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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Identification of the products of the haptoglobin-related gene.

A novel haptoglobin, which is undetectable using conventional peroxidase staining techniques, has been detected using immunoblotting. The behaviour of this novel haptoglobin after electrophoresis under native, denaturing and reducing conditions indicates that it is the product of the haptoglobin-related gene. This protein is detectable in normal human plasma.

A protective effect of apolipoprotein E e2 allele on dementia in Down's syndrome.

BACKGROUND: The apolipoprotein E (ApoE) e4 allele has been associated with an increased risk for Alzheimer's disease, whereas the e2 allele has been shown to be protective. Similar effects in Down's syndrome (DS) have been postulated but not yet demonstrated. METHODS: We obtained DNA from 221 DS individuals and from 162 population controls, and 77 DS children. Older DS subjects were evaluated for dementia and compared to age-matched DS controls. RESULTS: The DS sample with dementia (n = 31) had a significantly lower frequency of the ApoE e2 allele compared to age-matched nondemented DS controls (0% vs. 8.3%, p = .0136). The older DS population had a significantly lower frequency of ApoE e4 compared to population controls (11.7% vs. 20.6%, chi-square 8.9, p = .0028). CONCLUSIONS: The lower frequency of the e2 allele in demented DS subjects compared to age-matched nondemented DS controls suggests a protective effect for ApoE e2 in the development of dementia in DS. The lower frequency of ApoE e4 in our older DS sample compared to population controls points to a detrimental effect of the e4 allele on longevity.

No association between catechol-O-methyltransferase (COMT) gene polymorphism and attention deficit hyperactivity disorder (ADHD) in an Irish sample.

Pharmacological and biochemical studies have indicated that imbalances in dopaminergic transmission may contribute to the aetiology of attention deficit hyperactivity disorder (ADHD). The enzyme catechol-O-methyltransferase (COMT) plays a key role in the degradation of catecholamines such as dopamine, L-DOPA, adrenaline, and noradrenaline and therefore could be considered as a candidate locus for ADHD susceptibility. We hypothesised that a proportion of the genetic susceptibility to ADHD may be a consequence of dopamine depletion in the synapses due to high-level activity of the COMT gene (allele 1). Using the haplotype-based haplotype relative risk method and 94 affected children and their parents genotyped for COMT alleles, we found no significant differences in the frequency of the transmitted and nontransmitted alleles to ADHD cases from their parents. The absence of association between COMT alleles and ADHD indicated that this locus does not play a significant role or at least a role independent of other genes, in predisposing to ADHD in the Irish population.

HLA and schizophrenia: refutation of reported associations with A9 (A23/A24), DR4, and DQbeta1*0602.

The aim of this study was to test previous hypotheses of association between schizophrenia and human leukocyte antigen (HLA) specificities A9 (A23/24), DR4, and allele DQbeta1*0602. Using sequence-specific oligonucleotide probes, 256 familial schizophrenic patients and 261 unrelated controls were genotyped at polymorphisms for HLA-A, DRbeta1, and DQbeta1 loci. No significant (p < 0.05) differences in the allele frequencies between schizophrenics and controls were found, either when examining the sample as a whole or when subdivided by clinical subtype or gender. The present data do not support association between these HLA specificities and schizophrenia, and our review of previous studies suggests that reported associations may well be false positive results.

Schizophrenia and HLA: No association with PCR-SSOP typed classical loci in a large Irish familial sample.

Susceptibility to autoimmunity is strongly influence by genes clustered in the MHC region, particularly class I and class II antigens. It has been proposed that there is an immune component in the aetiology of schizophrenia, and the distribution of human leukocyte antigens (HLA) in schizophrenic patients and controls has been investigated in numerous studies. Positive associations have been reported between schizophrenia and the HLA-A1, A2, A9, B5, Cw4, and DR8 and negative associations with HLA-DR4 and HLA-DQbeta*0602. Small sample size, variable diagnostic methodology, unreliable laboratory and statistical procedures, and possible mismatching of cases and controls may have contributed to a lack of consistency of results to date. Therefore, in this investigation we used a large and carefully diagnosed homogeneous Irish familial schizophrenic sample compared with ethnically matched controls. All alleles were determined using polymerase chain reaction amplification followed by short specific oligoprobes. We found no evidence of association with 80 HLA alleles (some previously not examined) from 4 genes. Our data therefore do not support the involvement of these classical HLA loci in the aetiology of schizophrenia at least in these Irish families. The remaining classical HLA loci (HLA-B and HLA-C) should be typed when reliable DNA-based methods become available.

No association of the dopamine DRD4 receptor (DRD4) gene polymorphism with attention deficit hyperactivity disorder (ADHD) in the Irish population.

Attention deficit hyperactivity disorder (ADHD) is one of the most prevalent childhood-onset syndromes affecting 3%-6% of school-age children worldwide. Although the biological basis of ADHD is unknown, a dopaminergic abnormality has long been suggested. The dopamine D4 receptor gene (DRD4) has been mapped to chromosome 11p15.5 and has been implicated in predisposition to ADHD. Several independent genetic association studies have demonstrated increased frequency of the DRD4 7-repeat allele in ADHD cases compared with controls or excess transmission of the 7-repeat allele from parents to affected offspring. However, there have also been few negative studies. In this study we investigated 78 ADHD parent proband trios and 21 parent proband pairs for the transmission of the DRD4 alleles in HHRR and case control design. We found no significant differences in the frequency of the DRD4 alleles transmitted or not transmitted to ADHD cases from their parents nor when comparing case allele frequencies to ethnically matched controls. Therefore, it is unlikely that the DRD4 7-repeat allele is associated with ADHD in the Irish population.

Presenilin 1 and alpha-1-antichymotrypsin polymorphisms in Down syndrome: no effect on the presence of dementia.

As people with Down syndrome (DS) age, they are at greater risk for Alzheimer disease (AD) than the general population. It has been suggested that polymorphisms at the genes for presenilin-1 (PS-1) and alpha-1-antichymotrypsin (ACT) confer an increased risk for AD in the general population, and therefore potentially to AD in people with DS. We obtained DNA from 231 individuals with DS and 233 population controls. People with DS were evaluated for dementia. Allele frequencies at PS-1 and ACT polymorphisms in people with DS were compared to those in age-matched controls. There were no frequency differences between the control sample and DS sample for PS-1 or ACT alleles or genotypes. Similarly, there were no differences in allele frequencies between the demented and age-matched non-demented DS samples. However a higher frequency of PS-1 heterozygotes in the demented DS group was noted. We conclude that unlike the general population, neither PS-1 nor ACT polymorphisms appear to have a similar detrimental effect on dementia in DS. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 88:616-620, 1999.