Attention-Deficit Hyperactivity Disorder (ADHD) encompasses other symptoms besides inattention, hyperactivity, and impulsivity, such as language problems. ADHD can have a non-remitting course and is also found in older individuals, although there are no studies on language problems in elderly individuals with the disorder.To investigate the presence of language impairment in older adults with ADHD.Language impairment was investigated in three older ADHD adults, and compared with two matched control subjects using a narrative discourse task. The transcript discourses were evaluated based on the Trabasso Model for discourse analysis, and then processed by the Speech Graph Analysis software.Compared to control subjects, ADHD patient discourse had more Plot components and their networks exhibited more Edges. The patients had higher scores on the Narrative Inefficiency, Density and Diameter Indexes as well as on the Average Clustering Coefficient. The networks of control subjects were sequential, with little or no recursiveness, whereas those of ADHD subjects were convoluted.Our results suggest that language deficits described in children, adolescents and young adults with ADHD may persist in older adults with the disorder. All patients signed an informed consent approved by the Ethics Committee. The patients presented problems involving memory functioning . All patients described a history of inattention symptoms dating back to early adolescence (before the age of 12), lifelong problems with daily life management (suggestive of impaired executive functions) and persistent forgetfulness, which they considered to be worse now than before. The patients had never received a psychiatric diagnosis and were drug-naïve. None of the patients had impairment in their Activities of Daily Living (ADL) according to reliable collateral reports. All patients underwent the following evaluation: a) psychiatric, b) neurological, c) neuropsychological assessment, and d) neuroimaging. Neuropsychological testing comprised the following: a) auditory verbal memory , b) visual memory , c) working memory , d) attention (Digit Span and Five Digits Test), e) visuoconstructive skills (Block Design), f) executive functions , Five Digits Test, Trail Making B), and g) a language evaluation , Reading and Writing). The results from all patients were within normal range, except for the Trail Making B test of the male patient, but this result had no clinical correlate. The narrative discourse analysis was performed on a separate occasion. All MRI scan results were considered normal by a trained neuroradiologist.Patients reported no depressive symptomatology, and their scores on the Geriatric Depression Scale (GDS) were within normal range. None of the patients displayed any primary language deficit suggestive of Primary Progressive Aphasia (PPA). Also, they did not exhibit impaired performance on any of the cognitive domains in the neuropsychological evaluation; therefore, they did not fulfill DSM-5 diagnosis for Major Neurocognitive Disorder (dementia) or Mild Neurocognitive Disorder (MCI – Mild Cognitive Impairment). All patients received an ADHD diagnosis according to guidelines proposed by Kooij.,main plotline (the search for the pet frog and the events directly related to the aim of getting it back) and a secondary plotline . There are many advantages regarding this strategy: a) a book containing no words with a coherent story provides an ecological way of eliciting and accessing narrative discourse; b) the events (in both main and secondary plotlines) happen sequentially one at a time, allowing investigation of the flow of the narrative discourse; c) each event occurs just once in the story, so linearity is expected in narratives; d) the book has been previously used in many studies as visual stimuli for narrative analysis,,,,Narrative discourse can be distinguished from other types of discourse (such as spontaneous and descriptive) because it requires the individual to verbally reproduce a story while respecting the temporal and causal relationships among events. For this, we used visual stimuli from the book “Frog, Where are you?”The wordless picture book is presented to the patient who is given the following instruction: “Here is a picture book. It’s about a boy and his pets, a frog and a dog. You should look at each page, and then tell me the story. I’m going to record the story you produce. You can look through the whole book as many times as you want before we start. It is not necessary to memorize the book, you’ll keep it with you. You should try telling the best possible story, pretending that I don’t know it.”The narrative produced by the patient is first recorded, then transcribed and submitted for analysis as described below.,-primary events , and secondary events of the narrative (from the secondary plotline). The narrative is then transformed into a written sequence in order to be analyzed by software. We used the Speech Graphs Analysis (SGA) software program,-,,node, and the link between the story components as an edge.We used the previous studies of Slobin, Trabasso and Von der Broek, Stein and Gleinn, Nickels and Trabasso and SteinSpeech Graphs Analysis (SGA- http://neuro.ufrn.br/softwares/speechgraphs),,The For comparison, we show the graphs of matched control individuals without memory complaints and normal performance on Activities of Daily Living; they belonged to the same social class as the patients and were invited to participate by the research group. The quantitative data displayed here aims to show how specific parameters can be used to investigate difference between groups; the small sample size precluded statistical analysis, i.e., comparisons between groups.www.demneuropsy.com.br/).The graphs produced by SGA analysis from the narrative discourse of an ADHD patient, and one graph of a control individual are shown below  and 2; tDiameter of narrative discourses, as the Diameter is the length of the longest among the shortest path between the node pairs of a network, and here indicates the linearity of the narrative (Number of Plot Components (network nodes) was similar, two ADHD individuals clearly provided more narrative nuclei (as indicated by the Number of Plot Nuclei) when compared to controls, indicating the unnecessary use of more narrative components or nuclei in their narratives , narratives were convoluted in ADHD patients. Clinically, their discourse was poorly organized, redundant, often portraying a more descriptive than narrative discourse, with too many details and some embellishment. This last aspect can be quantitatively demonstrated by the arrative . AlthougAverage Coefficient Cluster (CC) indicates the occurrence of repeated nodes in the network and there are no repeated events in the book, positive non-zero CC values in all ADHD patients indicate inefficient or disorganized narratives (Since the rratives . In summThe ADHD patients were thoroughly investigated through clinical, neuropsychological and neuroimaging evaluation and we are therefore confident that their language deficits were unrelated to abnormal aging, albeit Mild Cognitive Impairment or Primary Progressive Aphasia. These results suggest that language deficits previously described in children, adolescents and adults with ADHD may persist into old age. Notably, the new methodology used in this study provides measurable results for language deficits previously described qualitatively.The small sample size precluded statistical comparisons; these preliminary results aim to demonstrate that quantitative analysis with specific scores can be useful in the investigation of language impairment in ADHD. Although individuals were not assessed by blinded examiners, their discourses were transcribed and analyzed by the software, making potential bias negligible.In conclusion, similar to that demonstrated in younger individuals with ADHD, our results suggest that language impairment is also present in elderly patients. Evaluations based on a more complex and quantitative analysis might provide a deeper understanding of the impact of ADHD on cognition. From a clinical perspective, language impairment can be objectively described to patients and may also be used to address treatment strategy efficacy. Further studies assessing narrative discourse involving a larger number of ADHD adults are warranted.


N = 391) and nonpsychiatric controls (N = 213). We tested for differentially methylated positions (DMPs) associated with both ADHD diagnosis and ADHD polygenic risk score, by using linear regression models including smoking, medication effects, and other potential confounders in our statistical models. Our results support previously reported associations between ADHD and DNA methylation levels at sites annotated to VIPR2, and identify several novel disease-associated DMPs (p < 1e–5), although none of them were genome-wide significant. The two top-ranked, ADHD-associated DMPs (cg17478313 annotated to SLC7A8 and cg21609804 annotated to MARK2) are also significantly associated with nearby SNPs (p = 1.2e–46 and p = 2.07e–59), providing evidence that disease-associated DMPs are under genetic control. We also report a genome-wide significant association between ADHD polygenic risk and variable DNA methylation at a site annotated to the promoter of GART and SON (p = 6.71E–8). Finally, we show that ADHD-associated SNPs colocalize with SNPs associated with methylation levels in saliva. This is the first large-scale study of DNA methylation in children with ADHD. Our results represent novel epigenetic biomarkers for ADHD that may be useful for patient stratification, reinforce the importance of genetic effects on DNA methylation, and provide plausible molecular mechanisms for ADHD risk variants.Epigenetic variation in peripheral tissues is being widely studied as a molecular biomarker of complex disease and disease-related exposures. To date, few studies have examined differences in DNA methylation associated with attention-deficit hyperactivity disorder (ADHD). In this study, we profiled genetic and methylomic variation across the genome in saliva samples from children (age 7–12 years) with clinically established ADHD ( A recent large genome-wide association study (GWAS) meta-analysis of ADHD identified the first genome-wide significant association with common DNA variants3. These data allow for a more precise estimate of the burden of ADHD genetic risk, which is associated with ADHD diagnosis and some of its mechanisms, such as poor executive function4.Attention-deficit hyperactivity disorder (ADHD) has a substantial heritable component, with genetic factors interacting with early-life environmental exposures to mediate risk8. Epigenetic processes, which act to dynamically control gene expression and are known to regulate key neurobiological and cognitive processes in the brain, represent both a potential clue to mechanisms and a possible source of novel biomarker discovery10. Epigenetic studies of mental disorders have focused primarily on DNA methylation, the best-characterized and most stable epigenetic modification. It acts to influence gene expression via physical disruption of transcription factor binding and the attraction of methyl-binding proteins that initiate chromatin compaction and gene silencing11. Epigenetic variation can be influenced by environmental exposures. In fact, many of the environmental risk factors associated with ADHD have been associated with changes in DNA methylation in peripheral tissues16. Epigenetic changes may also be induced by exposures related to the disease  or the pathological process itself. Although patterns of DNA methylation are cell-type specific, and generalization from peripheral tissue to brain mechanisms is not appropriate, disease-associated changes in peripheral DNA methylation might serve as useful biomarkers. For instance, DNA methylation changes in peripheral tissues may be indicative of early exposures  or clinical subtypes, or may be predictors of outcome or treatment response17. Importantly, both genetic and environmental etiologies can operate through epigenetic effects, and there is evidence that genetic effects on DNA methylation are relatively conserved across tissues19. Thus, an integrated genetic/epigenetic approach is strongly recommended for “second generation” studies of methylation biomarkers20. Combining genetic and epigenetic data might also nominate novel mechanistic pathways associated with disease as hypotheses for future study21.The specific mechanisms by which genetic risk factors influence ADHD are not known, although recent evidence supports a role for non-sequence-based  genomic variation in neuropsychiatric phenotypes26, and four studies targeting specific environmental exposures, such as malnutrition, in relation to ADHD symptoms, by using candidate and epigenome-wide association study (EWAS) approaches30. Three prior EWAS studies are relevant. The first and only study to examine ADHD cases directly, was a small study by our group31. That study of 105 children was able to replicate suggestive findings in MYT1L and VIPR2, also a top hit in an EWAS of environmental exposure28 and a recent twin study32. An EWAS of ADHD symptoms in adult population cohorts reported additional novel candidate sites for exploration, as well as noting that most top-ranked loci were driven by DNA methylation quantitative trait loci (mQTL)33. A third population-based EWAS study7 identified prenatal DNA methylation sites related to later ADHD symptom trajectories, though they did not replicate at the age of 7.DNA methylation studies relevant to ADHD or ADHD symptoms to date are limited. They include five candidate gene studies on relatively small samplesWe report here the first large-scale EWAS of children with ADHD and extend our previous study to incorporate analyses of genetic effects in the context of variable DNA methylation, exploring both mQTL and polygenic risk burden derived from GWAS. Given our focus on identifying biomarkers relevant to ADHD, we use DNA derived from saliva in an effort to minimize potential selection bias resulting from clinical differences in rates of child refusal of blood draws.In a case-finding procedure, families were recruited by soliciting community volunteers with public advertisements and mass mailings. The local Institutional Review Board approved the studies. Parents provided written informed consent; children provided written informed assent. All families completed a multi-informant, multi-method screening process to establish eligibility and diagnostic group assignment for ADHD, non-ADHD, as well as comorbid disorders .Current and lifetime prescription of any psychoactive medication, including any stimulant or non-stimulant preparation, was recorded and statistically controlled. A full frequency list of medications in the sample is given in Table lumi34 and minfi35 packages. Data were normalized with lumi34 using smooth quantile normalization. Cell-type profiles and proportions were calculated using reference-free cell-type prediction with the RefFreeEWAS36 package, and beta values were adjusted to account for cell-type proportions in each sample. Methylation values were set as missing if they deviated >4 times the interquartile range from the mean of each probe. Participant samples were further curated to restrict to unrelated children who met full criteria for non-ADHD or ADHD. We excluded a small number of children who only met criteria for the ADHD hyperactive profile, those with subthreshold ADHD , and those missing information about medication usage, resulting in a final sample of 604 unrelated children. For analyses examining the effects of the ADHD polygenic risk score, only children with European ancestry4 were included (N = 472). We removed probes for the following reasons  and analysis workflow is provided in Fig. ns Table : detecti4. Briefly, DNA was hybridized to the PsychCHIP_v1-1 , developed by Illumina, Inc. in collaboration with the Psychiatric Genetics Consortium (PGC). Genotypes were used to determine relatedness38 among samples and population stratification as previously described4. The PRS was constructed using the PGC + iPSYCH meta-analysis3 as the discovery dataset .Salivary DNA samples were genotyped, and the ADHD polygenic risk score (PRS) for each individual calculated as described previouslyhttps://github.com/pryabinin/ohsu_adhd_ewas). Briefly, linear models , the first three genomic principal components (PCs), medication usage (binary variable signifying the history of psychoactive or stimulant medication), and a maternal smoking score12, which are referred to below as the “standard covariates”. The first three genomic PCs were selected based on their association with self-reported ethnicity in our sample. The relationship between ethnicity and genomic PCs in our sample was verified using HapMap samples as a reference. In addition, for the PRS analysis, a covariate was included to account for the number of missing SNPs in the PRS calculation for each patient. Finally, due to sex differences in ADHD prevalence, salient clinical features39, and familial burden not explained by autosomal differences40, a sex interaction term was included for both ADHD status and ADHD PRS , as well as differentially methylated positions (DMPs) were evaluated using custom R scripts , bel41, using the results of each regression model separately. The following Comb-p parameters were used: seed p-value = 0.001, maximum distance between probes = 500 bp, and a minimum of three probes allowed in a DMR. This set of parameters is consistent with previous work42 in the field and recommendations from simulation experiments43.Testing for differentially methylated regions (DMRs) was performed using the Comb-p software tool44, where the methylation level at each DMP was regressed on the minor allele count of each SNP. Covariates for sex, age, and the first three genomic PCs were included in the model. For all DMPs with p < 1e–5 , all 6,374,797 autosomal SNPs were assessed .DNA methylation quantitative trait loci (mQTLs) were calculated using a linear, additive model implemented in the GEM package in R3, were evaluated. Statistical analyses were performed using summary data-based Mendelian randomization45 (SMR), as well as the Bayesian colocalization method in the coloc package46 in R.DNA methylation QTLs discovered in our cohort were examined for evidence of colocalization with ADHD-associated variants. All pairs of methylation probes and SNPs within each of 12 ADHD-associated regions Table , determiGenetica (Table https://github.com/bhattp09/genetica) that collates information from publicly available databases  and through the Weizmann Institute of Science (http://www.genecards.org/). Investigations into miRNA profiles were performed using the miRWalk tools47, and seed regions overlapping the hybridized probe sequence was confirmed using the UCSC Genome Browser48.Downstream interpretation of the associated DMPs was performed using ca Table , a customethylGSA package49 was used to investigate whether DMPs were enriched among genes involved in a particular biological function or process. methylGSA implements an unbiased test of enrichment, accounting for the number of methylation probes within each gene. The default significance threshold suggested by methylGSA (p ≤ 0.001) was used to select DMPs for the enrichment analyses. We first tested for enrichment among brain-expressed genes, using two gene sets defined by the Human Protein Atlas50 (https://www.proteinatlas.org/humanproteome/tissue/brain): genes with brain-enriched expression , and genes with brain-elevated expression . We next tested for enrichment among all Gene Ontology (GO) categories. GO categories enriched with a false discovery rate-adjusted p ≤ 0.05 were considered significant.The p-value threshold of 8.8e–8, which is consistent with a recent recommendation for the genome-wide significance threshold for analyses of EPIC array data51. Following other studies, a p-value threshold of 1e–5 was used to identify suggestive DMPs for preliminary interpretation to guide future hypotheses52. A more inclusive p-value threshold of 0.001 was used to select DMPs for gene set enrichment analyses as suggested by the MethylGSA R package49. In the DMR analysis, a Šidák-corrected p-value < 0.05 was used to determine significance. Finally, a Bonferroni-corrected p-value threshold of 1.8e–10 was used to identify significant mQTLs.Within each EWAS analysis, DNA methylation sites were considered significantly differentially methylated if they met a Bonferroni-corrected p-value < 8.8e–8 was calculated for all probes included in our analysis, following a previously described method51. Power calculations were done using each probe’s variance across all subjects, and the number for cases and controls in the experiment. The proportion of probes with a particular power threshold (ranging between 0 and 1) is shown in Fig. The statistical power to detect an effect size of 1% difference in DNA methylation between cases and controls at a p = 0.03), but the difference was not significant in the European-ancestry subsample used for the PRS analyses. As is common in studies of ADHD, the ADHD group had a slightly lower IQ , but both groups had mean IQ in the average range. The groups were of similar age, although age was included as a covariate as a precaution. Boys were overrepresented in the ADHD group and we adjusted all results for sex.Table 10. As expected, reported ages and predicted ages were highly correlated , providing reassurance as to the validity of the DNA methylation data obtained from saliva. There was no evidence for differential epigenetic age acceleration in the ADHD group (p = 0.98) . This result indicates that, as expected, ADHD is not associated with any systemic methylomic differences across probes included on the Illumina EPIC array.We calculated a predicted age for each child, based on age-associated DNA methylation sites98) Fig. . We alsoλ = 1.02  to the median expected log10)53. A Q–Q plot and Manhattan plot are presented in Fig. Differential DNA methylation between ADHD cases and controls at individual sites was investigated by regressing methylation levels at each probe on ADHD status (Eq. ) with oup = 8.8e–8), which corrects for all 568,281 probes tested. Table p < 1e–5. Methylation differences between ADHD cases and controls ranged between 0.3% and 1.4%. The top 100 probes associated with ADHD are provided in Table p = 1.54e–6), shows higher DNA methylation in ADHD cases compared with controls (Δβ = 0.93%) and is located in the promotor region of SLC7A8 , also with higher methylation (Δβ = 1.38%) in ADHD cases, is located in the 3′-UTR of MARK2 . A Q–Q plot and Manhattan plot are presented in Fig. p = 6.71E–8), characterized by reduced DNA methylation with higher PRS , when including ADHD status in the regression model, indicating that the effect is not driven by elevated polygenic burden in ADHD cases. This probe is located in a CpG island of a bivariate promoter between the GART and SON genes. None of the SNPs included in the PRS are direct mQTLs (see below) for cg15472673 , indicating that the association with the PRS is not driven by a simple genetic effect on DNA methylation. DNA methylation levels at 12 other probes were associated with the PRS at p < 1.0e–5, 10 of which showed increased methylation with higher PRS (Table We have previously shown that the ADHD PRS is associated with ADHD status (explaining ~4.5% of the variance) in this sampleion (Eq. ). Again,PRS Fig. . The assRS Table . A summaVIPR2 and MYT1L specifically in boys31, we first examined sex-by-diagnosis interaction effects among all probes annotated to these two genes (239 probes) at an adjusted significance threshold of p < 0.0002 (0.05/239). The two strongest sex-by-diagnosis interactions annotated to VIPR2 were for cg26975193 and cg20998127 (sex-by-diagnosis interaction p = 7.51e–6 and p = 0.000459). Supporting the finding from our previous study, males with ADHD had lower methylation compared with male controls at both sites . However, among females, ADHD cases had higher methylation levels than controls at cg26975193  and were not significantly different from controls at cg20998127 . To ensure replication of the hypomethylation effect in boys with ADHD reported by Wilmot et al.31, we removed the samples included in that previous report (n = 73 that survived QC for the current probe set). Effects among males were consistent at both sites .Because we previously reported an association between ADHD and DNA methylation at sites annotated to MYT1L, our previously reported finding did not survive multiple-testing correction , although the direction of effect at this site  was consistent with our previous data, even with prior samples (n = 73) removed . Likewise, no probes annotated to MYT1L showed main effects that passed the Bonferroni threshold . All results from the analysis of these genes are reported in Table For p < 1e–5).We next performed an EWAS of sex-specific DMPs associated with both ADHD and the ADHD PRS. No sex interactions were significant at the EWAS-wide threshold, although nine probes show sex-by-diagnosis interactions Table  and five41. A single significant DMR on chromosome 6 was identified (Šidák-corrected p = 3.4e–5), which contained eight probes associated with the ADHD PRS in a sex-specific manner  using the Comb-p software toolner Fig. . SpecifiSLC7A8 and cg21609804 annotated to MARK2, Table SLC7A8 probe (cg17478313) was associated with SNP rs7141505 (p = 1.2e–46), located in the gene’s promoter region. The MARK2 probe (cg21609804) was associated with an intronic SNP rs928948 (38 kb upstream of the CpG) at p = 2.07e–59  and genotype (p = 4.4e–45 and p = 8.9e–58) are associated with methylation levels when included together in the regression models. These results indicate that the mQTL effects are not simply due to allele frequency differences between ADHD cases and controls. An additional 279 SNPs are associated with methylation levels at these two DMPs (31 for cg17478313 and 248 for cg21609804), and pass our experiment-wide mQTL significance threshold (p < 1.8e–10).We identified methylation quantitative trait loci (mQTL) associated with all DMPs suggestively associated with (a) ADHD or (b) the ADHD PRS Table . DNA met–59 Fig. . Further–59 Fig. . Also, fp < 1e–5), significant mQTLs were found only for cg04453792 annotated to USP31 (p < 1.8e–10). All mQTL analysis results are provided in Table For the ADHD PRS, of all the associated DMPs , SLC7A8 (minimum p = 0.3), GART/SON (minimum p = 0.029), USP31 (minimum p = 0.069), or LOC100130015 (minimum p = 0.011) were seen in the GWAS meta-analysis, indicating there is no evidence of pleiotropy for these mQTLs.To determine whether mQTL variants associated with the top-ranked DMPs (for both ADHD and the ADHD PRS) were also implicated in GWAS for ADHD , we examined the results from the PGC ADHD GWAS meta-analysis15 Table . We asse3. Of the 12 ADHD-associated regions identified in the GWAS meta-analysis, 11 contained genome-wide significant cis-mQTLs in our cohort . SNP rs2279574 is a missense variant within DUSP6, and rs1656622 lies within an intron of SEMA6D. All significant mQTLs in the 12 ADHD-associated regions are included in Table To expand the investigation of pleiotropy, all mQTLs observed in our cohort (not just those associated with DMPs) were tested for colocalization with variants associated with ADHD in the recent ADHD GWAS meta-analysis conducted by the PGCrt Table . Evidencrt Table . For twort Table  and S10,p < 0.001, the recommended default in the methylGSA package)49 are annotated to 91 genes with elevated expression  in the brain . Several  or are involved in cell adhesion , both previously implicated in ADHD or other psychiatric traits58. No GO categories were significantly enriched with either ADHD-associated or ADHD PRS-associated DMPs after multiple testing correction. All enrichment analysis results are shown in Table For exploratory and hypothesis-generating purposes, we report gene set findings. DMPs nominally associated with the ADHD PRS , and provides new data for considering replications, or lack thereof, with previous epidemiological studies.This is the first large EWAS to examine well-characterized ADHD cases and controls, including more ADHD cases than any prior study of DNA methylation in ADHD. It is distinguished by the inclusion of polygenic risk effects—not previously studied in ADHD in an EWAS context—and the examination of sex effects. Furthermore, the inclusion of genetic effects, crucial for interpretation, has rarely been done in ADHD methylation studies. The findings make contributions pertaining to potential peripheral biomarkers for ADHD and ADHD genetic risk, provide additional evidence for the genetic regulation of many disease-associated methylation differences in ADHD, and suggest regulation of DNA methylation as a plausible mechanism for ADHD risk variants identified in GWAS. The study identifies suggestive new candidates to pursue  to detect methylation differences of 1% between cases and controls at ~68% of sites Fig. ; howeverVIPR2 in never-medicated boys31 was supported in a targeted analysis after removing children used in our prior report, although at a smaller effect size, suggesting that this sex-specific effect is reproducible. Sites annotated to VIPR2 have also been identified as differentially methylated in two other studies of ADHD32, but with effects varying in direction. Given that we observe a sex-specific effect in VIPR2  and other studies did not stratify by sex, it is possible that the inconsistencies across studies related to the direction of effect are due to differences in the sex ratio of the samples studied. Other differences across study samples, such as differences in age or specific environmental exposures, may also contribute to the inconsistent direction of effect. The role of VIPR2, a receptor for a small neuropeptide implicated in a wide range of biological functions, needs more exploration. Genetic variation in VIPR2 has been associated with schizophrenia60, and in one candidate gene study, with mood disorders61.With these strengths and limitations in mind, five main findings are noteworthy. First, our previous report of lower DNA methylation in SLC7A8 and MARK2). SLC7A8 is a sodium-independent solute transporter of neutral amino acids62 and plays a role in metal ion homeostasis and toxicity63 including mercury toxicity64. The DMP annotated to MARK2 is at the seed of two miRNAs, hsa-miR-1199-5p, and hsa-miR6751-3P, suggesting that differential methylation at this locus may indirectly affect gene expression through regulation of miRNAs. The full list of miRNA targets for this sequence using miRWalk47 is provided at https://github.com/pryabinin/ohsu_adhd_ewas. MARK2 is a regulator of cell polarity and microtubule dynamics66, is required for neurite outgrowth67, and has recently been implicated in both Alzheimer’s disease and bipolar disorder68.Second, we identified suggestive findings for a small number of DNA methylation sites that can provide possible biomarkers for ADHD and associated phenotypes. We note that the top two DMPs are both located in regulatory regions of their respective genes , is also an mQTL suggestively associated with methylation at sites within MARK2 (minimum mQTL p = 3.8e–8), indicating that the variant might act through the regulation of DNA methylation.Both of the top-ranked DMPs are also associated with nearby SNPs, indicating the complexity of the association between DNA methylation in these genes and ADHD. Of note, a variant in GART and SON, with higher PRS associated with reduced methylation. SON SNPs have been implicated in a GWAS for educational attainment70, which is noteworthy given the known relationship between ADHD and academic underachievement71, as well as the evidence for a shared genetic basis for ADHD and educational attainment72. De novo mutations in SON have been linked to intellectual disability, and gene expression studies suggest that it is a master regulator of genes involved in neurodevelopment73.Third, we identified one DMP significantly associated with polygenic risk for ADHD (ADHD PRS), and another 12 DMPs suggestively associated. The strongest association was at a site in the bivariate promoter for PSORS1C3, significantly associated with the ADHD PRS in a sex-specific manner. A previous study found that a DMR in this same region was hypomethylated in major depressive disorder (MDD) suicide cases74, although it did not assess sex-specific effects.Fourth, we found a single DMR, near Finally, we found significant evidence of colocalization for mQTLs observed in our cohort and ADHD-associated SNPs, suggesting that regulation of DNA methylation may be a mechanism by which many ADHD risk variants operate.33. That study of three population-based cohorts found three suggestively associated DMPs, none of which overlap with the DMPs reported here. The lack of overlap is not surprising, however, given the amount of heterogeneity across cohorts reported in the meta-analysis. That study also failed to see an overlap between ADHD-related DMPs and ADHD GWAS hits from the PGC. However, consistent with our findings, the study found cis-mQTLs for their top-ranked DMP, highlighting the importance of genetic effects.The small number of DMPs and DMRs we observe, and the small effect sizes, are consistent with a recent EWAS meta-analysis of ADHD symptoms in adults10. It also appears likely to be helpful in mapping potential causal routes for genetic influences on ADHD. Future work will examine sex and medication effects, moderators of change and/or stability of DNA methylation over time, map backward to environmental correlates, and evaluate the extent to which identified genes or systems show methylation effects earlier or later in development.In conclusion, our findings suggest that further study of DNA methylation in ADHD can be productive to identify biomarkers of illness, and potentially, disease mechanisms, although ultimately multi-position algorithms will likely be necessary to create clinically useful biomarkersManuscript SupplementSupplemental Table S5Supplemental Table S6Supplemental Table S7Supplemental Table S8Supplemental Table S9Supplemental Table S10


Attention deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder that is associated with risk-taking behaviors, poor self-control, and interpersonal difficulties. Affected individuals have an increased probability of involvement with the criminal justice system, contributing to a higher rate of arrest, and imprisonment compared with the general population; they are also inadequately treated once sentenced. Because prison staff play a central role in the identification of inmates with mental disorders, they could well be key to improving provision of care. There is however little knowledge of the conceptions, perceptions, and attitudes of prison staff toward ADHD. Such information could help to identify starting points for awareness training and further implementation of specific ADHD treatment. To bridge this gap, we undertook a study based on a qualitatively-driven mixed methods design, combining qualitative data collection in the form of narrative interviews with 19 prison staff from a Swiss correctional facility with quantitative data collection in the form of a survey that included the Attitudes toward Prisoners scale. The interviews were analyzed with QSR NVIVO 11 and a qualitative content analysis approach was used to evaluate findings. Prison staff were generally aware of ADHD and its symptomology, believing it to a be “real,” but “fashionable” disorder and favoring hereditary-genetic or biological explanatory models for its development. They viewed inmates with ADHD rather negatively, as complicating correctional efforts, and perceived them as sticking out, as tying up more resources and as frequently being involved in confrontations. Our findings suggest that difficulties in pragmatic aspects of communication and language comprehension may be perceived “as not listening or following instructions,” creating additional tensions. Consequently, inmates with ADHD are more often exposed to disciplinary sanctions, such as solitary confinement—an intervention deemed “necessary” by staff. Therefore, staff training on ADHD might need to cover evidence on adverse effects. Non-pharmacological interventions for treatment were preferred and considered to be highly efficacious. Skepticism toward pharmacological treatment prevailed, even when benefits from stimulant medication were described. Interestingly, this skepticism was not the result of negative experiences with the misuse and diversion of stimulants. Acceptance of multimodal treatment among prison staff may require customized strategies. Those 68% n = 6 of indin sample . While t68% n = 1 of indivn sample . Comparen sample . On the n sample –21. In an sample . This la 68% n = 6 of indin sample , 23. Onen sample . For exan sample . He basen sample . For exan sample . The lacn sample . On a din sample .It has long been understood that prison staff plays a central role in the identification and provision of care for inmates with mental disorders in general . In partHere, we propose to explore these gaps by undertaking a mixed method design study, which aims to qualitatively:Investigate whether prison staff perceive ADHD to be a mental disorder,Explore prison staff beliefs regarding the causes of ADHD,Elaborate on past experiences with inmates with a diagnosis of ADHD,Identify prison staff's view on the role of therapies in ADHD management during times of detention, and quantitatively:Evaluate prison staff's personal attitudes toward offenders and their potential for rehabilitation in general.This quantitative element was included in order to situate the narratives of this sample in the context of their overall attitudes toward offenders, thus providing a framework for interpreting the qualitative findings reported. It might be that this Swiss sample distinguishes itself from correctional officers in other jurisdictions, for example by having a far more positive attitude toward rehabilitation, which would construe a limitation and a further obstacle in the generalization of our findings.This study employed a qualitatively driven mixed methods design combining qualitative data collection in the form of narrative interviews with quantitative data collection in the form of a survey  to invesA modified site-based approach was used to identify and recruit study participants from Realta prison, located in the Canton of Grisons, Switzerland . It is oBecause we expected professional background, function and position, years of employment, gender and age to be important correlates of variation in attitudes and perceptions of prison staff toward mental disorders, these were the characteristics of interest when approaching a “gatekeeper.” The importance of gatekeepers in recruitment of participants from hard to reach communities and complex societies has been described elsewhere . In the To investigate whether prison staff perceive ADHD to be a mental disorder, to explore participants' beliefs regarding the causes of ADHD, to elaborate on past experiences with inmates with a diagnosis of ADHD and to identify prison staff's views on the role of therapies in ADHD management during times of detention, we conducted single, semi-structured, in-depth interviews lasting between 25 and 66 min, with an average duration of 51 min. We used a self-developed and flexible interview guide which also covered attitudes toward other mental disorders. Two female researchers (NF and AB) conducted the interviews. NF was at the time a Master's student at the Faculty of Human Sciences, Institute of Psychology, preparing a thesis under the supervision of ML, a forensic psychiatrist and faculty member of the medical school. AB, an attending physician at the Psychiatric University Hospital, Zurich who headed the specialized outpatient clinic for ADHD and had experience in qualitative interviews, trained NF and conducted the initial two interviews in the presence of NF. Subsequent interviews were all conducted by NF with regular feedback given by ML based on audiotapes and transcripts.The research team itself had gathered previous experience in employing qualitative research methodology on perceptions toward ADHD, SUD, and psychosis among the general population, medical and legal experts and affected individuals. Results have been reported elsewhere , 35–38.Before the interviews, participants had an understanding that NF had a background in psychology and that the research represented a collaboration between the Department for Justice, Health and Safety, Grisons, and several psychiatric institutions and that the research would address prison staff's experiences with inmates suffering from a wide variety of mental disorders.All interviews were conducted in Swiss German, an Alemannic dialect spoken in the German-speaking part of Switzerland and in some bordering Alpine communities. Participants were encouraged to speak this dialect in order to make them feel more comfortable. Open-ended questions and non-leading probes were used to encourage participants to speak freely and to elaborate on their statements. Paraphrasing and summarizing main points during the interviews helped minimize misunderstandings and clarify ambiguous statements. Interviews were—with the exception of the initial two interviews—conducted on a one-to-one basis and were digitally recorded. Field notes were taken afterwards.By grounding the questions in participants' practice experiences, and by reformulating the questions, we sought to avoid generalized responses. All interviews took place in an office of the correctional facility. The office was reserved for the interviews to ensure that interviewer and participants were undisturbed. Water was available as a refreshment. There were no repeat interviews.r = 0.82). To obtain biographical data and to conduct the ATP, a survey was set up employing REDcap software . After removing identifying information, each transcript was assigned a code number. The transcripts were not returned to the participants. Subsequently the transcripts were uploaded into QSR NVIVO 11 for Windows (see below).The procedure regarding the content analysis differed only slightly in comparison to previous studies by our research group and has been described in detail before . QualitaThe coding process ensured a systematic, comprehensive, and detailed reading of each interview transcript. First, the coders familiarized themselves with the transcripts in order to identify the different subjects of interest. After several interviews had been coded, the categories for the study were redefined, reviewed and revised in a consensual manner at meetings between AB and ML. When there was disagreement regarding the coded material, ML applied the final code. As a result of the coding process and for the purpose of this paper, four main categories were identified and selected: (a) personal stance toward ADHD, (b) explanatory models, (c) experiences with previously diagnosed inmates, and (d) attitudes toward necessary interventions. An overview of the categories is shown in To illustrate the categories and for reporting purposes, examples of coded quotations were chosen by AB and ML and translated from German into English by ML. Deepl Translator a machine translation service launched in August 2017 was used to support and simplify this translation process. Quotations were then improved by a bilingual German/English speaker (ML) and edited by an English native speaker (Heather Murray) to ensure readability for an international audience.t-tests and Bonferroni correction (p < 0.00138).The quantitative sociodemographic data were evaluated using SPSS version 24. Attitudes toward prisoners with and without a mental disorder were compared using paired sample The research was conducted in accordance with the 1964 Helsinki Declaration and was authorized by the independent ethics committee (IEC) of the faculty of Human Sciences of the University of Bern. All participants were assured confidentiality, and gave their written informed consent for the study and, specifically, for the digital recordings of the interviews.During this study the research team established contact with 21 subjects. Of these, one declined to participate. The barrier to participation for this potential participant could not be determined. Another potential subject who had initially agreed to participate was impeded due to an unexpected illness. The scheduling of the interviews proved to be complex, as the employee's absences and changes in work shifts needed to be considered. Despite careful planning, several appointments had to be rescheduled because of unexpected changes in the shift plan, which however did not lead to reduced participation. On the contrary, subjects showed great interest in the research topics. In total, 19 subjects provided their written, informed consent. All completed the interview. None of the participants withdrew their consent at a later time.n = 19) was composed of a higher percentage of male staff (73.3%) than of females (26.3%). The mean age of the participants was 50.4 years (± 10.6 years) with a mean of 15.9 years (± 11.6 years) of working experience in the correctional system. The vast majority of participants (84.2%) had completed vocational training outside the prison system and received a federal diploma before starting to train as correctional officers, as this is a prerequisite for an application to justice department authorities in most cantons .In qualitative research, saturation is commonly defined as the point when no new themes arise and was achieved in this study after 15 interviews. Because additional interviews were agreed upon prior to reaching saturation, these were followed through with.Unexpectedly and across all occupational groups, all participants had heard about the disorder and/or had had personal experience with inmates with ADHD, expressing themselves in a complex way on the topic . FurtherYes, what strikes me in the prison is that there are always psychologically remarkable inmates who cannot keep to the rhythm of everyday life. For example, I didn't know there were, uh, adults with ADHD. I didn't know that. And when you (meet) such prisoners later on, they attract attention when they are so hyperactive. But I did not know until then that this was due to ADHD. I didn't understand it, until I came to work here.ID 9In view of the reservations described in the literature about ADHD, it was important for us to depict in particular the general attitude and any skepticism of prison staff. For this purpose, we used follow-up questions that dealt with the characterization of ADHD as a “fashion diagnosis,” hence a diagnosis that is currently enjoying particular popularity. While participants considered the disorder to be “fashionable,” similar to the diagnosis of “burn-out,” the majority were of the opinion that the “phenomenon” existed, i.e., that there was a “true core” behind it. The interviewees considered the “accuracy” of expert diagnosis as the basis for initiating treatment to be a major problem; they thought that experts were not able to accurately differentiate between those who are impaired but “generally o.k.” and those requiring pharmaceutical treatment. In this context, comparisons to “past-times” were repeatedly made, arguing that, today, all those who could not “function” received a diagnosis, whereas “nervous-fidgety” behavior would just have been accepted by society in the past. Although these statements evoke the idea of “overdiagnosis,” such explicit wording was not used by our participants.Um, I think it's a really hard disease to diagnose, I strongly believe that. And I actually think that some, um, professionals can't really diagnose it clearly. But I think this phenomenon exists. It really does exist.ID 8Yeah, it is like “burnout”, it's fashionable. It's not easy in the professional world. But when such a hyperventilated  child tests the limits, I would claim that not everyone has the problem. There are certainly half of those with ADHD who actually have the disease and a dysfunction. But often it is also the case that someone acts out in the school yard, [thinking] when I act out, I'm accepted, then I'm somebody.ID 10Irrespective of reservations about the assessment of ADHD, the disorder was deemed to be a topic of increasing relevance, as more inmates were perceived to be affected and/or diagnosed. Our participants based these perceptions on their many years of experience in the correctional system. In this context, too, it became clear that skepticism about the “existence” of this disorder had generally diminished after joining the prison work force and gaining “hands on” experience.Yes, yes we have ADHD too. Also rising. (...) I think the whole thing will increase in the next few years. This is becoming more and more [common].ID 13In our sample almost all participants had developed explanatory models about the origin of ADHD . Only twMost frequently participants associated the development of ADHD with hereditary-genetic or, more generally speaking, with biological causes, indicating a clear preference for scientific explanations. Occasionally, birth complications (asphyxia) or consequences of alcohol consumption during pregnancy were mentioned.I think it's genetic, I think it's uh biologically phew yes I think genetic and biological above all. So I think the environment has less of an impact. I think the environment has an impact on how someone deals with it. But for the onset of ADHD, that does not arise from bad (…) social influence. Yes genetically, biologically, like that.”“ID 15Less frequently, prison staff identified environmental, parental, or social factors as the main reason for developing ADHD. Factors like increased media consumption, disproportionate use of computer and communication devices, and an overflow in day to day activities were mentioned. The main motive identified was “social pressure” as exemplified below:I think also familial probably. Their whole youth, their upbringing, school – all around. The whole environment that has developed differently now compared to some years ago, because the pressure from outside is greater and the parents, and many are children of foreigners, where this is just one problem and it goes wrong in many other directions. They do not learn German at home and that's an additional problem. So I think that's already a big part of the problem.”“ID 13Mystical explanatory models were adopted by one participant. This explanatory model was linked to a perceived simple solution for symptoms, in this case the consideration of “water veins” and “bed-positioning.”It may also have been experienced, perhaps even electro-smog in childhood, I could imagine. Or someone always lies over a water pipe as a child, a water vein …, that's it. I could imagine. The parents are desperate, go to the doctor, get medication, everything and, in the end, it would have been that one only had to change the [position of the] bed”“ID 11Our participant sample had gathered a great wealth of experience in dealing with detainees diagnosed with ADHD during various stages of incarceration and in a multitude of everyday prison life situations . AlthougIn addition, it must be noted that a majority of prison staff, irrespective of professional background and line of work, seemed to be aware of the mental health status of inmates, indicating a simplified passing on of medical information in this prison. This was justified by the necessities of a strongly labor-oriented correctional facility, in which some jobs assigned to inmates required, for example, the operation of heavy machinery. In this context, it was explained that medical information, such as prescription drug use, must be transparent to most staff.Analysis identified several important interwoven factors which, in the eyes of prison staff, complicated correctional efforts on an individual level and moreover impacted inmates collectively. Participants had experienced inmates with ADHD as “sticking out and standing out” and perceived them as loud, temperamental, fidgety, quickly frustrated, and verbally aggressive. It was commonly reported that this group of detainees were the ones who violated prison rules. In the eyes of the participants, they were the ones who wanted to leave the cell after lock-in, were unorganized and missed appointments, for example, in their work environment or in health services, and did so even when this meant incurring significant disadvantages. Other prison staff indicated that inmates with ADHD required longer and more repetitive instructions, more explanations and generally more attention compared to prisoners who did not have this disorder. In this context, some participants acknowledged different degrees of severity of the disorder, resulting in varying impairments of social functioning.Those who are always tingly, can't sit quietly, are always on the move – and that's difficult in the evening when they're locked up. In the first days these are the ones that constantly ring (the bell) and ask if they can be let out for a moment, because they have to run. But that, uh, yes in the night you have time to explain why it's not possible and why they have to stay in overnight (...) and that is enough even if you just take the 5 min to quickly talk to them. And then they have the feeling that “I'm not alone”, and at night it's very quiet in the prison, so it's possible. Just a few words can help. I have the feeling they need a bit more attention now and then, uh, by the fact that they can get medication from us of course, uh, there is automatically a short conversation and that every now and then I have the feeling they want that, too.ID IThe inmate with ADHD just stands out, he simply stands out among all the others. Or if he's burdened with that. He can't sit still and things just don't work out. Always compared to those who do not have the syndrome (...): Yes, and that they have no patience. Or that they overreact at work as well and also their temperament, I think that they are more quickly frustrated and become aggressive.ID 9A majority of prison staff suggested that inmates with ADHD often came into conflict with other inmates. Surprisingly, this was not only perceived to be the result of impulsive-uninhibited behavior targeted directly at other prisoners, e.g., in the sense of verbal or physical assaults, but was seen as a consequence of the increased social attention given by prison staff, which is envied by other inmates. Because inmates with ADHD demanded longer explanations, this time was then not available to other detainees. Unlike other more obvious debilitating mental disorders, such as schizophrenia, prisoners with ADHD are not perceived as mentally ill by fellow inmates, which is why behavioral abnormalities are less accepted and do not evoke sympathy.This is noticeable among prisoners with ADHD. The other prisoners, the collective body, see these prisoners less as ill. If someone has schizophrenia and is clearly behaving strangely, then the illness aspect is not questioned among inmates. But someone who takes up a lot of time, for example from the foreman, in consulting, that is seen critically. We have office hours and people can come up to the floor and, when the caregiver is free, they can come into the office and chat. You just take turns. And sometimes there are remarks to those with ADHD: “Oh you again, you have a torrent of words today.” And in fact, we really need more time with them. And sometimes this really causes friction among the detainees.I would actually say that these are not the ones that are considered sick. They are rather those who are considered to be buzzing and noisy, always scrounging and such.ID 9Yes, they're very, very, very difficult, how shall I say, to handle. They talk too much, they are, uh, restless and that makes it very difficult for the others to accept them as they are. I think it's generally difficult for these men in a community (laughs). I think.ID 12Almost all respondents experienced the care of detainees with ADHD as labor- and time-intensive, energy-consuming, and generally exhausting. While it was perceived as very important to establish structure in day-to-day routines and a schedule that is both organized and predictable, it was exactly those things that were found to be most challenging to implement, when dealing with this group of inmates. In this context it was often mentioned that inmates with ADHD were easily able to upset carefully crafted prior schedules, if these behaviors were not monitored and managed well.Yeah, um, I'd say it's the hustle and bustle. They are so difficult to handle, um, because you have to structure them well from the outside, because otherwise it will roll over you or make an hour out of 10 min.In order to deal with them, as an advisor or also in the security service, it is necessary to give really clear instructions. Make straightforward announcements, try to put things in order. Especially when the inmate talks a lot. Ask what's important now. They can be very big energy guzzlers. They can turn all your work upside down because everything is so insanely important, right now.ID 8Security staff in particular described in great detail the inability of inmates with ADHD to regulate themselves and to stop impulsive behavior, which quickly manifested itself in verbal lapses in times of frustration. In the eyes of security staff, this results in more disciplinary sanctions being imposed on inmates with ADHD, such as placement in solitary confinement. A few of the staff had noticed that such a massive restriction of freedom of movement among this group of prisoners only increased undesirable behavior, but deemed this to be a necessity in order to enforce prison rules and to ensure equal treatment for other inmates. In this context, some staff also indicated that, for those placed in solitary confinement, psychiatric support was not always available quickly enough.Yes, very time-consuming. ‘Cause this could go well for a moment, and then they’ll be back somewhere up on the roof. You have to get them back down and if it goes well, everything is fine, but then they do something stupid like using the word “asshole” and they are back in solitary confinement. Naturally that is not possible. That's not possible for others either. Then they are back in confinement and problems are inevitable. They want to get out, they want to smoke, they want “grass,” they want this and they want that, they are constantly on the “bell” yes, yes, it is time-consuming.ID 13Now, while we are talking, one's in solitary confinement for five or 6 days and he's already in trouble. We have to move him to another facility now, because it's no longer possible for us to keep him here. He also goes from zero to 100 in no time and won't calm down. He's a real “Ritalin-boy,” too. He really needs to be seen by a psychiatrist, but he is only here for 20 days and nobody is going to yank out a leg (“bust a gut”) over this. He will be in solitary confinement for another 10 days and then he will be released and we will not hear from him again for 1 or 2 years. Then he might return. If they are here for such a short term, there is no chance that a psychiatrist will see them. No chance.ID 13The vast majority of our sample were of the opinion that dealing with inmates with ADHD required a specific approach and had, over the years, themselves developed a variety of interventions that they deemed useful. Regarding treatment for ADHD, almost all interviewees expressed a strong preference for psycho-social interventions, behavioral measures and cognitive training .Prison staff specifically mentioned coaching, providing structure, positive reinforcement, time, and support when asked about necessary interventions. The meaning of support was interpreted heterogeneously. For example, one staff member reported that he specifically tried to fatigue prisoners with ADHD by pushing them to their physical limits. Interestingly, some staff also found that necessary interventions consisted of safeguarding and protecting affected inmates, and suggested establishing a specific ward for inmates with mental disorders.So I think, uh, interventions gonna have to take place more on the behavioral level. So they need clear structures, clear instructions, no arguing. So I think that is important with ADHD, for people with ADHD. (…) And today I think it's possible to change something, but it's, uh, I think a very big effort, in self-control. And although I think that a drug can be helpful, first and foremost it should be about cognitive and behavioral measures. In my eyes.ID 15With me it's good for them, because when they work with me, they can really give it a go. If I know that one of the detainees has ADHD, then I give him a job that normally two people do. “Put the three stones on the bus.” And if he can do that, that is a form of intervention (medication) without him knowing or realizing it. But that he is exhausted in the evening, that he is needed, that is also healthy.ID 10Although inmates with ADHD who were prescribed stimulants such as methylphenidate were perceived as more stable, easier to live with and better manageable for staff, almost all participants showed a certain reluctance toward the necessity of pharmaceutical interventions. None of them was completely against stimulant treatment; however, they clearly did not see it as a viable first-line treatment.Ritalin. If this is well-adjusted and they get it, then, uh, they work fine in here. But they're a little more demanding, I'd say on the average. So they need more attention from us and if they have the medication of course, then it's easy, then it's possible.ID 17Some of them are in need of protection. Sometimes you have to, uh, practice with them to get through everyday life here. Help them to acquire knowledge about the structures so that they can learn to move here. Um, they need treatment in the sense that they are medicated, that they are also under psychiatric care and observation, this support has to be guaranteed, so that changes are noticed.ID 8In the present qualitatively driven mixed methods study, we investigated the general view and awareness of ADHD held by Swiss prison staff, explored their beliefs regarding causes of this disorder, inquired about experiences with inmates with a diagnosis of ADHD and identified attitudes toward ADHD management during times of detention.Our qualitative findings on the subject of ADHD should be interpreted in the light of this sample's fairly negative attitude toward offenders in general, as measured by scores on the ATP. The mean score of prison staff participating in this study was 90.58 (SD = 16.804), which is very similar to scores reported for correctional officers from the USA (90.7), Norway (90) and the United Kingdom, but substantially lower than, for example, scores reported for graduate students in psychology working in the Alabama prison project (113.2) , 44, 45.Our results indicate that prison staff working closely with inmates during different stages of incarceration and in a multitude of prison life situations are aware of ADHD and—very specifically—its core symptomatology. In fact, ADHD as such, is an actively discussed topic among prison staff with various professional backgrounds and believed to be of increasing relevance. Interestingly, the skepticism of staff toward the existence of a “Fidgety Phil Syndrome” and the classification of this form of “behavioral abnormalities” as a mental disorder decreased when they started to work in a prison environment, a finding that even held true for individuals who had previously gained experience in general psychiatry. Several explanations for this finding seem plausible: Firstly, it may be simply more likely for staff to encounter individuals with ADHD in a correctional facility, considering that the prevalence of individuals with ADHD symptoms is higher by a factor of 5–10 compared to the general population . SecondlWith regard to a previous study among the general population from neighboring Germany, it can be said that our qualitative findings complement quantitative results. Speerforck et al. reported recently that more than 90% of the German general population had heard of the terms “attention deficit hyperactivity disorder” or “ADHD,” and 77% of those believed adult ADHD to be a real disease . In addiApart from the general awareness and a shared belief that ADHD is a “real disease,” prison staff were under the impression that there was a lack of reliability in assessment of ADHD by experts, especially in respect to the threshold for initiation of pharmaceutical treatment. Furthermore, participants labeled ADHD as a particular “fashionable” diagnosis, i.e., as a disorder that is “popular” for mental health experts to diagnose or for patients to receive. Repeated statements that “behavioral abnormalities were socially more acceptable in the past” and that today “it is simply a matter of making a diagnosis” can also be regarded as indicative for the belief that ADHD is inappropriately mis- and overdiagnosed. Although, according to the knowledge of the authors, there are no studies that have investigated this phenomenon quantitatively or qualitatively among prison staff, quantitative studies among other populations show that this belief is widespread. For example, a survey of Australian general public attitudes toward the acceptability of pharmaceutical treatment for ADHD found that 78% of participants held the belief that individuals are diagnosed with ADHD when they do not actually have the disorder, a stark contrast to current evidence indicating that the majority of patients are underdiagnosed and undertreated –59. StilN = 1,008) reported stressful life events, a pressure to perform and problems with parents or family as the top ranked causal beliefs among the German general population for adult ADHD . In addition, scores on the ATP scale suggest that this sample's general attitude toward offenders is comparable to those reported for correctional officers from other European jurisdictions and even some common law countries. Second, there are limitations associated with volunteer bias, to which most studies are also susceptible. In order to minimize possible bias of the gatekeeper in selecting participants, considerable effort was made to ensure recruitment of a sample that incorporated diversity, also in respect to interest in the research topic. As described in more detail above, study participation was carried out during regular working hours and was also counted as full working time for participants. Only one of the potential participants contacted declined to participate. The barrier to participation for this potential participant, however, could not be determined. As a qualitatively driven mixed methods design study, this study was not driven by a theoretical framework. Future studies on this subject could, however, use the insights gained here to pursue more focused research. We also recognize that the results may in part be specific to the Swiss legal and penal system. Nevertheless, the literature indicates that some perceptions and views identified in this study, such as a lack of knowledge on multimodal treatment options for ADHD and the skepticism toward the use of stimulant medication, have also been reported from other countries. Our findings provide several relevant insights into views held by prison staff on ADHD as a mental disorder and on individuals living with this disorder while being detained. Most importantly, our findings are based on prison staff's own reports identifying a range of experiences. These findings were not limited to predefined experiences, as might occur in a survey-based research. Furthermore, a written survey might have increased the likelihood of socially desirable responses.This research extends our understanding of members of prison staff's perceptions and explanatory models of ADHD, their experiences with inmates diagnosed with this highly prevalent neurodevelopmental disorder and their attitudes toward ADHD management during times of detention. Our results indicate that prison staff working with inmates during different stages of incarceration are aware of ADHD and its core symptomatology and believe it to a be “real disorder.” Simultaneously, participants labeled ADHD as a particularly “fashionable” diagnosis and thought that ADHD was inappropriately mis- and overdiagnosed. Unlike other populations, this sample favored hereditary-genetic or biological explanatory models for the development of ADHD over environmental and social-cultural explanations. Irrespective of professional background, staff generally viewed inmates with ADHD rather negatively, as complicating correctional efforts both on an individual level and for inmates collectively, and had had the experience that they more often came into confrontation with other prisoners. While this is in line with earlier reports, our findings suggest that this is not only due to physical aggression, but also to difficulties in pragmatic aspects of communication. It seems plausible that these impairments create additional tension between inmates, especially when one considers that prison staff voiced the opinion that inmates with ADHD are not perceived as mentally ill by fellow inmates. A recurring theme in the context was that inmates with ADHD are more often exposed to disciplinary sanctions, especially in one of their most controversial forms, namely placement in solitary confinement. While prison staff had experienced negative consequences of this form of punishment and believed it to be “counterproductive,” they deemed it to be “necessary to ensure equal treatment,” contrasting starkly with current evidence. In light of our findings, it may be useful during development of ADHD awareness courses to consider including material on the adverse effects of solitary confinement. It should not go unmentioned that, in the opinion of prison staff, detained individuals with ADHD required a specific form of “management” or “treatment” in everyday prison life. Non-pharmacological interventions were preferred because they were believed to be efficacious and were considered to be free of adverse effects—underlining the gap between evidence and public opinion. The skepticism toward pharmacological treatment remained, even when interviewees described benefits from stimulant medication and viewed inmates in treatment as more easily manageable. Interestingly, this was not the result of negative experiences with the misuse and diversion of stimulants, which was not reported. We are cautiously optimistic that at least in comparable penal institutions, staff may be open to the implementation of non-pharmacological treatments. In order to develop the acceptance of stimulant treatment for inmates with ADHD, it might be helpful to point to the successes of other pharmaceutical treatments for mental disorders that were once considered highly controversial, but are generally better accepted today by prison staff.Anna.Buadze@puk.zh.ch.Excerpts of the transcripts relevant to the study are available on substantiated request from the corresponding author. Requests to access the datasets should be directed to The studies involving human participants were reviewed and approved by the research was conducted in accordance with the 1964 Helsinki Declaration and was authorized by the independent ethics committee (IEC) of the faculty of Human Sciences of the University of Bern. All participants were assured confidentiality, and gave their written informed consent to participate in the study and, specifically, to digital recording of the interviews. The patients/participants provided their written informed consent to participate in this study.AB and NF: data gathering, evaluation of data. NF: development of topic guide, obtaining ethical approval. RS, SY, and AS: writing, revisions. AB and ML: conception, development of topic guide, evaluation of data, writing manuscript, revisions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Attention deficit hyperactivity disorder (ADHD) is the most common neurobehavioral disorder in childhood and can significantly affect a child’s personal and social development and academic achievement. Taking into account the model of attentional networks proposed by Posner et al., the aim of the present study was to review the literature regarding two main explicative models of ADHD, i.e., the inhibition model and the cognitive-energetic model, by discussing behavioral and neurological evidence of both models and the limitations of each model. The review highlights evidence that favors the energetic model and points to an unstable arousal as a potential pathogenetic factor in ADHD. Attention deficit hyperactivity disorder (ADHD) is one of the most prevalent psychiatric disorders . ADHD isAlthough the first case description of ADHD was reported in 1902 , the etiThe alerting network has been associated with the right frontal and parietal lobe, and the locus coeruleus, which provides the system with norepinephrine (NE) . The orithe inhibition model  on model , which sdel CEM; , which pBarkley’s model  proposesAccording to In addition to the incongruent results in EF, there are also studies that have failed to find significant group differences in inhibition , which iRegarding neurological evidence, we would expect individuals with ADHD to show abnormalities in the structure and function of brain areas involved in the executive attentional network  as well as in the dopaminergic system compared to typically developed peers. In a recent review published by lower level includes four general stages of computational mechanisms of attention: encoding, search, decision, and motor organization. The middle level comprises the energetic pools: arousal, effort, and activation. The arousal pool is defined as a phasic response that is time-locked to stimulus processing and is typically influenced by signal intensity and novelty, and it is behaviorally indexed by sleep–wake patterns. The effort pool is characterized by the energy necessary to meet with task demands, is located within the hippocampus, and seems to function by both exciting and inhibiting the other two energetic pools . The activation pool is defined as the tonic physiological readiness to respond. Finally, the upper level refers to the executive control system and is associated with planning, response inhibition, error detection and correction responses, WM, and flexibility, among other abilities.The CEM focuses on energetic factors as the most critical explanation of ADHD, proposing that the deficit of these factors leads to both inattention and hyperactivity symptoms. This model claims that the overall efficiency of information processing is determined by the interplay among three mechanisms at different levels, with top-down and bottom-up streams among them . The lowFollowing this approach, impairments observed in individuals with ADHD are expected at the three levels of the CEM. For instance, motor difficulties have been reported in children with ADHD , which mThe conception of ADHD as a result of a hypoaroused brain state has been supported by electrophysiological studies. Previous electroencephalographic (EEG) studies among children with ADHD have reported an increase in low-frequency power (predominantly in the theta band) and a decrease in high-frequency power  . SpecifiThe alerting system proposed by the inhibition model, proposed by the cognitive-energetic model, introduced by The exact etiopathogenesis of ADHD is still unclear. In this review, we discuss the behavioral and neurological previsions and evidence of two well-known models: NA and DM conceived this manuscript. NA and DM reviewed the literature. NS-P and DM wrote the first draft of the manuscript. DM, NA, and LF edited and provided intellectual input to the manuscript. All authors approved the final version of the manuscript for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bartonella bacteria have been associated with an increasingly wide range of human and animal diseases. These emerging pathogens have been identified as being globally dispersed. Ticks and small rodents are known hosts of Bartonella and play a significant role in the preservation and circulation of Bartonella in nature. This study investigated the occurrence of hoist spp. in ticks (Acari: Ixodidae) and plateau pikas (Ochotona curzoniae) in Shiqu County, which is located on the eastern Qinghai-Tibetan Plateau in China. Shiqu County is spread over approximately 26,000 km2, with an average altitude of above 4200 m and a vast area of pastureland.Dermacentor everestianus, 79.0%, 646/818; Haemaphysalis qinghaiensis, 21.0%, 172/818) were collected from yaks in 4 villages of Shiqu County. Only Bartonella melophagi was detected in tick samples, with a total prevalence of 30.1% (246/818). The infection rates of B. melophagi in ticks from Arizha, Maga, Derongma, and Changxgma were 4.8, 76.8, 12.5, and 18.0%, respectively. The infection rate of B. melophagi in Maga was higher (p < 0.01) than those in other villages. Regarding plateau pikas, the total infection rate of Bartonella spp. was 21.7% (62/286), with 16.7% (12/72), 30.9% (25/81), 13.8% (9/65), and 23.5% (16/68) in Arizha, Maga, Derongma, and Changxgma, respectively. Finally, B. queenslandensis and B. grahamii were detected in plateau pika. No significant difference was observed (p > 0.05) in the infection rates between these study sites.A total of 818 ticks (D. everestianus and H. qinghaiensis were found in Shiqu County with high infection of Bartonella spp. in the ticks and plateau pika. The threats of Bartonella species to public health should be closely monitored.To date, only  Bartonella genus currently includes 36 named and 17 Candidatus species [B. alsatica, B. bacilliformis, B. elizabethae, B. henselae, B. koehlerae, B. melophagi, B. quintana, B. rochalimae, B. tamiae, B. vinsonii subsp. berkhoffii, B. vinsonii subsp. arupensis, and B. washoensis [Bartonella, respectively. They play an essential role in the preservation and movement of Bartonella in nature within arthropod-mammal systems. Shiqu County has an area of approximately 26,000 km2, an average altitude of above 4200 m and a vast area of pastureland on the eastern Qinghai-Tibetan Plateau. Its population was estimated at 97,000, consisting of individuals with low education and poor health. Yaks, horses and Tibetan sheep are common livestock in Shiqu County; among the three, yak has the largest population  and severe tick infestation is often observed in yak. Apart from livestock, plateau pika (Ochotona curzoniae) has the largest population of local small rodents and closely interact with local people and livestock. The significance of ticks has long been recognized due to their ability to feed on a large range of host species and to transmit Bartonella pathogens that can infect a variety of vertebrate hosts, including humans. However, little information is known about bartonella and their hosts and vectors in Shiqu County. This study aims to prove the presence of Bartonella spp. in plateau pikas and ticks and provide preliminary results for establishing prevention and control measures for this tick-borne disease.The shoensis –8. TicksDermacentor everestianus  and Haemaphysalis qinghaiensis . Information on ticks and 16S rRNA sequences are included in Supplementary files A total of 818 ticks were collected from 4 villages in Shiqu County  was observed. The infection rates of Bartonella spp. in Arizha, Maga, Derongma, and Changxgma were 4.8, 76.8, 12.5, and 18.0%, respectively (Table Bartonella spp. in ticks was higher in Maga (p < 0.01) (marked with “*” in Table p > 0.05), although the infection rate of Bartonella in H. qinghaiensis (79.1%) was higher than that in D. everestianus (69.2%).Ticks were first screened for gltA gene, and gltA-positive samples were then screened for rpoB. Total infection rate of Bartonella spp. in plateau pikas was 21.7% (positive for both gltA and rpoB genes), with 16.7% (12/72), 30.9% (25/81), 13.8% (9/65), and 23.5% (16/68) in Arizha, Maga, Derongma, and Changxgma, respectively. No significant difference in infection rates was observed (p > 0.05) between these study sites.With regard to plateau pikas, spleen samples were first screened by PCR targeting the gltA and rpoB genes from ticks and pikas were sequenced and compared to each other. A total of seven unique sequences of gltA , with 100% coverage; the sequences MN056883 and MN056888 from plateau pikas were 97.03–100% identical to B. queenslandensis (MH748120), with 99–100% coverage; the sequences MN056884, MN056886, and MN056887 from plateau pikas were 100, 97.61, and 96.73% identical to B. grahamii (KT445918 and CP001562), with 100% coverage; and sequence MN056885 from plateau pikas was 98.81% homologous to B. rochalimae (KU292571), with 100% coverage. For the rpoB gene, sequences MN296287-MN296291 from ticks were 99.12–99.71% identical to B. melophagi (EF605288), with 99–100% coverage; sequences MN296286 and MN296294 from plateau pikas were 95.65–97.86% identical to B. grahamii (AB426697 and JN810811), with 100% coverage; and sequence MN296292 from plateau pikas was 99.69% homologous to B. queenslandensis (MH748136), with 100% coverage. However, the sequence MN296293 from plateau pikas was only 92.28 and 92.58% similar to Bartonella sp. (AB529489) and B. grahamii (AB426696), respectively, with 100% coverage.In this study, all amplicons of the Bartonella spp. species thresholds: gltA ≥ 96.0% and rpoB ≥ 95.4%) proposed by La Scola et al. [B. melophagi was detected in the tick samples  and D. everestianus . D. everestianus was reported only in northwestern China and Nepal [H. qinghaiensis has only been reported in China [Lepus oiostolus). All life stages of the tick can develop in sheep, goats, yaks, and cattle [D. everestianus ticks, H. qinghaiensis mostly performs its activity at low altitudes. Arizha, Changxgma, and Derongma are located in the subfrigid zone, whereas Maga village is located in the cold temperate zone. Due to the significant difference in altitude between Maga and the other three villages, H. qinghaiensis was only found in Maga.Two tick species were identified in this study: nd Nepal  at an alnd Nepal . Larvae nd Nepal , 11. Howin China –16 and id cattle . ContrarBartonella DNA, although in varying percentages and locations. A survey regarding ticks from 16 states of the United States revealed that the overall prevalence of B. henselae in Ixodes ticks was 2.5% [Bartonella spp.  were detected in 2.1% of I. ricinus, with the highest rate in ticks from Vienna (with an infection rate of 7.5%), and the prevalence was higher in adult ticks than in other life stages [Bartonella indicated that the overall presence of Bartonella in ticks (combining evidence from multiple surveillance studies) was approximately 15% [All types of ticks were found to contain was 2.5% . In Auste stages . Furthertely 15% . In our B. melophagi, a human bacterial pathogen, was first isolated from sheep blood in 2007 [B. melophagi was isolated from domestic sheep blood and sheep keds (Melophagus ovinus) from the southwestern United States [B. melophagi and that sheep ked is its main vector. The sheep ked (M. ovinus) is the most studied ked due to its veterinary importance and because of the economic losses caused by its infestation. In contrast to ticks, the whole life stages of sheep ked occur on the host, being strictly host dependent. In Shiqu, in addition to yak, which has the largest population among local livestock , Tibetan sheep  is the main source of income for local residents; Tibetan sheep are mainly raised for meat and fur. Due to the traditional lifestyle of Shiqu County, Tibetan sheep are in close contact with local residents , and severe sheep ked infection is often observed. Therefore, in the future, we believe that more in-depth studies are necessary to determine the precise role of sheep ked and Tibet sheep in the transmission of Bartonella in Shiqu County. in 2007 , and the in 2007 . Recentld States , indicatB. melophagi detected in D. everestianus and H. qinghaiensis was reported; this is the first molecular evidence of B. melophagi in Shiqu County. However, there is no current evidence supporting the ability of these ticks to transmit B. melophagi to livestock or humans. To address this issue, experiments should be performed to assess the ability of D. everestianus and H. qinghaiensis to transmit B. melophagi in the future.In this study, the first DNA of Bartonella infection has been mostly reported in Rodentia [Lagomorpha. Until now, there has been only one report of Bartonella infection in plateau pikas, with a positive rate of 18.99% [Bartonella strains have been obtained, and most of them are closely related to B. taylorii and B. grahamii [B. grahamii, a pathogenic strain in humans, was detected in all four villages, while B. queenslandensis was detected only in Maga. In Shiqu, plateau pikas, which has largest population of local small rodents, are in close contact with local people and livestock and can be infested with fleas and ticks, implicating them in the transmission of Bartonella spp. In China, Bartonella infections among humans have been mainly reported in the central plain area, including Jiangsu, Zhejiang, Anhui, and Hubei provinces. No cases or suspected cases have been reported in the Qinghai-Tibetan Plateau. Therefore, the relationship between plateau pikas and the transmission of Bartonella should be further studied. A thorough analysis with controlled experiments should be conducted to determine the exact routes of transmission between plateau pikas, transmission between plateau pikas and their vectors, and transmission from plateau pikas to humans and livestock.Rodentia –38, and f 18.99% . A totalgrahamii . Based oBartonella spp. in D. everestianus and H. qinghaiensis ticks sampled from yaks in Shiqu County. In this region, key mammalian tick hosts are domesticated yaks and wild mammals such as rodents and plateau pikas. A more comprehensive study of Bartonella pathogens to further assess the prevalence of Rickettsia spp. in other livestock and wildlife hosts from Shiqu County should be performed in the future.In summary, we have shown, for the first time, a high prevalence of This study was conducted in Shiqu County , Sichuan Province, China  as previously described [gltA-positive samples were further analyzed with PCR targeting rpoB (379 bp) [B. henselae preserved in the laboratory) and a negative control (nuclease-free water). The observed bands were purified using the QIAquick Gel Extraction Kit and sent for sequencing . The obtained sequences were analyzed by employing Bioedit v.7.0.2 and were subjected to nucleotide BLAST search through the NCBI database. Sequences with ≥95% quality cover and identity were considered positive for Bartonella spp. and were compared with validated Bartonella species in GenBank/EMBL/DDBJ through the Clustal X program . Clones with gltA and rpoB sequences that shared ≥96.0% and ≥ 95.4% similarity with the validated species, respectively, were considered the same species [Ticks were sectioned longitudinally; one section was used for DNA extraction. For all spleen samples, an average of 30 mg of tissue was used. The total DNA of all samples was extracted using the TIANamp Genomic DNA Kit  for tick molecular identification and characterization of escribed . All glt379 bp) . All pri bp . AllgltA and rpoB sequences of Bartonella using the Kimura two-parameter model with partial gap deletion and a cutoff of 95% site coverage. The evolutionary distance was calculated, and bootstrap analysis with 1000 iterations was carried out with MEGA6 [Bartonella spp. prevalence between different sampling locations, plateau pikas, and tick species. A p-value of < 0.05 was considered significant.For phylogenetic analysis, neighbor-joining phylogenetic trees were constructed based on the th MEGA6 . SPSS19.Additional file 1. Sequences of the gltA gene.Additional file 2. Sequences of the rpoB gene.Additional file 3. Adult specimen of H. qinghaiensis. A Dorsal view; B. Ventral view.Additional file 4. Adult specimen of D. everestianus. A Dorsal view; B. Ventral view.Additional file 5. Sequences of 16S rRNA (H. qinghaiensis).Additional file 6. Sequences of 16S rRNA (D. everestianus).Additional file 7. Tick collection information.Additional file 8. Information of tick species and frequency of the sequences.