This is one of the first studies to relate gene expression in peripheral blood to neuropsychiatric symptoms using whole genome expression arrays. The expression of many genes correlated with the IA, HI scales or both. This finding supports the concept that the pathophysiology of ADHD and/or its subtypes likely involves the interaction of multiple genes. Additionally, the genes that correlated with both IA and HI (common IA-HI genes) may provide a molecular correlate of the combined symptoms in ADHD, as well as facilitate an understanding of the association between IA and HI symptoms. Given the small number of participants, the results are preliminary and will need to be confirmed in subsequent studies. This study did not test whether the genes identified could be used to distinguish individuals with ADHD of the predominantly IA, predominantly HI, or combined types. The current study identified genes that correlated with IA, HI scales or both across all of the participants with TS. These genes might be useful in identifying ADHD phenotypes but future studies with a much larger cohort would be needed to address this question.
How gene expression in blood might correlate with ADHD symptoms
One of the questions this study raises is how RNA expression in peripheral blood cells might correlate with IA or HI symptoms that are thought to be mediated by central nervous system pathways. First, many of the neurotransmitters and receptors expressed in brain are also expressed in peripheral leukocytes [7, 10–12]. Factors that affect neurotransmitters and receptors that mediate symptoms in brain may affect the same neurotransmitters and receptors in leukocytes. Such factors that might affect gene expression in both blood and brain and affect IA and HI symptoms include catecholamines, stress hormones, chemokines and cytokines. In addition, peripheral leukocytes that might be involved in the pathogenesis of TS can signal to neurons via the endothelial cells at the blood brain barrier (BBB). For example, it has been shown that up-regulation of choline acetyltransferase (ChAT) and acetylcholine (ACh) receptor expression in T and B cells  can signal via the BBB endothelial cells to neurons in brain , a pathway that could modulate ADHD symptoms. Finally, neurons in brain can signal to leukocytes in blood via the endothelial cells at the BBB. For example, neuronal release of catecholamines can signal to BBB endothelial cells which can change adhesion molecule expression on the endothelial cells that then signal to leukocytes. These mechanisms are hypothetical since the current studies cannot gauge what the relationship between blood and brain gene expression might be, particularly given the different genetic influences in blood compared to brain. Though the exact mechanism is unknown, the correlation of gene expression in blood with IA, HI behaviors or both may provide unique insights into pathogenesis of ADHD symptoms.
Common IA-HI associated genes
Most of the top pathways associated with the common IA-HI genes in participants with TS were immune-related including IL-4 Signaling, B cell receptor signaling, T cell receptor signaling, and glucocorticoid receptor signaling. Glucocorticoid release, which is mediated by the hypothalamic-pituitary-adrenal axis, could affect IA and HI symptoms and gene expression of leukocytes . Network analysis showed the common IA-HI genes were associated with cell death, behavior, as well as nervous system development and function (Figure 2). Imaging studies in ADHD [2, 27] have suggested many brain structures associated with cognitive/attention networks display functional abnormalities. These interacting neural regions included the dorsal anterior mid cingulate cortex, dorsolateral prefrontal cortex, ventrolateral prefrontal cortex, parietal cortex, striatum and cerebellum . These brain network changes could be associated at least in part with the molecular network changes noted here (Figure 2).
The neurotransmitter genes SLC6A2 and GRIN2B observed in the common IA-HI gene list have been associated with ADHD. SLC6A2 is a norepinephrine transporter that has been studied in ADHD due to the fact that drugs that block the norepinephrine transporter are efficacious in treating ADHD [17, 28]. SNPs in the SLC6A2 gene have been associated with ADHD . Glutamatergic signaling pathways also represented candidate susceptibility genes. Thus, three SNPs in the GRIN2B gene were associated with ADHD, and quantitative trait analyses showed associations of these markers with both the IA and HI symptom dimensions of ADHD. Disruption of GRIN1 (2A-D), another glutamate receptor subunit gene, leads to significant alterations in cognitive and/or locomotor behavior including impairments in latent learning, spatial memory tasks and hyperactivity .
One of the top canonical pathways over-represented in HI-candidate genes was the role of NFAT in the regulation of the immune response and natural killer cell signaling. This is consistent with a previous report of natural killer cell genes being differentially expressed in TS patients diagnosed with ADHD . Other HI-candidate genes were associated with integrin and growth hormone signaling. Recent Genome Wide Association Studies (GWAS) studies found that basic biological processes, especially integrin signaling, are involved in ADHD pathophysiology .
The neurotransmitter-related genes COMT, DRD2, MAOA and SLC6A4 were also included in the HI-candidate gene list and have been previously associated with ADHD [17, 29] . DRD2, COMT and MAOA are catecholaminergic genes. SLC6A4 is a serotonin transporter that transports the neurotransmitter serotonin from synaptic clefts into presynaptic neurons. MAOA is a mitochondrial enzyme which degrades norepinephrine, dopamine and serotonin . COMT also catalyzes degradation of catecholamines including dopamine, norepinephrine and epinephrine. The DRD2 dopamine receptors mediate the effects of dopamine in the indirect basal ganglia pathway. The density of DRD2 receptors is highest in the basal ganglia, and HI is related to excessive dopamine activity in the basal ganglia [29, 30].
Genes expressed in blood that correlated with IA symptoms and have been previously associated with ADHD included DRD1, MOBP, FOXP and FADS2. DRD1 is most abundant in the prefrontal cortex (PFC) which is believed to be critical for regulating attention, motivational behavior and emotion. Either too little or too much DRD1 receptor stimulation impairs PFC function . In addition, genetic studies have suggested an association between DRD1 with the ADHD IA symptoms in particular .
GWAS have suggested that SNPs in the FOXP1 and MOBP genes are associated with ADHD . FOXP1 is a FOX transcription factor family member. FOX transcription factors regulate tissue- and cell type-specific gene transcription during both development and adulthood. Another family member FOXP2 is involved in developmental speech and language disorders and directly regulates targets related to neural development and synaptic plasticity and developmental disorders like autism and schizophrenia .
This study only addressed gene expression correlated with the ADHD symptoms (IA and HI) in participants with TS, and did not consider other co-morbidities like tic severity or obsessive-compulsive symptom severity. It is not known if the genes associated with IA and HI symptoms in the TS subjects could be replicated in general populations of children with ADHD. Given that many genes overlapped between IA and HI symptoms in subjects with TS, some of these might also overlap in subjects with ADHD without TS.
Two participants who had been previously prescribed medication were included in the current study. To determine if these subjects might have biased the results, our Principal Components Analysis (not shown) revealed that there were no outliers in the gene expression data, suggesting these two individuals did not significantly bias the correlations observed. Moreover, our previous studies including these individuals did not show them to be outliers with regard to fMRI findings or alternative splicing [10, 13]. Nevertheless, the fact that prior medications might affect blood gene expression should be addressed in future research.
The largest limitation of the study is that, in spite of many genes being correlated with HI and/or IA symptoms, no gene passed multiple comparison correction testing using the Benjamini-Hochberg False Discovery rate (FDR<5%), and none of the genes were confirmed using an independent method such as RT-PCR. Thus, a future confirmatory study likely including RT-PCR and possibly corrections for blood cell types in a much a larger sample size will be needed to validate the genes reported here.
Genetic studies have shown that of the many genes involved in ADHD, a given gene may only contribute a small percent to the symptoms [5, 6, 17]. This could explain the modest association between a single gene and ADHD symptoms. Thus, pathways identified in this study are likely to be more reproducible in follow up studies rather than individual genes. Importantly, a gene co-expression analysis did validate these pathway-related ADHD genes. Moreover, our gene-gene correlation results demonstrate that the multiple probesets targeting a specific gene on the Affymetrix human U133 arrays were highly correlated each other (Additional file 6 Table S3). The validity of the findings is also supported by the fact that 27 genes that correlated with IA and/or HI scales have been reported in previous genetic studies of ADHD (Table 2).