The association with depressive symptoms was mediated in part by peer problems and academic attainment which accounted for 14.68% and 20.13% of the total effect, respectively. Childhood ADHD is associated with increased risk of later depression. The relationship is mediated in part by peer relationships and academic attainment. This highlights peer relationships and academic attainment as potential targets of depression prevention and intervention in those with ADHD. Future research should investigate which aspects of peer relationships are important in conferring later risk for depression.There is increasing evidence that childhood Attention-Deficit Hyperactivity Disorder (ADHD) elevates risk of later depression, but the mechanisms behind this association are unclear. We investigated the relationship between childhood ADHD symptoms and late-adolescent depressive symptoms in a population cohort, and examined whether academic attainment and peer problems mediated this association. ALSPAC  is an ongoing prospective longitudinal population-based UK cohort that has collected data since September 1990. 2950 individuals with data on parent-reported ADHD symptoms in childhood (7.5 years) and self-reported depressive symptoms in late adolescence (17.5 years) were included in analyses. 2161 individuals with additional data at age 16 years on parent-reported peer problems as an indicator of peer relationships and formal examination results  as an indicator of academic attainment were included in mediation analyses. Childhood ADHD symptoms were associated with higher depressive symptoms (The online version of this article (10.1007/s00787-019-01463-w) contains supplementary material, which is available to authorized users. Major Depressive Disorder (MDD) is a leading cause of disability worldwide . The incTwo possible factors linking ADHD and depression are difficulties in peer relationships and academic attainment. Difficulties with peer relationships have been shown to be important in depression aetiology –16 and chttps://www.alspac.bris.ac.uk), a large prospective birth cohort in South-West England. Pregnant women in Bristol with a due date between April 1991 and December 1992 were approached to participate. This resulted in 14,541 pregnancies enrolled in the study and 13,988 children alive at 1 year. The children’s development has been followed regularly since birth largely via questionnaires and face-to-face assessments. The methodology and sample are described elsewhere, but the population is generally representative of UK children [https://www.bristol.ac.uk/alspac/researchers/our-data/). This study is based on a primary sample of 2950 participants for whom data were available on childhood ADHD symptoms, depressive symptoms in late adolescence, and covariates . Mediation analyses included 2161 participants where data were additionally available on peer problems and academic attainment at 16 years.This sample was derived from the Avon Longitudinal Study of Parents and Children (ALSPAC)   ADHD symContinuous and binary depression variables were used as the outcome in this study to give an indication of the impact of ADHD on depressive symptoms and on the odds of reaching a clinical cut-point for depression. For the continuous outcome, ‘depressive symptom score’ was indicated by the self-rated Short Moods and Feelings Questionnaire (SMFQ) at 17 years and 6 months. The SMFQ is a 13-item questionnaire designed to cover core depressive symptomology with a three-point response scale for each question of ‘Not true’ (0), ‘Somewhat true’ (1), or ‘True’ (2) summed to generate a maximum score of 26 . For theMediator variables were measured at age 16 years. This ensured a time lag between exposure (age 7.5), mediator (age 16) and outcome (age 17.5) as recommended for mediation analysis .The 5-item Peer Problem subscale of the mother-completed Strengths and Difficulties Questionnaire (SDQ) at 16 years was used . Items sAcademic attainment was assessed by performance in formal examinations at the end of secondary school at 16 years . GCSEs are graded from A* (highest grade achievable) to U (lowest grade achievable). A total GCSE and equivalents point score was calculated by summing individual point scores for each GCSE and equivalent qualification grade achieved  .Analyses were adjusted for mother’s socioeconomic status according to occupation and maternal age at birth to account for sociodemographic factors associated with depression . These wChildhood ADHD symptoms were standardised, so that a unit increase was equivalent to a standard deviation unit increase. Linear regression was used to examine the association between standardized childhood ADHD symptoms and continuous depressive symptom score at 17.5 years. Logistic regression was used to examine the association between standardised childhood ADHD symptoms and depression assessed using the binary SMFQ clinical cut-point at 17.5 years. To examine whether sex influenced the association between ADHD symptoms and depression, an interaction term of ADHD symptoms and sex was regressed against depressive symptoms. The Wald test was then used to test whether the model with the interaction term was significantly different to the model without the interaction term. Regressions were also repeated using an exposure variable of ADHD diagnosis.Peer problems and GCSE result scores at 16 years were tested as mediators of the association between childhood ADHD symptoms and adolescent depressive symptoms in two single mediator models. A ‘potential outcomes’ causal mediation framework was used with STATA commands ‘medeff’ and ‘medsens’ , 46. MedAs previous work has found victimization to mediate the association between ADHD symptoms and subsequent depression , we addiThree sensitivity tests were conducted to check for the effect of timing of variables. Use of mediator data collected at an earlier time point than depression data does not ensure that these mediators actually preceded depressive symptoms. Thus, mediation analyses were repeated adjusting for depressive symptoms at 14 years (prior to the measurement of mediators) to account for depression occurring earlier in adolescence (Supplement 1). Furthermore, although academic attainment data were not available at an earlier age than 16 years, we repeated the peer relationship mediation analysis using peer problem and depression data collected at earlier time points. We used SDQ peer problems data collected at 9.5 years  and SMFQ depressive symptoms at age 13 years (Supplement 2). In addition, we checked whether observed associations between ADHD and adolescent depression were due to ‘pre-existing’ childhood emotional problems occurring approximately contemporaneously with ADHD. This could confound the direct association between ADHD and depressive symptoms. Therefore, emotional problems as indicated by the SDQ emotional problems subscale at 8 years   were adjTo address the possibility of bias due to non-random missing data, all analyses were repeated with Inverse Probability Weighting (IPW) applied. IPW is a reliable technique for handling missing data, particularly in longitudinal studies where participants can have missing values on multiple variables . Weightsn = 14) met the criteria for DSM-IV ADHD diagnosis at 7.5 years. DSM-IV ADHD symptoms  were used to define the primary ADHD exposure variable. 17.32% (n = 511) met the clinical cut-point for depression at 17.5 years . As expected, the majority of those with ADHD were male (85.71%) and the majority of those reaching the binary cut-point for clinically significant depressive symptoms were female (66.73%). Table In the 2950 participants  with complete data, 0.47% . There was no significant interaction between ADHD symptoms and sex in predicting depressive symptom score , p = 0.32), though the relationship was slightly stronger in females  than males .Standardised childhood ADHD symptoms predicted the continuous outcome of adolescent depressive symptom score (p < 0.001). This remained the case when adjusting for socioeconomic factors, sex and childhood emotional problems , p = 0.16), though the association was slightly stronger in females  than males . Clinical diagnosis of ADHD  was also associated with increased odds of clinically significant depressive symptoms at 17.5 years .Childhood ADHD symptoms predicted the binary outcome of clinically significant depressive symptoms at age 17.5 years , accounting for 12.50% of the association.Peer relationships at 16 years mediated the association between childhood ADHD symptoms and late-adolescent depressive symptoms, accounting for 14.68% of the total effect Table . Peer prAcademic attainment mediated the association between childhood ADHD symptoms and late-adolescent depressive symptoms, accounting for 20.13% of the total effect .One of the advantages of the potential outcomes approach to mediation is that it allowed testing of potential unobserved confounding of the mediator–depression relationship. The ‘medsens’ test indicated that the product of observed variance in mediator and outcome that would need to be explained by an unmeasured confounder for mediation effects to disappear ranged from approximately 0.005–0.008 (Supplement 6). To aid interpretation of this coefficient, we compared it with the estimate for a measured confounder—socioeconomic status of mother. The estimate for socioeconomic status of mother on mediation by academic attainment was 0.0002. The coefficient for unobserved confounding required to eliminate the observed mediated effects is, therefore, considerably larger than that present for the measured confounder of socioeconomic status. Thus, it seems unlikely that unobserved confounding would account for the observed results.b = 0.55, SE = 0.12, p < 0.001) and with the binary measure of clinically significant depressive symptoms in late adolescence . Mediation results with IPW applied are shown in Supplement 7.Results remained very similar when IPW was applied to analyses. Childhood ADHD symptoms remained associated with the continuous late-adolescent depressive symptom score  and academic attainment. This indicates that the development of interventions targeting the impact of children’s ADHD on their peer relationships and academic attainment may have the added benefit of reducing depression risk, in addition to treating core ADHD symptoms. For instance, a number of existing psychosocial interventions available for those with ADHD that target social skills and academic skills –68 may hFor future research, a greater understanding of the specific components of peer relationship difficulties in those with ADHD that confer depression risk would allow further insight into the most appropriate targets for intervention.We show in a longitudinal population cohort that childhood ADHD symptomology is associated with increased risk of clinically significant depressive symptoms in late adolescence. This adds to a growing body of research highlighting the need for depression monitoring in young people with ADHD. We found that the association is mediated in part by peer problems and academic attainment. This highlights these areas as potential targets for depression prevention and intervention in children with ADHD.Supplementary file1 (DOCX 75 kb)Below is the link to the electronic supplementary material.


R) revealed higher magnitude among all the study districts, the smallest being 2.02 (DMg) and 1.41 (DMn) and considerable range of variations . Among the study districts, Dinsho consisted the highest on-farm diversity estimate  followed by Goba and Sinana . Estimate of the landrace evenness (E) also showed the highest magnitude (>0.95) except in Agarfa district (0.77). The result suggests potentiality of the areas and wide cultivation of majority of the landraces in the villages. However, nowadays, only 14 landraces are under cultivation and the remaining 11 are totally eroded from the district(s) constituting the highest (56.0%) combined genetic erosion suggesting loss of important agronomic traits and, thus, a major bottleneck for further improvement and conservation plans. Thus, attention should be payed to conserving the landraces for better further use.Barley landraces is among the major cereal crops grown in Ethiopian highlands including Bale highlands. However, in recent days, the crop is highly declining to the extents of total loss. This study was, therefore, aimed at assessing the extents of its on-farm diversity and genetic erosion from Bale highlands, Ethiopia. Data were generated from twelve administrative districts and analyzed considering important ecological and genetic erosion models. A total of 25 distinct (at least in naming) barley landraces with varying distribution patterns have been identified in the areas. Landrace richness ( Genetic erosion refers to loss of genetic variability over space and time , 2. It cin situ conservation of genetic resources especially in areas of domestication or origin, where diversity of genetic resources is concentrated, is very essential [One of the negative consequences of genetic erosion is that it increases susceptibility to biotic and abiotic stresses  and, henssential , 7. Likessential , 9. Keepssential , 11.Hordeum vulgare L.) landraces are among those crops regardless of their valuable and distinct agronomic traits [ex situ conditions at the Ethiopian Biodiversity Institute (EBI), formerly established as the Plant Genetic Resources Centre of Ethiopia (PGRC/E) through the International Board on Plant Genetic Resources in 1974 ). If well managed, however, it will become important sources of breeding and improvement because of their unique and long-term adaptive and evolutionary potentials to different biotic and abiotic stresses.However, on-farm genetic resource conservation and research activities targeting improvement of indigenous crops received less attention in several countries , 13. Ethc traits . Their cc traits –20. AtteBale highlands are one of the potential agricultural areas in the country and are well known in higher diversity and wider cultivation of Ethiopian barley landraces. However, in recent days, both the diversity and cultivation are highly declining to the extents of total loss of some previously important landraces from areas where they had been widely cultivated and most are pushed to marginal growing conditions. The decline in diversity and production is attributed to several and interrelated factors such as widespread adoption of modern and exotic varieties, recent climate change that resulted in habitat destruction and recurrent drought, and advancements in agricultural technology including a shift towards using mechanized farming that totally favored crops like wheat that are largely produced in bulk for commercial and industrial purposes. In addition, lack of research activities targeting its breeding and improvement and lack of well-documented study on the extents of its genetic erosion has hampered its conservation and improvement programs , 21–23. The study was carried out in Bale zone, Southeast Ethiopia. Robe, the zonal city is 430 km from Addis Ababa, the capital of Ethiopia with 7° 08′ N (latitude), 39° 59′ E (latitude). The study encompassed 12 potential barley growing districts in the zone . The disUntil recently, one of the major bottlenecks in assessing the extents of on-farm genetic diversity and genetic erosion in a given crop species is lack of standardized and well-developed methodology. The problem is partly attributed to the relatively longer time required to gather adequate data, the multidisciplines required to end up with a concrete justification, and the dynamicity of genetic diversity itself in time and space , 24, 25.Ethiopia is among the countries facing the problem and has very scarce, if not none, detailed baseline information on the extents of on-farm genetic diversity and genetic erosion in indigenous food crops and their wild relatives . Barley The present study was conducted considering the concepts revealing a strong linkage between farmer's traditional knowledge and extents of on-farm genetic diversity (genetic erosion) and the large concordance between local (vernacular) names given to landraces and their genetic distinctiveness –30. AccoA community based cross-sectional research design was employed focusing on selected farmers' districts and kebeles within a district that were identified after a rapid preliminary informal survey and discussions with the zonal and district agricultural bureau experts. The information was gathered from both primary and secondary sources on 2019/20.Sample size was determined using a standard formula suggested by Freund and Williams  considerN = 0.25∕SE2 = 400, where SE (standard error) = 2.5%, which means 0.025, SE2 (standard error square) = 0.000625, and N is the sample size.Accordingly,  The total sample was fairly distributed to the study districts assuming equal coverage to obtain a relatively balanced response and conclusion.Valuable data were collected through a questioner, household interview, and focus group discussions (FGD) using key informants and personal observations at barley fields.Afan Oromo' and ‘Amharic' and distributed to the selected 400 household heads. The household heads were purposively selected based on the preliminary survey and documents from district agricultural offices. In addition, all the required age groups and sexes including elder women household heads were intentionally involved to guarantee good coverage of the required diversity in indigenous knowledge.Questionnaire method was used with the intention of setting information from a wide range of sources (respondents) regarding the indigenous knowledge and practices involved in barley landraces farming, management, conservation, and utilizations in the study areas. The questionnaire was written in English and translated into local languages such as ‘Interview questions were used to substantiate the information generated through the questionnaires. In this regard, semistructured questions that address matters regarding the barley landraces currently or used to be cultivated, extents of their production relative to other cereal crops, production challenges, and major utilizations were presented. The key informants were carefully selected from the household heads of both sexes and different age groups involved in the questionnaire method based on their willingness and rich practical knowledge on barley production, conservation, and utilization in the areas.Focal group discussions were carried out with selected barley growing elders and experts to complement the information obtained from individual farmers and to minimize missing data. The key informants involved were well-recognized elder farmers aged 50 or more and spent their entire lives in the localities and were engaged in barley farming and seed selection. Open group discussions regarding the reasons why barley landraces are left marginalized, main factors for the current decline in production of the landraces, and their general views regarding the benefits of the landraces were presented. Finally, after thorough discussion, consolidated ideas were noted.Agricultural extension experts and development agents (DAs) at all the selected districts and Peasant Association levels, as well as experienced researchers at Sinana Agricultural Research Institute, the regional institution located in the study zone and mandated for research on barley and wheat, were consulted to cross-check whether the landraces identified by the local farmers were really landraces or improved varieties. Furthermore, secondary data from the Ethiopian Biodiversity Institute (EBI) and barley researchers were used to validate the landraces and screen the improved and exotic varieties released through the formal system.Descriptive statistics regarding sociodemographic characteristics of the respondents were analyzed using Minitab version 19. The extents of on-farm genetic diversity in the crop were analyzed by using the different ecological models that have been adapted to species diversity. Accordingly, Margalef's, Menhinick's, Shannon-Weaver, and Simpson's diversity  indices were employed assuming the landraces as distinct species , and thuR) among the twelve districts was compared by using Margalef's (DMg) and Menhinick's (DMn) indices as follows:Computationally, landrace richness  (L is the number of landraces in each study district and R designates the number of records for each landrace.where E) was determined as a measure of Shannon-Weaver information index (GDs) and was given by E = GDs/lnL, where L is the total number of landraces cited in each study district and GDs is a measure of Shannon-Weaver information index and given byPi is the proportional abundance of the ith landraces and given by . Its magnitude is greater or equal to 0.Similarly, landrace evenness (D) by taking into account the frequency of occurrence of each recorded farmer landrace (assuming as a distinct species) in the total districts. It is given byPi2 is the squared proportion of landrace i to the total records.Spatial diversity or abundance of the landraces was computed using Simpson's index  and it was calculated over a period of twenty to thirty years following  which isSupplementary TableSummary of the respondent's sociodemographic characters was presented in Most of the respondents (88.8%) used to grow barley for more than a couple of decades (greater than 40 years old) revealing their rich knowledge and behavioral practices in barley landrace production. Hence, it suggests appropriateness of the study population in providing sufficient and valuable information regarding the landraces under cultivation or used to be cultivated over the last couple of decades (20 to 30 years ago) along with the main challenges of production and main utilizations.Larger proportion of the respondents (333 or 83.25%) had less than primary school education but they have rich indigenous knowledge-based agricultural practices and, thus, long been involved in barley landrace selection, conservation, and maintenance processes. Similarly, regardless of dominance in Oromo ethnic group that are Muslims, the ethnic and religious heterogeneity in the area has also played a great role in the selection and conservation of some landraces for their specific endue qualities.Indigenous knowledge-based vernacular (common) names given to genetic resources is among the indicators of genetic distinctiveness, and names are usually assigned from the points of view of their distinct characteristic features, specific or special end-use qualities, or other at least locally important attributes , 27, 28.Kolo' (roasted barley grain). Similarly, Kubsa was named to indicate its high flour quality.According to the farmers consulted, different characteristic features of the landraces or associated features that the farmers thought important for identification and showing distinctiveness had been used for assigning the names. For example, Kasale, Muga, Bira Adi, and Bira Dima were named to give emphasis to their seed colors such as deep black, faded black, white, and reddish colors, in that order, and thus to distinguish them from other related landraces. Butuji, and Barasdad were given names to reveal the short and strong stem length that could resist lodging. Farasgama, Kinkicho, Mukura, and Kate were named from the points of view of short spike length. Aruso Bale, Aruso Balticha, and Aruso Limat were given a common name ‘Aruso' to show their suspected area of origin in ‘Arsi,' one of the administrative zones in Oromia region that is adjacent to the study area, Bale zone. The second names, Bale, Balticha, and Limat were given to avoid confusion and show their distinctiveness from each other. Haji Yune and Kabe were named after a well-known farmer called Haji Yune and Kabe who were pioneer in cultivating the landraces. End-use qualities were also used in naming the landraces that have distinctive and specific utilizations. For example; Senef Gebs were named to show the ease of dehulling while preparing ‘Kolo or roasted barley grain, Kasale and Mugaare used for makingTella, local fermented beer or Kenetto, local nonfermented beverage. Shewayrga et al. [Sorghum bicolor L. (Moench)) landraces from Northeastern Ethiopia. Similarly, Tsegaye and Berg [Some of the special attributes of the landraces were noted and accordingly, most of them had white seed color revealing their end-use qualities related preferences by the local community assuming that food and beverage products produced from white seeded barley bear appealing color. However, being white does not imply their prevailing nutritional content and yet no supporting report in Ethiopia or elsewhere in the world. Another special feature common to most of the landraces is that they are two rowed representing the intactness of those original and ancestral landraces as suggested by . As a rea et al.  reportedand Berg  noted a vice versa. On the other hand, some of the landraces, for example, Kasale, Senef Gebs, Wadago, and Samareta were commonly cited in all the study districts (the first two) except one (the second two) with different proportions. Haji Yune, Gomboba, Bira Adi, Aruso Bale, and Muga are also common to most of the study districts suggesting their wide use for preparing different cultural and religious dishes in Bale highlands. However, some of the landraces had a very restricted distribution because of their localized end-use qualities. For example, Muga is restricted to Dinsho district where it is largely used for making Tella  and Keneto, local unfermented and nonalcoholic drink prepared during cultural and religious ceremonies. Similarly, Kinkicho, Mukura, and Kate are each restricted to only two of the districts suggesting their restricted end-use qualities such as Shorbaor Soup (Kinkich), Besso, or a recipe prepared from fine ground flour (Mukura), and Kita or unleavened and thicker pancake (Kate) that are common to the districts. All the remaining landraces had distribution frequency of three to six districts. The trend suggests artificial selection pressure targeting their end-use qualities and purposeful maintenance of some landraces to use for the intended purpose. There have been similar reports from all the study districts and even from across the barley growing regions of the country [Distribution patterns of the 25 barley landraces recorded in the present study were presented under Tables  country , 35, 36.Marka , Injera (leavened thin pancake), Kita (unleavened thick pancake). Similarly, selection was being made towards favoring landraces with a relatively good yield, bearing higher resistance to environmental stress, and shorter length of maturity to supplement the food shortage gap in extreme highland and degraded areas where wheat production is not suitable. In this regard, Aruso Balticha and Kasale are preferred because of their preferred quality in resisting extreme climatic conditions. As a result, on-farm diversity of the landraces is narrowing, and a larger number of the landraces have been neglected and eventually eroded despite their important agronomic traits and contribution to further targeted breeding. Tadesse and Asres [In general, the distribution patterns observed is a result of selection pressures that are directly or indirectly related to exploiting and excelling local utilizations of the crop. The selection process largely targeted favoring those landraces that bear higher market demand and good flour quality to meet the demand of household consumption, religious, or cultural ceremonies. For example; Senef Gebs has more market demand and helpful in generating cash to several youngsters in the localities; Kasaleis maintained for its religious and cultural related end-use qualities; Samareta and Wadago bear good flour quality because of their white seed color that is favored by the local communities in making nd Asres  reportedR) computed in terms of Margalef's (DMg) and Menhinick's (DMn) indices is presented in DMg) and 1.41 (DMn) in Gololcha and Gura Damole districts, respectively, and considerable range of variations . The result suggests potentiality of Bale highlands in harboring larger number of barley landraces in broad sense and important agronomic traits (associated genes) in narrow sense that could be used for further breeding and conservation actions.Summary of the interlandrace diversity or landrace richness  followed by Goba and Sinana districts . The result suggests that these districts largely relied on barley cultivation because of their larger topography that is highly rugged, degraded, and not suitable for cultivation of other cereal crops. Thus, one can imagine how important the landraces are in supporting the livelihood of subsistence farmers living in extreme conditions. Gura Damole and Gololcha districts each scored relatively lower diversity estimate  implying dominance of wheat production in the areas. However, such a high diversity index sometimes might not necessarily indicate the extent of genetic diversity and possible importance of the crop since some districts may cultivate only limited barley landrace but in a large land mass that could constitute a very low diversity index.With regard to the study districts considered, Dinsho consisted the highest on-farm diversity estimate  , centralE) (Extent of diversity estimate in terms of evenness index (E)  also shoD) considering occurrence of the landraces in one or more of the target locations is presented in Genetic abundance which was determined using Simpson's diversity index (injera (leavened thin pan cake type), dabo , and Kolo (roasted barley grain). In recent days, they are marginally cultivated on a very small plot of land mainly for making Tela  and Keneto . Thus, special measures need to be considered in widening their cultivation and end-use qualities taking into account their nutritional contents before they completely erode.However, regardless of the abundance, some landraces are highly marginalized and unlike others, cultivated only in small quantities for their specific end-use qualities. For example, the black seeded landraces called Kasale and Muga are currently at high risk of extinction because of only their seed color that, according to farmers, does not seem appealing for making food items such as local Temporal genetic diversity and extents of genetic erosion in the barley landraces considered from Bale highlands is presented in The result suggests that though Bale highland is containing higher spatial barley genetic resources, the diversity is in a rapid shift towards declining integrity, and consequently, a larger number of genetic resources have been eroded and kept eroding through time. Such pronounced germplasm loss through time is resulting in loss of important agronomic traits and, thus, a major bottleneck for further improvement and conservation plans.Kolo' (roasted barley grain) which is sold in markets and along road sides. Bira Adi is mainly used as malt barley to brewery factories in the country and, thus, the main source of family income. Similarly, Aruso Balticha, Kasale, and Gomboba are preferred for their special end-use qualities in preparing foods and beverages for cultural and religious ceremonies.On the other hand, those integral landraces have continuously been cultivated and maintained because of their relatively high grain yield, unique end-use qualities, wide adaptation to the changing environmental conditions, local market price, and wide ethnobotanical uses. For example, Senef Gebs is maintained because of its significant contribution in generating cash to the youngsters as it is mainly used to prepare ‘Some of the landraces that have totally been lost had high abundance as indicated in Kasale, and pushing them closer to total extinction. In general, in order to exploit the maximum benefits from the crop and to meet future food needs for the world's rapidly growing human population, policymakers and researchers should pay attention to on-farm conservation and enhancement of the farmers' varieties.Farmers' varieties (landraces) are genetic stocks for improvement and maintenance. Ethiopia is one of the potential countries in harboring farmers' varieties including cultivated barley genetic resources. Bale highlands is among the potential areas in the country and could be the hotspots for the barley landrace conservation and breeding as evidenced from the present study that revealed a large number of (25) distinct landraces, at least in naming. Furthermore, the patterns of on-farm genetic diversity in the landraces and the study districts observed signal the rich genetic resource in the country and could assure the country's being one of the centers of origin and/or diversity for cultivated barley genetic resource since extents of genetic diversity is one of the indicators of areas of origin or diversity for a given genetic resource. However, in recent days, the landraces are declining both in terms of their cultivation and number . As a result, the landraces are suffering from extreme genetic erosion (56.0%) that eventually signposts a rapid loss of important agronomic traits. Likewise, specific end-use quality-based artificial selection pressure imposed by local farmers is marginalizing some of the landraces, for example,


Background. This cross-sectional study aimed to evaluate stress and immune biomarkers in saliva samples of attention-deficit/hyperactivity disorder ADHD compared to healthy non-ADHD children. Material and methods. A total of 132 children under 11 years old (8.5 ± 1.1) enrolled in a cross-sectional study: with confirmed ADHD (n = 60) and healthy controls (n = 72). The clinical evaluation included physical measurements  and unstimulated saliva collection and measurements of free cortisol, salivary alpha-amylase (sAA), and secreted immunoglobulins  with quantitative assay (ELISA) analysis. Unpaired t-test, Welch test, or Mann–Whitney U test were applied for group comparisons when appropriate, and the correlation between variables was analyzed with Spearman’s rank coefficient. Results were considered significant at p < 0.05. Results. In the ADHD group, body weight (p ≤ 0.01), BMI (p ≤ 0.009), and hip circumference (p ≤ 0.001) significantly differed, while waist size and BMI z-score did not (p > 0.05). Significant elevation of the salivary sAA (p = 0.03), sIgA (p = 0.02), and IgM (p ≤ 0.001) biomarkers were detected, without differences in the morning cortisol (p > 0.05). Significant correlations between cortisol and BMI, hip size, and IgA, as well as between IgG and sAA and IgA were obtained. Conclusions. Saliva can be used to monitor ADHD status with regard to biomarkers indicating the hypothalamus–pituitary–adrenal axis, as HPA axis, and sympathetic activity. The results indicate that morning collection of saliva in contrast to unchanged salivary cortisol, may evaluate mentioned above system dysregulations by measurements of sAA and immunoglobulins among ADHD children.  A dental office is a frequently visited health-care facility by children and adolescents under 18 years of age. This allows easy contact with children and may also help in monitoring mental stress during the developmental period of life among healthy or unhealthy subjects. Diagnosing and managing attention-deficit/hyperactivity disorder (ADHD) in childhood are exceptional achievements for medical clinicians . ResearcThe salivary glands work together with the neuroendocrine system, which makes saliva sensitive to stress or mood behaviors . ValuablMany studies have described a significant effect of stress on the activation of the hypothalamic–pituitary–adrenal (HPA) axis, resulting in increased cortisol production ,7. CortiCurrently, it is proposed that the HPA axis and the activity of the sympathetic nervous system (SNS) have to be monitored in parallel during mental stress ,14,15. SJust as salivary cortisol and amylase have been studied quite frequently, the salivary immunoglobulins of the immune system, such as IgA, IgG, and IgM, have been unexplored in ADHD . The oraThe intention was to select children with ADHD from one part of the area, the western sector of the country . The recIn one agglomeration, four primary schools were randomly selected from opposite geographic regions––eastern, western, northern, and southern. In the schools, the project was proposed to parents of children aged 6 to 11. The researchers offered a free child-development analysis and dental examination at the dental office. Four hundred willing parents were enrolled from four schools to have their children examined. Regarding the inclusion criteria , 150 chiThe cross-sectional project involved non-invasive analysis methods such as salivary sampling and questionnaires to confirm or deny a diagnosis of ADHD. The study plan was correct according to the standards of Good Clinical Practice (GCP) and had a positive opinion of the local Ethics Committee of the Medical University of Poznań (Resolution No. 26/18) . All parThe anthropometric assessment of a child’s development consisted of body measurements, such as height, waist, hip circumference, and body weight. The standing body height was measured with a SECA 216 wall-mounted stadiometer with an accuracy of 0.1 cm. The body weight of children was assessed in light clothing on a digital scale with an accuracy of 0.1 kg. The waist circumference was determined at the midpoint between the lower edge of the costal arch and the upper iliac crest using a metric tape . The hipAll participants in both groups underwent the same procedures in the dental office to avoid any bias that could affect salivary flow during the examination. The patients were examined in the same season of the calendar year  and at the same time in the morning, i.e., between 9:00 and 10:00. The same examiner took the saliva samples. All patients were examined 60 min after breakfast and oral hygiene procedures. Unstimulated total saliva was spat into sterile containers for 15 min. Children were instructed to move their body to forward position and focus on the spitting activity. The first portion of the saliva was collected in a separate container and then allowed to accumulate. It was then spit out into a sterile disposable tube . Immedia2 = 0.998, p < 0.001) [The concentration of free cortisol in the saliva was quantitatively determined by the ELISA method using the ELISA Kit DES6611 for the In Vitro Diagnostic (IVD) . The standard curve ranged from 0 to 30 ng/mL, the intra- and inter-assay variability coefficient was assessed to be below 6%, and the standard curve was statistically significant (r< 0.001) ,32. 2 = 0.982, p < 0.001) [The sAA concentrations were quantitatively determined by enzyme-linked immunosorbent assay (ELISA) using the ELISA Kit DEEQ6231 for the In Vitro Diagnostic (IVD) . The standard curve ranged from 0 to 500 U/mL, the intra- and inter-assay variability coefficients were assessed to be below 5%, and the standard curve was statistically significant (r< 0.001) .2 = 0.995, p < 0.001) [Salivary sIgA concentrations were quantitatively determined by the ELISA method using the ELISA Kit DEXK276 for the In Vitro Diagnostic (IVD) . The standard curve ranged from 0 to 400 µg/mL, the intra- and inter-assay variability coefficient were assessed to be below 5%, and the standard curve was statistically significant (r< 0.001) ,35.2 = 0.999, p < 0.001) [Salivary IgG concentrations were quantitatively determined by the ELISA method using the ELISA Kit E0544h for research use only (RUO) . The standard curve ranged from 0 to 5000 pg/mL, the intra-assay variability coefficient was <4.4%, and the inter-assay variability was <7.8%, respectively (r< 0.001) ,37. 2 = 0.986, p < 0.001) [Salivary IgM concentrations were quantitatively determined by the ELISA method using the ELISA Kit E0543h for research use only (RUO) . The standard curve ranged from 0 to 500 ng/mL, the intra-assay variability coefficient was <6.2%, and the inter-assay variability was <9.1%, respectively (r< 0.001) . All ELISA tests were performed according to the manufacturer’s instructions without any modifications. All samples and standards were run in duplicate, and the mean value of the two assays was used for statistical evaluation. Optical density was read with a spectrophotometric plate reader  for a wavelength of 450 nm ± 10 nm. A four-parameter algorithm (four-parameter logistic) was used to assay the concentration in the tested samples.p < 0.05. Statistical analyses were performed with STATISTICA v13 software .The analyzed data were expressed as mean ± standard deviation, median, and minimum and maximum values as appropriate. The normality of the distribution was tested using the Shapiro–Wilk test, and the equality of variances was checked using Levene’s test. A comparison of two unpaired groups was performed using the Mann–Whitney U-test. The relationship between variables was analyzed with Spearman’s rank correlation coefficient. All results were considered significant at p > 0.05). The ADHD participants weighed 10.5% more than those in the control group (p ≤ 0.01), as body weight (p ≤ 0.01), BMI (p ≤ 0.009), and hip circumference (p ≤ 0.0002) were statistically different between patients and controls, but not the BMI z-score or waist size (p > 0.05)  . The mea > 0.05) . p = 0.03), sIgA (p = 0.02), and IgM (p = 0.0000001) levels in the ADHD group (p = 0.739)  . s = −0.35; p = 0.01), hip size , and IgA . IgG was correlated to alpha-amylase  and IgA   . s = −0.308; p = 0.01)  . The selected salivary biomarkers were chosen by evaluating previous studies on salivary stress and immune biomarkers and ADHD. However, there are limited results in the scientific literature, mostly connected with cortisol and sAA but not IgA, IgG, or IgM . Saliva Nevertheless, determinations in saliva open many paths due to the non-invasiveness and repeatability of the measurements ,51.The results in this study of an increased sAA concentration compared to cortisol found raised some questions and notes. In the analyses of other authors, the sAA was collected during acute and chronic stress stimuli, and higher concentrations were obtained ,53.In one ADHD study, a lower evening and diurnal sAA secretions in response to stress were noted . It shouLactobacillus [In the present study, all immune biomarkers, IgA, IgG, and IgM, were significantly increased in the salivary samples of ADHD children. The level of secretory IgA was markedly higher among ADHD children than in those in the control group and correlated to free cortisol and IgG levels in the saliva. This finding shows that sIgA levels may be linked to the stress response and may also confirm a link to HPA axis hyperactivity. According to the current literature, an increase in IgA in oral saliva may also indicate a greater hypersensitivity of the oral mucosa and susceptibility to infectious diseases . This inbacillus . Additiobacillus . It coulbacillus . Our research suggests many directions in which salivary enzymes and immunoglobulins may be analyzed. Their concentrations in saliva are essential also for stimulating dental caries development in the oral cavity. Our previous study showed high dental caries in primary and permanent dentition among ADHD children . AdditioOur results showed a significant increase in body mass, hip circumference, and BMI with correlation to cortisol. Comparative research also indicates excessive body mass or BMI compared to pediatric norms or control groups ,60,61,62This cross-sectional study aimed to evaluate stress and immune biomarkers in salivary samples of children with attention-deficit/hyperactivity disorder (ADHD) compared with those of healthy, non-ADHD children.In our opinion, saliva may be a promising material for non-invasive ADHD monitoring. The results indicate that morning collection of saliva in contrast to unchanged salivary cortisol, may evaluate HPA and SNS dysregulations by measurements of sAA and immunoglobulins among ADHD children.


Bartonella infections in small mammals in the Qaidam Basin, western China, could provide a scientific basis for the control and prevention of Bartonella infections in humans. Accordingly, in this study, small mammals were captured using snap traps in Wulan County and Ge’ermu City, Qaidam Basin, China. Spleen and brain tissues were collected and cultured to isolate Bartonella strains. The suspected positive colonies were detected with polymerase chain reaction amplification and sequencing of gltA, ftsZ, RNA polymerase beta subunit (rpoB) and ribC genes. Among 101 small mammals, 39 were positive for Bartonella, with the infection rate of 38.61%. The infection rate in different tissues (spleens and brains)  and gender  of small mammals did not have statistical difference, but that in different habitats had statistical difference . Through genetic evolution analysis, 40 Bartonella strains were identified , including B. grahamii (30), B. jaculi (3), B. krasnovii (3) and Candidatus B. gerbillinarum (4), which showed rodent-specific characteristics. B. grahamii was the dominant epidemic strain (accounted for 75.0%). Furthermore, phylogenetic analysis showed that B. grahamii in the Qaidam Basin, might be close to the strains isolated from Japan and China. Overall, we observed a high prevalence of Bartonella infection in small mammals in the Qaidam Basin. B. grahamii may cause human disease, and the pathogenicity of the others Bartonella species needs further study, the corresponding prevention and control measures should be taken into consideration.Investigation of the prevalence and diversity of  Bartonella is a genus within the Bartonellaceae family in the Alphaproteobacteria class. Bartonella species are small, intracellular, vector-borne hemotropic gram-negative bacteria, some of which can infect a variety of mammals and cause human Bartonellosis1. The Bartonella species and their respective reservoir hosts are increasing constantly, and over 40 species and subspecies of Bartonella have now been detected in domestic and wild animals including cats, dogs, rodents, cattle, deer, bats, and so on2. Several Bartonella species were recognized as human pathogens, such as B. bacilliformis3, B. quintana4, B. henselae5, B. elizabethae6, B. clarridgeiae7, B. koehlerae8, B. vinsonii subsp. arupensis9, B. vinsonii subsp. berkhoffii10, B. grahamii12, B. rochalimae13, B. tamiae14, B. ancashensis15, B. washoensis16, and so on, which could cause endocarditis, myocarditis, neuroretinitis, meningitis, splenomegaly, lymphadenopathy and neurologic disorders in humans., particularly rodents are considered natural reservoirs of many Bartonella species. Previous studies have reported that the infection rate of Bartonella is as high as 70% in rodents worldwide17, including in America18, Asia19, Africa20, and Europe21. Furthermore, many pathogenic Bartonella can be detected in rodents, which indicated that investigating the epidemiological characteristics of Bartonella in rodents is of great significance for the prevention and control of human Bartonellosis.Small mammalsBartonella species in Ochotona curzoiae in the Qinghai-Tibet Plateau, with a positive rate of 18.99%22. However, few systematic investigations of Bartonella species in small mammals have been reported in the Qinghai-Tibet Plateau. The Qaidam Basin, located in the Haixi Mongolian Tibetan autonomous prefecture, northwest of Qinghai Province and northeastern Qinghai-Tibet Plateau, is the highest basin in China, with an altitude between 2600 and 3000 m; nearly 20 species of rodents have been reported to inhabit this area23. Furthermore, the Qaidam Basin, is a famous tourist attraction, attracting many visitors each year; therefore, the prevention and control of rodent-related pathogenic microorganisms are important for public health.In the previous study, our team had detected Bartonella species in small mammals (including rodents and Ochotona curzoiae) from the Qaidam Basin. Our findings provided insights into the distribution of Bartonella in small mammals and the resulting public health threat in this region.Accordingly, in this study, we investigated the prevalence and genetic diversity of Cricetulus longicaudatus (46), Mus musculus (17), Phodopus roborovskii (11), Meriones meridians (11), Ochotona curzoniae (10), Allactaga sibirica (3), Cricetulus migratorius (2) and Microtus oeconomus (1). The geographical distribution of the trapped small mammals was shown in Fig. In total, 101 small mammals were captured from six trapping sites, including 78 in Wulan County (01-78 QHWL) and 23 in Ge’ermu City (79-101 QHGEM). All captured small mammals were identified morphologically into eight species, including Bartonella isolation, and the pure colonies obtained were then confirmed by polymerase chain reaction (PCR) amplification of partial gltA gene (379 bp). In total, 39 small mammals, classified into four species , were positive for Bartonella infection; 21 were positive in both the brain and spleen, 12 were positive in the spleen, and six were positive in the brain, with an overall infection rate of 38.61% (39/101). The positive rates in different tissues were not statistically significant  .There were 97 spleens and 74 brains of 101 small mammals (excluded uncollected and contaminated specimens) used for Bartonella infection rate of 40.00% (18/45). Twenty small mammals of three species were captured in forests, with an infection rate of 60.00% (12/20). Additionally, 10 small mammals of one specie were captured in meadows, with no Bartonella infection, and 26 small mammals of four species were captured in semi-desert areas, with an infection rate of 34.62% (9/26). Thus, the infections rates in different habitats were significantly different  , and all isolates clustered into four clusters, i.e., clusters I to IV , neighbor-joining (NJ), minimum-evolution (ME), and unweighted pair-group method with arithmetic mean to construct phylogenetic trees and obtained the same results; thus, the ML method was used for further analyses. Phylogenetic trees were constructed based on the DNA sequences of the concatenations of bC genes 483 bp, aB. grahamii, B. jaculi, B. krasnovi, and Candidatus B. gerbillinarum were prevalent in the Qaidam Basin, and that B. grahamii was the dominant Bartonella species. Bartonella was not detected in the small mammals from two of six trapping sites, and the distribution of Bartonella species showed obvious geographical differences. Moreover, B. grahamii and B. jaculi were distributed in Wulan County, whereas Candidatus B. gerbillinarum and B. krasnovii were distributed in Ge’ermu City , Canada (48–90%), Netherlands (72%), etc17. In addition, the infection rate was significantly different in different habitats , with the highest infection rates observed in forests.Bartonella make it difficult to culture in vitro28. Generally, the spleen tissue is used in Bartonella culture. In this study, brain tissue was successfully used for Bartonella isolation for the first time. Additionally, we found that the positive rates in different tissues (spleens and brains) of small mammals did not differ significantly. Moreover, we detected two Bartonella species in the same small mammal, i.e., B. krasnovii in the spleen and Candidatus B. gerbillinarum in the brain, indicating the complexity of Bartonella infection in small mammals. A previous study indicated that there might be a high Bartonella coinfection rate in rodents29. Therefore, in order to further explore the coinfection of Bartonella species, additional studies of multi-tissue culture, multi-clone detection, and multiple PCR detection by using well-defined species or genotype PCR primer sets are needed29.The nutritional requirements of Bartonella species have been implicated as the causative agents of human disease. Here, we obtained four Bartonella species in rodents, B. grahamii, B. jaculi, B. krasnovii and Candidatus B. gerbillinarum. Especially, B. grahamii was detected in Cricetulus longicaudatus specificly, B. jaculi was detected in Allactaga sibirica specificly, Candidatus B. gerbillinarum and B. krasnovii were detected in Meriones meridianus specificly, indicating rodent-specific characteristics. B. grahamii was the dominant epidemic strain circulating in the Qaidam Basin, which was associated with neuroretinitis and cat scratch disease (CSD) in immunocompromised people12, suggesting that Bartonella species detected in Cricetulus longicaudatus may have the ability to cause human disease. It was reported that Bartonella species antibodies and DNA were detected in cerebrospinal fluid of cats and dogs31, suggesting the possible relationship between Bartonella infection and central nervous system disease. Bartonella isolation from the rodent brain supported this observation. Until now, B. henselae and B. quintana were reported to cause central nervous system infection33, however, the effects of B. grahamii on the central nervous system needs further investigation.Several rodent-associated gltA sequences collected from the GenBank, B. grahamii from Cricetulus longicaudatus in the Qaidam Basin was mainly clustered with B. grahamii from Apodemus speciosus in Japan34 and Ochotona curzoniae in China22. Some B. grahamii prevalent in the Qaidam Basin might have high homology with strains from Japan, which was consistent with the previous study34. B. jaculi, Candidatus B. gerbillinarum and B. krasnovii have only been reported in Eygpt35 and Israel29, which pathogenicity was not clear and needs further study. However, in this study, Bartonella infection was not detected in Ochotona curzoniae, possibly because of the differences in sampling sites and the small number of samples collected, which need further investigation.Through phylogenetic analysis based on Bartonella species in small mammals from the Qaidam Basin. Four Bartonella species were detected in rodents, among which B. grahamii was the dominant strain, and potentially pathogenic to humans. Additionally, Bartonella were isolated from rodent brains for the first time, and two Bartonella species were detected in different tissues of the same rodent, which indicated the complexity of Bartonella infection and the necessity for multi-tissue culture, multi-clone detection and multiple PCR detection. Our results raise the potential threats to public health by the Bartonella species, and surveillance of Bartonella in animals and investigation of suspected clinical cases in humans need to be strengthened in the Qaidam Basin.This study provided a better understanding of the prevalence and genetic diversity of This study was approved by the Ethics Committee of Chinese Center for Disease Control and Prevention (No: ICDC-2015001). Small mammals were sampled with the help of Wulan Center for Disease Control and Prevention, and Ge’ermu Center for Disease Control and Prevention. All animals were treated according to the Guidelines of Regulations for the Administration of Laboratory Animals  and the Guidelines for Treating Animals Kindly from Ministry of Science and Technology of the People’s Republic of China. All efforts were made to minimize discomfort to the animals.36, small mammals were captured using snap traps in a diversity of habitats in July 2018 in Wulan County  and Ge’ermu City , Qinghai Province. And the small mammal species were determined morphologically. The spleens and brains were harvested under sterile conditions from each animal after euthanasia, and all samples were stored in liquid nitrogen until use.According to the previous study2. Pure colonies of Bartonella species were obtained according to previous methods22.Approximately 20 mg of each spleen and brain sample from the small mammal was homogenized by adding 200 μL sterilized trypsin soy broth , plated onto two trypsin soy agars containing 5% (vol/vol) defiber sheep blood (BD Biosciences), and incubated at 37 °C in an atmosphere containing 5% CO22. PCR was performed to detect the Bartonella citrate synthase (gltA) gene. Then, gltA positive strains were evaluated for amplification of ftsZ, RNA polymerase beta subunit (rpoB) and ribC genes. DNA amplification was performed in 50 μL mixtures containing 25 μL 2 × TransTaq-T PCR SuperMix , 22 μL double-distilled H2O, 1 μL (10 μmol/L) of each primer . The expected PCR products were purified using the QIAquick PCR Purification Kit  according to the manufacturer’s protocols, and then sequenced using specific primers for gltA, ftsZ, rpoB and ribC with an Applied Biosystems 3730 xl Genetic Analyzer .Crude DNA was extracted according to the previous methodBartonella species sequences in the GenBank using the BLAST program at the National Center for Biotechnology Information Website (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic tree was created using the maximum-likelihood method with MEGA version 7.0, and bootstrap values were calculated with 1000 replicates42 . Brucella abortus was used as the outgroup.The sequences generated in this study were submitted to the GenBank (accession numbers MT815283-MT815438). The nucleotide sequence homology was blasted against reported Bartonella in different habitats, genders and tissues of small mammals were analyzed using the Chi-square test. All data were analyzed using SPSS 22.0 . P < 0.05 was considered statistically significant.The positive rates of All the authors consent to publish the article in its present form.


HGB8P were identified in two independent EA ADHD patients. A meta-analysis of the two ethnicities for differential ADHD recurrent variants compared to controls shows a small number of overlaps. These results suggest that rare recurrent variants in noncoding regions may be involved in the pathogenesis of ADHD in children of both AA and EA ancestry; thus, WGS could be a powerful discovery tool for studying the molecular mechanisms of ADHD.Attention-deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder with poorly understood molecular mechanisms that results in significant impairment in children. In this study, we sought to assess the role of rare recurrent variants in non-European populations and outside of coding regions. We generated whole genome sequence (WGS) data on 875 individuals, including 205 ADHD cases and 670 non-ADHD controls. The cases included 116 African Americans (AA) and 89 European Americans (EA), and the controls included 408 AA and 262 EA. Multiple novel rare recurrent variants were identified in exonic regions, functionally classified as stop-gains and frameshifts for known ADHD genes. Deletion in introns of the protocadherins families and the ncRNA  Attention-deficit hyperactivity disorder (ADHD) is a common neurological disorder resulting in significant life-long impairments . PreviouThe patients were recruited by the Center for Applied Genomics (CAG) at The Children’s Hospital of Philadelphia (CHOP), which has established the world’s largest pediatric biobank with full electronic medical record (EMR) coupling. There were 205 ADHD cases, including 116 African Americans  and 89 European Americans , and 670 controls, including 408 AA and 262 EA, whose samples were whole genome sequenced. The AA cases included 27 patients of attention deficit disorder without hyperactivity and 89 patients of attention deficit disorder with hyperactivity. The EA cases included 34 patients of attention deficit disorder without hyperactivity and 55 patients of attention deficit disorder with hyperactivity. The data is available at the database of Genotypes and Phenotypes (dbGaP) portal with the accession number phs001165.The whole genome sequencing (WGS) using the Illumina platform and average depth coverage is 30× for the genome region. Each lane or sub-lane of data was aligned to GRCh37-lite with bwa v0.7.10 . The unaA total of 875 WGS samples were processed and annotated by Illumina Edico Genome’s DRAGEN  platform. The DRAGEN platform uses a field-programmable gate array (FPGA) to provide hardware-accelerated implementations of genome pipeline algorithms, including BCL conversion, compression, mapping, alignment, sorting, duplicate marking, and haplotype-based variant calling. ANNOVAR , and theThe variant is considered the “same” if the mutation is at the same genomic locus with the same alternative allele. SNVs or indels that occurred more than once in ADHD and had less than 1% frequency in controls were marked as recurrent variants of interest and moved forward in analysis if they had an MAF lower than 1% in the NHLBI 6500 exome sequencing project (6500 ESP) , 1000 Gep-value was based on using the Benjamini–Hochberg method. The impact of mutations were predicted and annotated by multiple tools, including SIFT [The enrichment analysis was based on DAVID  bioinformatics platform  and the ing SIFT , LRT [16ing SIFT , Mutatioing SIFT , Mutatioing SIFT , FATHMM ing SIFT , RadialSing SIFT , and cliing SIFT . The numNUDT19 and PCOLCE2 were predicted as deleterious by LRT and MutationTaster, meanwhile rare recurrent variants in OR2D3, TAS2R14, POLN, ZNF880, ZNF433, and GLG1 were predicted as deleterious by MutationTaster. For EA, the variant rs371526758 (chr1: 10042426-G-A) in NMNAT1 was predicted as deleterious by LRT and MutationTaster and reported as “pathogenic” in the ClinVar database. This variant resulted in a stop codon at exon 5 of NMNAT1 in two EA ADHD patients. Previous studies show that mutations in NMNAT1 cause Leber congenital amaurosis and white matter disorders [FRY gene was predicted to be deleterious by SIFT, LRT, and MutationTaster in EA.We identified 1815 zero-in-control rare recurrent variants in the AA cases and 1244 in the EA cases . For AA,isorders . AnotherATXN2 gene. ATXN2 is associated with multiple neurological diseases, and the variant is one of the top independent signal in our study. The frameshift insertion at the first exon of ATXN2 is seen in two independent individuals. Six AA individuals contain rare recurrent frameshift variants at exon 3 and 62 for ABCA13, which were annotated as ADHD-related genes by Genecards [DGKQ is a reported gene in Parkinson’s disease [DGKQ at exon 5. FMNL2 has been identified in patients with depression [CHD7, the causative gene for CHARGE syndrome, where many patients also show ADHD behavior [HSD3B1 is related to the ADHD pathogenesis in rat [UNC5B is an ADHD-associated GWAS locus [p-value = 0.018).Beside stop-gain mutations, there are 31 rare recurrent frameshift variants in AA and 34 in EA . Multiplenecards  and is ienecards . DGKQ is disease , and fivpression  and is tpression . For thes in rat  and a tos in rat . UNC5B ip-value = 8.7 × 10−5), DEVELOPMENTAL , PSYCH , and NEUROLOGICAL  through DAVID, but not in any biological processes or pathways. In contrast, 581 ncRNAs in EA show significant enrichments in biological processes of homophilic cell adhesion via plasma membrane adhesion molecules  and in calcium ion binding . There are 1221 transcripts with intronic indels in EA, and they also show enrichment in homophilic cell adhesion via plasma membrane adhesion molecules . Although the distributions of intronic rare recurrent variants are similar in AA and EA  transcripts contain zero-in-control rare recurrent variants for AA and 581 for EA. The ncRNAs in AA show enrichment of specific disease classes, including METABOLIC , and reveals enrichment for disease classes such as neurological and developmental through DAVID  were identified in previous studies for ADHD [Meta-analysis was applied when combing the two ethnicities together as described in the Methods section. No exonic zero-in-control rare recurrent variants passed the threshold ntiation b. There ntiation , and 8 ofor ADHD . These rThe glutamatergic neurotransmitter system plays an important role in ADHD, and the mGluR activator fasoracetam showed promising results when applied in clinical trials of adolescents harboring mutations in mGluR network genes . Based oADHD is a common, inheritable neurodevelopmental disorder largely of unknown etiology. Previous studies suggest that ADHD is more likely to be regulated and impacted at the level of biological pathways instead of an individual gene ,39. RarePrevious studies are highly focused on SNVs in coding regions; on the other hand, noncoding genomic structural variations and noncoding genes have been shown to play important roles in many human diseases, including other neurodevelopmental diseases such as autism and intellectual disability . AnotherATXN2 in AA and UNC5B in EA) have been reported as ADHD associated in previous studies [We uncovered multiple novel rare recurrent variants in coding regions that present a plausible role in the pathogenesis of ADHD and further supported by their absence in population databases and non-ADHD controls. These include 12 vs. 9 stop-gain rare recurrent variants and 31 vs. 34 rare recurrent frameshifts variants in AA and EA patients, respectively. Two genes  and neuronal differentiation . The neurogenesis-related genes have higher portion of known ADHD genes (18%), including CTNNA2, one of the top hits of ADHD GWAS studies associated with the metabotropic glutamate receptor (mGluR) pathway [The most significant finding in this study is the impact of noncoding rare recurrent variants, especially intronic indels, on biological processes and pathways for ADHD. While intra-genomic indels may affect the transcriptional machineries , these regulatory effects tend to be less severe than the functional effects of intronic indels by causing intron retention and loss-of-function . The funHGB8P. PCDHs are critical for the formation and function of neural circuits, including dendrite arborization, axon outgrowth and targeting, synaptogenesis, and synapse elimination, and epigenetic regulation of clustered PCDHs has been shown to play roles in brain developments. The molecular and functional interaction between protocadherin-γC5 and GABAA receptors was proven in rats [GRM7 at chr3:7515182. Another notable observation is that ADHD patients with a CNTN4 mutation phenotype tend to be more severe, with a higher prevalence of emotional dysregulation and demonstrate enriched response to fasoracetam. We uncovered two nonsynonymous SNVs in CNTN4, one at chr3:3084045-A-T exon 21 in EA and another one at chr3:3030048-G-A exon 14 in AA, both predicted as deleterious based on prediction tools. Although no rare recurrent variants were identified in CNTN4, we found another gene, CNTN6, from the Contactins family that contains an intronic indel, a rare recurrent variant that passed the significant threshold of meta-analysis at chr3:1136572 (2-bp deletion).In two independent EA patients, we identified a four-base intronic deletion at chr5:140806031 covering 18 genes of the protocadherin (Pcdhs) families A and B, while the locus is also the exonic region for noncoding RNA,  in rats . GABAA h in rats , suggest in rats . We idenIn summary, we conducted a whole genome analysis in ADHD patients using whole genome sequencing data that takes noncoding regions and ethnicity factors into consideration. The results show that rare recurrent indels in noncoding regions, especially introns, may play an important role in the development and progression of ADHD, suggesting that WGS may be a powerful tool to discover novel molecular mechanisms of ADHD. Additionally, our study highlights that genomic-level population differences exist between European Caucasian and African American patients. This study focused on zero-in-controls rare variants. As a major weakness of this approach, the individual rare variants presented in this study lack statistical significance after correction for multiple tests due to their low frequencies. The individual rare recurrent variants presented in this study as compelling ADHD candidates warrant further study in larger-scale studies.


The Attention Deficit Hyperactivity Disorder (ADHD) Teen Integrative Data Analysis Longitudinal (TIDAL) dataset integrates data from four randomized trials.N = 854; 72.5% male, 92.5% racial/ethnic minority, ages 10–17) were assessed three times across 12 months. Data includes parent, self, and teacher ratings, observations, and school records. The battery was harmonized using an Integrative Data Analysis (IDA) approach to form variables that assign unique values to all participants.Participants with ADHD (The data will be used to investigate: (1) profiles that organize the heterogeneous population into clinically meaningful subgroups, (2) whether these profiles predict treatment response, (3) heterogeneity in treatment response and variables that predict this response, (4) how treatment characteristics and adjunctive supports predict treatment response, and (5) mediators of treatment and whether these mechanisms are moderated by treatment characteristics.The ADHD TIDAL Dataset will be openly shared with the field to maximize its utility. The teenage years are a critical period for ADHD intervention. Longitudinal studies indicate a close relationship between adolescent functioning and ADHD persistence into adulthood –3. MoreoDespite their efficacy, implementation and utilization of adolescent ADHD treatments are poor –14. ThusThe clinical profiles of adolescents with ADHD are highly heterogeneous –17; yet,Pursuing these aims require person-level approaches  . As the N = 854; ages 10–17) [To increase data resources in the field of adolescent ADHD, we constructed the ADHD Teen Integrative Data Analysis Longitudinal (ADHD TIDAL) dataset. The ADHD TIDAL dataset will be openly shared with the field. We integrated data from four randomized trials (s 10–17) –29 that s 10–17)  frameworIntegrative data analysis is a relatively new technique that allows researchers to pool raw data from multiple studies to produce cumulative scientific knowledge . IDA difTo conduct an analysis on the combined dataset, the same variables must be present in some form in all studies. The four studies were conducted by the same investigators, so many measures of interest are common across studies   and requIDA allows for either random or fixed effects models, depending on the number of studies and whether a study is conceptualized as a random sample from the population of interest. Fixed effects IDA conceives of each study as a known, specific sample from the population and can be conducted with as few as two studies. Random effects IDA conceives of each study as a random sample from the population and requires a minimum of 20 to 30 studies. The ADHD TIDAL dataset includes four studies, so all analyses are conducted within a fixed-effects IDA framework. This means that dummy codes indicating study membership are included in each analysis to account for differences between studies. We interpret all results within a fixed effects IDA framework indicating that: (1) we can only make inferences back to the specific studies, not to similar studies on this population and (2) we cannot fully disaggregate some between- and within-study effects, due to study-specific code variables in analyses .Our four studies were chosen for the IDA because they shared common characteristics that promoted successful harmonization. From 2010 to 2019, the research team conducted seven longitudinal treatment outcome studies of psychosocial treatment for adolescents with ADHD. We sought to include studies in the IDA that: (1) included participants from the large local school district with standardized attendance, grades, and disciplinary data, (2) possessed inclusion criteria that all participants meet DSM criteria for ADHD during a comprehensive psychiatric evaluation that included a structured parent interview   and pareIn study A (see Table Study B (see Table N = 123) were randomly assigned to STAND or STAND-G using a stratified randomization procedure within study wave [Middle or high school students with ADHD (see Table udy wave . Study eN = 278) who were incoming patients at four community mental health clinics. Over 3 years, treatment was provided by agency employees who were randomly assigned to receive STAND training and supervision or treat cases using UC practices. Adolescents were also randomized to STAND vs. UC. Retention was 99.3% at post-treatment and 97.5% at follow-up (data from at least one informant).This trial  is an advantage when trying to find relationships between variables. Particularly in studies of clinical populations, restricted range can reduce statistical power and impede detection of relationships between variables. The larger, more heterogeneous combined sample improves statistical power. In addition, traditionally under-represented groups  and behaviors  are well-represented in the IDA sample.While the four studies are similar in scope, they also differ in several ways. Study B includes school-referred youth, study D includes patients in community agencies, and studies A and C included patients at a university clinic. Studies A and B utilize a no treatment control group; studies B, C, and D compare active treatments, including therapist-selected intervention in community mental health . Study B included summer treatment, while studies A, C, and D included treatment delivered at various points during the school year.N = 854) are presented in Tables Demographic characteristics of the full sample  is manualized and consists of eight 90-min weekly sessions attended by the adolescent and parent . ParentsThe 8-week STP-A  includesUsual Care (UC) therapists at community mental health agencies were instructed to treat study cases using usual procedures in the agency and the treatments they believed would be most effective. They received weekly supervision from agency supervisors according to typical agency practices. Complex analyses of UC practices have been proposed as a future direction; at present, UC psychotherapy for adolescent ADHD remains a black box.In Study A, a treatment as usual comparison group was offered no treatment by the study team. In Study B, an untreated comparison group was followed in the fourth year of the study. In these conditions, participants were permitted to pursue naturalistic treatment in their communities.Table Adolescent Academic Problems Checklist (AAPC) measure observable secondary-school specific organization, time management, and planning (OTP) problems and are validated for use in samples of adolescents with ADHD [The self, parent, and teacher-report versions of the 24-item ith ADHD . The AAPith ADHD . One itestrongly agree) to 5 (strongly disagree) [The parent and teen Conflict Behavior Questionnaire-20 (CBQ-20) assessed the quality of the parent-teen relationship. Respondents were asked to rate statements on a five-point scale from 1 (isagree) . The CBQisagree) .not at all present), 1 (just a little), 2 (pretty much), or 3 (very much). To calculate an index of symptom severity the average level (0–3) of each item on the ADHD subscales is obtained. The psychometric properties of the DBD rating scale are very good, with support for internally consistent subscales [In the DSM-IV-TR era, the parent and teacher Disruptive Behavior Disorders Ratings Scale (DBD-RS)  measuredubscales .not at all) to 3 (very much). Symptom severity is the mean level (0–3) of ADHD subscale items. Psychometric properties of the measure are very good, with empirical support for internally consistent IN and HI subscales [In the DSM-5 era, IN and HI were measured using a DSM-5 ADHD Rating Scale completed by adolescents, parents, and teachers . Respondubscales . The DSMubscales .The parent-reported Child Behavior Checklist (CBCL) and Youth Self Report (YSR) were administered as broadband youth psychopathology scales . These sObservations of planner use assessed the degree to which students recorded homework assignments. Percentage of classes in which homework was recorded (or some indication of no homework) was calculated for the last 5 days of school. Planner use was calculated as the mean of daily scores. Photocopies or screenshots were obtained to document use. If the adolescent did not record any homework, he/she received a score of zero. This measure demonstrates high inter-rater reliability  . ObservaReport cards were obtained directly from the school district at the end of each academic quarter. GPA for each quarter was calculated by converting academic grades  to a 5-point scale . Grades were not weighted for the difficulty of the class. GPA provides an objective and ecologically valid measure of school performance that is meaningful to parents and schools. The average grade on each completed assignment was also calculated. Assignments included any mandatory academic work turned in by the student except for tests, quizzes, and exams . Extra credit assignments and class participation were not counted towards this average. Missing assignments were also not weighted in the average. The average grade on each test, quiz, or exam was calculated. The percentage of assignments turned in calculated by dividing turned-in assignment count by the total number of assignments due.The school district provided records of student disciplinary incidents at the end of each year. Counts of each type of disciplinary incident  were calculated and coded according to Robb and colleagues . Minor dThe Wechsler Abbreviated Scale of Intelligence (WASI or WASI-II) was administered to participants as an index of IQ. Full-scale IQ was measured using a composite score from the Matrix Reasoning and Vocabulary subtests (Full-2) or all four subscales (Full-4) of the Wechsler Abbreviated Scale of Intelligence-2nd Edition (WASI-II) . The WASThe Impairment Rating Scale (IRS) was administered to parents and teachers (IRS) . ParentsThe DISC is a structured interview that was administered to assess ADHD, ODD, and CD diagnoses. The DISC queries the presence of each symptom . The ADHD module contains supplemental probes for symptom-specific impairment . SymptomThe PAMS is a 16-item checklist that measures the frequency of adaptive and maladaptive parental involvement strategies related to adolescent OTP skills . ParentsCaregiver Strain Questionnaire (CSQ) [Parent strain stemming from the parent-adolescent relationship was measured by the 21 item re (CSQ) . The parThe Adult ADHD Self-Report Scale (ASRS) measured parental ADHD . EighteeThe Symptom Checklist-90-Revised (SCL-90-R) is a 90-item broadband scale of adult psychopathology that measures nine symptom domains using a 5-point Likert scale . The SCLThe Patient Health Questionnaire-9 depression scale has excellent internal reliability as well as criterion and construct validity . ParentsParents completed the World Health Organization (WHO) Quality of Life Questionnaire, a multidimensional profile of quality of life for cross cultural use. The English version is self-administered and covers 25 facets of quality of life within six broad domains. It captures positive and negative aspects of quality of life and possesses strong psychometric properties .Harmonization of measures followed methods of Bauer (2017)  and CurrParent depression was assessed by the SCL-90 (studies A and B), PHQ-9 (study C), and the WHO QOL (study D). Specific items from the SCL-90 (13 of 90 items) and the WHO QOL (5 of 26 items) that reflected symptoms of major depression were selected; the PHQ-9 measures the nine DSM symptoms of major depression and all items were included. Each participant’s item scores were combined and recoded to reflect endorsement of the nine DSM-5 major depression symptoms. Nonlinear moderated factor analysis (NLMFA) was used to determine item-specific characteristics  within the IRT framework, how those item characteristics vary across study, and to provide each participant with a common-scale score of depression . This haAdolescent ADHD symptoms were measured using DSM-IV criteria in studies A and B and using DSM-5 criteria in studies C and D; both parent and teacher report of adolescent ADHD symptoms were collected. The DSM-IV-TR criteria  omits corresponding adolescent/adult specifiers added to the DSM-5 ADHD criteria . For each participant, item scores were recoded to reflect endorsement the 18 ADHD symptoms. Since ADHD symptoms are grouped into IN and HI, a two-factor model was used. Nonlinear moderated factor analysis (NLMFA) was used to determine item-specific characteristics  within the IRT framework, how those item characteristics vary across study, and to provide each participant with common-scale scores of IN and HI ADHD symptoms . These hhttps://nda.nih.gov).Our research team has several analyses planned using the ADHD TIDAL dataset. However, numerous opportunities for data analysis exist beyond our specific aims. We believe that pursuing personalized medicine questions for adolescents with ADHD will provide useful information that promotes improved treatment engagement and response—leading to meaningful changes in long-term outcomes. We invite additional research teams to utilize the ADHD TIDAL dataset, which is publicly available for use at the National Institute of Mental Health, National Data Archive  will be used to identify treatment-relevant phenotypes and environmental factors based on relevant individual  and family context variables . Variables that demonstrate superior psychometric properties when modeled as an observed variable will not be modeled in the context of the latent profiles.In a second aim, we will examine whether baseline latent profile and observed variables predict treatment engagement and response. The first analyses will describe who is most at risk for treatment disengagement . Finally, we will examine whether baseline latent profile and observed variables predict treatment response, with primary outcomes  as the distal outcomes.Our third aim will examine heterogeneity in treatment response over time. This aim will identify latent, unobserved groups of individuals who differ from one another in terms of their outcome  trajectory over time. This analysis will allow us to examine this heterogeneity and determine if clinical profile, family context, adjunctive supports , and treatment characteristics  predict treatment response.Our fourth aim will be to identify key treatment mediators and moderators of the relationship between treatment group and change in key outcomes . Potential mediators of the treatment effect on outcomes include teen organization skills, parent contingency management, parent-teen conflict, and parental well-being. Potential moderators of the treatment effect on outcomes include individual, treatment, and family variables (as noted above).N = 854, 9 treatment groups , and 3 measurement occasions, the required effect size is approximately d = 0.12, a very small effect. In previous analyses of individual studies included in this IDA, we found effect sizes of d = 0.5 or higher for moderation of the treatment effect on ADHD symptoms . For the mediation analyses, we used the tables generated by Fritz and MacKinnon [N = 854 and using the preferred method of bootstrap confidence intervals, we have greater than 80% power to detect even small effects for both the treatment to mediator and mediator to outcome slope. In previous analyses of individual studies included in this IDA, we found effect sizes of d = 0.5 or higher for the moderating effect of treatment on ADHD symptoms .Given the fixed sample size, we present sensitivity analyses that provide the smallest effect that can be detected, rather than power analyses. For latent profile analyses and growth mixture models proposed in aims 1 through 3, simulation studies indicate that the Bayesian Information Criterion and bootstrap likelihood-ratio test perform best at determining the correct number of classes , 60. TheacKinnon . With a The ADHD TIDAL dataset is suitable for examining treatment outcome questions that expand on those noted above by selecting independent and dependent variables, moderators, and mediators that our team did not incorporate into our planned analyses. Given the broad age range of participants, cross-sequential analyses with the ADHD TIDAL dataset could reveal important information about the nature of ADHD symptoms and related impairments. The samples demographic diversity also may support research questions related to gender, cultural, or socioeconomic differences in the expression of ADHD. The broad range of data available in the ADHD TIDAL dataset also may help research teams estimate effect sizes for power analyses and conduct pilot analyses prior to data collection studies, as well as further integration with existing datasets.


Higher hyperactive ADHD symptom scores instead were associated with lower socio-economic status . These results highlight the importance of monitoring and supporting the development of very preterm children throughout the school years, as subthreshold ADHD symptoms represent risk factors for psychosocial problems and for receiving a future clinical diagnosis of ADHD.This study investigated the association between attention-deficit/hyperactivity disorder (ADHD) symptomatology in preschool-aged children who were born very preterm (<33 weeks) and cognitive outcomes, clinical risk and socio-demographic characteristics. 119 very preterm children who participated in the Evaluation of Preterm Imaging Study at term-equivalent age were assessed at a mean age of 4.5 years. Parents completed the ADHD Rating Scale IV, a norm-referenced checklist that evaluates ADHD symptomatology according to diagnostic criteria, and the Behavior Rating Inventory of Executive Function-Preschool version. Children completed the Wechsler Preschool and Primary Scales of Intelligence and the Forward Digit Span task. Longitudinal data including perinatal clinical, qualitative MRI classification, socio-demographic variables and neurodevelopmental disabilities were investigated in relation to ADHD symptomatology. All results were corrected for multiple comparisons using false discovery rate. Results showed that although the proportion of very preterm children with clinically significant ADHD did not differ from normative data after excluding those with neurodevelopmental disabilities, 32.7% met criteria for subthreshold ADHD inattentive type and 33.6% for combined type, which was higher than the expected 20% in normative samples. Higher ADHD symptom scores  were associated with greater executive dysfunction . Higher inattentive ADHD symptom scores were associated with lower IQ (ρ = -0.245,  Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental condition which affects 5–7% of children, depending on the source of information used to reach a diagnosis , 2. ChilAccording to the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-V), ADHD is characterized by two main symptom presentations, inattention and hyperactivity-impulsivity . AlthougADHD symptoms in very preterm children exhibit specific cognitive correlates that are not observed in term-born children with ADHD , and resThe majority of studies to date in very preterm samples have focused on school age, when ADHD is typically diagnosed. However, the importance of detecting early risk factors associated with ADHD is well recognised . AttentiHere we investigate ADHD symptoms, and their cognitive and clinical correlates in preschool-aged children who were born very preterm. We hypothesised that higher ADHD symptoms in very preterm children would be associated with lower executive functions and intelligence, even in the absence of a clinical diagnosis. We further hypothesised that inattentive ADHD symptoms would be associated with perinatal clinical adversity rather than environmental risk.Study participants were 119 very preterm born children who were recruited at birth in 2010–2013 as part of the Evaluation of Preterm Imaging Study (e-Prime Eudra: CT 2009-011602-42) from hospitals within the North and Southwest London Perinatal Network . Infantshttp://imd-by-postcode.opendatacommunities.org), which provide a proxy for socio-economic status.Perinatal clinical and socio-demographic data were collected with permission from the Standardized Electronic Neonatal Database and included gestational age at birth, sex, days of mechanical ventilation, days of parenteral nutrition, intrauterine growth restriction , mother’s age when leaving full time education and Index of Multiple Deprivation (IMD) score, determined by parents’ postcode at the time of infant’s birth  system sited on the neonatal intensive care unit using an eight-channel phased array head coil. Multiple MR sequences were acquired as detailed in  T2-weighThe ADHD Rating Scale-IV (ADHD-RS-IV)  was usedth percentile of the sex-matched normative score distribution, as recommended by Power and colleagues [th percentile was also used to include sub-threshold symptoms.Scores were defined as clinically significant when equal or greater than the 90lleagues . The 80tWe investigated a range of cognitive outcomes that are widely studied in the preterm literature. General intellectual development was assessed with the Wechsler Preschool and Primary Scales of Intelligence battery (WPPSI-IV) . Age corPhonological short term memory was assessed using the digit span forward test, using an adapted version of the Wechsler Intelligence Scale for Children IV digit span task . InhibitMotor impairment was determined on the basis of scores below 2 standard deviations on the Gross Motor Scale of the Bayley Scales of Infant Development-III  obtainedChildren with at least one of the following were regarded as having a neurodevelopmental disability (NDD): neuromotor impairment , diagnosis of ASD and/or GDD, sensory or/and speech problems.SPSS version 22.0  and Matlab  were used for analyses. Binomial tests were used to compare the proportion of very preterm children with clinically significant  levels of behavioural problems against age-matched US population norms. Analyses were run before and after exclusion of children with NDD.To assess the prevalence of inattention and hyperactivity/impulsivity symptoms in very term children, a data-driven clustering procedure was also used on the ADHD-RS-IV scores to fractionate the sample into groups, defined by the imbalance between their inattentive and hyperactive/impulsive symptoms, similar to . An ExpeThe associations between ADHD symptoms, IQ and neurocognitive skills  were investigated using non-parametric Spearman’s rank-order correlation. Analyses were run after exclusion of children with NDD and p values were always corrected for multiple comparisons using a false discovery rate (FDR) approach controlling alpha error to 5% . Spearmath centile of the sex-matched normative score distribution.Comorbidities between NDD and behavioural problems were defined by having NDD plus ADHD symptoms above the 90th centile cut-off, 25.0% had hyperactivity/impulsivity and 58.3% had combined symptoms. When using the 80rd percentile cut-off and excluding children with NDD, 32.7% of very preterm children had inattentive symptoms and 33.6% had combined symptoms, which was higher than the expected 20% in normative samples.The proportion of very preterm children with clinically significant inattentive (20.2%), hyperactive/impulsive (16.0%) and combined symptoms (16.8%) was higher than the expected 10% in normative samples of predominantly term-born individuals , 12 with high levels of both inattentive and hyperactive/impulsive symptoms (10.1%), 44 with medium levels of both symptoms (37%), 50 with low levels of both symptoms (42%) and no child with mainly hyperactive/impulsive symptoms see .p = 0.082), hyperactive/impulsive  and combined ADHD symptom scores , even after exclusion of children with NDD.Mann-Whitney tests showed no sex differences in inattentive  but not with hyperactive and combined ADHD symptom scores . Higher ADHD symptom scores  were associated with poorer inhibitory self-control, flexibility and emergent metacognition scores, as shown in After excluding children with NDD, higher inattentive ADHD symptom scores were associated with lower full-scale IQ  and more days on parenteral nutrition (ρ(119) = 0.222, p = 0.015). All results survived FDR correction. There was no evidence of differences in ADHD symptom scores according to severity of perinatal brain lesions . When analyses were repeated after excluding children with NDD, results remained unchanged. 45.5% of children with NDD also had major lesions on neonatal MRI .Higher inattentive ADHD symptom scores were associated with more days on mechanical ventilation (ρ(119) = 0.196, p = 0.004 and ρ(119) = 0.198, p = 0.031, respectively). Children belonging to the 5th IMD quintile, reflecting the most deprived socio-economic group, had a z score of 0.438, while those children belonging to the 1st IMD quintile, reflecting the least deprived socio-economic group, had a z score of -0.433. After FDR correction, these associations remained unaltered. Similar findings persisted when children with NDD were excluded from the analysis.Hyperactive/impulsive and combined ADHD symptom scores were correlated with higher IMD scores which reflect lower socio-economic status (ρ(119) = 0.259, Although the proportion of very preterm children with clinically significant ADHD did not differ from normative data after excluding those with neurodevelopmental disabilities, 32.7% met criteria for subthreshold ADHD inattentive type and 33.6% for combined type, which was higher than the expected 20% in normative samples. These results highlight the importance of monitoring and supporting the development of very preterm children throughout the school years, as subthreshold ADHD symptoms have been associated with an increasing risk of psychosocial problems and of receiving a future diagnosis of the clinical disorder –37.In addition to the high subthreshold ADHD inattentive symptom prevalence, our results showed that inattention was a core deficit in this sample of very preterm children. K-means clustering identified a group of very preterm children with predominantly inattentive or combined symptoms, but did not identify a subgroup of children with predominantly hyperactive/impulsive ADHD symptoms. This is in contrast with what described by who similarly used k-means clustering in a sample of 165 children diagnosed with ADHD and were able to parse children based on the three typical ADHD clinical presentations .Psychiatric risk in extremely preterm children has been found to be higher in those with coexisting neurodevelopmental disabilities , althougInattentive ADHD symptoms were associated with lower IQ, which has been studied as a longitudinal predictor of educational outcomes . There iPrevious findings in older preterm children suggested that a core executive difficulty underlined their inattentive problems , 46, 47,invasive ventilation via an endotracheal tube (reflecting respiratory failure). In a larger sample of infants the current study participants were drawn from, these two risk factors were independently associated with lower fractional anisotropy values throughout the white matter at term equivalent age, reflecting alterations in brain development [The current finding of a positive correlation between inattentive ADHD symptoms and perinatal medical complications suggests that preterm-born pre-schoolers display a phenotypic profile that differs from that observed in the general population, with aetiological underpinnings in the clinical risk associated with preterm birth. This was assessed using markers of general infant health: number of days of parenteral nutrition (reflecting gut failure) and number of days spent on elopment . Howeverelopment . These delopment , 53. TheThe idea that perinatal events can be implicated in the aetiology of psychiatric disorders has long since been proposed  and seveThe early identification of a preterm profile characterised by inattentive ADHD symptomatology associated with lower IQ and perinatal clinical risk suggests the need for early screening and educational interventions aimed at improving specific attention problems in very preterm children, many of whom exhibit academic difficulties at school age and are three times more likely to have special educational needs than their term-born peers . AttentiEnvironmental variables including socio-economic status have been studied as risk factors for ADHD , althougLimitations of this study include the lack of a locally matched control group. This could have influenced the results, possibly underestimating the level of problems experienced by very preterm children . It alsoIn summary, this study shows that already during preschool age very preterm children are at increased risk of sub-threshold ADHD symptoms, especially the inattentive type. Inattentive ADHD symptoms were associated with intellectual functioning at time of assessment, and with perinatal clinical risk, supporting the hypothesis of a possible neurobiological origin of ADHD, via alteration of white matter microstructural properties  that canS1 Dataset(CSV)Click here for additional data file.