Impact of autism-associated genetic variants in interaction with environmental factors on ADHD comorbidities: an exploratory pilot study.

Attention-deficit/hyperactivity disorder (ADHD) is determined by genetic and environmental factors, and shares genetic risk with ASD. Functional single-nucleotide polymorphisms of the metabotropic glutamatergic signaling pathway are reported to increase the risk for ASD. The aim of this pilot study was to explore the main effects of respective ASD variants as well as their interaction effects with well-replicated ADHD environmental risk factors on the risk for ADHD, ADHD symptom severities, and comorbidities. We included 318 children with ADHD, aged 5-13 years, and their parents (N = 164 trios, N = 113 duos, N = 41 singletons). Interaction of ASD risk variants CYFIP1-rs7170637, CYFIP1-rs3693, CAMK4-rs25925, and GRM1-rs6923492 with prenatal biological and lifetime psychosocial risk factors was explored in a subsample with complete environmental risk factors (N = 139 trios, N = 83 duos, two singletons) by transmission disequilibrium test and stepwise regression analyses. We identified nominally significant (alpha < 0.05) GxE interactions of acute life events with CYFIP1-rs3693 on ADHD diagnosis (p = 0.004; fdr = 0.096) but no significant association of any single marker. Further results suggest that the risk for comorbid disruptive disorders was significantly modulated by GxE interactions between familial risk factors and CAMK4-rs25925 (p = 0.001; fdr = 0.018) and prenatal alcohol exposure with CYFIP1-rs3693 (p = 0.003; fdr = 0.027); both findings survived correction for multiple testing (fdr value < 0.05). Nominal significant GxE interactions moderating the risk for anxiety disorders have also been identified, but did not pass multiple testing corrections. This pilot study suggests that common ASD variants of the glutamatergic system interact with prenatal and lifetime psychosocial risk factors influencing the risk for ADHD common comorbidities and thus warrants replication in larger samples.

Haplotype co-segregation with attention deficit-hyperactivity disorder in unrelated German multi-generation families.

Complex disorders have proved to be elusive in the search for underlying genetic causes. In the presence of large multi-generation pedigrees with multiple affected individuals, heritable familial forms of the disorders can be postulated. Observations of particular chromosomal haplotypes shared among all affected individuals within pedigrees may reveal chromosomal regions, in which the disease-related genes may be located. Hence, the biochemical pathways involved in pathogenesis can be exposed. We have recruited eight large Attention Deficit-Hyperactivity Disorder (ADHD, OMIM: #143465) families of German descent. Densely spaced informative microsatellite markers with high heterozygosity rates were used to fine-map and haplotype chromosomal regions of interest in these families. In three subsets and one full family of the eight ADHD families, haplotypes co-segregating with ADHD-affected individuals were identified at chromosomes 1q25, 5q11-5q13, 9q31-9q32, and 18q11-18q21. Positive LOD scores supported these co-segregations. The existence of haplotypes co-segregating among affected individuals in large ADHD pedigrees suggests the existence of Mendelian forms of the disorder and that ADHD-related genes are located within these haplotypes. In depth sequencing of these haplotype regions can identify causative genetic mechanisms and will allow further insights into the clinico-genetics of this complex disorder.

Biological and psychosocial environmental risk factors influence symptom severity and psychiatric comorbidity in children with ADHD.

Attention-deficit/hyperactivity disorder (ADHD) is a genetically as well as environmentally determined disorder with a high rate of psychiatric comorbidity. In this study, non-genetic biological and psychosocial risk factors for ADHD symptom severity and comorbid disorders were assessed in 275 children with ADHD, aged 5-13 years, mean age 9.7 (SD 1.9). Pre-/perinatal biological and lifetime psychosocial risk factors as well as data on parental ADHD were obtained. A different pattern of risk factors emerged for inattentive and hyperactive-impulsive ADHD symptoms. Inattentive symptoms were strongly influenced by psychosocial risk factors, whereas for hyperactive-impulsive symptoms, predominantly biological risk factors emerged. Hyperactive-impulsive symptoms also were a strong risk factor for comorbid oppositional defiant (ODD) and conduct disorder (CD). Smoking during pregnancy was a risk factor for comorbid CD but not ODD and further differential risk factors were observed for ODD and CD. Comorbid anxiety disorder (AnxD) was not related to ADHD symptoms and additional biological and psychosocial risk factors were observed. This study adds to the body of evidence that non-genetic biological and psychosocial risk factors have an impact on ADHD symptom severity and differentially influence comorbid disorders in ADHD. The findings are relevant to the prevention and treatment of ADHD with or without comorbid disorders.

Risk factors of autistic symptoms in children with ADHD.

Autistic symptoms are frequently observed in children with attention-deficit/hyperactivity disorder (ADHD), but their etiology remains unclear. The main aim of this study was to describe risk factors for increased autistic symptoms in children with ADHD without an autism or autism-spectrum diagnosis. Comorbid psychiatric disorders, developmental delay, current medication, prenatal biological and postnatal psychosocial risk factors as well as parental autistic traits were assessed in 205 children with ADHD. Linear regression models identified maternal autistic traits, current familial risk factors and hyperactive symptoms as predictors of autistic symptoms in children with ADHD. Findings are indicative of possible genetic as well as environmental risk factors mediating autistic symptoms in children with ADHD. An additional validity analysis by ROC, area under the curve (AUC), suggested a cut-off of 11 to differentiate between ADHD and high-functioning ASD by the Social Communication Questionnaire (SCQ).

Attention-deficit/hyperactivity disorder phenotype is influenced by a functional catechol-O-methyltransferase variant.

The catechol-O-methyltransferase gene (COMT) plays a crucial role in the metabolism of catecholamines in the frontal cortex. A single nucleotide polymorphism (Val(158)Met SNP, rs4680) leads to either methionine (Met) or valine (Val) at codon 158, resulting in a three- to fourfold reduction in COMT activity. The aim of the present study was to assess the COMT Val(158)Met SNP as a risk factor for attention-deficit/hyperactivity disorder (ADHD), ADHD symptom severity and co-morbid conduct disorder (CD) in 166 children with ADHD. The main finding of the present study is that the Met allele of the COMT Val(158)Met SNP was associated with ADHD and increased ADHD symptom severity. No association with co-morbid CD was observed. In addition, ADHD symptom severity and early adverse familial environment were positive predictors of lifetime CD. These findings support previous results implicating COMT in ADHD symptom severity and early adverse familial environment as risk factors for co-morbid CD, emphasizing the need for early intervention to prevent aggressive and maladaptive behavior progressing into CD, reducing the overall severity of the disease burden in children with ADHD.

Glucocorticoid receptor variants in childhood attention-deficit/hyperactivity disorder and comorbid psychiatric disorders.

Stress results in a variety of neuroendocrine, immune and behavioral responses and represents a risk factor for many disorders. Following exposure to stress, glucocorticoids are secreted from the adrenal cortex and act via the ligand-activated glucocorticoid receptor (GR). Several polymorphisms of the GR-encoding gene NR3C1 have been described and functionally investigated. However, the impact of these variants on complex diseases such as Attention-Deficit/Hyperactivity Disorder (ADHD) is still unclear. In this study, 251 children with ADHD, 19 affected and 35 unaffected siblings, and their parents were included in a family-based association study assessing seven common variants of NR3C1 (TthIIII_rs10052957; NR3C1-I_rs10482605; ER22/23EK_rs6189/rs6190; N363S_rs56149945; BclI_rs41423247; GR-9beta_rs6198). A four-marker haplotype (TthIIII-NR3C1-I-ER22/23EK) was nominally associated with ADHD. In addition, in index children with ADHD, associations with comorbid disorders, inattentive and hyperactive-impulsive symptoms were explored. N363S minor allele carriers were more likely to show comorbid conduct disorder (CD). In our study, NR3C1 variants moderately affected ADHD and had a significant effect on comorbid CD. Therefore, NR3C1 as an important gene of the hypothalamic-pituitary-adrenal axis seems to be particularly relevant for the pathophysiology of ADHD combined with comorbid CD. For a deeper understanding, investigations in larger samples of healthy, ADHD and CD individuals are warranted.

Empirically Determined, Psychopathological Subtypes in Children With ADHD.

OBJECTIVE: The aim of this study was to empirically determine subgroups of ADHD defined by specific patterns of psychopathology. METHOD: A clinical sample of 223 children with ADHD, aged 5 to 14 years, was examined with the Child Behavior Checklist (CBCL). In addition, comorbid psychiatric disorders, psychosocial risk factors, and socioeconomic status were assessed. RESULTS: Cluster analysis of CBCL subscales yielded a solution with four distinct subgroups. While "externalizers" showed a high rate of comorbid oppositional defiant disorder (ODD) and conduct disorder (CD), "obsessive-compulsives" exhibited thought problems, low rates of comorbid CD, and high symptoms of inattention. "High psychiatric symptom carriers" had high rates of familial risk factors, acute life events, comorbid ODD, and CD. "Low psychiatric symptom carriers" also scored low in all other variables studied. CONCLUSION: Children with ADHD can be divided into four subgroups according to their CBCL-based psychopathology, and these subgroups differ in their risk factor profiles.

High loading of polygenic risk for ADHD in children with comorbid aggression.

OBJECTIVE Although attention deficit hyperactivity disorder (ADHD) is highly heritable, genome-wide association studies (GWAS) have not yet identified any common genetic variants that contribute to risk. There is evidence that aggression or conduct disorder in children with ADHD indexes higher genetic loading and clinical severity. The authors examine whether common genetic variants considered en masse as polygenic scores for ADHD are especially enriched in children with comorbid conduct disorder. METHOD Polygenic scores derived from an ADHD GWAS meta-analysis were calculated in an independent ADHD sample (452 case subjects, 5,081 comparison subjects). Multivariate logistic regression analyses were employed to compare polygenic scores in the ADHD and comparison groups and test for higher scores in ADHD case subjects with comorbid conduct disorder relative to comparison subjects and relative to those without comorbid conduct disorder. Association with symptom scores was tested using linear regression. RESULTS Polygenic risk for ADHD, derived from the meta-analysis, was higher in the independent ADHD group than in the comparison group. Polygenic score was significantly higher in ADHD case subjects with conduct disorder relative to ADHD case subjects without conduct disorder. ADHD polygenic score showed significant association with comorbid conduct disorder symptoms. This relationship was explained by the aggression items. CONCLUSIONS Common genetic variation is relevant to ADHD, especially in individuals with comorbid aggression. The findings suggest that the previously published ADHD GWAS meta-analysis contains weak but true associations with common variants, support for which falls below genome-wide significance levels. The findings also highlight the fact that aggression in ADHD indexes genetic as well as clinical severity.

Candidate system analysis in ADHD: evaluation of nine genes involved in dopaminergic neurotransmission identifies association with DRD1.

OBJECTIVES: Several pharmacological and genetic studies support the involvement of the dopamine neurotransmitter system in the aetiology of attention-deficit hyperactivity disorder (ADHD). Based on this information we evaluated the contribution to ADHD of nine genes involved in dopaminergic neurotransmission (DRD1, DRD2, DRD3, DRD4, DRD5, DAT1, TH, DBH and COMT). METHODS: We genotyped a total of 61 tagging single nucleotide polymorphisms (SNPs) in a sample of 533 ADHD patients (322 children and 211 adults), 533 sex-matched unrelated controls and additional 196 nuclear ADHD families from Spain. RESULTS: The single- and multiple-marker analysis in both population and family-based approaches provided preliminary evidence for the contribution of DRD1 to combined-type ADHD in children (P=8.8e-04; OR=1.50 (1.18-1.90) and P=0.0061; OR=1.73 (1.23-2.45)) but not in adults. Subsequently, we tested positive results for replication in an independent sample of 353 German families with combined-type ADHD children and replicated the initial association between DRD1 and childhood ADHD (P=8.4e-05; OR=3.67 (2.04-6.63)). CONCLUSIONS: The replication of the association between DRD1 and ADHD in two European cohorts highlights the validity of our finding and supports the involvement of DRD1 in childhood ADHD.

Genome-wide analysis of copy number variants in attention deficit hyperactivity disorder: the role of rare variants and duplications at 15q13.3.

OBJECTIVE: Attention deficit hyperactivity disorder (ADHD) is a common, highly heritable psychiatric disorder. Because of its multifactorial etiology, however, identifying the genes involved has been difficult. The authors followed up on recent findings suggesting that rare copy number variants (CNVs) may be important for ADHD etiology. METHOD: The authors performed a genome-wide analysis of large, rare CNVs (<1% population frequency) in children with ADHD (N=896) and comparison subjects (N=2,455) from the IMAGE II Consortium. RESULTS: The authors observed 1,562 individually rare CNVs >100 kb in size, which segregated into 912 independent loci. Overall, the rate of rare CNVs >100 kb was 1.15 times higher in ADHD case subjects relative to comparison subjects, with duplications spanning known genes showing a 1.2-fold enrichment. In accordance with a previous study, rare CNVs >500 kb showed the greatest enrichment (1.28-fold). CNVs identified in ADHD case subjects were significantly enriched for loci implicated in autism and in schizophrenia. Duplications spanning the CHRNA7 gene at chromosome 15q13.3 were associated with ADHD in single-locus analysis. This finding was consistently replicated in an additional 2,242 ADHD case subjects and 8,552 comparison subjects from four independent cohorts from the United Kingdom, the United States, and Canada. Presence of the duplication at 15q13.3 appeared to be associated with comorbid conduct disorder. CONCLUSIONS: These findings support the enrichment of large, rare CNVs in ADHD and implicate duplications at 15q13.3 as a novel risk factor for ADHD. With a frequency of 0.6% in the populations investigated and a relatively large effect size (odds ratio=2.22, 95% confidence interval=1.5­3.6), this locus could be an important contributor to ADHD etiology.

Genome-wide copy number variation study associates metabotropic glutamate receptor gene networks with attention deficit hyperactivity disorder.

Attention deficit hyperactivity disorder (ADHD) is a common, heritable neuropsychiatric disorder of unknown etiology. We performed a whole-genome copy number variation (CNV) study on 1,013 cases with ADHD and 4,105 healthy children of European ancestry using 550,000 SNPs. We evaluated statistically significant findings in multiple independent cohorts, with a total of 2,493 cases with ADHD and 9,222 controls of European ancestry, using matched platforms. CNVs affecting metabotropic glutamate receptor genes were enriched across all cohorts (P = 2.1 × 10(-9)). We saw GRM5 (encoding glutamate receptor, metabotropic 5) deletions in ten cases and one control (P = 1.36 × 10(-6)). We saw GRM7 deletions in six cases, and we saw GRM8 deletions in eight cases and no controls. GRM1 was duplicated in eight cases. We experimentally validated the observed variants using quantitative RT-PCR. A gene network analysis showed that genes interacting with the genes in the GRM family are enriched for CNVs in ∼10% of the cases (P = 4.38 × 10(-10)) after correction for occurrence in the controls. We identified rare recurrent CNVs affecting glutamatergic neurotransmission genes that were overrepresented in multiple ADHD cohorts.

Case-control genome-wide association study of attention-deficit/hyperactivity disorder.

OBJECTIVE: Although twin and family studies have shown attention-deficit/hyperactivity disorder (ADHD) to be highly heritable, genetic variants influencing the trait at a genome-wide significant level have yet to be identified. Thus additional genomewide association studies (GWAS) are needed. METHOD: We used case-control analyses of 896 cases with DSM-IV ADHD genotyped using the Affymetrix 5.0 array and 2,455 repository controls screened for psychotic and bipolar symptoms genotyped using Affymetrix 6.0 arrays. A consensus SNP set was imputed using BEAGLE 3.0, resulting in an analysis dataset of 1,033,244 SNPs. Data were analyzed using a generalized linear model. RESULTS: No genome-wide significant associations were found. The most significant results implicated the following genes: PRKG1, FLNC, TCERG1L, PPM1H, NXPH1, PPM1H, CDH13, HK1, and HKDC1. CONCLUSIONS: The current analyses are a useful addition to the present literature and will make a valuable contribution to future meta-analyses. The candidate gene findings are consistent with a prior meta-analysis in suggesting that the effects of ADHD risk variants must, individually, be very small and/or include multiple rare alleles.

Meta-analysis of genome-wide association studies of attention-deficit/hyperactivity disorder.

OBJECTIVE: Although twin and family studies have shown attention-deficit/hyperactivity disorder (ADHD) to be highly heritable, genetic variants influencing the trait at a genome-wide significant level have yet to be identified. As prior genome-wide association studies (GWAS) have not yielded significant results, we conducted a meta-analysis of existing studies to boost statistical power. METHOD: We used data from four projects: a) the Children's Hospital of Philadelphia (CHOP); b) phase I of the International Multicenter ADHD Genetics project (IMAGE); c) phase II of IMAGE (IMAGE II); and d) the Pfizer-funded study from the University of California, Los Angeles, Washington University, and Massachusetts General Hospital (PUWMa). The final sample size consisted of 2,064 trios, 896 cases, and 2,455 controls. For each study, we imputed HapMap single nucleotide polymorphisms, computed association test statistics and transformed them to z-scores, and then combined weighted z-scores in a meta-analysis. RESULTS: No genome-wide significant associations were found, although an analysis of candidate genes suggests that they may be involved in the disorder. CONCLUSIONS: Given that ADHD is a highly heritable disorder, our negative results suggest that the effects of common ADHD risk variants must, individually, be very small or that other types of variants, e.g., rare ones, account for much of the disorder's heritability.

Cortisol awakening response in healthy children and children with ADHD: impact of comorbid disorders and psychosocial risk factors.

Attention-deficit/hyperactivity disorder (ADHD) is one of the most common child psychiatric disorders. Previous studies have reported a blunted cortisol response to challenging situations and a decreased cortisol awakening response (CAR) in children with ADHD. As ADHD often is comorbid with oppositional defiant disorder (ODD), conduct disorder (CD), or anxiety disorder (AnxD), and changes in hypothalamic-pituitary-adrenal (HPA) axis activity have also been reported for these disorders, the present study aimed to compare the CAR in children with ADHD with and without comorbid disorders. Data on the CAR were obtained in 128 children with ADHD (aged 6-13 years) and in 96 control children (aged 6-12 years). Children with ADHD+ODD showed an attenuated CAR (area under the curve, AUC) compared to children with ADHD without ODD/CD and control children. Findings point towards either disinhibition or pervasive underarousal in children with ADHD+ODD, and seem to be specific for children with ADHD+ODD, as the attenuated CAR-AUC was not observed in children with ADHD without comorbid disorders or children with ADHD+CD or ADHD+AnxD. In addition, current adverse parenting conditions, family conflicts, and acute life events were associated with mean increase in CAR, emphasizing the role of psychosocial risk factors in mediating HPA axis activity in children with ADHD.

Functional analysis of a potassium-chloride co-transporter 3 (SLC12A6) promoter polymorphism leading to an additional DNA methylation site.

The human potassium-chloride co-transporter 3 (KCC3, SLC12A6) is involved in cell proliferation and in electro-neutral movement of ions across the cell membrane. The gene (SLC12A6) is located on chromosome 15q14, a region that has previously shown linkage with bipolar disorder, schizophrenia, rolandic epilepsy, idiopathic generalized epilepsy, autism and attention deficit/hyperactivity disorder. Furthermore, recessively inherited mutations of SLC12A6 cause Andermann syndrome, characterized by agenesis of the corpus callosum, which is associated with peripheral neuropathy and psychoses. Recently, we have demonstrated the association of two G/A promoter polymorphisms of SLC12A6 with bipolar disorder in a case-control study, and familial segregation of the rare variants as well as a trend toward association with schizophrenia. To investigate functional consequences of these polymorphisms, lymphocyte DNA was extracted, bisulfite modified, and subsequently sequenced. To investigate SLC12A6 promoter activity, various promoter constructs were generated and analyzed by luciferase reporter gene assays. We provide evidence that the G- allele showed a significant reduction of reporter gene expression. In human lymphocytes, the allele harboring the rare upstream G nucleotide was found to be methylated at the adjacent C position, possibly accountable for tissue-specific reduction in gene expression in vivo. Here we demonstrate functionality of an SNP associated with psychiatric disease and our results may represent a functional link between genetic variation and an epigenetic modification.