New Insights into the Pathogenesis and Pharmacogenomics of Attention Deficit Hyperactivity Disorder

performance of cognitive tasks. Reductions in volume have been observed in the total cerebral volume, the prefrontal cortex, the basal ganglia (striatum), the dorsal anterior cingulate cortex, the corpus callosum and the cerebellum. Hypoactivation of the dorsal anterior cingulate cortex, the frontal cortex and the basal ganglia have also been reported. Caudate volume is reduced in association with externalizing disorders of childhood/adolescence. Working memory deficits appear in familial high-risk offspring and those with externalizing disorders of childhood. There are specific white matter abnormalities in patients with ADHD. Different ADHD subtypes may have some overlapping microstructural damage, but they may also have unique microstructural abnormalities. ADHD-I is related to abnormalities in the temporo-occipital areas, while the combined subtype (ADHD-C) is related to abnormalities in the frontal-subcortical circuit, the frontolimbic pathway, and the temporo-occipital areas. An abnormality in the motor circuit may represent the main difference between the ADHD-I and ADHD-C subtypes [7].


Introduction
Attention deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder in which genomic, epigenomic and environmental factors are involved. ADHD is one of the most prevalent psychiatric disorders in children, affecting 8-12% of schoolage children. The worldwide-pooled prevalence of mental disorders is 13.4% (anxiety disorder, 6.5%; depressive disorder, 2.6%; ADHD, 3.4%; any disruptive disorder, 5.7%) [1]. ADHD is the most frequently diagnosed neurodevelopmental disorder, with 6.4 million children and adolescents diagnosed with ADHD as of 2011 in the USA, and a current economic burden estimated in the region of $77 billion in the USA alone. 3.5 million children and adolescents are taking medication for ADHD [2]. Increasing numbers of adult ADHD patients are reported. Incidence increases exponentially; 40.4% of all patients have another psychiatric diagnosis before being diagnosed with ADHD; afterwards, 17.4% receive other diagnoses. Diagnoses contraindicating stimulants were found in 25.8% of the patients with other diagnoses before (10.5% of total) and in 40.0% (6.9% of total) after a diagnosis of ADHD. There is an increasing incidence and instability in the diagnosis of ADHD [3]. The prevalence of adult ADHD is estimated to be 3.8% in some regions. Men, when compared with women, are more likely to have ADHD (5.5% men vs. 2% women).
Biomarkers to characterize the ADHD phenotype include clinical data, psychometric assessment, laboratory analysis, brain neuroimaging, brain electrophysiology, and genomic, proteomic and metabolomic profiles [4]. These biomarkers are essential for defining the phenotypic features of the disease and for monitoring therapeutics (efficacy and safety issues) [5,6].
ADHD is associated with hypofunctional medial prefrontal cortex and orbitofrontal cortex. This network involves the lateral prefrontal cortex, the dorsal anterior cingulate cortex, the caudate nucleus and putamen. Abnormalities affecting other cortical regions and the cerebellum are also currently seen. Anatomical studies suggest widespread reductions in volume throughout the cerebrum and cerebellum, while functional imaging studies suggest that affected individuals activate more diffuse areas than controls during the Candidate genes for ADHD focused on genes involved in the dopaminergic neurotransmission system, such as DRD4, DRD5, DAT1/SLC6A3, DBH, and DDC. Genes associated with the noradrenergic (NET1/SLC6A2, ADRA2A, and ADRA2C) and serotonergic systems (5-HTT/SLC6A4, HTR1B, HTR2A, TPH2) have also received considerable interest. Additional candidate genes related to neurotransmission and neuronal plasticity that have been studied less intensively include SNAP25, CHRNA4, NMDA, BDNF, NGF, NTF3, NTF4/5, GDNF [13][14][15] (Table 1).
A meta-analysis for 8 common variants located in 5 top candidate genes for ADHD (BDNF, HTR1B, SLC6A2, SLC6A4 and SNAP25) revealed that a major part of the previously postulated associations were inconsistent in the pooled odds ratios. There is a weak significant association with a SNP located in the 3' UTR region of the SNAP25 gene (rs3746544, T allele). In addition to the low coverage of genetic variability given by these variants, phenotypic heterogeneity between samples (ADHD subtypes, comorbidities) and genetic background may explain these differences. Previously proposed cumulative associations with common polymorphisms in SLC6A4 and HTR1B genes were not supported [16]. However, the contribution of several candidate genes has been supported by other meta-analyses (DRD4, DRD5, DAT1, HTR1B and SNAP25), whereas others indicate that little evidence supports an important role for the 'classic' ADHD genes, with possible exceptions for SLC9A9, NOS1 and CNR1 [9]. Several genomewide linkage studies have been conducted and, although there are considerable differences in findings between studies, several regions have been supported across several studies (bin 16.4, 5p13, 11q22-25, 17p11) [14]. Linkage studies have been successful in identifying loci for adult ADHD and led to the identification of LPHN3 and CDH13 as novel genes associated with ADHD across the lifespan [15].
Major neuropsychiatric disorders are highly heritable, with mounting evidence suggesting that these disorders share overlapping sets of molecular and cellular underpinnings. A study screening the degree of genetic commonality across six major neuropsychiatric disorders, including ADHD, anxiety disorders, autistic spectrum disorders, bipolar disorder, major depressive disorder, and schizophrenia, identified a total of 180 genes on the basis of low but liberal GWAS p-values. 22% of genes overlapped two or more disorders. The most widely shared subset of genes -common to five of six disorders -included ANK3, AS3MT, CACNA1C, CACNB2, CNNM2, CSMD1, DPCR1, ITIH3, NT5C2, PPP1R11, SYNE1, TCF4, TENM4, TRIM26, and ZNRD1. Many of the shared genes are implicated in the postsynaptic density, expressed in immune tissues and co-expressed in developing human brain. Two distinct genetic components were both shared by each of the six disorders; the 1st component is involved in CNS development, neural projections and synaptic transmission, while the 2nd is implicated in various cytoplasmic organelles and cellular processes. Combined, these genetic components account for 20-30% of the genetic load. The remaining risk is conferred by distinct, disorder-specific variants [17]. About 45 of the 85 top-ranked ADHD candidate genes encode proteins that fit into a neurodevelopmental network involved in directed neurite outgrowth. Data on copy number variations in patients with ADHD and data from animal studies provide further support for the involvement of this network in ADHD etiology [18]. What remains unknown is whether candidate genes are associated with multiple disorders via pleiotropic mechanisms, and/ or if other genes are specific to susceptibility for individual disorders. Meta-analyses (1,519 meta-analyses across 157 studies) examining specific genes and specific mental disorders that have core disruptions to emotional and cognitive function and contribute most to burden of illness such as major depressive disorder, anxiety disorders (including panic disorder and obsessive compulsive disorder), schizophrenia, bipolar disorder and ADHD, identified 134 genes (206 variants) as significantly associated risk variants. Null genetic effects were also reported for 195 genes (426 variants). 13 [19,20].
Data from the Psychiatric Genomics Consortium [11] including 896 ADHD cases and 2,455 controls, and 2,064 parent-affected offspring trios, provided sufficient statistical power to detect gene sets representing a genotype relative risk of at least 1.17. Although all synaptic genes together showed a significant association with ADHD, this association was not stronger than that of randomly generated gene sets matched for the same number of genes. Given current sample size and gene sets based on current knowledge of genes related to synaptic function, the results reported by Hammerschlag et al. [21] do not support a major role for common genetic variants in synaptic genes in the etiology of ADHD. However, haplotypes co-segregating with ADHD-affected individuals were identified at chromosomes 1q25, 5q11-5q13, 9q31-9q32, and 18q11-18q21 in the German population [22].
Rare copy number variations (CNVs), such as chromosomal deletions or duplications, have been implicated in ADHD and other neurodevelopmental disorders. To identify rare (frequency ≤ 1%) CNVs that increase the risk of ADHD, Jarick et al. [23] performed a whole-genome CNV analysis based on 489 young ADHD patients and 1285 adult population-based controls and identified one significantly associated CNV region. In tests for a global burden of large (>500 kb) rare CNVs, they observed a nonsignificant 1.126-fold enriched rate of subjects carrying at least one such CNV in the group of ADHD cases and rare CNVs within the parkinson protein 2 gene (PARK2) with a significantly higher prevalence in ADHD patients than in controls. The PARK2 locus (chr 6: 162 659 756-162 767 019) harbored three deletions and nine duplications in the ADHD patients and two deletions and two duplications in the controls. CNVs at the PARK2 locus were found in four additional ADHD patients and one additional control. Mutations and CNVs in PARK2 are known to be associated with Parkinson's disease. 57 large, rare CNVs were identified in children with ADHD and 78 in controls, showing a significantly increased rate of CNVs in ADHD. This increased rate of CNVs was particularly high in those with intellectual disability. An excess of chromosome 16p13.11 duplications was noted in ADHD. CNVs identified in ADHD were significantly enriched for loci previously reported in both autism and schizophrenia [24].
It is commonly believed that the symptoms of ADHD are closely associated with hypo-function of the dopaminergic system. Dopamine D2 receptor activation decreases the excitability of dopamine neurons, as well as the release of dopamine. Several genes associated with the catecholaminergic system including the dopamine receptor genes (DRD4 and DRD5), the dopamine transporter gene, and the gene for dopamine beta-hydroxylase, which catalyzes conversion of dopamine to norepinephrine are associated with ADHD. ADHD is believed to be a result of abnormalities in the frontal regions of the brain, particularly the prefrontal cortex and associated subcortical structures and circuits. Underpinning these abnormalities are disturbances of catecholamine neurotransmission. Patients with ADHD have depleted levels of dopamine and norepinephrine thought to be largely the result of dysfunction of their respective transporter systems [25]. Gene and genome-wide association studies have suggested that serotoninergic gene variants are associated with increased risk of ADHD. A chronic deficit of serotonin (5-HT) at the synapse may trigger symptoms of ADHD. Serotonin through the orbitofrontal-striatal circuitry may regulate behavioral domains of hyperactivity and impulsivity interacting with abnormal dopaminergic neurotransmission in ADHD. Selective serotonin re-uptake inhibitors, L-tryptophan (the amino acid precursor of 5-HT), and non-stimulant drugs acting on the 5-HT system are modestly effective in some ADHD cases [26]. Balance between excitatory glutamate and inhibitory GABA neurotransmitter is essential and critical for proper development and functioning of brain. GABAergic (gamma aminobutyric acid) and glutamatergic interneurons maintain excitability, integrity and synaptic plasticity. Loss of inhibitory GABA and glutamate-mediated hyper-excitation may contribute to the development of autism spectrum disorder and ADHD [27].

Proteomics and Metabolomics
Proteomics and metabolomics are still immature disciplines in ADHD. Proteomic biomarkers can be used for distinguishing between comorbid psychiatric disorders in clinical setup as well as their potential for understanding mechanisms underlying the disorders and in discovery of new treatment strategies. Metabolomics, a highthroughput investigatory strategy developed in recent years, can offer comprehensive metabolite-level insights that complement protein and genetic findings [28][29][30].

Treatment
The therapeutic strategies for the treatment of ADHD can be classified into 6 categories: (i) stimulants, (ii) non-stimulants, (iii) psychotropics, (iv) combination therapies, (v) multimodal interventions, and (vi) non-pharmacological treatments. Efficacious and well-tolerated medications are available for the treatment of ADHD (methylphenidate, ethylphenidate, lisdexamfetamine, atomoxetine, metadoxine, guanfacine) [31][32][33][34][35][36][37][38][39]. Stimulants such as methylphenidate (MPH) and amphetamines are the most widely used medications approved by the US Food and Drug Administration (FDA) for the treatment of ADHD in children. Many studies have reported the long-term efficacy and tolerability of immediate-release formulations of MPH. The disadvantages of such formulations include the need for multiple daily dosing and a potential for abuse. The efficacy and tolerability of dexmethylphenidate, the active D-isomer of MPH, in an extended-release formulation have also been reported. An extended-release formulation of mixed amphetamine salts that is dosed once daily has been found to be efficacious and well tolerated. The non-stimulant atomoxetine has been reported to be well tolerated and efficacious, although it may not be as effective as stimulants; this formulation is, however, less likely than stimulants to be associated with abuse and diversion. The pro-drug stimulant, lisdexamfetamine dimesylate, was developed to provide a long duration of effect that is consistent throughout the day, with a reduced potential for abuse. Currently available treatments for ADHD in children are efficacious and well tolerated, but many of them are limited by the requirement for multiple daily dosing, the presence of unwanted effects, and abuse potential [38].
In an US cohort, 77.8% of subjects were treated with stimulants; boys were 1.8 times more likely than girls to be treated. The median age at initiation (9.8 years), median duration of treatment (33.8 months), and likelihood of developing at least one side effect (22.3%) were not significantly different by gender. Overall, 73.1% of episodes of stimulant treatment were associated with a favorable response. The likelihood of a favorable response was comparable for boys and girls. Treatment was initiated earlier for children with either ADHD combined type or ADHD hyperactive-impulsive type than for children with ADHD predominantly inattentive type and duration of treatment was longer for ADHD combined type. There was no association between DSM-IV subtype and likelihood of a favorable response or of side effects. Dextroamphetamine and methylphenidate were equally likely to be associated with a favorable response, but dextroamphetamine was more likely to be associated with side effects [39].
Some studies indicate that parents of children with ADHD prefer to avoid stimulant medications in favor of behavioral or psychosocial interventions, while others report that parents see medication as a preferred treatment [40]. In general, only 50% of patients with ADHD receive pharmacological treatment [2].

Pharmacogenetics
There are few studies devoted to the pharmacogenetics of ADHD which might provide conclusive results with practical application in the clinical setting [41][42][43][44][45][46]; however, if compared with other brain disorders, ADHD pharmacogenetics has been relatively well documented [47].
The genes involved in the pharmacogenomic response to anti-ADHD drugs fall into five major categories: (i) genes associated with the pathogenesis of ADHD (disease-specific genes, pathogenic genes); (ii) genes associated with the mechanism of action of drugs (mechanistic genes); (iii) genes associated with drug metabolism (metabolic genes); (iv) genes associated with drug transporters; and (v) pleiotropic genes involved in multifaceted cascades and metabolic reactions ( Table 2).

Dextroamphetamine
Pathogenic genes associated with dextroamphetamine include CSNK1E and SLC6A3. Mechanistic genes are ADRA1A, ADRA1B, FOS, SLC6A2, SLC6A3, and SLC18A2. Dextroamphetamine is a major substrate of CYP2D6 and a minor substrate of COMT, and an inhibitor of MAOA and MAOB enzymes. Genes involved in the transport of dextroamphetamine include the protein products of the SLC6A2, SLC6A3, SLC6A4, and SLC18A2 genes [41,47] (Table 2).

Atomoxetine
Atomoxetine is a major substrate of CYP2D6, a minor substrate of CYP2C19, a moderate inhibitor of CYP2D6 and CYP3A4, and a weak inhibitor of CES1, CYP1A2, CYP2C9, CYP2D6, and SLC6A2. The pathogenic genes involved in the effects of atomoxetine are ADRA2C, DRD4, SLC6A2, and SLC6A3; and it's most important mechanistic gene is SLC6A2. SLC6A2 and SLC6A3 participate in the transport of atomoxetine [41,47] (Table 2).

Guanfacine
Guanfacine is a substrate of ABCB1 and CYP3A4. ADRA1B and ADRA2A are pathogenic genes involved in guanfacine effects, and ADRA2A is the most important mechanistic gene. ABCB1 is a fundamental transporter for guanfacine intro the BBB [41,47] (Table  2).

Conclusions
Although pharmacological and alternative treatments have been used in children with ADHD to ameliorate their behavioral symptomatology, at the present time pharmacological treatment with stimulants, non-stimulant medications and psychotropic drugs appears to be the most effective form of therapeutic intervention, not devoid of side-effects. Recent advances in drug development and pharmacogenomics predict a better future in terms of novel therapeutic options in order to avoid the still unknown long-term consequences derived from the chronic administration of conventional drugs on brain, cardiovascular, metabolic and endocrine functions. It is important to assume that by trial-and-error, without information on the pharmacogenetic profiles of ADHD patients, only 30% of the children receive the appropriate medication at the right dosage [47]. In this regard, the introduction of pharmacogenetic procedures in clinical practice is the best option for the optimization of therapeutics while reducing costs and adverse drug reactions.