Many psychiatric disorders show gender differences in prevalence. Recent studies suggest that female patients diagnosed with anxiety and depression carry more genetic risks related to attention-deficit hyperactivity disorder (ADHD) compared with affected males.In this register-based study, we aimed to test whether female patients who received clinical diagnoses of anxiety, depressive, bipolar and eating disorders are at higher familial risk for ADHD and other neurodevelopmental disorders, compared with diagnosed male patients.We analysed data from a record-linkage of several Swedish national registers, including 151 025 sibling pairs from 103 941 unique index individuals diagnosed with anxiety, depressive, bipolar or eating disorders, as well as data from 646 948 cousin pairs. We compared the likelihood of having a relative diagnosed with ADHD/neurodevelopmental disorders in index males and females.Female patients with anxiety disorders were more likely than affected males to have a brother with ADHD (odd ratio (OR) = 1.13, 95% CI 1.05–1.22). Results for broader neurodevelopmental disorders were similar and were driven by ADHD diagnoses. Follow-up analyses revealed similar point estimates for several categories of anxiety disorders, with the strongest effect observed for agoraphobia . No significant associations were found in individuals with depressive, bipolar or eating disorders, or in cousins.These results provide modest support for the possibility that familial/genetic risks for ADHD may show gender-specific phenotypic expression. Alternatively, there could be gender-specific biases in diagnoses of anxiety and ADHD. These factors could play a small role in the observed gender differences in prevalence of ADHD and anxiety. Not only are psychiatric disorders moderately to highly heritable, there is also growing evidence of a substantial component of shared genetic risks across many disorders.9 It is plausible that the genetic liability for psychiatric problems could manifest in a gender-specific manner, partly explaining the observed prevalence differences.Certain psychiatric disorders are diagnosed more commonly in male patients (for example attention-deficit hyperactivity disorder (ADHD) and other neurodevelopmental disorders, such as autism),11 These findings support the possibility of gender-specific manifestation of biological risk, where the same risk variants are associated with different psychiatric disorders in males (for example ADHD) and females (for example anxiety/depression). However, these studies included fewer than 400 affected children, many of whom were young, not having passed through the risk period for depression, which limited the generalisability of the results to early-onset anxiety and depression.Indeed, in children diagnosed with anxiety or depression , girls have on average a higher burden of common (i.e. polygenic risk) and rare (i.e. copy number variant) genetic risks implicated in ADHD .In the current study, our aim was to examine a much larger population sample of children and adults, clinically diagnosed with a wider range of psychiatric disorders . We used a different design to that used in previous research, to test for converging evidence using diverse approaches with different strengths and limitations. We used information from a national population register to define diagnoses based on diagnostic codes from specialist in-patient and out-patient records to examine potential gender-specific effects in real-life diagnostic practices. We used nationwide data on biological relatives to define genetic liability for psychiatric disorders based on familial risk (which encompasses both common and rare inherited variants). We hypothesised that female patients who have received clinical diagnoses of anxiety, depressive, bipolar and eating disorders are at higher familial risk for ADHD and other neurodevelopmental disorders, compared with male patients who have received these diagnoses.12 The linkage was approved by the regional ethics review board in Stockholm, Sweden. The requirement for informed consent was waived because the study was register based and individuals were not personally identifiable. We used information from the Medical Birth Register,13 Total Population Register,14 National Patient Register (NPR),15 Multi-Generation Register16 and Cause of Death Register.17 We identified all individuals who were living in Sweden and were born between 1964 and 2008, and had received an ICD-10 diagnosis in any of the four categories of psychiatric disorders of interest 18 between 1997 and 2013. These dates were selected as the NPR was launched in 1964, the ICD-10 was in use from 1997 and follow-up was until the end of 2013, meaning all index individuals were at least 5 years old. To control for changing diagnostic practices over time, we only considered diagnoses based on ICD-10 and not previous versions of the ICD for the four diagnostic categories of interest. Thus, index patients were identified based on available information from 1997 to 2013, when they were aged between 5 and 49 years old.We used a data linkage of several Swedish registers based on each individual's personal identification number.19 and the ICD-10 (1997 onwards) in Sweden and would be at least 5 years old by the end of the available follow-up period. Full siblings were identified based on having the same biological parents but not being born in the same month. First cousins were identified based on having the same maternal or paternal biological grandparents but not the same parents.We retained only index individuals with both known biological parents linked in the Multi-Generation Register and at least one full sibling (excluding twins) or first cousin born between 1987 and 2008. These dates were selected to capture all relatives who would have been children during the usage of the ICD-9 (1987–1997)15 The majority of the diagnoses were obtained from only out-patient consultations (77%) or a combination of in- and out-patient information (93.5%). To improve identification of individuals diagnosed with eating disorders without recorded diagnoses in the NPR, diagnoses were also obtained from the Cause of Death Register (based on ICD-10 codes) and the treatment quality registers (based on DSM-IV criteria)20. See Data on clinical diagnoses were obtained from the Swedish NPR, which contains information on in-patient psychiatric care (1987–2013) and specialist out-patient consultations with doctors (2001–2013). It includes specialist diagnoses according to ICD-9 and ICD-10, with ongoing updates and validation of the NPR.24 Given that the typical onset of these disorders is in mid-childhood/early adolescence, we excluded index individuals who only received diagnoses at a very young age and never again . Using these cut-offs, n = 575 unique index individuals were excluded from sibling-pair analyses and n = 507 from cousin-pair analyses. We also defined variables for age at first recorded diagnosis for each disorder category.For primary analyses, specific diagnoses for female-biased psychiatric disorders were collapsed into four binary variables: any anxiety disorder, any major depressive disorder (MDD), any bipolar disorder and any eating disorder. This was deemed justifiable given the ICD groupings, as well as evidence of diagnostic fluidity, comorbidity and shared genetic influences across diagnostic subtypes.Information on presence of ADHD and neurodevelopmental disorders was also obtained from the NPR for both index individuals and their relatives (i.e. siblings and cousins). A broader group of neurodevelopmental disorders was defined as presence of one or more of the following disorders: ADHD, autism spectrum disorder, motor disorders, tic disorders, intellectual disability, communication disorders, specific intellectual disorders and dyslexia. Both ICD-9 and ICD-10 codes were used see  to decreP < 0.0031.Logistic regression analyses were used to test for an association between familial risk of ADHD/neurodevelopmental disorders and the gender of index individuals diagnosed with anxiety disorders, MDD, bipolar disorders or eating disorders. The exposures were presence of diagnosed: (a) ADHD or (b) any neurodevelopmental disorders, in the sibling  and the dependent variable was the gender of the diagnosed individual . All analyses were stratified by the gender of the sibling, to prevent confounding from potential gender-specific diagnostic biases in relatives. These primary analyses were adjusted for multiple testing using a stringent Bonferroni correction (0.05/16 tests); the main results are evaluated against a threshold of As the data included non-independent observations, a sandwich estimator was used to cluster the observations by family and derive robust standard errors. The following covariates were included in analyses: birth year of the index individual, birth year of the relative, age at first diagnosis of the disorder of interest  in the index individual, and presence of comorbid ADHD/neurodevelopmental disorders (depending on the exposure) in the index individual. All analyses were run using Stata version 15.1.Follow-up analyses were used to determine whether results for the broader group of neurodevelopmental disorders differed when ADHD was not considered as part of this broad group. Next, we stratified the index individuals based on presence of comorbid ADHD, to determine whether the results differed in individuals with comorbid diagnoses , compared with those without comorbid ADHD.For significant primary associations, follow-up analyses tested whether any specific ICD diagnostic codes were driving observed association signals.To test whether the degree of familial relatedness had an impact on the results, we analysed observations from cousin pairs. Full siblings share on average 50% of their genomes whereas first cousins share 12.5% of their genomes and siblings share a greater degree of their environmental exposures than cousins do, so familial effects are expected to be stronger in siblings.https://doi.org/10.1192/bjo.2020.47 for details of sample sizes after applying these exclusions.Several sensitivity analyses were conducted. First, we used more stringent diagnostic definitions to exclude index individuals with only one recorded diagnosis within each category . ADHD diagnoses were also only considered as present if they were diagnosed at least twice. This allowed us to investigate whether diagnostic uncertainty could explain the observed results. Second, we excluded all individuals who were aged <18 years at the end of the follow-up period (i.e. those born in 1996 or later) to ensure that all individuals had lived through the main risk period of getting diagnosed with ADHD. Third, the analyses were repeated including only siblings who were born in Sweden and still living there by the end of the follow-up period (end of 2013). Observations from siblings who had migrated or died were excluded. This was done to account for possible uncertainty and missing information during the available follow-up period. See supplementary Table 1 available at The primary cohort included a total of 103 941  unique index individuals who were affected with at least one of the female-biased psychiatric disorders of interest and had at least one full sibling; each sibling pair was treated as one observation, yielding 151 025 sibling pairs. For the analyses of cousin pairs, the total sample consisted of 646 948 cousin-pair observations from 171 247 unique index individuals .As expected, there were more females in the primary cohort for each of the diagnostic categories , with a The four diagnostic categories were frequently comorbid, with 30.9% of the participants receiving a diagnosis from multiple categories throughout the follow-up period; see supplementary Table 2. Female patients were more likely to have multiple diagnoses than male patients . The most common pair of comorbid conditions were anxiety disorders and MDD, in both genders.When the main analyses were repeated for exposure to risk of broad neurodevelopmental disorders not including ADHD, the effect sizes decreased, the confidence intervals increased and there were no associations for any of the diagnostic categories (supplementary Table 3). Thus, all further analyses focused on just ADHD diagnosis as the exposure.n = 7327, OR = 1.20, 95% CI 1.05–1.37), P = 0.0066], with a smaller, non-significant effect in those without comorbid ADHD . In contrast, the analysis of sisters with ADHD showed no association with gender in index individuals with anxiety and comorbid ADHD , yet an association in those without comorbid ADHD . No associations were seen for the other disorders (supplementary Table 4).Stratifying the index individuals based on presence of comorbid ADHD yielded significant associations between having a brother with ADHD and female anxiety in those with comorbid ADHD (The primary association seen in the context of anxiety was followed up to determine whether any specific type(s) of anxiety disorders were driving the association signal. Supplementary Table 5 displays detailed numbers for the ICD-10 anxiety categories. The following ICD codes, each affecting at least 3% of the individuals with anxiety, were examined: agoraphobia (F40.0), social phobias (F40.1), panic disorder (F41.0), generalised anxiety disorder , mixed anxiety and depressive disorder (F41.2) and unspecified anxiety disorder (F41.9). The latter category was only considered as present if none of the other categories of anxiety examined were present. Compared with the primary analyses, these follow-up analyses showed similar point estimates for several categories  in the analyses of brothers, albeit the confidence intervals overlapped with 1 for most categories, except agoraphobia: OR = 1.64, 95% CI 1.12–2.39; see The estimated magnitude of effect sizes for the association between having a cousin with ADHD and the gender of index individuals with anxiety were lower than seen in the sibling-pair analyses and non-significant . No other associations were significant in the cousin analyses (supplementary Table 7).Sensitivity analyses were used to examine the stability of the results; see supplementary Table 1 for sample sizes and female:male ratios. Of note, the exclusions for these analyses were biased towards excluding more index males , as evidenced by comparing the sample sizes and female:male ratios in Supplementary Table 8 displays the results of the sensitivity analyses aiming to account for diagnostic uncertainty. The estimated association between female anxiety and having a male sibling with ADHD was lower with a wider confidence interval than in the primary analysis .Restricting the analyses to observations where both individuals were aged ≥18 years old at the end of the follow-up period  also decreased this association compared with the primary analyses  (supplementary Table 9). Although the confidence intervals still overlapped with 1 for the association between females with bipolar disorders and having a brother with ADHD, the point estimate increased slightly when analysing only adults , compared with the full analysis .Excluding observations with incomplete information during follow-up did not substantially affect the results (supplementary Table 10). The sensitivity analyses did not reveal any consistent patterns of association results in individuals diagnosed with MDD, bipolar or eating disorders.The aim of this study was to test the hypothesis that female patients diagnosed with anxiety, depressive, bipolar and eating disorders are more likely to have a relative with ADHD or another neurodevelopmental disorder compared with affected male patients. We found modest support for this hypothesis in individuals diagnosed with anxiety disorders. Although the effect sizes in the analyses of brothers of those with bipolar disorders and sisters of those with eating disorders were similar to those observed for anxiety, they were statistically non-significant, perhaps because of the lower power associated with the lower prevalence of these disorders. No associations were seen for MDD, despite a similar sample size to that available for anxiety disorders.11 Notably, there were relatively few MDD diagnoses in these previous studies, because of the young sample. In the current study, we find supporting evidence in adults. Although non-significant, the point estimates in the sensitivity analyses of only adults were not that different for anxiety  and MDD  and higher for bipolar disorders , suggesting that differences in age at diagnosis could explain the discrepant results observed for anxiety and MDD in the full cohort.The results observed for anxiety are consistent with two studies of Swedish children, which found that females with anxiety and/or depression had a higher burden from genetic variants implicated in ADHD.25 On the other hand, another study of this UK population found no gender interaction with polygenic risk scores for ADHD and risk of anxiety, MDD and bipolar disorders,26 which is consistent with our results for MDD and bipolar disorders but inconsistent with the results for anxiety. Our results suggest that the age of diagnosed individuals may have an impact on the observation of an association between familial/genetic risks and gender differences in psychiatric disorders.Our non-significant results for MDD are inconsistent with a recent UK population study of middle-aged adults, which found weak evidence to suggest that rare neuropsychiatric copy number variants may be associated with depression in females.3 Further work is needed to determine whether only certain anxiety subtypes show gender-specific associations with ADHD risk.Follow-up analyses for the association between ADHD in brothers and anxiety in females revealed broadly consistent estimates for several categories of anxiety, albeit only the association in agoraphobia was statistically significant. Although agoraphobia typically onsets in early adulthood, it may be preceded by transient and subthreshold childhood anxiety symptoms.The association in individuals with anxiety was seen for observations from brothers but not sisters. This may be because sisters are less likely to be diagnosed with ADHD, therefore, decreasing the likelihood of observing the same association as seen in the analysis of brothers. However, in index individuals without comorbid ADHD, the estimate for the association between ADHD in sisters and female anxiety increased. In contrast, there was a larger estimated effect of brother's ADHD on female gender in those with comorbid ADHD, with a smaller effect in those without ADHD. This suggests that gender-specific effects may differ depending on the presence of comorbid diagnoses.27 suggesting that genetic factors are likely to be of greater importance in explaining the familial effect we observed. Thus, the results imply that in individuals diagnosed with anxiety disorders, females are more likely to be at familial (likely genetic) risk for ADHD than males, consistent with previously observed molecular genetic effects.11The observed association in anxiety was not significant in the cousin-pair analyses, despite the larger sample size. We could not account for the possibility that shared environmental effects or gene × environment correlations also contributed . However, twin studies have shown a limited role for shared environmental effects in ADHD,11One possible interpretation of these results is that there is gender-specific manifestation of risk factors, where females at genetic risk for psychiatric problems may be more likely to develop, or be diagnosed with, anxiety rather than ADHD or both anxiety and ADHD, whereas at-risk males may be more likely to have ADHD than anxiety. The results suggest that comorbid ADHD and anxiety in females may also be linked to familial risk of ADHD. Although our stratified analyses suggested that the association between brother's ADHD and female anxiety was somewhat stronger in individuals with comorbid ADHD, the confidence intervals overlapped with those in individuals without comorbid ADHD and the opposite pattern was seen for sisters. In previous work, accounting for parent-reported ADHD symptoms did not explain the increased ADHD genetic burden in females with anxiety and depression.v. 43.5% of males). Also, this is plausible given that inattentive ADHD symptoms (which are likely to be less easily identified than hyperactive symptoms) are more common in females29 and the inattentive subtype of ADHD may be underdiagnosed when following ICD criteria. Also, the current diagnostic criteria for ICD and DSM are primarily based on symptoms typically observed in males and may therefore miss symptoms more characteristic of female ADHD.31 It is notable that in index individuals with comorbid ADHD and anxiety in our study, females were diagnosed with ADHD later than males  years; males: mean 18.3 (s.d. = 6.4) years; OR = 1.03, 95% CI 1.02–1.03).The mechanisms for how different psychopathology could emerge in males and females are unknown but could potentially involve hormonal differences and/or gender-specific parenting and social influences. Alternatively, there could be gender-specific diagnostic biases in clinical practice, such as diagnostic overshadowing, where females at genetic risk for psychiatric problems who are presenting with complex psychopathology may be more likely to receive a diagnosis of anxiety instead of ADHD or both anxiety and ADHD, whereas males may be more likely to be diagnosed with ADHD. In this case, females being diagnosed with anxiety could indeed have symptoms of ADHD that are either being missed, do not meet diagnostic symptom criteria or are only diagnosed later. Indeed, in the group of index individuals with comorbid anxiety and ADHD, females were more likely than males to be diagnosed with anxiety before ADHD  is incomplete in the register as these conditions are commonly diagnosed by educational psychologists. This, together with the lower sample size for neurodevelopmental disorders beyond ADHD may have had an impact on the observed results. Further work is needed to determine whether the results of this study extend to a broader range of neurodevelopmental disorders.Our results examining a broader group of neurodevelopmental disorders showed that the observed effect was limited to when ADHD was included in the definition of neurodevelopmental disorders. This suggests that the results may not apply to neurodevelopmental disorders beyond ADHD, despite shared genetic risks reported across these disorders.The results need to be interpreted in light of several limitations. Our effect sizes are rather modest. Furthermore, despite including all individuals in Sweden diagnosed with ICD-10 clinical diagnoses of anxiety disorders, MDD, bipolar and eating disorders, there were relatively few individuals, particularly males, with bipolar disorders and eating disorders. As a result of the use of real-life clinical data, we may have missed individuals who are affected with psychiatric disorders but do not have a recorded diagnosis in the register; as such, our results relate to patterns of diagnoses in individuals who come to clinical attention, who may be the most severely affected. To minimise the possibility of missing information on ADHD in relatives, we applied more stringent exclusion criteria, and this did not substantially have an impact on the results.Our inclusion and exclusion criteria, particularly for several of the sensitivity analyses, resulted in more index males being excluded, which may have had an impact on the representativeness of the data. Finally, because of the selection of the primary cohort on the basis of having full siblings, the results are not representative of individuals with no siblings; therefore, stoppage effects, which may operate when parents do not have further children (for example in the presence of psychiatric difficulties), could have an important impact that we were not able to investigate as part of this study design. Ultimately, a variety of study designs will be needed to determine whether and to what extent gender-specific manifestation of psychiatric risks and gender-specific diagnostic biases may explain the widespread gender differences in prevalence of psychiatric disorders.In short, we report that females with diagnosed anxiety are more likely to have a brother with ADHD, compared with males diagnosed with anxiety. This suggests that familial/genetic risk factors for ADHD may be more likely to manifest as anxiety in females compared with males. This association between familial risk for ADHD and gender of the person affected by a psychiatric disorder does not surpass statistical significance in the context of depression, bipolar disorders or eating disorders, showing limited support for gender-specific effects. Our results support previous work by suggesting that gender-specific manifestation of familial/genetic risk or gender-specific diagnostic biases may play a small role in explaining the gender differences seen in the prevalence of ADHD and anxiety. Further research using molecular genetic approaches will be needed to confirm and extend these results. The results highlight the importance of studying gender differences in prevalence to better understand psychiatric disorders with a view towards improving clinical practice to more effectively target interventions to diagnose and help affected individuals.


In Study 2, the impulsivity measure predicted the 8-year-olds’ ADHD diagnoses over and above concurrent ADHD symptoms (χ2(1) = 10.0, p = 0.002, OR = 5.0, 95% CI: 1.8–14.0). The impulsivity measure showed good concurrent and predictive validity. The measure can be useful for the early identification of children at risk for developing ADHD and externalizing disorders.Impulsivity, comprising deviations of brain-based bottom-up and top-down control processes, has been regarded as a crucial, early emerging marker of a developmental pathway to attention-deficit/hyperactivity (ADHD) and externalizing disorders. In two independent studies , we analyzed the concurrent and predictive validity of a task-based neuropsychological impulsivity measure for preschool children. The sample of Study 1 comprised 102 3–5-year-old children (46% boys). In Study 2, 138 children (59% boys) with elevated ADHD symptoms were recruited and assessed at the ages of 4–5 and 8 years. In both studies, preschool impulsivity was measured by a summary score of neuropsychological tasks on approach motivation and hot inhibitory control. For Study 1, the impulsivity measure was significantly associated with symptoms of ADHD and oppositional defiant disorder (ODD) (χ Several developmentally oriented etiological models on attention-deficit/hyperactivity disorder (ADHD) and externalizing disorders assume that impulsivity constitutes a core deficit which appears early in development. The trait impulsivity model, for example, postulates that impulsivity is a crucial vulnerability factor associated with the development of ADHD and subsequent externalizing disorders, such as oppositional defiant disorder (ODD), conduct disorder (CD), and substance use disorder ,2. It isImpulsivity is a complex construct which can be defined as the tendency to show stimulus-provoked automatic behaviors and non-reflective approach reactions in reward-related contexts, based on a preference for smaller immediate rewards over larger delayed rewards . This chIn recent years, many studies have analyzed the associations of ADHD and ODD and CD (diagnoses and symptoms) with performance in neuropsychological tasks and reward-related functions. With regard to ADHD, meta-analyses of these studies found medium-sized differences between children with ADHD and healthy comparison groups in tasks capturing the preference for an immediate reward  in middle and late childhood and adolescence  and poorGiven the centrality of early emerging impulsivity symptoms in etiological models in ADHD and ODD (the assumption that these symptoms constitute an early vulnerability marker) and the seemingly good validity of neuropsychological measures on hot IC and behavioral approach tendencies in preschool years, it seems worthwhile to analyze whether these measures allow for the early identification of children at risk of developing ADHD and ODD. Longitudinal research on the issue, however, is scarce. Marakovitz and Campbell  followedStudy 1 : The impulsivity score discriminates between children showing high vs. low ADHD and ODD symptoms;Study 2 : In a sample of preschool children at risk for ADHD, the impulsivity score predicts an ADHD diagnosis at school age and adds incremental predictive validity over and above concurrent (preschool) parent- and teacher-reported ADHD symptoms.Sample characteristics , the measurement of ADHD outcomes (symptoms vs. diagnosis), and the psychometric properties of the neuropsychological tasks might have contributed to the different findings. Moreover, exclusively hot IC tasks were used. It could be worthwhile to add tasks concerning behavioral approach tendency. A measure which validly captures early task-based impulsivity would be of high utility. Such a measure would not only allow analyses of developmental trajectories to ADHD and ODD but, moreover, could improve the early identification of children at risk. Therefore, we aimed to analyze the concurrent and predictive validity of a neuropsychological measure on hot IC and approach tendency  in preschool ages (1) in a cross-sectional study involving a community-based sample of preschool children, where the concurrent validity of the task-based impulsivity measure was analyzed, and (2) in a longitudinal study involving a high-risk sample of children with elevated ADHD symptoms, where the predictive validity of the impulsivity score was analyzed. The following hypotheses are tested:To further explore the validity of the impulsivity score, we aim to identify an optimal cut-off for the discrimination between children with and without high symptoms of ADHD and ODD in Study 1. In Study 2, this cut-off score is used to predict the school age ADHD diagnosis.The sample comprised 102 parent–child pairs recruited from child care centers. Children were aged 3–5 years . The following exclusion criteria were applied: IQ < 80, sensory handicaps, motor disabilities, chronic diseases, any medication of the child, and insufficient knowledge of the German language in either the parent or the child. ADHD and ODD symptoms. Symptoms of ADHD and ODD were assessed using the hyperactivity/inattention subscale and the Conduct Problems Scale of the Strengths and Difficulties Questionnaire (SDQ) , completImpulsivity score. Two tasks on hot IC and one task on behavioral approach tendency were conducted. In the Cookie Delay Task by Kochanska , the chiIn the Gift Wrap Task by Kochanska , the chiThe Stranger-with-Toys Task by Asendorpf  is a meaDescriptive statistics of the variables in Study 1 are reported in Following Breaux et al. , to simpIn a preliminary analysis, we calculated the unadjusted correlations of the impulsivity score (and the three single tasks) with the parent- and teacher-reported ADHD and ODD scores. To identify potential confounding variables, we analyzed whether the age and gender of child or parental education level were significantly associated with the ADHD, ODD, or impulsivity scores. In further analyses, we adjusted for those variables which showed significant associations. To test our hypotheses, hierarchical logistic regression models  were usep = 0.034) and parental education level  showed significant associations with some of these variables. We therefore adjusted for the gender of the child and parental education level in the logistic regression analyses.The age of the child was not associated with the SDQ Hyperactivity/Inattention score, the SDQ Conduct Problems score, the ADHD or ODD groups, or the impulsivity score (r between −0.01 and 0.14). The gender of the child . Controlling for the gender of the child and parental education level in the logistic hierarchical regression models did not change these results . The sensitivity and specificity were 0.56 and 0.61 for the ADHD groups and 0.73 and 0.61 for the ODD groups.n = 85, 59% boys) with elevated ADHD symptoms were recruited from child care centers. At recruitment, parents completed an ADHD screening questionnaire (see below). Children who scored in the upper quartile  were considered. Exclusion criteria from the study sample were an IQ below 80, chronic diseases involving brain functions, any continuous pharmacological treatment, and insufficient German language skills of the parent or child. A sample of n = 138 four-to-five-year-old  children (Examinations were carried out at the child care centers by two trained investigators. The first investigator worked with the child while the second investigator scored the child’s performance in the tasks. The investigators were unaware of the child’s ADHD or ODD symptom scores.t-scores between −1.17 and 1.30), the gender of the child (χ2(1) = 0.29), and paternal education level (χ2(3) = 0.30). However, children who did not attend the 8 year assessment had mothers with lower education levels (χ2(3) = 8.48, p = 0.037). The 8 year assessment was carried out at our lab. An investigator (psychologist), who was blind to all data of the first assessment wave, conducted the clinical interview with the mother.A total of 122 children and their parents took part in the second assessment wave at 8 years . The 17 children who dropped out from the assessment did not differ from the remainder with respect to parent- or teacher-rated ADHD and ODD symptoms, the age of the child in months  of the Diagnostic System for Psychiatric Disorders (DISYPS-II) . The parImpulsivity. Impulsivity was measured according to the procedure described above (Study 1). In the present study, inter-rater reliability for the three neuropsychological tasks was checked in 20% of cases and proved to be very good .ADHD and ODD symptoms and diagnoses. At the age of 8 years, the ADHD and ODD/CD diagnostic modules of the Child and Adolescent Psychiatric Interview (CAPA) by Angold et al. [n = 29 (24%) received an ADHD diagnosis. d et al.  were cond et al. . Of the The ADHD and ODD symptoms of the child were additionally assessed by parent and teacher questionnaires. Parents and school teachers completed the FBB-ADHS of the DISYPS-III . The quep < 0.001 and 0.80, p < 0.001; ODD score: parent SDQ–Conduct Problems scores, teacher SDQ–Conduct Problems scores, CAPA-ODD score: r between 0.30, p = 0.004 and 0.51, p < 0.001).Descriptive statistics of the variables in Study 2 are reported in In a preliminary analysis, the unadjusted correlations of the impulsivity score (and the single tasks) with all ADHD and ODD scores (symptom scores and diagnoses) were calculated. Hierarchical logistic regression models were used to test whether the impulsivity score predicted the 8 year ADHD diagnosis. In the first regression model (Model 1), the gender of the child and parental education level were controlled ). In a second regression model (Model 2), we additionally controlled for the Wave 1 mother- and teacher-reported ADHD symptom scores (Step 1) in order to test whether the impulsivity score contributed to the prediction of the Wave 2 ADHD diagnosis over and above the concurrent ADHD symptoms. Finally, ROC analysis on the prediction of the Wave 2 ADHD diagnoses was used to validate the impulsivity cut-off score identified in Study 1. Logistic regression analyses on the prediction of ADHD diagnosis (Models 1 and 2) by the impulsivity cut-off score were conducted to explore the predictive effects.Preliminary analyses. The impulsivity score was significantly associated with the Wave 1 and 2 ADHD and ODD scores .D scores . ChildreIncremental predictive validity of the impulsivity score. The impulsivity score significantly predicted the ADHD diagnosis over and above the gender of the child and maternal education level  as in Study 1: sensitivity for the prediction of the 8 years ADHD diagnosis was 0.76, and the specificity was 0.70. The impulsivity cut-off score significantly predicted the Wave 2 ADHD diagnosis (Models 1 and 2). The effect sizes (odds ratios) were of medium to large magnitudes and indicated 6.6-fold . In a high-risk sample, Breaux et al.  adjustedWe found that a neuropsychological impulsivity measure improved the identification of children at risk of developing ADHD until school age. High impulsivity added 10% of the explained variance in the school-age ADHD diagnoses over and above the preschool ADHD symptoms, the gender of the child, and the parental education level. In future research, it would be worthwhile to further evaluate  and improve  the neuropsychological impulsivity measure. Such a measure can be useful from a clinical perspective, enabling children who are actually at risk of developing ADHD to be identified early on and receive support and treatment. False positive decisions could be avoided in those who are likely to outgrow their behavior problems.The presented research on the validity of our neuropsychological impulsivity summary score has several strengths, such as the use of two independent samples, the use of different, well-validated measures of ADHD and ODD symptoms as criterion variables, and a theory-driven selection of neuropsychological tasks. Limitations, however, might be seen in the rather small sample sizes of the two studies and the inclusion of just three neuropsychological tasks. Our primary intention was to conduct cross-validation of the link between the neuropsychological impulsivity measure and ADHD and ODD. Though the sample sizes of the two studies were rather limited, the result that the measurement adjusted on the basis of the first study showed good predictive validity, and the second study points to the generalizability of the link. As already mentioned, an extension of the impulsivity measure by further neuropsychological tasks on hot IC or approach tendency could increase the reliability and validity of measurements. Furthermore, it could be worthwhile to substitute the Gift Wrap Task with another task, due to the difficulty to conduct the task’s second part outside of a video lab.To conclude, in preschool age trait impulsivity can be validly measured by a neuropsychological task-based measure. The measure proved to be useful for ADHD risk prediction. In longitudinal research on developing ADHD and externalizing disorders the impulsivity measure might be useful for an unbiased, valid assessment of an indicator of the early emerging vulnerability.


Individuals with ADHD are at increased risk for poor occupational outcomes. Educational attainment and psychiatric comorbidity may be important contributing factors for these outcomes, but the role of these factors is not well characterized. This study aimed to investigate the associations between ADHD and occupational outcomes, and to examine the influence of educational attainment, comorbid developmental disorders and intellectual disability on these associations.We linked the Swedish population graduating from compulsory school 1998–2008 (N = 1.2 millions) to population-wide register-based data on clinical psychiatric diagnoses and medications, objective annual measures of educational, and occupational outcomes. Individuals were followed for between 6 to 16 years after graduation.Individuals with ADHD had annually on average 17 percent lower income, ratio = 0.83 (95% CI 0.83–0.84), 12.19 (11.89–12.49) more days of unemployment, and a higher likelihood of receiving disability pension, odds-ratio = 19.0 (18.4–19.6), compared to controls. Comorbid diagnoses of intellectual disability and developmental disorder explained most of the association between ADHD and disability pension, while lifetime educational attainment partially explained associations between ADHD and all occupational outcomes. Analyses of occupational trajectories found that income was lower and unemployment elevated relative to controls with the same educational attainment. Higher educational attainment correlated with higher income similarly among individuals with ADHD and controls after accounting for individual background factors.The occupational burden associated with ADHD is substantial. Comorbid developmental disorders, intellectual disability and educational difficulties  from childhood to adulthood are important factors to consider when designing interventions to improve occupational outcomes in individuals with ADHD. Attention-deficit/hyperactivity disorder (ADHD) is a prevalent, childhood-onset neurodevelopmental disorder affecting around 5 percent of children , and 2.8Studies based on ADHD symptoms have found negative associations with income, and positive associations with welfare dependence, work disability, and unemployment –11. ReseOverall, the current knowledge on ADHD and occupational outcomes is based on cross-sectional, often self-reported measures, of ADHD and occupational outcomes. We advance this field using longitudinal, individually linked, register-based measures of occupational outcomes covering the entire Swedish population of compulsory school graduates 1998–2008. These data comprised 1.2 million individuals, including 28.9 thousand diagnosed with ADHD, which contributed 12.4 million yearly observations of occupational outcomes among controls and 285 thousand among individuals with ADHD to our analyses. We addressed three questions regarding ADHD and occupational outcomes. First, we estimated the association between ADHD and occupational outcomes. Second, we explored the role of developmental disorder and intellectual disability, and lifetime EA, in explaining these associations. Third, we examined the influence of EA on occupational trajectories before and after an educational completion while accounting for unobserved individual characteristics, including genetic factors.We extracted data on all 1,221,453 individuals graduating from Swedish compulsory school between 1998 and 2008 from the National School Register (NSR). With few exceptions, these individuals were born 1982–1992 as compulsory school graduation in Sweden usually takes place the year individuals turn 16. These individuals were then linked to demographic information in the Total Population Register (TPR), and annual measures of occupational outcomes and EA from 1998 to 2013 in the Longitudinal Integration Database for Health Insurance and Labor Market Studies (LISA). LISA covers the entire Swedish population aged 16–65. The National Patient Register (NPR) and the Prescribed Drug Register (PDR) were used to gain information on medical diagnoses and dispensed medication respectively. The NPR includes principal and secondary in/outpatient diagnoses (outpatient data since 2001) coded using the International Classification of Diseases (ICD). The PDR includes information on the Anatomical Therapeutic Chemical classification system (ATC) for each medication dispensed since July 2005. The Regional Ethics Committee in Stockholm, Sweden, approved this study (diary number 2013/862-3115). Research using anonymized Swedish register data does not require informed consent from participants.Income was indexed by disposable income (100s of Swedish crowns) equal to the difference between income from assets  and liabilities . We inflated income to 2019 prices, and converted to euro   = 0.83), 11.59 more days per year of unemployment, and a 18.99 times higher odds, risk difference (RD) = +11.0%, of disability pension, compared to individuals without ADHD , and more than halved the odds-ratio of disability pension to 5.68, RD = +3.0%, while unemployment was marginally affected. Adjusting for lifetime secondary and tertiary EA influenced the income ratio, and the risk of disability pension similar to adjusting for comorbid ID/DD, but also attenuated the unemployment difference to 8.33 days .residual panels of The The modeled differences between individuals with and without ADHD are plotted in the lower panels (“modeled”) of While these results show lower income and higher unemployment among individuals with ADHD relative to controls, the extent to which EA influences occupational trajectories within these groups is less clear. The sensitivity analyses of income trajectories concurred with the overall patterns, but suggested smaller differences between individuals with and without ADHD for the models including a lagged outcome only, while models including individual fixed-effects only suggested stronger differences .Using virtually the entire Swedish population of 1,196,744 individuals graduating compulsory school 1998 to 2008 with up to 16 years of follow-up, this study identified several important associations between ADHD and occupational outcomes, and their interplay with EA and comorbid DD/ID. The association between ADHD and lower income, higher unemployment, and a higher risk of disability pension, was not entirely explained by comorbid DD/ID or EA. Lifetime EA explained part of the associations between ADHD, income and unemployment respectively, while comorbid DD/ID were important determinants of the elevated risk of disability pension in ADHD. Notably, when accounting for unobserved individual characteristics, the longitudinal effects of ADHD on income and unemployment within the same EA, were roughly similar in magnitude relative to controls in all educational strata . This suggests that important differences in occupational outcomes between individuals with and without ADHD are driven by other factors than EA. These results are of importance for policy makers and other stakeholders, both in terms of awareness of occupational functioning amongst individuals with ADHD, and regarding expectations on education as a determinant of future occupational performance among individuals with ADHD.Our findings regarding the crude associations between ADHD and occupational outcomes are consistent with previous research using similar definitions of ADHD, and research exploring associations with other psychiatric conditions and behavioral problems , 39–41. Three important patterns emerged regarding the role of EA for the association between ADHD and occupational outcomes. First, we found that adjusting for lifetime EA decreased the income difference between individuals with and without ADHD, which is consistent with a recent molecular genetic study reporting that the association between the polygenic risk of ADHD and income was mediated by EA by 14–58 percent , and a nFinally, these patterns challenge the idea that post-compulsory EA itself explains occupational differences between individuals with and without ADHD. Factors in ADHD that correlate with occupational outcomes, such as genetic predispositions  and deveOur study strengthens the existing knowledge on ADHD and occupational outcomes, and also extends the knowledge base by proposing a longitudinal role of EA for occupational outcomes in ADHD. Contrary to many prior studies on this topic, we had access to a large sample of individuals with ADHD in combination with objective measures of both occupational outcomes and EA. Nevertheless, we would like to highlight several important limitations.First, the generalizability of our results may be sensitive to the presence of pharmacologically treated ADHD. In our sample, 83 percent of individuals with ADHD had at some point been dispensed an ADHD medication. While the comparison group here is small, we find that individuals with ADHD and a dispensed ADHD medication had slightly lower income , lower unemployment , and higher risk of receiving disability pension  compared with those without such a dispensation . A more Second, the rates of clinically diagnosed ADHD in our source population increased from 1.7 percent among 1998 compulsory school graduates to 3.6 percent among those graduating in 2008 . The cliThird, as our unemployment measure only captures individuals actively seeking employment through the official employment agency, correlations between ADHD and other means of employment seeking may bias our estimates.Fourth, regarding generalizability of our results, access to education is facilitated by tuition-free universities and state-provided financing for students in Sweden. Such policies, and labor market structures , are important variables to consider when comparing our findings to that of other countries.ADHD is associated with poor occupational outcomes, and differences in psychiatric comorbidity and EA partly explain these associations. Although higher EA is associated with higher income and lower unemployment among individuals with ADHD over time, disparities between individuals with and without ADHD are persistent, and exist regardless of EA. When designing policies to improve occupational outcomes for individuals with ADHD, educational interventions should likely be weighed against alternatives  while considering the impact of factors such as age, comorbid intellectual disability and developmental disorders.S1 File(DOCX)Click here for additional data file.S1 Method(DOCX)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.


Tourette syndrome (TS) is a neurodevelopmental disorder characterized by motor and vocal tics. Co-occurrence of attention-deficit/hyperactivity disorder (ADHD) or obsessive–compulsive disorder (OCD) is very frequent in the pediatric population as well as the presence of an impairment of the executive functions. The aim of our study was to investigate motor timing, that is, the temporal organization of motor behavior, in a pediatric population of Tourette patients. Thirty-seven Tourette patients (divided in 22 “pure” Tourette patients and 15 with ADHD) were compared with 22 healthy age- and gender-matched subjects. All subjects underwent a neuropsychiatric screening and were tested for their planning and decision-making abilities by using a standardized test, such as Tower of London (ToL). Two experimental paradigms were adopted: finger-tapping test (FTT), a free motor tapping task, and synchronization–continuation task. An accuracy index was calculated as measure of ability of synchronization. We found that “pure” TS as well as TS+ADHD showed lower scores in the FTT for the dominant and non-dominant hands than controls. Moreover, in the synchronization and continuation test, we observed an overall lack of accuracy in both TS groups in the continuation phase for 2,000 ms , interestingly, with opposite direction of accuracy index. Thus, “pure” TS patients were classified as “behind the beat,” whereas, TS+ADHD as “ahead of the beat.” The performance in the finger tapping was inversely correlated to ToL total scores and execution time, whereas we did not find any correlation with the accuracy index of the synchronization and continuation test. In conclusion, here, we explored motor timing ability in a childhood cohort of Tourette patients, confirming that patients exhibit an impaired temporal control of motor behavior and these findings may be explained by the common underlying neurobiology of TS and motor timing. Tourette syndrome (TS) is a childhood-onset neurodevelopmental movement disorder clinically characterized by the presence of multiple motor tics and one or more phonic/vocal tics that last for more than 1 year . Pure TSvia a decision mechanism to previously important duration codes held in reference memory. Functional imaging studies in humans and lesional studies in animals pointed out the role of basal ganglia nuclei and cortico-striato-thalamo-cortical (CSTC) circuitry  and “pure” TS (n = 22).Thirty-seven patients with TS diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders (DSM)-5 criteria were prospectively recruited from the outpatient Movement Disorder Clinic of Bambino Gesù Children's Hospital of Rome. Inclusion criteria were  a defineParticipants were tested individually in a quiet room in the Movement Disorder Clinic of the Department of Neuroscience; tests administration was performed with clinical feasibility to avoid fatigue, with frequent breaks and chats with the children along the evaluation, in order to make them feel more comfortable and increase the focus on the activities. Each session entirely lasted from 2 to 3 h. For the computer-based tests, we used a 17-inch laptop for the presentation of stimuli and recording the responses by the participants. The LCD screen had a resolution of 1,440 × 900 pixels and a refresh rate of 60 Hz. A standard Italian keyboard was used as response keys. The background luminance of the screen was a mid-level gray measured at a viewing distance of 65 cm.Raven's Progressive Matrices) and (Tower of London [ToL] test) was performed. All TS patients were tested for tics severity and impairment using a questionnaire interview, the Yale Global Tic Severity Scale (YGTSS). For a detailed description of the tests, see All subjects underwent a structured neuropsychological evaluation. They were tested by either a neuropsychologist or a child neuropsychiatrist. We performed a screening for neuropsychiatric comorbidities using parent report questionnaire to assess ADHD, obsessions and compulsions disorder, anxiety disorder, mood disorder, and conduct disorder . Children's Yale–Brown Obsessive–Compulsive Scale (CY-BOCS) was used for obsessions and compulsions severity score. Furthermore, a structural neuropsychological evaluation including  a non-veces) and  a spatia®) downloaded on a laptop computer. Inquisit is a software widely used for experimental psychology (see www.millisecond.com for the latest research papers with Inquisit); in the Millisecond® website, a test library is provided with downloadable tests (and scripts) including those used for this study. For the SM tones at 500 Hz and 70 dB, sound pressure level (SPL) (duration 50 ms) were delivered through headphones. Subjects performed a single hand task.To assess motor timing, we used the finger-tapping test (FTT) and the sensorimotor test (SM), performed using the Inquisit 5.0 software , adapted from the original Finger Tapping Oscillation Test part of Halstead's test battery of 1947 and later modified  implementing a Paced Motor Timing procedure described by Wittmann et al.  expressed as mean (±SD) of time intervals in ms between taps as measure of tapping speed for both Condition A and Condition B. For Condition A, we also considered the invalid responses (IRs) as the number of motor reactions (responses after 120 ms from the onset of tone) per trial as a further measure of synchronization ability. For Condition B, we considered TI expressed as mean (±SD) of time intervals in ms between taps. Additionally, for both Conditions A and B, we calculated an accuracy index (Δi) as follows: subjective time (TI)/t where subjective time was the TI produced experimentally by the subject, and t is the objective time, i.e., the base interval set . This index provided the directionality of the tapping performance, being >0 if the subject was behind the beat and <0 if the subject is ahead of the beat; moreover, it was a direct measure of the magnitude of the error in reproducing the corresponding time interval. To complete the two subtests, it takes approximately 6 min. Δi was used in all statistical analysis performed.We used a script available from Millisecond Softwaren et al. . In the n et al. : (a) in t test (where appropriate) and analysis of covariance (ANCOVA) (considering “age” as covariate) with Bonferroni post-hoc test for comparison between the groups, at 95% CI. For finger-tapping results, we used a two-way ANOVA, in which GROUP was analyzed as between-subjects factor, and HAND (DH vs. n-DH) as within-subjects factor. Post-hoc comparisons were performed using the Bonferroni post-hoc test. Linear least-square regression and generalized linear model (GLM) analyses were used in the selection of predictors. GLM estimation was performed by stepwise method with 95% CI. Significance was assigned for p < 0.05. All analyses were performed using SYSTAT software version 13.0 for Windows.Results are reported as mean ± SD. Normal distribution was assessed by Shapiro–Wilk test and Levine test for normality. Differences between means were assessed using unpaired two-tailed n = 22) and 40% with ADHD comorbidity . Male to female ratio was 3:1 in both groups. All patients and healthy controls did not take any medication. Participants' ages range between 8 and 14 years old, and no statistical significance was found between the groups . IQ levels were 108.3 ± 13.4 in the “pure” TS group, 110.1 ± 12.1 in the TS+ADHD group, and 114.5 ± 11.7 in the control group, showing no statistical significance between the groups . Total mean score of the YGTSS rating scale for all TS was 41.3 ± 15.4 (maximum score 100). The two TS groups did not differ for YGTSS scores . Total mean score of the CY-BOCS rating scale for all TS patients was 8.6 ± 1.3 (maximum score 40); particularly, the range of severity of patients with obsessions and compulsions was subclinical in 32/37 (86.5%), mild in 2/37 (5.4%), moderate in 1/37 (2.7%), and severe in 2/37 (5.4%). CBCL and CPRS scores showed statistical differences between “pure” TS and TS+ADHD in several domains including those related to ADHD comorbidity. Twenty-two pediatric healthy controls and 37 TS patients were included in the analysis. In the patients' group, 60% had a diagnosis of TS without any comorbid condition . In particular, we found a lower mean number of taps in both the Tourette groups, “pure” TS and TS+ADHD, respectively, 56.29 ± 5.78 (SD) (p = 0.03 post-hoc Bonferroni test) and 54.94 ± 7.03 (SD) (p < 0.01 post-hoc test) vs. controls, 63.28 ± 8.42 (SD). No statistical difference was found between “pure” TS and TS+ADHD. In non-DH trials, the “pure” TS group differed statistically from controls , whereas no difference was found between controls and TS+ADHD (48.97 ± 8.70 vs. 55.81 ± 7.99). Considering in the model “HAND” as factor within each level (GROUP), we found that both controls and “pure” TS patients showed a statistically significant reduction with non-DH compared with DH . We did not find any difference in TS+ADHD between DH and non-DH.In the FTT, DH as well as non-DH were tested. There was a statistical significance difference in mean number of taps  performed with DH between the three groups  for the interval (t) tested, 1,000 ms, all groups did not differ in a statistical manner. Δi1,000 values were 1.04 ± 0.04 for controls, 1.02 ± 0.05 for “pure” TS, and 1.02 ± 0.06 for TS+ADHD, indicating a good synchronization accuracy (Δi = 1). Mean number of IRs in each group per trial (reactions) confirmed the data. For the interval t, 2,000 ms, we did not find any statistical difference between the groups . Mean number of IRs confirmed the lack of difference between the groups. unpaced signals), Δi1,000 did not differ significantly between the three groups . Conversely, in the task for 2,000 ms, both TS groups showed a statistical difference compared with controls , adjusted for age; in particular, “pure” TS showed a Δi2,000 value >1 , indicating a direction of synchronization “behind the beat,” whereas TS+ADHD' Δi2,000 was <1 , placing these patients significantly “ahead of the beat.” A statistical significance was found comparing “pure” TS and TS+ADHD (p < 0.001). Data are summarized in In the SM task, we tested the ability of subjects to synchronize their taps with pacer signals with or without played beep . An accuracy index (Δi) was calculated as described in the Materials and Methods section to directly assess the accuracy of synchronization along with the directionality (earlier or later) of tapping. Additionally, in Condition A, we considered IRs as adjunctive measures of synchronization. In Condition A (p < 0.001) for the total score, with no effect of age in the model. As regards the total execution time, we found a statistical difference between the three groups. Post-hoc analysis showed a statistical significance between TS+ADHD and controls (p < 0.001), whereas no differences were found between “pure” TS and controls. Additionally, TS+ADHD patients showed statistically lower mean solution time values than controls (p = 0.045). No differences were found in the time of first move between the groups.We measured planning and decision-making abilities using the standardized ToL test. Results are summarized in p > 0.05), suggesting that tic severity did not impair per se motor performance. IQ and YGTSS did not correlate also with accuracy index for sensorimotor continuation test . We addressed the interplay between motor timing and ToL (planning and decision-making abilities). We used a GLM to statistically test this hypothesis. In the first model, we assigned as dependent variable the “mean of finger tapping” (DH) of FTT and as independent variables “groups” , the mean execution time, the total score, and interactions between them . We found that FTT scores were statistically correlated to group as expected, whereas in the “group × mean execution time” interaction, finger tapping scores and execution time were inversely related (p < 0.001). In the second model, we set as dependent variable the Δi2,000 and as independent factors the mean execution time, the total score, and interactions between them. We did not find any significant correlation.Comparing the finger tapping scores with the IQ and tics severity (YGTSS), we did not find any correlation , classifying “pure” TS as “behind the beat” and TS+ADHD as “ahead of the beat,” on the basis of their accuracy index. We also found that there is a lack of correlation between the timing accuracy and the tics severity scores (YGTSS), as well as with IQ scores. Martino et al.  recentlyADHD comorbidity is considered a confounding factor generating conflicting results in TS studies . On the review, see  for their planning and decision-making abilities by performing a standardized task, such as ToL test, as measure of their “executive” function. We found that the pure TS and TS+ADHD groups compared with the control group showed lower scores in the total score, whereas the total execution time was lower in the TS+ADHD group. Planning skills refer to the capacity to organize cognitive and motor behavior in order to perform different steps needed to reach a goal . TermineHowever, we did not find any correlation between continuation ability and ToL performance, suggesting that not all organization of motor timing is dependent from planning ability, and this is true for both groups independently from ADHD co-occurrence.Finally, some limitations of the study need to be addressed. First, note the small sample size of our cohort and, in particular, of the TS+ADHD group. Second, as a pure ADHD group is lacking, our results cannot be readily transferred to ADHD patients. Finally, although all our efforts have been made to minimize confounding factors, we cannot exclude intrinsic  bias in the selection of patients.In conclusion, we demonstrated that children with TS show an impaired motor timing organization in the free tapping and in the continuation task (tempo synchronization). Moreover, findings in TS+ADHD confirm that, at least in part, TS groups differ, signifying that ADHD co-occurrence should be considered in all studies involving TS to not exclude possible confounding effect.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Bambino Gesù Local Ethical Committee. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.FG, CP, and AC contributed to the conception and design of the study. CP, FG, and LD organized the database. FG and AC performed the statistical analysis. FG wrote the first draft of the manuscript. FG, AC, PC, and FV wrote sections of the manuscript and critically discussed the final version. All authors contributed to manuscript revision, read and approved the submitted version.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. The handling Editor declared a shared affiliation, though no other collaboration, with some of the authors FG, LD, and PC. The reviewer TS declared a shared affiliation, with no collaboration with some of the authors FG, LD, and PC to the handling Editor.


Chronic obstructive pulmonary disease (COPD) is an incurable and debilitating chronic disease characterized by progressive airflow limitation associated with abnormal levels of tissue inflammation. Therefore, stem cell-based approaches to tackle the condition are currently a focus of regenerative therapies for COPD. Extracellular vesicles (EVs) released by all cell types are crucially involved in paracrine, extracellular communication. Recent advances in the field suggest that stem cell-derived EVs possess a therapeutic potential which is comparable to the cells of their origin.t test was used to compare between 2 groups. Comparison among more than 2 groups was done using one-way analysis of variance (ANOVA). Data presented as median ± standard deviation (SD).In this study, we assessed the potential anti-inflammatory effects of human umbilical cord mesenchymal stem cell (hUC-MSC)-derived EVs in a rat model of COPD. EVs were isolated from hUC-MSCs and characterized by the transmission electron microscope, western blotting, and nanoparticle tracking analysis. As a model of COPD, male Sprague-Dawley rats were exposed to cigarette smoke for up to 12 weeks, followed by transplantation of hUC-MSCs or application of hUC-MSC-derived EVs. Lung tissue was subjected to histological analysis using haematoxylin and eosin staining, Alcian blue-periodic acid-Schiff (AB-PAS) staining, and immunofluorescence staining. Gene expression in the lung tissue was assessed using microarray analysis. Statistical analyses were performed using GraphPad Prism 7 version 7.0 . Student’s Both transplantation of hUC-MSCs and application of EVs resulted in a reduction of peribronchial and perivascular inflammation, alveolar septal thickening associated with mononuclear inflammation, and a decreased number of goblet cells. Moreover, hUC-MSCs and EVs ameliorated the loss of alveolar septa in the emphysematous lung of COPD rats and reduced the levels of NF-κB subunit p65 in the tissue. Subsequent microarray analysis revealed that both hUC-MSCs and EVs significantly regulate multiple pathways known to be associated with COPD.In conclusion, we show that hUC-MSC-derived EVs effectively ameliorate by COPD-induced inflammation. Thus, EVs could serve as a new cell-free-based therapy for the treatment of COPD.The online version contains supplementary material available at 10.1186/s13287-020-02088-6. These cells, in turn, release multiple inflammatory cytokines, proteinases, and chemokines that together contribute to the degeneration of lung parenchyma . The approved protocols for the animal study were based on the Guidelines for the Care and Use of Animals for Scientific Purposes  rats (250–350 g) aged 8–9 weeks (ses (USM ) which wn = 6): naive (untreated group), CS (injury group), CSSH , hUC-MSC-EVs (hUC-MSC-EVs-treated group), hUC-MSCs (hUC-MSC-treated group), and hUC-MSC-CM (hUC-MSC-conditioned media-treated group). All groups except naive were exposed to sidestream cigarette smoke for 15 min per session, 6 cigarettes for 2 sessions, 7 days a week, for 12 weeks in a smoking chamber -accredited laboratory in Animal Research Facilities, Advanced Medical and Dental Institute (IPPT), Universiti Sains Malaysia. The experimental procedure was conducted as previously described by Zheng et al.  with sli6), hUC-MSC-EVs isolated from 2.5 × 106 hUC-MSCs, and hUC-MSC-CM concentrated from 2.5 × 106 hUC-MSCs were used in the experiment. The naive and CS groups were euthanized on day 85; meanwhile, the rest of the groups were euthanized on day 99. Rats were euthanized by using intravenous injection of pentobarbital (200 mg/ml) .Treatments were given via intratracheal delivery in 150-μl vehicle (1xPBS) on day 85 post-cigarette induction. Rats were anaesthetized intravenously by using ketamine (50 mg/kg) and xylazine (5 mg/kg). hUC-MSCs  was collected from the rat tail vein and placed into a 1-ml EDTA tube  and subjected to whole blood count using the Cell Dyn Hematology Analyzer .Lm), which measures the enlargement of the alveolar space. Measurement was done by using × 40 objective and × 10 eyepiece, and photos of the sections were taken and superimposed with 30 × 30-μm grid. Ten pictures of 2 slides per animal with a total of 5 animals per group were captured. The number of alveolar intercepts along the gridline was counted and calculated based on the following formula as described previously  staining was performed for the analysis and scoring of peribronchial and perivascular inflammation, alveolar inflammation, and emphysema. Meanwhile, Alcian blue-periodic acid-Schiff (AB-PAS) staining was performed for the analysis of goblet cell count. Scoring of inflammation within the airway was conducted using a semi-quantitative analysis. Slides were blindly coded before a pathologist scored the tissues. The inflammation scoring was performed using the scale of 0 to 3 based on the presence and intensity of inflammatory cell infiltration in the peribronchial and perivascular areas. Two slides were analysed per animal, with a total of 5 animals per group. The score was done according to the following parameters: 0, no inflammation detected; 1, occasional cuffing with inflammatory cells; 2, most bronchi and vessels are surrounded by a thin layer of inflammatory cells (1–5 cells thick); and 3, most bronchi and vessels are surrounded by a thick layer of inflammatory cells (> 5 cells thick). Alveolar inflammation scoring was done by grid on tissue section photos captured by fluorescence microscopy . One hundred points were counted on random areas on the slides. Ten areas were analysed on 2 slides per animal with a total of 5 animals per group. Goblet cells were counted using light microscopy . Five hundred cells were counted, and the number of goblet cells was divided by the total cells to get a percentage of goblet cells. One slide per animal with a total of 5 animals per group was assessed. Emphysema was evaluated by using the mean linear intercept (N = number of lines across the photographed areaL = length of the line across the photographed aream = number of interceptst test to identify significant changes (p < 0.05) between the sample and control groups. Genes with p < 0.05 and fold change > 2.0 were filtered as significantly regulated. Volcano plot, heat map, principal component analysis, Venn diagram, and pathway analysis were generated using the Gene Spring software. Gene Ontology analysis using Panther (www.pantherdb.org) was used to classify differentially expressed genes (DEG) by its functional role. GO terms with p < 0.05 was considered significantly enriched by DEG.RNA extraction was performed on 30 mg of rat lung from the naive, CS, hUC-MSCs, and hUC-MSC-EVs groups using the RNeasy Mini Kit  following the manufacturer’s instructions. The purity and concentration of RNA were measured by NanoDrop ND1000 . RNA integrity was determined by Agilent RNA 6000 Nanokit . cDNA was synthesized and hybridized at 65 °C for 17 h and viewed using the Agilent SureScan Microarray Scanner . Comparison between different sample datasets was normalized and analysed using the Gene Spring software. The sample datasets were subjected to Immunofluorescent staining was performed to study the expression of NF-κB subunit p65. Briefly, tissue sections were deparaffinized in xylene and rehydrated in graded ethanols. The tissues were blocked with 5% goat serum for 30 min and incubated with primary antibody mouse monoclonal NF-κB-P65 (F-6)  1:200 for 1.5 h in room temperature. After washing with PBS, slides were incubated with secondary antibody Alexa Fluor 555 goat anti-mouse IgG (H+L)  and counterstained with DAPI 1:2000 in 1xPBS, and viewed under the IX71 Fluorescence Microscope .p ≤ 0.05, whereas p ≤ 0.001 was considered to be highly significant.Statistical analyses were performed using GraphPad Prism 7 version 7.0 . Comparison among more than 2 groups was done using one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. Data presented as mean ± standard deviation (SD). Differences are considered to be statistically significant when Mesenchymal stem cells that were isolated from the human umbilical cord blood were subjected to flowcytometry and differentiation analysis. hUC-MSCs were positive for CD73, CD90, CD105, and CD166 and negative for CD34, CD45, CD31, and HLA DR DP DQ Table .Table 1hUC-MSC-EVs were isolated by differential centrifugation to remove cell debris and apoptotic bodies. hUC-MSC-EVs pellet suspended in 1xPBS was characterized based on morphology, size distribution, and protein marker expression. Energy-filtered transmission electron microscopy examination showed hUC-MSC-EVs were rounded in shape with an average size of 200 nm Fig. a. Westerp < 0.05), whereas a slight non-significant decrease in response to hUC-MSC-EVs and hUC-MSC-CM Fig. To study the effect of hUC-MSC-EVs on the circulating immune cells, the peripheral blood was collected and subjected to full blood count. Figure p < 0.001, ***p < 0.0001)  Fig. c, d.FigThe accumulation of immune cells  in the lung is a key marker for the development of chronic inflammation in COPD. Figure p < 0.05) as compared to CS and self-healing (CSSH), and far better than the groups that received treatments with hUCMSC-EVs and hUCMSC-CM  reduction in the mean linear intercept of the alveolar pores and restoration of tissue was observed following treatment with hUC-MSC-EVs. Meanwhile, a non-significant reduction was observed in hUC-MSCs and hUC-MSC-CM.To study the effect of CS and treatment intervention on emphysema, the mean linear intercept of the alveolar pores were measured on H&E histological slides Fig. a. Quantip65 is a subunit of the prototypic, pro-inflammatory transcription factor NF-κB. Translocation of p65 to the nucleus of a cell is indicative of the cell pro-inflammatory response. To study the translocation of p65 into the nuclei of cells, IHC stained and imaged lung tissue sections were quantified following CS and treatment intervention. A significant increase in the percentage of p65-positive cells was observed in the CS group. Following 2 weeks of self-healing, a significant reduction of p65 was observed. Treatment with hUC-MSC-EVs, hUC-MSCs, and hUC-MSC-CM further reduced the p65 expression in the CS-exposed lung Fig. .Fig. 9Ip < 0.05) are considered significant for further investigation to understand the biological, cellular, and molecular functions. CS exposure was shown to lead to a total of 17,689 DEG, while treatment with hUC-MSC-EVs and hUC-MSCs is shown to have led to 15,160 and 23,485 DEG, respectively  in the CS, hUC-MSC-EVs, and hUC-MSCs groups, but high variation was observed in the naive group . Differentially expressed genes (DEG) which has been upregulated or downregulated more than the two fold difference  and treatment (hUC-MSCs and hUC-MSC-EVs) groups. Table Our study aimed to determine the effects of hUC-MSC-EVs in comparison with hUC-MSCs for the treatment of COPD. The therapeutic potential of MSCs and MSC-derived secreted factors have been widely demonstrated in various diseases, including rheumatoid arthritis, asthma, and Crohn’s disease , 64, 77.Meanwhile, the differentiation analysis showed the ability of hUC-MSCs to differentiate into adipocyte, osteocyte, and chondrocyte. hUC-MSC-EVs isolated from hUC-MSCs showed a rounded morphology with an average of 153 nm in diameter, and protein analysis showed a positive marker for CD63 exosomal marker. Following 12 weeks of CS exposure, the evidence of accumulation of inflammatory cell infiltrated in peribronchial and perivascular tissues as well as the parenchyma, goblet cell hyperplasia, expression of p65, and the development of emphysema, was consistent to that of previously published studies , 94 indiCigarette smoke is the leading risk factor of COPD, with over 80% of all COPD cases attributed to cigarette smoking. Therefore, cigarette smoke is widely employed by the researchers to develop the in vivo COPD model over other inducers such as biomass fuel, lipopolysaccharide, and elastase , 31, 39.COPD is characterized by airway and parenchymal inflammation that leads to mucus overproduction and emphysema, although these characteristics may not be present in all patients, as the emphysematous lung only occurs in 20% of all COPD patients , 14. NevAirway inflammation begins with the disruption of the airway and vascular function, allowing infiltration of immune cells in the lung , 70. In −/−) had shown significantly protected from the development of emphysema. Shapiro and colleagues further proved that the synergistic effects of neutrophil and macrophage are required to enhance the potency of both cells. The absence of NE causes the tissue inhibitors of metalloproteinases (TIMPs) to inhibit the action of macrophage elastase. Likewise, the absence of macrophage elastase caused an increase in α-1 anti-trypsin, a major inhibitor of NE. Thus, the presence of both neutrophils and macrophages is an important factor in the development of emphysema [The accumulation of immune cells in the alveolar walls are prerequisite for the development of emphysema. Neutrophil elastase (NE) was reported to induce the epithelial apoptosis and emphysema; meanwhile, excessive MMP-9 released by macrophage can result in permanent alveolar destruction , 41. Shamphysema .CS exposure also causes mucus overproduction, although the symptoms may not be present in all COPD patients . The mec+ T cell count. However, the group did not measure the accumulation of immune cells in the peribronchial and parenchyma areas [MSCs have been actively investigated as a potential therapy for COPD. Clinical studies measuring C-reactive protein in COPD patient revealed the benefit of MSC administration in mitigating the inflammation . In the ma areas . Apart fma areas . Reducedma areas . In the ma areas . Also, Mma areas . In agreTo date, there are no treatment options available to regenerate the lung damage in emphysema. However, stem cell-based therapy demonstrates a promising regenerative capability to restore the function of the damaged lung. MSCs and MSC-CM are shown to restore the lung function by mitigating the apoptosis in the emphysematous lung . This anOn the other hand, relatively few studies were conducted to decipher the effects of MSC-derived EVs in the emphysematous lung. The study by Kim et al.  comparedTo date, relatively few studies are examining the effect of MSCs in reducing the mucus overproduction. Although there are reports stating that mucus could be mitigated with the administration of MSCs, in-depth analysis of the mechanism involves remaining unknown , 61. BesOur microarray analysis aimed to determine the pathways associated with COPD and gene expression profile in our COPD model. We also seek to understand how the treatment with hUC-MSC-EVs and hUC-MSCs can change the gene expression profile and pathways in COPD model. Our DEG analysis of microarray data revealed the importance of p50, p65, and PRKCZ in our animal model. Twelve weeks of CS exposure significantly upregulated p50, p65, and PRKCZ, and the treatment with hUC-MSC-EVs significantly downregulated the expression of these genes. Immunohistochemistry staining on p65 confirms the significant upregulation of p65 protein in the CS group and significant downregulation of p65 when treated with hUC-MSC-EVs, hUC-MSCs, and hUC-MSC-CM. Our study was also revealed that p50, p65, and PRKCZ were involved in many pathway regulations that include the TNF-α NF-κβ signalling pathway, IL-2 signalling pathway, oxidative stress, oestrogen signalling pathway, and IL-4 signalling pathway.The expression of PRKCZ and NF-κβ play a vital role in inflammation and thus the pathogenesis of COPD. PRKCZ is upstream of NF-κβ, phosphorylating p65 at serine 311 to promote the acetylation of Lysine 310, thus activating the κβ transcription . Mice deCigarette smoke activates NF-κβ within 1 h of exposure to the lung thus causing inflammatory reactions which increase white blood cell count, lymphocyte count, and granulocyte count , 26. DatOur study revealed the downregulation of PRKCZ, p65, and p50 expression when treated with hUC-MSC-EVs. p50 regulated 18 pathways in the hUC-MSC-EVs group, while PRKCZ and p65 regulated 10 pathways suggesting the vital role of the NF-κβ pathway in hUC-MSC-EVs therapeutic effects in our model. Downregulation NF-κβ subunit by hUC-MSC-EVs can affect multiple pathways in our model, thus reducing the inflammation. MSC-EVs have been shown to decrease the expression of NF-κβ in an in vitro model of cystic fibrosis and experimental colitis , 98. MSCOur study had successfully isolated the hUC-MSC-EVs from hUC-MSCs. Twelve weeks of CS exposure induced the inflammation and increased goblet cell count and emphysema in the rat model. The treatment with hUC-MSC-EVs, hUC-MSCs, and hUC-MSC-CM decreased the inflammation in the lung and decreased the goblet cells, and destruction of the lung in a rat model of COPD similar to hUC-MSCs. hUC-MSC-EVs reduced the inflammation in part by the expression of PRKCZ, and NF-κβ subunits p65 and p50, which regulates many genes responsible for innate and adaptive immune response. Confirmation study using immunofluorescence on p65 showed a similar result as microarray analysis of DEG. Taken together, there are still limited data demonstrating the regenerative and the anti-inflammatory effects of MSC-EVs to mitigate the inflammation in COPD. More studies should be conducted to decipher the anti-inflammatory effects of MSC-EVs as a whole, as well as exosomes, and microvesicles as different particle might exhibit different therapeutic effects. Determination of cargo content of MSCEVs responsible for the anti-inflammatory effects and the mechanism of action of the cargo content of MSC-EVs can provide a clear with the ways toward the goal of using hUC-MSCs as a new treatment for COPD.Additional file 1: Suppl 1. GO Slim analysis of the biological process in CS (A), hUC-MSCs group (B) and hUC-MSC-EVs (C) groups.Additional file 2: Suppl 2. Significantly regulated pathway in CS-induced inflammation, hUC-MSC-EVs and hUC-MSCs. Thirty-eight pathways are significantly regulated in CS. 58 pathways are significantly regulated in hUC-MSC-EVs. Only 17 pathways are significantly regulated in hUC-MSCs. Pathway with p < 0.05 is considered as significantly regulated.


The Prandtl number is evaluated for the three-dimensional hard-sphere and one-component plasma fluids, from the dilute weakly coupled regime up to a dense strongly coupled regime near the fluid-solid phase transition. In both cases, numerical values of order unity are obtained. The Prandtl number increases on approaching the freezing point, where it reaches a quasi-universal value for simple dielectric fluids of about ≃1.7. Relations to two-dimensional fluids are briefly discussed. We evaluate the magnitude of the Prandtl number for two model fluids in three dimensions with very different pairwise interaction potentials across their corresponding phase diagrams. The hard-sphere (HS) fluid represents the steepest pairwise interaction while the one-component plasma (OCP) model represents the opposite limiting case of very soft long-range interaction.r, is infinitely large at e is the electric charge [The HS model is widely used to approximate structural and transport properties of simple gases, fluids and solids . The intc charge ,5,6. Thic charge ,8,9. BotT is the temperature in energy units  . In the operties . The fluϕ=π/ρσ3, whers [Γ≃174 ,13.Transport properties of the HS and OCP fluids are very well investigated in classical MD simulations. Extensive data on the self-diffusion, shear viscosity, and thermal conductivity coefficients have been published and discussed in the literature. For the HS system, we use the recent comprehensive simulation study by Pieprzyk et al. ,15. For U is the internal energy, P is the pressure, and In addition to viscosity and thermal conductivity data obtained previously in numerical simulations we need the specific heat values to calculate the Prandtl number . The folFor HS fluids, the internal energy is only due to kinetic contribution, te (EoS) :(3)PρT=1For the OCP we use a simple EoS proposed in Ref. , based oa, which exactly compensates the particle charge. These compound charge-neutral spheres are not overlapping and the system energy is just the sum of the energies of the individual spheres. The latter can be easily calculated via purely electrostatic arguments. The last two correction terms in  ,12,24, wThe subsequent calculation is straightforward and the results are presented in In Similarly, in the strongly coupled OCP  systems is not sufficient to perform a detailed comparison similar to that in three dimensional (3D) systems, presented above. The problem remains rather controversial. The absence of valid transport coefficients in 2D systems was predicted based on the analysis of the velocity autocorrelation function and of the kinetic parts of the correlation functions for the shear viscosity and the heat conductivity . HoweverOur present knowledge allows us to make only a rough estimate. According to the data shown in Figure 3 of Ref. , the she λ≃ρΩEa2  as freezrom Ref. ). As a rExperimental measurement of the Prandtl number  was performed in a complex plasma subject to a laser-driven flow . The repulations . The higulations ,39. ThisThe main results can be summarized as follows: (i) the steepness of the pairwise interatomic interaction in simple fluids does not affect considerably the magnitude of the Prandtl number; (ii) for simple pairwise repulsive interactions in 3D the Prandtl number is about unity and increases in the dense fluid regime; (iii) it reaches a quasi-universal value of


Having features of Attention Deficit Hyperactivity Disorder (ADHD) is associated with challenges with emotional regulation and cognitive function. Heritability for ADHD in adults is estimated to be 30%. The degree to which genetic risk for ADHD can be modified by protective factors, such as strong personal relationships and pursuing more education, to result in better emotional and cognitive outcomes at later ages is not well understood. We evaluated these relationships in a population-representative sample of older adults in the U.S. Health and Retirement Study, with 9,003 European Americans , and 1,622 African Americans . Outcomes included validated scales for psychological resilience, life satisfaction, depressive symptoms (DepSx), cognitive functioning, and impairment, assessed between 2008-2012. A genetic risk score for ADHD (GRS-ADHD) was calculated from a genomewide-scan, using a mixed ancestry sample. We used multivariable linear and logistic regression models, adjusted for age, gender, and genetic ancestry. We found a protective effect such that stronger personal relationships in adulthood reduced the inverse relationship between the GRS-ADHD and resilience and life satisfaction in later age (interaction p’s<.004 in EAs), but not with DepSx. In contrast, strong parental relationships in childhood attenuated the association between the GRS-ADHD and later life DepSx (interaction p’s<.007 in EAs and AAs) only. Education did not modify, but mediated the main effect of the GRS-ADHD on cognitive abilities and impairment in EAs and AAs. Findings have implications for later age health for those at greater genetic risk for ADHD.