performed. Polygenic risk score (PGRS) analysis revealed that ADHD case-control status was explained by genetic risk for ADHD, but no other major psychiatric disorders. Logistic regression analysis was performed genome-wide to test the association between SNPs and ADHD case-control status. We observed a genome-wide significant association (p = 3.15E-08) between ADHD and rs6686722, mapped to the Tenascin R (TNR) gene. Members of this gene family are extracellular matrix glycoproteins that play a role in neural cell adhesion and neurite outgrowth. Suggestive evidence of associations with ADHD was observed for an additional 111 SNPs (⩽9.91E-05). Although intriguing, the association between DNA variation in the TNR gene and ADHD should be viewed as preliminary given the small sample size of this discovery dataset.

Genome-wide association study reveals novel genetic locus associated with intra-individual variability in response time.

Intra-individual response time variability (IIRTV) is proposed as a viable endophenotype for many psychiatric disorders, particularly attention-deficit hyperactivity disorder (ADHD). Here we assessed whether IIRTV was associated with common DNA variation genome-wide and whether IIRTV mediated the relationship between any associated loci and self-reported ADHD symptoms. A final data set from 857 Australian young adults (489 females and 368 males; Mage = 22.14 years, SDage = 4.82 years) who completed five response time tasks and self-reported symptoms of ADHD using the Conners' Adult ADHD Rating Scale was used. Principal components analysis (PCA) on these response time measures (standard deviation of reaction times and the intra-individual coefficient of variation) produced two variability factors (labelled response selection and selective attention). To understand the genetic drivers of IIRTV we performed a genome-wide association analysis (GWAS) on these PCA-derived indices of IIRTV. For the selective attention variability factor, we identified one single-nucleotide polymorphism (SNP) attaining genome-wide significance; rs62182100 in the HDAC4 gene located on chromosome 2q37. A bootstrapping mediation analysis demonstrated that the selective attention variability factor mediated the relationship between rs62182100 and self-reported ADHD symptoms. Our findings provide the first evidence of a genome-wide significant SNP association with IIRTV and support the potential utility of IIRTV as a valid endophenotype for ADHD symptoms. However, limitations of this study suggest that these observations should be interpreted with caution until replication samples become available.

The role of cadherin genes in five major psychiatric disorders: A literature update.

Converging evidence from candidate gene, genome-wide linkage, and association studies support a role of cadherins in the pathophysiology of five major psychiatric disorders including attention deficit hyperactivity disorder, autism spectrum disorder (ASD), schizophrenia (SCZ), bipolar disorder (BD), and major depressive disorder (MDD). These molecules are transmembrane proteins which act as cell adhesives by forming adherens junctions (AJs) to bind cells within tissues. Members of the cadherin superfamily are also involved in biological processes such as signal transduction and plasticity that have been implicated in the etiology of major psychiatric conditions. Although there are over 110 genes mapped to the cadherin superfamily, our literature survey showed that evidence of association with psychiatric disorders is strongest for CDH7, CHD11, and CDH13. Gene enrichment analysis showed that those cadherin genes implicated in psychiatric disorders were overrepresented in biological processes such as in cell-cell adhesion (GO:0007156 & GO:0098742) and adherens junction organization (GO:0034332). Further, cadherin genes were also mapped to processes that have been linked to the development of psychiatric disorders such as nervous system development (GO:0007399). To further understand the role of cadherin SNPs implicated in psychiatric disorders, we utilized an in silico computational pipeline to functionally annotate associated variants. This analysis yielded eight variants mapped to PCDH1-13, CDH7, CDH11, and CDH13 that are predicted to be biologically functional. Functional genomic evaluation is now required to understand the molecular mechanism by which these variants might confer susceptibility to psychiatric disorders.

Allelic variation in dopamine D2 receptor gene is associated with attentional impulsiveness on the Barratt Impulsiveness Scale (BIS-11).

OBJECTIVES: Previous studies have postulated that noradrenergic and/or dopaminergic gene variations are likely to underlie individual differences in impulsiveness, however, few have shown this. The current study examined the relationship between catecholamine gene variants and self-reported impulsivity, as measured by the Barratt Impulsiveness Scale (Version 11; BIS-11) Methods: Six hundred and seventy-seven non-clinical adults completed the Barratt Impulsiveness Scale (BIS-11). DNA was analysed for a set of 142 single-nucleotide polymorphisms (SNPs) across 20 autosomal catecholamine genes. Association was tested using an additive regression model with permutation testing used to control for the influence of multiple comparison. RESULTS: Analysis revealed an influence of rs4245146 of the dopamine D2 receptor (DRD2) gene on the BIS-11 attention first-order factor, such that self-reported attentional impulsiveness increased in an additive fashion with each copy of the T allele. CONCLUSIONS: These findings provide preliminary evidence that allelic variation in DRD2 may influence impulsiveness by increasing the propensity for attentional lapses.