The application of human pluripotent stem cells to model the neuronal and glial components of neurodevelopmental disorders.

Cellular models of neurodevelopmental disorders provide a valuable experimental system to uncover disease mechanisms and novel therapeutic strategies. The ability of induced pluripotent stem cells (iPSCs) to generate diverse brain cell types offers great potential to model several neurodevelopmental disorders. Further patient-derived iPSCs have the unique genetic and molecular signature of the affected individuals, which allows researchers to address limitations of transgenic behavioural models, as well as generate hypothesis-driven models to study disorder-relevant phenotypes at a cellular level. In this article, we review the extant literature that has used iPSC-based modelling to understand the neuronal and glial contributions to neurodevelopmental disorders including autism spectrum disorder (ASD), Rett syndrome, bipolar disorder (BP), and schizophrenia. For instance, several molecular candidates have been shown to influence cellular phenotypes in three-dimensional iPSC-based models of ASD patients. Delays in differentiation of astrocytes and morphological changes of neurons are associated with Rett syndrome. In the case of bipolar disorders and schizophrenia, patient-derived models helped to identify cellular phenotypes associated with neuronal deficits (e.g., excitability) and mutation-specific abnormalities in oligodendrocytes (e.g., CSPG4). Further we provide a critical review of the current limitations of this field and provide methodological suggestions to enhance future modelling efforts of neurodevelopmental disorders. Future developments in experimental design and methodology of disease modelling represent an exciting new avenue relevant to neurodevelopmental disorders.

Rare DNA variants in the brain-derived neurotrophic factor gene increase risk for attention-deficit hyperactivity disorder: a next-generation sequencing study.

Attention-deficit hyperactivity disorder (ADHD) is a prevalent and highly heritable disorder of childhood with negative lifetime outcomes. Although candidate gene and genome-wide association studies have identified promising common variant signals, these explain only a fraction of the heritability of ADHD. The observation that rare structural variants confer substantial risk to psychiatric disorders suggests that rare variants might explain a portion of the missing heritability for ADHD. Here we believe we performed the first large-scale next-generation targeted sequencing study of ADHD in 152 child and adolescent cases and 188 controls across an a priori set of 117 genes. A multi-marker gene-level analysis of rare (<1% frequency) single-nucleotide variants (SNVs) revealed that the gene encoding brain-derived neurotrophic factor (BDNF) was associated with ADHD at Bonferroni corrected levels. Sanger sequencing confirmed the existence of all novel rare BDNF variants. Our results implicate BDNF as a genetic risk factor for ADHD, potentially by virtue of its critical role in neurodevelopment and synaptic plasticity.

Separating the wheat from the chaff: systematic identification of functionally relevant noncoding variants in ADHD.

Attention deficit hyperactivity disorder (ADHD) is a highly heritable psychiatric condition with negative lifetime outcomes. Uncovering its genetic architecture should yield important insights into the neurobiology of ADHD and assist development of novel treatment strategies. Twenty years of candidate gene investigations and more recently genome-wide association studies have identified an array of potential association signals. In this context, separating the likely true from false associations ('the wheat' from 'the chaff') will be crucial for uncovering the functional biology of ADHD. Here, we defined a set of 2070 DNA variants that showed evidence of association with ADHD (or were in linkage disequilibrium). More than 97% of these variants were noncoding, and were prioritised for further exploration using two tools-genome-wide annotation of variants (GWAVA) and Combined Annotation-Dependent Depletion (CADD)-that were recently developed to rank variants based upon their likely pathogenicity. Capitalising on recent efforts such as the Encyclopaedia of DNA Elements and US National Institutes of Health Roadmap Epigenomics Projects to improve understanding of the noncoding genome, we subsequently identified 65 variants to which we assigned functional annotations, based upon their likely impact on alternative splicing, transcription factor binding and translational regulation. We propose that these 65 variants, which possess not only a high likelihood of pathogenicity but also readily testable functional hypotheses, represent a tractable shortlist for future experimental validation in ADHD. Taken together, this study brings into sharp focus the likely relevance of noncoding variants for the genetic risk associated with ADHD, and more broadly suggests a bioinformatics approach that should be relevant to other psychiatric disorders.

The molecular genetic architecture of attention deficit hyperactivity disorder.

Attention deficit hyperactivity disorder (ADHD) is a common childhood behavioral condition which affects 2-10% of school age children worldwide. Although the underlying molecular mechanism for the disorder is poorly understood, familial, twin and adoption studies suggest a strong genetic component. Here we provide a state-of-the-art review of the molecular genetics of ADHD incorporating evidence from candidate gene and linkage designs, as well as genome-wide association (GWA) studies of common single-nucleotide polymorphisms (SNPs) and rare copy number variations (CNVs). Bioinformatic methods such as functional enrichment analysis and protein-protein network analysis are used to highlight biological processes of likely relevance to the aetiology of ADHD. Candidate gene associations of minor effect size have been replicated across a number of genes including SLC6A3, DRD5, DRD4, SLC6A4, LPHN3, SNAP-25, HTR1B, NOS1 and GIT1. Although case-control SNP-GWAS have had limited success in identifying common genetic variants for ADHD that surpass critical significance thresholds, quantitative trait designs suggest promising associations with Cadherin13 and glucose-fructose oxidoreductase domain 1 genes. Further, CNVs mapped to glutamate receptor genes (GRM1, GRM5, GRM7 and GRM8) have been implicated in the aetiology of the disorder and overlap with bioinformatic predictions based on ADHD GWAS SNP data regarding enriched pathways. Although increases in sample size across multi-center cohorts will likely yield important new results, we advocate that this must occur in parallel with a shift away from categorical case-control approaches that view ADHD as a unitary construct, towards dimensional approaches that incorporate endophenotypes and statistical classification methods.

Alpha-2A adrenergic receptor gene variants are associated with increased intra-individual variability in response time.

Intra-individual variability in response time has been proposed as an important endophenotype for attention deficit hyperactivity disorder (ADHD). Here we asked whether intra-individual variability is predicted by common variation in catecholamine genes and whether it mediates the relationship between these gene variants and self-reported ADHD symptoms. A total of 402 non-clinical Australian adults of European descent completed a battery of five cognitive tasks and the Conners' Adult ADHD Rating Scale. Exclusion criteria included the presence of major psychiatric or neurologic illnesses and substance dependency. A total of 21 subjects were excluded due to incomplete data or poor quality cognitive or genotyping data. The final sample comprised 381 subjects (201 males; mean age=21.2 years, s.d.=5.1 years). Principal components analysis on variability measures yielded two factors (response selection variability vs selective attention variability). Association of these factors with catecholamine gene variants was tested using single-step linear regressions, with multiple comparisons controlled using permutation analysis. The response selection variability factor was associated with two ADRA2A single-nucleotide polymorphisms (SNPs) (rs1800544, rs602618), p corrected=0.004, 0.012, respectively, whereas the selective attention variability factor was associated with a TH SNP (rs3842727), p corrected=0.024. A bootstrapping analysis indicated that the response selection variability factor mediated the relationship between the ADRA2A SNP rs1800544 and self-reported ADHD symptoms. Thus this study finds evidence that DNA variation in the ADRA2A gene may be causally related to ADHD-like behaviors, in part through its influence on intra-individual variability. Evidence was also found for a novel association between a TH gene variant and intra-individual variability.

Neurodevelopmental and neuropsychiatric disorders represent an interconnected molecular system.

Many putative genetic factors that confer risk to neurodevelopmental disorders such as autism spectrum disorders (ASDs) and X-linked intellectual disability (XLID), and to neuropsychiatric disorders including attention deficit hyperactivity disorder (ADHD) and schizophrenia (SZ) have been identified in individuals from diverse human populations. Although there is significant aetiological heterogeneity within and between these conditions, recent data show that genetic factors contribute to their comorbidity. Many studies have identified candidate gene associations for these mental health disorders, albeit this is often done in a piecemeal fashion with little regard to the inherent molecular complexity. Here, we sought to abstract relationships from our knowledge of systems level biology to help understand the unique and common genetic drivers of these conditions. We undertook a global and systematic approach to build and integrate available data in gene networks associated with ASDs, XLID, ADHD and SZ. Complex network concepts and computational methods were used to investigate whether candidate genes associated with these conditions were related through mechanisms of gene regulation, functional protein-protein interactions, transcription factor (TF) and microRNA (miRNA) binding sites. Although our analyses show that genetic variations associated with the four disorders can occur in the same molecular pathways and functional domains, including synaptic transmission, there are patterns of variation that define significant differences between disorders. Of particular interest is DNA variations located in intergenic regions that comprise regulatory sites for TFs or miRNA. Our approach provides a hypothetical framework, which will help discovery and analysis of candidate genes associated with neurodevelopmental and neuropsychiatric disorders.

Human amygdala volume is predicted by common DNA variation in the stathmin and serotonin transporter genes.

Despite the relevance of changes in amygdala volume to psychiatric illnesses and its heritability in both health and disease, the influence of common genetic variation on amygdala morphology remains largely unexplored. In the present study, we investigated the influence of a number of novel genetic variants on amygdala volume in 139 neurologically healthy individuals of European descent. Amygdala volume was significantly associated with allelic variation in the stathmin (STMN1) and serotonin transporter (SLC6A4) genes, which have been linked to healthy and disordered affective processing. These results were replicated across both manual and automated methods of amygdala parcellation, although manual tracing showed stronger effects, providing a cautionary note to studies relying on automated parcellation methods. Future studies will need to determine whether amygdala volume mediates the impact of stathmin and serotonin transporter gene variants on normal and dysfunctional emotion processing.

Dopamine transporter genotype predicts behavioural and neural measures of response inhibition.

The ability to inhibit unwanted actions is a heritable executive function that may confer risk to disorders such as attention deficit hyperactivity disorder (ADHD). Converging evidence from pharmacology and cognitive neuroscience suggests that response inhibition is instantiated within frontostriatal circuits of the brain with patterns of activity that are modulated by the catecholamines dopamine and noradrenaline. A total of 405 healthy adult participants performed the stop-signal task, a paradigmatic measure of response inhibition that yields an index of the latency of inhibition, termed the stop-signal reaction time (SSRT). Using this phenotype, we tested for genetic association, performing high-density single-nucleotide polymorphism mapping across the full range of autosomal catecholamine genes. Fifty participants also underwent functional magnetic resonance imaging to establish the impact of associated alleles on brain and behaviour. Allelic variation in polymorphisms of the dopamine transporter gene (SLC6A3: rs37020; rs460000) predicted individual differences in SSRT, after corrections for multiple comparisons. Furthermore, activity in frontal regions (anterior frontal, superior frontal and superior medial gyri) and caudate varied additively with the T-allele of rs37020. The influence of genetic variation in SLC6A3 on the development of frontostriatal inhibition networks may represent a key risk mechanism for disorders of behavioural inhibition.

Polymorphisms of the steroid sulfatase (STS) gene are associated with attention deficit hyperactivity disorder and influence brain tissue mRNA expression.

Previous studies in animals and humans have implicated the X-chromosome STS gene in the etiology of attentional difficulties and attention deficit hyperactivity disorder (ADHD). This family based association study has fine mapped a region of the STS gene across intron 1 and 2 previously associated with ADHD, in an extended sample of 450 ADHD probands and their parents. Significant association across this region is demonstrated individually with 7 of the 12 genotyped SNPs, as well as an allele specific haplotype of the 12 SNPs. The over transmitted risk allele of rs12861247 was also associated with reduced STS mRNA expression in normal human post-mortem frontal cortex brain tissue compared to the non-risk allele (P = 0.01). These results are consistent with the hypothesis arising from previous literature demonstrating that boys with deletions of the STS gene, and hence no STS protein are at a significantly increased risk of developing ADHD. Furthermore, this study has established the brain tissue transcript of STS, which except from adipose tissue, differs from that seen in all other tissues investigated. © 2010 Wiley-Liss, Inc.

Evidence that genetic variation in the oxytocin receptor (OXTR) gene influences social cognition in ADHD.

Some children with ADHD also have social and communication difficulties similar to those seen in children with autistic spectrum disorders and this may be due to shared genetic liability. As the oxytocin receptor (OXTR) gene has been implicated in social cognition and autistic spectrum disorders, this study investigated whether OXTR polymorphisms previously implicated in autism were associated with ADHD and whether they influenced OXTR mRNA expression in 27 normal human amygdala brain samples. The family-based association sample consisted of 450 DSM-IV diagnosed ADHD probands and their parents. Although there was no association with the ADHD phenotype, an association with social cognitive impairments in a subset of the ADHD probands (N=112) was found for SNP rs53576 (F=5.24, p=0.007) with post-hoc tests demonstrating that the AA genotype was associated with better social ability compared to the AG genotype. Additionally, significant association was also found for rs13316193 (F=3.09, p=0.05) with post-hoc tests demonstrating that the CC genotype was significantly associated with poorer social ability than the TT genotype. No significant association between genotype and OXTR mRNA expression was found. This study supports previous evidence that the OXTR gene is implicated in social cognition.

Haplotype association of calpain 10 gene variants with type 2 diabetes mellitus in an Irish sample.

BACKGROUND: Calpain 10 (CAPN10) gene may contribute to the pathogenesis of type 2 diabetes mellitus (T2DM). AIM: To examine the contribution of four CAPN10 gene variants to T2DM risk in an Irish sample. METHODS: Genotyping of marker 19 insertion-deletion (ins/del) and three CAPN10 variants, rs3792267, rs3749166 and rs5030952 at the CAPN10 gene was performed in 236 T2DM subjects and 120 controls. Allelic, genotypic and haplotype comparisons were conducted between the groups. RESULTS: In the examined markers, no significant differences were observed although the deletion/deletion allele tended to be more common in T2DM subjects (chi(2) = 3.2, P = 0.07). A significant overrepresentation of a haplotype comprising (rs3792267), (19) and rs3749166 (chi(2) = 5.3, P = 0.021) was seen in T2DM subjects. Two protective haplotypes were detected: (G-ins-G) of (rs3792267), (19) and rs3749166 (chi(2) = 6.7, P = 0.009) and (ins-G-C) of (19), (rs3749166) and rs5030952 (chi(2) = 8.5, P = 0.003). CONCLUSIONS: CAPN10 gene variants may affect T2DM susceptibility in the Irish population.

Functional analysis of intron 8 and 3' UTR variable number of tandem repeats of SLC6A3: differential activity of intron 8 variants.

Association studies have found that variation in the dopamine transporter gene (SLC6A3) is important in the susceptibility to attention-deficit hyperactivity disorder (ADHD) and response to methylphenidate treatment. An understanding of the biological mechanisms underlying these associations is still inconclusive. We assessed the relative activity of variable number tandem repeat (VNTR) alleles of SLC6A3 under basal and stimulated cellular conditions, as well as in the presence of pharmacological blockade of the dopamine transporter using gene-reporter constructs. The intron 8 VNTR 5-repeat allele is more active than the 6-repeat allele. In the presence of forskolin, both alleles were significantly induced. Blockade of the dopamine transporter did not influence activity of either allelic construct. No difference in activity between 9- and 10-repeat alleles of the 3'-untranslated region VNTR was observed under any experimental condition. These data suggest that the intron 8 VNTR is a functional variant with an ADHD susceptibility allele having reduced activity. The lack of enhanced allele-specific activity in response to treatment regimes suggests that differential activity under basal conditions is the primary mode of action.

ADHD and DAT1: further evidence of paternal over-transmission of risk alleles and haplotype.

We [Hawi et al. (2005); Am J Hum Genet 77:958-965] reported paternal over-transmission of risk alleles in some ADHD-associated genes. This was particularly clear in the case of the DAT1 3'-UTR VNTR. In the current investigation, we analyzed three new sample comprising of 1,248 ADHD nuclear families to examine the allelic over-transmission of DAT1 in ADHD. The IMAGE sample, the largest of the three-replication samples, provides strong support for a parent of origin effect for allele 6 and the 10 repeat allele (intron 8 and 3'-UTR VNTR, respectively) of DAT1. In addition, a similar pattern of over-transmission of paternal risk haplotypes (constructed from the above alleles) was also observed. Some support is also derived from the two smaller samples although neither is independently significant. Although the mechanism driving the paternal over-transmission of the DAT risk alleles is not known, these finding provide further support for this phenomenon.

Replication of a rare protective allele in the noradrenaline transporter gene and ADHD.

Replication is a key to resolving whether a reported genetic association represents a false positive finding or an actual genetic risk factor. In a previous study screening 51 candidate genes for association with ADHD in a multi-centre European sample (the IMAGE project), two single nucleotide polymorphisms (SNPs) within the norepinephrine transporter (SLC6A2) gene were found to be associated with attention deficit hyperactivity disorder (ADHD). The same SNP alleles were also reported to be associated with ADHD in a separate study from the Massachusetts General Hospital in the US. Using two independent samples of ADHD DSM-IV combined subtype trios we attempted to replicate the reported associations with SNPs rs11568324 and rs3785143 in SLC6A2. Significant association of the two markers was not observed in the two independent replication samples. However, across all four datasets the overall evidence of association with ADHD was significant (for SNP rs11568324 P = 0.0001; average odds ratio = 0.33; for SNP rs3785143 P = 0.008; average odds ratio = 1.3). The data were consistent for rs11568324, suggesting the existence of a rare allele conferring protection for ADHD within the SLC6A2 gene. Further investigations should focus on identifying the mechanisms underlying the protective effect.

Association of the steroid sulfatase (STS) gene with attention deficit hyperactivity disorder.

Attention deficit hyperactivity disorder (ADHD) is the most common behavioral disorder affecting children worldwide. The male bias in the prevalence of the disorder, suggests that some susceptibility genes may lie on the X chromosome. In this study we present evidence for a role of the X-linked steroid sulfatase (STS) gene and neurosteroids in the development of ADHD. Previously it has been observed that probands with ADHD have lower serum concentrations of the neurosteroids DHEA, which is synthesized from DHEA-S by STS. In further support, boys that suffer from XLI, a skin disorder caused by the deletion of the STS gene, have higher rates of ADHD, in particular the inattentive subtype. In a moderately sized sample of ADHD families (N = 384), we genotyped seven single nucleotide polymorphisms, tagging the entire gene. TDT analysis of the data yielded two polymorphisms that were significantly associated with ADHD (rs2770112-Transmitted: 71 Not Transmitted; 48; rs12861247-Transmitted: 43 Not Transmitted: 21), located towards the 5' end of the gene (P < 0.05). We conclude that the STS gene may play a role in susceptibility for ADHD, and that the neurosteroids pathways should be investigated further to access their potential contribution in susceptibility to the disorder.

Differential dopamine receptor D4 allele association with ADHD dependent of proband season of birth.

Season of birth (SOB) has been associated with attention deficit hyperactivity disorder (ADHD) in two existing studies. One further study reported an interaction between SOB and genotypes of the dopamine D4 receptor (DRD4) gene. It is important that these findings are further investigated to confirm or refute the findings. In this study, we investigated the SOB association with ADHD in four independent samples collected for molecular genetic studies of ADHD and found a small but significant increase in summer births compared to a large population control dataset. We also observed a significant association with the 7-repeat allele of the DRD4 gene variable number tandem repeat polymorphism in exon three with probands born in the winter season, with no significant differential transmission of this allele between summer and winter seasons. Preferential transmission of the 2-repeat allele to ADHD probands occurred in those who were born during the summer season, but did not surpass significance for association, even though the difference in transmission between the two seasons was nominally significant. However, following adjustment for multiple testing of alleles none of the SOB effects remained significant. We conclude that the DRD4 7-repeat allele is associated with ADHD but there is no association or interaction with SOB for increased risk for ADHD. Our findings suggest that we can refute a possible effect of SOB for ADHD.

Apolipoprotein E promoter polymorphisms (-491A/T and -427T/C) and Alzheimer's disease: no evidence of association in the Irish population.

BACKGROUND: Alzheimer's Disease (AD) is a progressive neurodegenerative disorder with as yet poorly understood aetiology. Both environmental and genetic factors have been implicated as predisposing factors. The APOE e4 allele is an established genetic susceptibility factor for AD for several populations including the Irish. Polymorphisms (-491A/T and -427T/C) at the promoter region of the APOE gene are postulated to affect the expression of the gene through differential binding of transcription factors. AIMS: Two APOE promoter polymorphisms (-491A/T and -427T/C) are examined for possible association with AD. METHODS: Using a case-control study design, a sample of 112 Irish late onset Alzheimer's (LOAD) patients and 107 ethnically matched controls were investigated for association with the above polymorphisms. CONCLUSIONS: No evidence of association between any of the examined markers and AD was observed. Haplotype analysis using markers -491A/T and -427T/C in conjunction with the APOE (Hha I) polymorphism revealed significant associations of three haplotypes with AD. However, this association was mainly due to the highly significant association of the APOE e4 allele with AD and not of the promoter variants.

Dopaminergic genotype biases spatial attention in healthy children.

In everyday life, our sensory system is bombarded with visual input and we rely upon attention to select only those inputs that are relevant to behavioural goals. Typically, humans can shift their attention from one visual field to the other with little cost to perception. In cases of 'unilateral neglect', however, there is a persistent bias of spatial attention towards the same side as the damaged cerebral hemisphere. We used a visual orienting task to examine the influence of functional polymorphisms of the dopamine transporter gene (DAT1) on individual differences in spatial attention in normally developing children. DAT1 genotype significantly influenced spatial bias. Healthy children who were homozygous for alleles that influence the expression of dopamine transporters in the brain displayed inattention for left-sided stimuli, whereas heterozygotes did not. Our data provide the first evidence in healthy individuals of a genetically mediated bias in spatial attention that is related to dopamine signalling.

Tryptophan hydroxylase 2 (TPH2) gene variants associated with ADHD.

Genetic and pharmacological studies have emphasised the role of serotonin 5-hydroxytryptamine (5-HT) as a possible etiologic factor in the development of attention-deficit hyperactivity disorder (ADHD). Tryptophan hydroxylase (TPH) is a rate-limiting enzyme in the biosynthesis of serotonin from tryptophan. Originally, the TPH gene was thought to be widely expressed, but a second form of TPH, TPH2, was recently identified and the TPH2 gene was found to be solely expressed in the brain. We examined eight single nucleotide polymorphisms (SNP) in the TPH2 gene for association with ADHD in 179 Irish nuclear families. Transmission disequilibrium test analysis revealed significant association between the T allele of marker rs1843809 with the disorder (chi2=12.2, P=0.0006, OR=2.36). Stratifying data by the sex of the transmitting parent showed that this association was enhanced when paternal transmission was considered (OR=3.7). In addition, several haplotypes (all including the associated marker) were associated with ADHD. These preliminary findings suggest that TPH2 is a susceptibility locus for ADHD. Further confirmation, preferably from different ethnic groups, is required to firmly implicate TPH2 in the pathophysiology of ADHD.

Association of the paternally transmitted copy of common Valine allele of the Val66Met polymorphism of the brain-derived neurotrophic factor (BDNF) gene with susceptibility to ADHD.

Attention deficit hyperactivity disorder (ADHD) is a common, highly heritable, neurodevelopmental disorder with onset in early childhood. Genes involved in neuronal development and growth are, thus, important etiological candidates and brain-derived neurotrophic factor (BDNF), has been hypothesized to play a role in the pathogenesis of ADHD. BDNF is a member of the neurotrophin family and is involved in the survival and differentiation of dopaminergic neurons in the developing brain (of relevance because drugs that block the dopamine transporter can be effective therapeutically). The common Val66Met functional polymorphism in the human BDNF gene (rs 6265) was genotyped in a collaborative family-based sample of 341 white UK or Irish ADHD probands and their parents. We found evidence for preferential transmission of the valine (G) allele of BDNF (odds ratio, OR=1.6, P=0.02) with a strong paternal effect (paternal transmissions: OR=3.2, P=0.0005; maternal transmissions: OR=1.00; P=1.00). Our findings support the hypothesis that BDNF is involved in the pathogenesis of ADHD. The transmission difference between parents raises the possibility that an epigenetic process may be involved.