Lung adenocarcinoma (LUAD) is one of the major types of lung cancer. Tumor-infiltrating immune cells (TIICs) are positively associated with overall survival (OS) in LUAD. The SPARC/osteonectin, cwcv and kazal-like domains proteoglycan 2 (SPOCK2) is a complex type of secreted proteoglycan involved in forming a protective barrier against viral infection. The purpose of this study was to investigate the relationship between SPOCK2 and TIICs and the prognostic role of SPOCK2 in LUAD. The GEPIA2, GEO, CPTAC, and HPA databases were analyzed to examine both the mRNA and protein expression of SPOCK2 in LUAD. GEPIA2 and the Kaplan-Meier Plotter (KM Plotter) were used to evaluate the prognostic value of SPOCK2 in LUAD patients. TCGA data were examined for a correlation between SPOCK2 expression and clinical characteristics. Gene enrichment analyses were performed to explore the underlying mechanism of SPOCK2 based on LinkedOmics. RegNetwork was used to predict the regulators of SPOCK2. The correlation between SPOCK2 and TIICs, including immune infiltration level and relative proportion was investigated via TIMER. KM Plotter was also used to evaluate the prognostic role of SPOCK2 expression in LUAD with enriched and decreased TIIC subgroups. We found SPOCK2 was significantly downregulated in LUAD compared with that in non-tumor controls and was correlated with clinical parameters. Moreover, a high SPOCK2 expression level predicted better survival. The SPOCK2-associated regulatory network was constructed. SPOCK2 influenced the TIIC infiltration level and relative proportion in LUAD. Furthermore, a high SPOCK2 expression level was associated with a favorable prognosis in enriched CD4 + T cells and macrophage subgroups in LUAD. In conclusion, a high level of SPOCK2 expression predicted favorable prognosis and was significantly correlated with TIICs in LUAD. Therefore, the expression of SPOCK2 may affect the prognosis of LUAD partly due to TIICs. Lung cancer has become the most common cancer type and causes the largest number of cancer-related deaths in the world . Lung adSPARC (osteonectin), cwcv and kazal-like domains proteoglycan 2 (SPOCK2) is known as a secreted protein that is acidic and cysteine-rich, playing a significant role in the development and progression of ovarian cancer, endometrial cancer, and prostate cancer . It was We first analyzed the differential mRNA expression of SPOCK2 between LUAD and normal lung tissues in the Gene Expression Profiling Interactive Analysis (GEPIA2) and Gene Expression Omnibus (GEO) databases. We also explored SPOCK2 protein expression via the Clinical Proteomic Tumor Analysis Consortium (CPTAC) and Human Pathology Atlas Project (HPA) databases. GEPIA2 and Kaplan-Meier (KM) Plotter were employed as online bioinformatics tools to study the prognostic correlation between SPOCK2 expression and LUAD. We observed SPOCK2 was related to tumor stage (TNM classification) in LUAD using The Cancer Genome Atlas (TCGA) data. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted to assess the potential role of SPOCK2 in LUAD. Furthermore, the correlation between SPOCK2 and TIICs in LUAD was investigated in TCGA and GEO databases via TIMER. We also used the KM Plotter to examine the prognostic role of SPOCK2 with enriched and decreased TIIC subgroups. Our findings may shed light on the mechanism and role of SPOCK2 in LUAD.1  were performed using the GraphPad Prism 7 software . Differences between the two groups were calculated by unpaired t-tests .Correlation assessment were carried out using SPSS version 17.0 . The associations between SPOCK2 expression and the clinicopathological parameters of the LUAD patients were analyzed using the chi-squared test. Bivariate correlations between study variables were assessed with the Spearman’s rank correlation coefficient. P < 0.05, P < 0.001, We analyzed SPOCK2 mRNA expression in LUAD and normal lung tissue RNA sequencing data from the TCGA database via GEPIA2. The results revealed that the SPOCK2 mRNA expression level was lower in LUAD than in non-tumor lung tissue (P = 0.001), clinical stage (P = 0.037), T classification (P < 0.001) and N classification (P = 0.012). However, SPOCK2 expression was not associated with sex (P = 0.111) or metastasis (P = 0.755) (P = 0.001) and clinical stage (P = 0.015), T classification (P = 0) and N classification (P = 0.001) (P (HR) = 0.038) in LUAD. Further, we used the KM Plotter to assess the prognostic value of SPOCK2 in LUAD and we found the similar result   . Similar= 0.001) . Then, t= 0.001)  and we f 0.0047) . These rp = 5.45e–73), GIMAP8 , and GIMAP7 , which are regulators of lymphocyte survival and homeostasis  and negatively (right) correlated with SPOCK2 were shown in the heatmap . The reseostasis . The reseostasis  and KEGGeostasis  analysiseostasis .P = 1.45e–21), CD8 + T cells , CD4 + T cells , macrophages , neutrophils , and dendritic cells . These results indicated that SPOCK2 was important in regulating immune infiltration in LUAD. In addition, we observed via the TIMER database that SPOCK2 expression was strongly associated with the immunomarkers of TIICs and that these correlations remained unchanged after tumor purity correction  rate of LUAD. Therefore, we used TIMER to analyze possible associations between SPOCK2 expression and the TIIC infiltration level in LUAD. As shown in rrection .Further, we tried to examine whether the tumor immune microenvironment was different in LUAD patients with high SPOCK2 levels compared to those with low levels. The 106 LUAD samples from GSE37745 were divided into 2 groups based on median expression value of SPOCK2 expression value, with 53 samples in the high expression group and 53 samples in the low expression group. We used CIBERSORT by TIMER to examine the proportions of 22 types of immune cells by analyzing the gene expression profiles of these samples. We found that the proportions of several subtypes of T cell and activated lymphocyte subsets were significantly increased in the SPOCK2 high expression group compared with those in the SPOCK2 low expression group, including CD8 + T cells, activated memory CD4 + T cells, regulatory T cells, M1 macrophages, activated myeloid dendritic cells, and neutrophils . TogetheWe confirmed that SPOCK2 expression was positively correlated with favorable prognosis and TIICs in LUAD. Therefore, we speculated that the expression of SPOCK2 affects prognosis partly due to TIIC infiltration. We conducted prognostic analysis based on the SPOCK2 expression in enriched or decreased immune cell subgroups via KM Plotter. We observed that high expression levels of SPOCK2 in enriched CD4 + T cell (HR = 0.59) and enriched macrophage (HR = 0.57) cohorts were associated with a better prognosis, while there was no significant association among decreased subgroups . These rDue to the poor outcomes reported in the latest cancer statistics released in 2019, there is an urgent need to identify novel prognostic markers for LUAD . Cancer In this study, we attempted to systematically explore the expression, prognostic value, correlation with TIICs, and potential mechanism of SPOCK2 in LUAD. In recent studies, SPOCK2 has been shown to be highly expressed in ovarian cancer  and lowlTo elucidate the molecular mechanisms underlying the role of SPOCK2 in LUAD, we explored the function of SPOCK2 and its coexpressed genes using LinkedOmics via GO and KEGG analysis with clusterProfiler. Most of the GO and KEGG categories were enriched in regulating lymphocytes, especially T cells based on LinkedOmics. TFs  and miRNSPOCK2 was reported to enhance anti-viral ability by inhibiting the cellular attachment and entry of the influenza virus . In addiWe found that SPOCK2 was positively associated with the TIIC infiltration level among TCGA data, using TIMER. Moreover, the association between SPOCK2 expression and the marker genes of TIICs validated the role of SPOCK2 in LUAD tumor immunity.TIIC subpopulations are different among cancer patients. For example, M1 macrophages and activated TIICs correlate with a relatively better prognosis . LikewisPrognostic analysis of SPOCK2 expression levels based on immune cells in LUAD was performed using the KM Plotter. We observed that a high SPOCK2 expression level in the enriched CD4 + T cells and macrophage subgroups in LUAD was associated with a favorable prognosis. T cells and macrophages are closely associated with clinical outcome in LUAD  and our To the best of our knowledge, the present study confirmed for the first time that SPOCK2 greatly affects LUAD prognosis. We found that a high expression level of SPOCK2 favored better survival in LUAD and correlated with TIICs. Therefore, SPOCK2 may affect prognosis partly due to its relationship with TIICs. Nonetheless, there were some limitations in our study. More LUAD patient samples are needed to confirm the prognostic value of SPOCK2, and the function of SPOCK2 in TIIC regulation in cancers as well as its influence on the response to immunotherapy should be verified in future clinical trials. In conclusion, SPOCK2 may be a useful biomarker and therapeutic target for LUAD prognosis and treatment, respectively.http://www.ncbi.nlm.nih.gov/geo/) and TCGA (http://cancergenome.nih.gov) repositories.The datasets GSE32863, GSE37745 and TCGA-LUAD for this study can be found in the GEO (QL: conceptualization. JZ: data curation, writing-original draft preparation. MC: methodology. JG, RZ, and TD: software. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although the number of adults with attention deficit hyperactivity disorder (ADHD) has increased considerably in recent years, there are few qualitative investigations of the experiences of adults with adult-diagnosed ADHD in Japan. This study aimed to explore in depth the diagnosis-related experiences and needs of such adults.Participants were 12 psychiatric outpatients aged 23–55 years diagnosed with ADHD during adulthood. Individual semi-structured interviews were conducted to examine participants’ experiences of receiving, and subsequently coping with, an ADHD diagnosis. A thematic analysis of the interview data was performed.Six themes emerged: difficulties in accepting the diagnosis, interest in ADHD, feelings of relief, identity concerns, dealing with symptoms, and acceptance of ADHD. Despite initial negative reactions, participants were willing to learn about the disorder, spending time seeking ADHD-related information and sharing it with loved ones. Participants felt relieved after the diagnosis, as they realized why they had experienced long-term problems and incorrect labeling. However, participants also had identity concerns. They gradually began to accept their ADHD symptoms and deal with them better.The results suggest that, when treating individuals with adult-diagnosed ADHD, it is important to promote self-understanding and reduce negative attitudes toward ADHD; to provide appropriate, brief, evidence-based information about ADHD; and to give individuals sufficient time to think about their ADHD symptoms, how they have affected their daily lives, and how to cope with them in the future. Attention deficit hyperactivity disorder (ADHD) is a chronic neurodevelopmental disorder characterized by the following behaviour-related symptoms: inattention, hyperactivity, and impulsivity . AlthougThree types of pharmacotherapy have been approved for adults with ADHD in Japan: atomoxetine, which has been available since 2012, methylphenidate extended-release tablets since 2013, and guanfacine since 2019. However, unlike other countries with existing clinical guidelines for adults with ADHD, such as the UK, Canada, and Australia –8, thereThus far, several qualitative studies to explore the experiences of adults with ADHD have been conducted in European countries. Matheson et al. revealed the burdens experienced by adults with ADHD such as difficulties to access services and barriers to continue treatment . SchreveCompared with qualitative studies of adults with ADHD in European countries as described above, to the best of our knowledge, there are no qualitative descriptive studies exploring the experiences of adults with adult-diagnosed ADHD in Japan. This study aimed to explore and better understand the diagnosis-related experiences and needs of this group.A qualitative approach using semi-structured interviews was used to enable a detailed, in-depth exploration of individuals’ experiences and needs. Qualitative techniques are particularly useful in improving knowledge of poorly understood areas of health care .Participants due to attend follow-up appointments at three psychiatric services in Tokyo during the research period of June 2016 to December 2016 were invited to participate in the study. The inclusion criteria were outpatients (i) aged 20 years or older and (ii) who had received a diagnosis of ADHD during adulthood (aged 17 years or over). The exclusion criteria were (i) not fluent in Japanese, (ii) refusal to provide written informed consent, and (iii) unable to participate in the interview owing to severe symptoms. The researcher, who was totally independent from the psychiatric services, recruited the participants. A purposive sampling strategy was used. Outpatients who regularly visited the three psychiatric services were approached following the inclusion and exclusion criteria described above. Immediately after the routine appointments, the researcher fully explained the aim and overview of this study to potential participants using a written document. Participation was voluntary. Once the potential participants received the explanation, they had sufficient time outside the services to consider whether or not to agree to take part in. Thereafter, consent was taken in the next appointment. A gift voucher of 1000 Japanese yen was given to the person who participated in this study.Participants were 12 individuals diagnosed with ADHD during adulthood. The mean age at the interview was 36.5 years and the mean age when ADHD was diagnosed was 33.5 years. Table How did you feel when ADHD was first diagnosed?What did you do after the diagnosis?Did your feelings about the diagnosis change with time; if so, in what way?The researcher (YA), who had received prior training in qualitative methodology, conducted the semi-structured interviews in Japanese. YA is an assistant professor in the Department of Psychiatric Nursing with experience in psychiatric care, and has published qualitative research on experiences of people with mental health conditions in international journals. YA is a native Japanese speaker and familiar with Japanese culture. Face-to-face interviews were conducted in the consultation room as a private space immediately following their routine appointments. An interview guide was used containing the following questions:The full interview guide is available in Additional file A thematic data analysis, following the six phases proposed by Braun and Clarke , was useSix themes and nine subthemes describing the experience of receiving a diagnosis of ADHD during adulthood were derived from the interview data. The six themes were as follows: difficulties in accepting the diagnosis, interest in ADHD, feelings of relief, identity concerns, dealing with symptoms, and acceptance of ADHD.The following storyline of participants’ experiences emerged from the analysis. Immediately following diagnosis with ADHD, participants were upset and found it difficult to accept the diagnosis. Despite this initial negative reaction, however, participants were willing to learn about the disorder, spending time seeking information about ADHD and sharing it with loved ones. They experienced some relief through information seeking and sharing, as they realized why they had been struggling with problems and had labelled themselves incorrectly for a long time. At the same time, the more they learned about ADHD, the more confused they felt about their identity. Although participants experienced mental turmoil, they gradually became better at dealing with ADHD symptoms by acquiring coping skills, taking ADHD medication, and adjusting their work environment. Participants also became more accepting of the inevitability of the ADHD symptoms.The themes are described below with illustrative quotes from the participants.“My ADHD diagnosis was quite sudden and I did not know what was what anymore, because I did not know anything about ADHD.” .“I thought ADHD was a disease of young boys! Why me? You must be joking!” .Immediately after ADHD diagnosis, many participants became upset and felt unable to accept the diagnosis. Several reasons for this were expressed. First, participants did not have sufficient knowledge about ADHD. Some participants felt confused and unclear about what they should do, as they had never heard of ADHD. Although some participants had heard of ADHD, some interviewees thought that it only occurred in childhood and did not know that ADHD symptoms could continue into adulthood.I disliked the word ‘developmental disorder’. It seemed to indicate lack of development and looking stupid.” .““[People with developmental disorder] seem to be odd. I was shocked and felt disgusted that I was also one of those oddballs.” .Self-stigma about developmental disorders made it difficult for participants to accept the ADHD diagnosis. Even those not familiar with ADHD had heard of developmental disorder. Many participants had negative attitudes and biased views toward developmental disorders. Once they had been diagnosed with ADHD and realized that it was a developmental disorder, their latent stigmas started to emerge and reflect back as self-stigmas.Like with my asthma, I carefully looked up ADHD because I was worried about being able to understand the situation if something bad happened.” .““I really spent time looking up ADHD on the Internet.” .All participants attempted to obtain information about ADHD and its treatments after ADHD diagnosis. Participants picked up pamphlets from the waiting room in the service clinic, purchased books about ADHD, and looked up the term “ADHD” on the Internet at home. They were interested in ADHD and actively sought information about the disorder. Most participants wanted to learn what ADHD was and how to cope with it.“I talked to my mother a lot about my ADHD and revealed it to my boss, too.” .“I asked my friend, who is receiving medication for psychiatric problems, ‘What is your experience like?’” .During information seeking, many participants were willing to share information about the disorder and its treatment with their loved ones. The family members of some participants also actively gathered information about ADHD so they could discuss the disorder and its treatments. Some participants talked to friends or acquaintances who were receiving psychiatric treatment. All participants sought to deepen their knowledge about ADHD and share it with others close to them.“Something unknown and unidentified, which I have had for a long time, has been cleared up. I feel so much better knowing about ADHD.” .“It was just like all the precursors of my story had converged.” .Along with increased knowledge about the characteristics and symptoms of ADHD, participants gradually began to understand that they had experienced ADHD symptoms since childhood. Many felt relieved when they realized that their symptoms were at the root of problems and difficulties they had had or sensed. They became aware of why they had experienced long-term problems throughout their lives.“I was relieved, as I knew that my label of a sloppy person or a lazy person was inappropriate.” .Many participants had felt different from others for a long time. Participants’ friends and social acquaintances had often cautioned them about their behaviours. Participants had then been identified (and had labelled themselves) as inferior to their classmates.“Although I had been dealing with my difficulties as indications of other diseases for a long while, all my past experiences could be explained by ADHD.” .Moreover, those who had sought psychiatric treatment previously had been diagnosed with different mental disorders. One participant explained that she had been diagnosed with other diseases like schizophrenia, bipolar disorder, panic disorder, and sleeping disorder before the ADHD diagnosis. She now realized that all her previous disorders had been caused by ADHD symptoms, although each disease had been treated separately.“Well then, ‘Who am I really?’ I thought.” .“The more I learned about ADHD, the more I doubted who I was … Are my assumptions about myself so far, all wrong? I started to feel anxious.” .Despite feeling emotional relief, participants experienced doubt or confusion about their identity. As they realized that their ADHD symptoms accounted for their traits and behaviours toward others, participants began to ask themselves “Who am I really?” One participant felt that the impression of himself he had had since childhood had been shattered, because the ADHD symptoms were the real cause of his character. These concerns about self-identity generated anxiety, as participants felt that ADHD symptoms defined, and perhaps dominated, their lives. Another participant stated “I am myself,” even after his ADHD diagnosis. He could not imagine himself without the ADHD symptoms. He now perceived his traits as ADHD symptoms, although they had always been part of him.“I knew getting distracted easily was one of the symptoms and I tried to deal with it.” .Gradually, many participants acquired their own coping behaviours and skills to deal with ADHD symptoms. In seeking information about ADHD and how to cope with it, they looked back over their lifestyle and acquired skills and considered how these expressed their symptoms. For instance, participants reported the following coping skills: taking notes, even about “small stuff”; writing down any ideas that “enter my head” before speaking; listening to others until they have finished speaking; and setting an alarm 15 min before they had to leave the house as a reminder. The ADHD diagnosis gave participants the opportunity to acquire more relevant and useful coping skills.“Depending on circumstances, it is better to have no ADHD symptoms. So, I started to take the ADHD medication as a tool to deal with my ADHD symptoms.” .Taking medication was another way of dealing with ADHD symptoms and some participants felt quite positive about taking medications. Most were willing to obtain information about appropriate medication from websites, books, and other people (such as family members and friends) in addition to receiving information from their psychiatrists. One participant reported that the psychiatrist’s explanation had not been very clear and so he found it difficult to understand the positive and negative aspects of medication treatment even though he was willing to receive it.“I decided I was not going to deal with it just by myself anymore, and I let my boss know about my ADHD. My boss reminds me about due dates and informs me about my daily schedule.” .Participants felt that it was important not only to learn new coping skills for daily life or to control symptoms by medication, but also to adjust their working environment to suit their talents and symptoms. The symptoms of ADHD can be influenced, for better and worse, by the environment or the attitudes of others; therefore, some participants disclosed their ADHD diagnosis to their boss and colleagues at work and accepted the accommodations offered to them for their symptoms. Participants who had consulted a psychiatrist about maladaptation to the workplace found it easy to disclose their ADHD symptoms to others.“My prejudice about ADHD has decreased over the last year.” .“It is a disease, unavoidable, not my fault, and not just me … Knowing it is a brain disease made me stop resisting it.” .Participants finally recognized that their ADHD diagnosis was inevitable and undeniable. Prejudices and negative attitudes toward ADHD gradually disappeared and participants became more willing to accept the diagnosis. Many participants had previously expressed negative judgments about their problematic behaviours, such as agitated behaviour in public, incoherent or disruptive talking in front of others, and being untidy. Subsequently, they stopped blaming themselves and struggling with such symptoms. Participants started to move beyond their negativity and began to accept ADHD.Figure The spiral indicates that the process was not always straightforward: participants sometimes felt they were moving forward, but at other times felt they were going backwards before they reached an acceptance of their condition.To our knowledge, this is the first study to investigate diagnosis-related experiences of adults diagnosed with ADHD during adulthood in Japan.In addition to feeling emotional relief, participants also experienced identity confusion, which is in accord with findings from some European studies. Hansson Halleröd et al. found that participants who received an ADHD diagnosis during adulthood described how their diagnosis explained a previously inexplicable life history, but also caused devaluation and identity concerns . Young eAs participants reflected back on their lives and recognized that their ADHD symptoms now made complete sense, they felt relieved. This suggests that if a psychiatrist identifies ADHD symptoms, it is important that individuals are informed immediately although notification of an ADHD diagnosis may not necessarily occur in a psychiatric setting in Japan . It is iImmediately after receiving the ADHD diagnosis, participants thought about negative images and stigmatizing attitudes toward this developmental disorder. This seems unique to this study, as previous research has not reported such a finding. Several studies demonstrated that ADHD is strongly associated with stigmatization , 21. WeiThis study had several limitations. First, the sample may have been biased as participants were recruited from a population that regularly had psychiatric consultations. Second, the period between the ADHD diagnosis and the interview varied between individuals from less than 1 year to nearly 10 years (see Table Adults diagnosed with ADHD during adulthood in Japan experienced difficulties in accepting their diagnosis, interest in ADHD, feelings of relief, identity concerns, dealing with symptoms, and acceptance of their condition. When treating adult-diagnosed individuals, it is necessary to supplement the ADHD diagnosis with appropriate evidence-based information; promote self-understanding and reduce stigmatized attitudes toward ADHD; and provide individuals with sufficient time to think about ADHD symptoms, to consider how they may have affected daily life, and to develop ways to cope with ADHD in the future.Additional file 1. Interview Guide.


Attention-deficit and hyperactivity disorder (ADHD) is a widespread neurodevelopmental disorder in children and adolescents, persisting into adulthood in a majority of them. ADHD and substance use disorders (SUDs) commonly co-occur in the clinical adult population. The higher-than-normal prevalence rates of SUDs in people with ADHD indicate increased risk for developing SUD. This narrative review deals with the question of whether or not adults with both disorders should be treated with methylphenidate (MPH), addressing specific issues surrounding this form of treatment. MPH is considered as first-line pharmacotherapy for ADHD. However, because of its stimulant-like reinforcing properties, MPH has a significant addictive potential to which persons with SUDs are especially susceptible. Appropriate treatment is therefore complex. Because of concerns about misuse and diversion of MPH medication, clinicians may be reluctant to use MPH to manage ADHD symptoms in these patients. However, it is essential to diagnose and treat ADHD adequately as appropriate therapy reduces the impairments, as well as the risk of developing comorbid disorders and poor treatment response. MPH should not be deprived of these patients because of the risk for misuse, especially as several strategies can be applied to minimize this risk. To conclude, carefully applied guideline-based diagnostics to clarify the potential presence of ADHD as well as a responsible prescription practice in a well-defined therapeutic setting with reliable monitoring of medication intake and regular consultations are essential conditions for a safe and proficient MPH treatment of ADHD in patients with SUD. The prevalence of attention-deficit and hyperactivity disorder (ADHD) in children and adolescents is estimated to be between 2 and 7% , wherebyADHD is a multifactorial, clinically heterogeneous neurodevelopmental disorder  caused bThe long-prevailing view that ADHD is a disorder of childhood and adolescence, phasing out in adulthood, has been refuted –16. EvenAlthough the neurobiological functional deficits remain the central problem for all age groups, the severity and course of the disorder in adults are extremely heterogeneous . ImpairmThis article briefly reviews existing literature on methylphenidate (MPH) treatment of adult ADHD in persons with substance use disorders (SUDs), especially with opioid use disorder (OUD). It takes up on specific problems, such as misuse and diversion of MPH, which are associated with MPH therapy in these patients. It also aims to give recommendations on diagnostics, therapy, and treatment settings, when dealing with these issues.This narrative review is predominantly based on a MEDLINE database search. Additionally, importance was given to include guidelines for clinical practice, such as NICE guidelines  or conseCo-occurring ADHD and SUDs are routinely encountered in clinical settings . StudiesThe relationship between ADHD and SUD is complex. As psychoactive substances reduce, imitate, or aggravate the symptoms of ADHD, differentiating between both disorders is challenging, which impedes the research in this field , 33. The2/D3 receptor availability in the midbrain, caudate nucleus , and hypothalamus ] and are, at least in part, still present in adulthood. The diagnostic process should be performed, if possible, under abstinent conditions. Also, the individuals in OAT should be well-adjusted to the medication ].Compared to children and adolescents, there is a great backlog in the diagnostics and therapy of ADHD in adults . To diagdication , so thatICD-10 offers the possibility to code the presence of ADHD, the criteria for adults are not explicitly mentioned. Diagnosis based on guidelines includes the developmental psychopathology of ADHD by stating the collection of retrospective symptoms as an integral part of the diagnosis.To ensure a careful and comprehensive evaluation for possible ADHD, a guideline-based diagnostic procedure is recommended in current clinical practice . AlthougDSM-IV/DSM-5) is recommended. In patients with SUD, a thorough assessment of the current SUDs is essential to evaluate the influence of substance use on ADHD symptoms (According to NICE guidelines , the diasymptoms .DSM-IV (The next step is to collect a complete medical history and to perform a physical examination to exclude organic causes, such as thyroid disease, seizure disorder, or sleep disorder. To assess former and current symptoms, parents/siblings, and other important persons of trust should be interviewed. Validated structured interviews, such as Conners' Adult ADHD Diagnostic Interview for DSM-IV  or the DDSM-IV  can be uIn a following step, disorder-relevant symptoms and their manifestation in individuals are assessed by using standardized methods for detailed assessment. For this purpose, there are several validated instruments available in different languages, such as Adult ADHD Self-Report Scale and the short version for screening, Wender-Utah-Rating-Scale for childhood symptoms, or Conners' or Brown's scale for current symptoms. The Conners' or Brown's scale is recommended to assess the magnitude of the impairments caused by ADHD. As they are normed , a severFinally, a neuropsychological assessment provides relevant information about the general level of cognitive performance and existing neurocognitive deficits. Mainly neuropsychological tests aiming at attentional and executive functions as well as tests to measure various aspects of intelligence are used.The decision for a therapy, respectively, or the choice of a specific treatment depends on the severity of the current situation, mental and social impairments, present comorbid disorders, the relevance of the symptoms in performing daily routine tasks, and existing resources . IdeallyMPH is a centrally acting psychostimulant  that is MPH unfolds its stimulant, indirectly sympathomimetic effects by inhibiting presynaptic reuptake of dopamine and noradrenaline . Unlike Neuropharmacobehavioral studies of injected MPH and cocaine revealed not only similarities, but also significant differences between the two substances –69. For The misuse liability of MPH is well-known. In the literature, mostly intravenous and nasal administrations, which led to substance- or administration-specific complications, are described as cases of misuse . ComplicMisuse and diversion are inherent risks of prescribing controlled substances, and a substantial minority of patients with prescribed MPH will misuse their own prescription or divert their medications to others . In receIt has not yet been clarified to what extent ADHD treatment with MPH contributes to the development of SUDs. Current evidence is based on secondary analyses as prospective studies, to our knowledge, are still missing. The existing results are inconsistent; however, they do not indicate that MPH contributes to the development of SUDs. On the contrary, treating ADHD patients with MPH may reduce their risk for SUD , 85, 86.The question is whether adults with both disorders should be treated with MPH despite its misuse potential and the risks connected to it. In due consideration of the scientific literature, the benefits of ADHD treatment with MPH outweigh its risks; therefore, in our opinion, treatment with MPH should be considered as first-line treatment also in adults with ADHD and SUD. However, these risks should be seriously taken into account when planning the therapy. Furthermore, a reasonable therapeutic setting should be arranged with patients for the prescription of MPH. Additionally, patients should be closely monitored for possible side effects and misuse/diversion of MPH.d-amphetamine (In any case, a verified guideline-based diagnosis is the basic requirement for the treatment of ADHD. Insights from this diagnostic procedure can be used to plan the treatment and to determine the indication of therapy offers . The trehetamine . Also, nhetamine . Atomoxehetamine . HoweverIn general, extended-release formulations of MPH are recommended for the treatment of adult ADHD , which mSC, JS, MV, and KD did the literature review and wrote the draft. MW and KD commented on the first draft. SB, MW, and KD commented on the second draft. All authors commented on the final manuscript, which was completed by SC, JS, MV, SB, MW, and KD.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Age-matched children without ADHD (n = 43) served as controls. All plasma and urine metabolites were determined by gas chromatography-mass spectrometry. We observed higher plasma nitrite and lower plasma ADMA concentrations in the ADHD children. MPH-treated ADHD children had higher plasma nitrite concentrations than MPH-untreated ADHD children. As NOS activity is basally inhibited by ADMA, MPH treatment seems to have decreased the inhibitory potency of ADMA. Percentiles of systolic blood pressure were higher in MPH-treated ADHD children. The underlying mechanisms and their implications in the MPH therapy of pediatric ADHD with MPH remain to be elucidated in larger cohorts.Attention deficit hyperactivity disorder (ADHD) is a common pediatric psychiatric disorder, frequently treated with methylphenidate (MPH). Recently, MPH’s cardiovascular safety has been questioned by observational studies describing an increased cardiovascular risk in adults and blood pressure alterations in children. We considered members of the L-arginine (Arg)/nitric oxide (NO) pathway as possible early cardiovascular risk factors in pediatric ADHD children. They include the NO metabolites, nitrite and nitrate, the NO precursor Arg, and asymmetric dimethylarginine (ADMA), an endogenous NO synthase (NOS) inhibitor and a cardiovascular risk factor in adults. We conducted a prospective clinical trial with 42 ADHD children (aged 6–16 years) with ( Attention deficit hyperactivity disorder (ADHD) is a common psychiatric disorder in childhood, globally affecting about 2%–7% of all children . AlthougThe L-arginine (Arg)/nitric oxide (NO) pathway is virtually present in all cell types, including endothelial, neuronal, and immune cells. Arg is the substrate of all known NO synthase (NOS) isoforms. This enzyme family converts Arg to NO and L-citrulline . The phyN-guanidine (NG) methylated Arg analogs NG-methyl-L-arginine (L-NMMA), asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) are inhibitors of NOS .Statistical analyses were performed using the statistical software package IBMp = 0.34 and p = 0.39, respectively). The anthropometric characteristics of the ADHD children of the +MPH and −MPH groups showed a minor but statistically significant difference with respect to the head circumference, which was smaller by 1.7% in the +MPH group. SBP percentiles were by 29% higher in the +MPH group compared to the −MPH group (t (39) = 1.58; p = 0.03, Regarding age or gender distribution, no differences were seen between the +MPH and −MPH groups . Age (9.p = 0.001). There was no difference between −MPH and +MPH for age-corrected ADMA plasma concentrations of the ADHD children (see p = 0.006). This difference persisted after correction of the plasma nitrite concentration by age of the ADHD children  and +MPH  groups.The plasma concentrations of the analytes are displayed in dren see . The pladren see . The conThe age-corrected ADMA and nitrite plasma concentrations of the ADHD children were divided into two groups according to their IQ values ranging between 50 and 100  and IQ values above 100 . We found no interaction of MPH and IQ with regard to age-corrected nitrite plasma concentrations, but we found an interaction for age-corrected ADMA plasma concentrations . WithoutCreatinine-corrected urinary nitrate and nitrite concentrations in the control group did not differ from those in the −MPH and +MPH groups . Also, tNO3) to plasma nitrite (PNO2) concentration, i.e., PNOxR and the molar ratio of urinary nitrate to urinary nitrite  did not differ between the ADHD groups  of nitrate (FENO3) was about 15% in both ADHD groups. The fractional excretion of nitrite (FENO2) was 0.6% in the −MPH group and 0.4% in the +MPH group  than of eNOS activity (IC50 ≈ 12 µM) and has been suggested to be a modulator of nociception [In both ADHD groups, we measured almost the same ADMA concentrations in plasma and urine, respectively. The ADMA plasma concentration in the ADHD children is on average by 70 nm lower than in the non-ADHD children of the same age. Obviously, MPH treatment has no effect on ADMA synthesis, metabolism, and excretion. Higher ADMA concentrations may increase the extent of the intrinsic inhibition rate of NOS activity , while liception . In addiiception . Hence, The results of our study may suggest that NO synthesis is elevated in neuronal cells due to locally decreased ADMA concentrations, eventually enhancing sensory hyper-responsiveness in a midbrain sensory structure (superior colliculus). This might be an underlying mechanism of distractibility in ADHD which increases inattention and distractibility in pediatric ADHD . AmphetaNG) groups of certain Arg residues in the dopamine transporter (DAT) of rat striatum [NG-dimethylated DAT, i.e., ADMA-DAT, could attenuate the interaction of MPH with DAT. Whether MPH inhibits the activity of PRMT towards DAT and other proteins including histones remains to be investigated. Another reason for diminished ADMA concentrations in ADHD patients could be an elevated hydrolysis of ADMA by DDAH. In vitro studies suggest that DDAH activity is increased by the inflammatory cytokine IL-1β [MPH has been shown to interact with guanidine (striatum . Methylane IL-1β . Howeverne IL-1β . That thne IL-1β , it doesp = 0.047, In addition to being an endogenous inhibitor of NOS activity, ADMA is supposed to exert NO-independent effects . Thus, AIn our study, we found that ADHD children chronically treated with MPH had closely comparable DBP values, but their SBP values were higher compared to the ADHD children not treated with MPH. It is worth mentioning that the SBP values of ADHD children acutely treated with MPH were also higher than those of ADHD children treated with placebo . TherefoA limitation of our study is the relatively small number of newly diagnosed and MPH-treated ADHD children. A longitudinal study with a larger group of newly diagnosed ADHD children and MPH-medicated ADHD children could help to elucidate the relevance of lower plasma ADMA and higher plasma nitrite concentrations in ADHD. Such studies should also address potential associations of circulating ADMA and nitrite with clinical and intellectual measures including IQ. Additionally, we cannot exclude that children with medication suffered from a severe form of ADHD, which could have caused selection bias. In an ongoing study, we are investigating the Arg/NO pathway in adult ADHD subjects. It is conceivable that parameters of the Arg/NO pathway, notably circulating ADMA and nitrite, could serve as biomarkers for the generally assumed cardiovascular risk in ADHD after long-term MPH therapy. In our study we did not measure additional biochemical parameters such as cGMP as a major of NO-dependent activation of guanylyl cyclase. Although several pathways may contribute to cGMP, its measurement in plasma and urine may provide valuable information about the biological activity of the Arg/NO pathway in ADHD. Our ADHD children aged six to 13 years had lower plasma ADMA and higher plasma nitrate and nitrite concentrations than age-matched children without ADHD. ADHD itself was associated with lower ADMA synthesis and higher NO synthesis. MPH did not change the plasma and urinary ADMA concentration, suggesting no effect of MPH on whole-body ADMA synthesis in pediatric ADHD. MPH treatment was associated with higher plasma nitrite concentrations in the ADHD children, suggesting an induction of NOS expression and/or activity in NOS-expressing cells. This might reflect a compensatory mechanism to antagonize SBP-enhancing effects of MPH. The Arg/NO pathway is altered in newly diagnosed ADHD children. MPH medication enhances NO synthesis apparently independent of ADMA, a potent endogenous inhibitor of nNOS. Further clinical studies are urgently needed to elucidate the underlying mechanism and the cardiovascular safety of MPH in ADHD.


Despite several additive manufacturing techniques are commercially available in market, Fused Deposition Modeling (FDM) is increasingly used by researchers and engineers for new product development. FDM is an established process with a plethora of advantages, but the visible surface roughness (SR), being an intrinsic limitation, is major barrier against utilization of fabricated parts for practical applications. In the present study, the chemical finishing method, using vapour of acetone mixed with heated air, is being used. The combined impact of orientation angle, finishing temperature and finishing time has been studied using Taguchi and ANOVA, whereas multi-criteria optimization is performed using the Technique for Order of Preference by Similarity to Ideal Solution (TOPSIS). The surface finish was highly responsive to increase in temperature while orientation angle of 0° yielded maximum strength; increase in finishing time led to weight gain of FDM parts. As the temperature increases, the percentage change in surface roughness increases as higher temperature assists the melt down process. On the other hand, anisotropic behaviour plays a major role during tensile testing. The Signal-to-noise (S/N) ratio plots, and ANOVA results indicated that surface finish is directly proportionate to finishing time because a longer exposure results in complete layer reflowing and settlement. Additive manufacturing is a digital fabrication process used for the fabrication of tailor-made parts utilizing principle of layer-by-layer material addition . The proThe thermoplastic part material and support material in the form of thin wire is fed by rollers into heated nozzle which partially melts the wire. As the nozzle moves in a pre-decided path in X, Y and D directions, the thermoplastic layers are deposited on table. This layer by layer deposition of plastic material created an actual product in extremely small duration  depending upon size and shape. The overhanging parts are made of support material which can be simply cleaned after manufacturing . DespiteChohan et al.  exploredTakagishi and Umezu  studied To overcome such problems, an innovative experimental set-up has been developed for surface improvement of FDM components through hot air mixed with acetone vapours . Although experiments have been performed using in-house developed apparatus , the impA vapour finishing test-rig have designed and fabricated which has provision to alter temperature of chemical fumes. Moreover, the innovative apparatus has been designed to avoid wastage of chemical vapours during loading and unloading of FDM Parts, as shown in For present experimentation total twenty-seven parts are fabricated to carry out nine experiments taking three repetitions for respectively trials to evade random error. The specimens are fabricated at 0°, 45° and 90° orientation angle as previous literature revels that these settings have maximum impact on surface finish and tensile strength. After fabrication, the printed specimens are taken out of print bed carefully and treated in ultrasonic bath for cleaning and removal of support structure.The average surface roughness of FDM parts was calculated through Profile Measurement Equipment “SJ-210” . The average-roughness (Ra) is specifically examined for current investigation and observations were taken normal to deposition direction. The measurements were recorded using stylus tip angle of 60 °C tip and radius 2μm moving at 0.75 mN. The Gaussian filter was used for filtering the surface roughness profiles at cut off length 0.025 cm and 0.25 cm transverse length as per ISO 4287 . The surThe mass of samples has been calculated before and after finishing using Citizen digital weighing apparatus  having readability upto 0.0001 g and maximum limit of 220 g. During weight and roughness measurements, three readings were recorded, each pre- and post-finishing, and the average of three readings was taken as the final value. Moreover, surface-roughness was deliberately evaluated at the same location before, and after, the finishing process to avoid random errors. For Taguchi analysis, percentage-variation in weight and surface-roughness are used as response parameters and calculated as following Equation (1):After finishing process, the peak tensile strength of parts was measured using Universal Tensile Tester  with 500 mm/min cross head movement speed as per ASTM 638-14 standards  as shownFor percentage-variation in surface-roughness and tensile strength, the “larger is better” characteristic is required and thus, Signal to Noise Ratio (SN Ratio) is computed  using EqWhereas, the “smaller is better” characteristic is required for percentage change in weight and SN Ratio is calculated in Equation (3):In above equation, n represents number of replicated experiments while i is response which may vary from 1 to n.The micrographs were obtained to ascertain the manifestations occurring at micro scale on the surface and in cross-section of FDM parts. The micrographs are retrieved using Scanning Electron Microscope (SEM) apparatus . Tensile strength determines the maximum stress that a material can withstand without breaking. Hence, the images of failure point are taken at high vacuum mode, 10 KV accelerating voltage, 12 mm. working distance and double sided carbon tape. Initially, the site of the break point, five millimeters of the cross section was cut and subjected to gold coating to make it conductive. The coated parts were then placed in a vacuum chamber where the scanning of the parts at different magnification takes place.The Taguchi L9 orthogonal-array along with Signal to Noise (SN) Ratio of leading and trailing observations of surface-roughness, tensile strength and weight has been calculated. It can be clearly noticed that the percentage-variation in average surface roughness (%∆Ra) varies from 64.12% to 87.59% after vapor finishing. The maximum contribution (84.88%) of finishing temperature has been observed at 95% confidence level. The higher temperature ensures effective meltdown of upper layers of ABS polymer which intensified the surface enhancement phenomenon after parts are cooled. On the other hand, orientation angle does not affect the surface roughness as vapours could not to reach inside layers of printed polymer parts. Similarly, finishing time does not influence the surface finishing phenomenon as hot vapours instantly melted the upper layers after exposure. The measurements of surface-roughness also indicated that the percentage change in surface-roughness through vapor finishing is significantly more than other mechanical finishing techniques as well as pre-processing techniques . P-values and F-values. It can be deduced from F-values and P-values of ANOVA table that orientation angle significantly affects the tensile strength at 95% confidence level. The internal polymer bead deposition angle enhances the strength of parts in certain direction due to phenomenon of anisotropy in FDM parts.The tensile strength results were retrieved and percentage contribution of each parameter has been displayed in In case of weight measurements, the average weight of FDM samples has increased even after cooling down the parts for 24 h after chemical vapor exposure. In present experimentation, the percentage gain in weight ranges from 0.14% to 0.40%, for all the experiments. On other hand, 45° orientation have least strength due to weak bonding between layers fabricated at this angle. Although peak strength achieved at 90° orientation angle is greater than 45°, but due to change in deposition strategy, it could not yield maximum strength. Furthermore, the weight of FDM samples is directly proportional to finishing time, which is quite evident because with an increase in finishing time results in a higher exposure, which resulted in more absorption of vapors inside the FDM parts which resulted in weight gain. On the other hand, finishing temperature and orientation angle have least impact on the weight gain.Although, an optimization study has been conducted to analyze the impact of individual parameter on response, there is requirement for a multi-criteria optimization, which would yield the best results for every response. The present work objects to evaluate the optimum settings of FDM and finishing processes which would equally balance all the responses. Therefore, TOPSIS tool has been used to identify the optimized parameter settings was sub sequentially proposed . As suggInitially, the values of normalized matrix for experimentation abbreviated as rij are calculated using Equation (4) given below:In second step, the weights of response parameters are multiplied with respective columns of normalized matrix. In present work, the equal importance has been given to each response parameter. The values of normalized matrix weightage normalized matrix have been shown in The positive-ideal  and negative-ideal  can be written as Equations (5), and (6), respectively, ℇThe evaluated values of positive-ideal (best) and negative-ideal (worst) results are displayed in Euclidean distance concept is utilized to evaluate the value of positive and negative separation measures. Hence, Equations (7) and (8) are used to compute the separation measures,The solutions calculated after implementing afore-mentioned equations have been shown in The ranking of alternates is done based on the relative closeness-value. The relative closeness of alternative Aj is written in Equation (9): Finally, Equation (9) has been used for calculation of relative closeness along with ranking for each experimental result has been shown in The optimization study has been exclusively conducted to ascertain the effect of combined operating parameters of FDM and vapour finishing process utilizing dedicated apparatus which creates controlled environment for vapour finishing. An apparatus has been prepared which utilizes air at variable temperature mixed with chemical vapours. TOPSIS have utilized for multi-objective optimization of process parameters which is highly effective tool used for industrial applications.The study has been conducted on dedicated vapor finishing apparatus which utilized influence of hot chemical vapors mixed with heated air for surface enhancement of ABS parts.The finishing temperature has a significant impact on surface finish, while orientation angle and finishing time were significant parameters for tensile strength and weight of FDM parts respectively.It was found that higher temperature resulted in better finish due to instant meltdown of upper plastic layers.Orientation angle of 0° led to highest value of tensile strength as layers are deposited in horizontal plane. Moreover, higher exposure duration induced permanent weight gain due to increase in absorption of vapours.Since each response parameter is impacted differently by input parameters, multi-criteria optimization tool TOPSIS has been utilized to identify optimal settings.The optimum parameter settings can be implemented to improve the surface-quality of FDM parts which can be utilized for end-use products and for rapid tooling applications.The study aims to investigate the impact of the orientation angle, finishing temperature and time on surface quality, tensile strength and weight gain of FDM parts during surface finishing process. A dedicated experimental set-up has been designed and made with provision of temperature control to investigate the impact of operating parameters on surface finish, tensile strength and weight of ABS parts. TOPSIS has been applied for multi-criteria optimization of FDM components to acquire single set of process parameters for mass production of finished FDM parts. Moreover, the optimal-set of process parameters for FDM and vapour finishing can be used for mass finishing of customized products. The study can be further broadened to evaluate the impact of process-parameters on dimensional accuracy, compressive strength and wear behaviour. Following conclusions are derived:


Recent case–control genetic studies of attention deficit hyperactivity disorder (ADHD) have implicated common and rare genetic risk alleles, highlighting the polygenic and complex aetiology of this neurodevelopmental disorder. Studies of other neurodevelopmental disorders, such as autism spectrum disorder (ASD), Tourette disorder, developmental delay/intellectual disability and schizophrenia indicate that identification of specific risk alleles and additional insights into disorder biology can be gained by studying non-inherited de novo variation. In this study, we aimed to identify large de novo copy number variants (CNVs) in children with ADHD. Children with a confirmed diagnosis of ADHD and their parents were genotyped and included in this sample. We used PennCNV to call large (>200 kb) CNVs and identified those calls that were present in the proband and absent in both biological parents. In 305 parent–offspring trios, we detected 14 de novo CNVs in 13 probands, giving a mutation rate of 4.6% and a per individual rate of 4.3%. This rate is higher than published reports in controls and similar to those observed for ASD, schizophrenia and Tourette disorder. We also identified de novo mutations at four genomic loci  that have previously been implicated in other neurodevelopmental disorders, two of which (16p13.11 and 22q11.21) have also been implicated in case–control ADHD studies. Our study complements ADHD case–control genomic analyses and demonstrates the need for larger parent–offspring trio genetic studies to gain further insights into the complex aetiology of ADHD. Recent case–control genome-wide studies point to the involvement of thousands of relatively common single-nucleotide polymorphisms2, very rare protein-truncating sequence variants and likely pathogenic missense mutations3, as well as large, rare copy number variants (CNVs)6. Although all of these classes of variant are associated with ADHD risk, robustly implicating specific risk alleles is an ongoing process. For instance, only a handful of CNV loci 5 have been individually implicated in risk of ADHD and given the large size of these loci, the specific genes involved are yet to be pinpointed. Studies of de novo mutations using parent–offspring trios provide an especially powerful approach to gene discovery because the background rate in unaffected individuals is low, and therefore if an elevated rate in cases is demonstrable, such mutations are likely pathogenic and can provide important insights into disease biology7.Attention deficit hyperactivity disorder (ADHD) is a highly heritable (~70%), common and impairing neurodevelopmental disorder with a complex genetic architecture12. Individuals with ASD have a rate of de novo CNVs that is between three- and fivefold higher than in unaffected siblings or control individuals13. Individuals with simplex Tourette disorder have a similar rate of de novo CNVs to probands with ASD14 as do individuals with schizophrenia7. Of these neurodevelopmental disorders, the rate of de novo CNVs is highest in individuals with DD/ID, at ~10%15.De novo likely damaging mutations, that include CNVs which intersect genes, have been identified for other neurodevelopmental disorders, such as autism spectrum disorder (ASD), Tourette disorder, developmental delay/intellectual disability (DD/ID) and schizophreniaN = 173) that was only slightly elevated above previous reports of de novo rates in controls16. In this study, we set out to characterise large de novo CNVs in parent–offspring trios of probands diagnosed clinically with ADHD.Trio-based genetic studies of ADHD are lacking. There has been only one small study and it reported a rate of de novo CNVs (1.7% or three probands out of 17) by trained, supervised psychologists. Full-scale IQ was assessed using the WISC-IV18. A proportion of the probands in the sample (N = 136 or 44.6%) have been analysed in previous case–control CNV studies5. Approval for the study was obtained from the North West England and Wales Multicentre Research Ethics Committees. Written informed consent to participate was obtained from parents and children aged 16 years and older and assent was gained from children under 16 years of age.Children with ADHD, aged 5–17 years old, were recruited from UK child and adolescent mental health and paediatric clinics. ADHD diagnoses based on DSM-IV or DSM-III-R were confirmed via a semi-structured research diagnostic interview  were used for CNV calling.DNA was obtained from blood and oragene  samples for ADHD probands and both biological parents. In all, 19 and nearby calls were merged if the CNVs were separated by <50% of their combined length. CNVs were removed if they were called using <10 probes, had size <50 kb, probe density <20 kb/probe or a PennCNV confidence score <10. Samples were removed if they had log R ratio standard deviation > 0.3, B allele frequency (BAF)-drift >0.01 or waviness factor > 0.05. In addition, we applied further, more stringent criteria in ADHD probands and excluded individuals with extreme scores on QC metrics (>4 SD than the sample mean) as follows: if they had an apparently excessive CNV load (number of segments > 50.8 or length in kilobases (KB) > 16593) or poor probe variance (BAF-SD > 0.063). These additional steps were performed to ensure that potential de novo CNVs would be of high quality, whereas being conservative and not removing a whole trio based on reduced data quality in just one parent. In total, N = 305 complete trios passed all of the above QC and were kept for analyses. After these steps, only CNVs of size >200 kb were retained for analysis, to ensure accuracy of calls.CNVs were called using PennCNVCNVs were then annotated as “transmitted”, “non-transmitted”, or “likely de novo” based on presence or absence of a call made in either parent and/or proband. All CNVs that were flagged as likely de novo were visually examined in the offspring and both confirmed biological parents and only clear-cut, un-ambiguous de novo calls were retained for analyses, minimising the false positive rate at the expense of potentially missing real calls. We did not perform validation, as the traces were definitive  that passed QC, we detected a total of 14 de novo CNVs (N = 9 that were >500 kb and N = 5 that were between 200 and 500 kb) in 13 ADHD probands, with one individual having two CNVs that were >500 kb. Two of the de novo CNV carriers were female and the rest were male. The overall mutation rate was 4.6% (14/305) and the per individual rate was 4.3% (13/305). Although we did not have population controls available in this study, a recent study of control trios using similar methodology reported a de novo mutation rate of 1.0% for CNVs >200kb24; thus the rate in ADHD was over four times higher than this comparable control rate. The mutation rate of de novo deletions was 3.3% and the rate of de novo duplications was 1.3%.In the The mean IQ of de novo CNV carriers was 83.6 (SD = 13.0) and the mean IQ of the rest of the sample was 85.2 (SD = 13.5), with 3 (25.0%) of the de novo CNV carriers (with non-missing IQ) having comorbid ID (IQ < 70), and 26 (9.6%) of the rest of the sample having comorbid ID. The group of de novo CNV carriers was too small to perform a statistical comparison. Information on age of the parents at birth of their children was available for only 10 of the de novo CNV carriers  = 24.5(3.4); paternal age mean (SD) = 27.1(5.8)) and the means of the rest of the samples were: maternal age mean(SD) = 26.9(5.8); paternal age mean(SD) = 29.8(6.8). As with IQ, the sample was too small for a robust statistical comparison.2 or the list of genes implicated by recent sequencing studies of ASD, ADHD and DD/ID23. However, four of the de novo CNVs have previously been robustly implicated in other neurodevelopmental disorders22, two of which have also been implicated previously in case–control CNV studies of ADHD6, as summarised in Table 25 and a de novo duplication at 15q13.126 have also previously been observed in people with schizophrenia. Given that a proportion of the current sample have previously been included in published case–control CNV studies5, we determined that eight of the de novo loci in our study overlap at least partially (≥50% overlap) with a previously reported CNV in a person with ADHD, with six of these eight de novo CNV carriers having taken part in the previous case–control studies. On the other hand, six of the loci in our study  are in novel regions and have not been previously reported in individuals diagnosed with ADHD in these large case–control studies.Table 5, six were de novo CNV carriers, giving a de novo CNV rate of 4.4%. Of the samples not previously published, six were de novo CNV carriers, giving a comparable de novo CNV rate of 4.1%.Of the samples included in previously published studies14, and somewhat higher than observed for schizophrenia7. Notably, the rate we observed is substantially higher than that which has been reported for controls24 and the previous and only trio-based study of ADHD that observed an overall rate of 1.7%16. Aside from the lower power of this previous, smaller study, one potential explanation for the difference is that, unlike Lionel and colleagues16, we did not exclude those with an IQ < 70. However, in the current study, only three of the 13 individuals who were carrying de novo CNVs had an estimated IQ < 70, with one further individual who had missing IQ data. The de novo CNV rate in children with ADHD without ID (IQ > 70) was 3.5% in our study, compared to the 1.7% rate previously observed16.In this study, we present observations on de novo CNVs from the largest published trio-based study of ADHD to date. The overall mutation rate for de novo CNV carriers was 4.6%, which is a similar rate to that observed for two other DSM-5 childhood neurodevelopmental disorders: ASD and Tourette disorder3 or implicated in ADHD based on a recent, large case–control GWAS2. However, several of the CNVs have been implicated by previously published ADHD case–control CNV association studies (which will include inherited and de novo CNVs); these include the robustly implicated 16p13.11 duplication and the 22q11.2 duplication6.The identified CNVs affected a total of 262 genes. None of the genes spanned by the de novo CNVs have been highlighted by the only large exome sequencing study of ADHD to date27. Similar findings have emerged from a sequencing study of ADHD, which highlighted strong overlap in the genes implicated in ADHD and ASD3, whereas GWAS demonstrate a moderate (rg = 0.36) genetic correlation between ADHD and ASD28, and a previous CNV study suggested that the same biological pathways are impacted by CNVs in ADHD and ASD29. Our study adds to the growing body of literature supporting the relatively recent reconceptualization of ADHD as a neurodevelopmental disorder, as evidenced by its DSM-5 definition.The overlap of de novo CNV loci in the regions 15q13.1–13.2, 16p13.11, 16p12.2 and 22q11.21 with loci previously implicated in ASD, DD/ID, Tourette disorder and schizophrenia is consistent with the notion that neuropsychiatric CNVs often have associations with a broad range of neurodevelopmental and psychiatric phenotypes22, whereas deletions at this locus (not observed here) are associated with schizophrenia as well as other neurodevelopmental impairments21.However although there is overlap in implicated CNV regions , whereas the parental DNA samples were more likely to come from blood . Although the QC metrics were somewhat poorer for the saliva samples, given the careful QC protocol and examination of all the traces , the rate of de novo CNVs in this study is unlikely to be overstated. Collecting blood samples from children with severe neurodevelopmental disorders poses a challenge for genetic research studies. Furthermore, although the comparison of the results of this study to previously published studies is limited by such differences in source of DNA, different genotyping arrays and variable CNV-calling protocols, our focus on well-defined large, rare CNV loci is a robust approach that is unlikely to be biased towards detecting false positives, but may have resulted in missing valid de novo CNVs in the sample.16 and the current study) and there have been no large, trio-based sequencing studies of ADHD. To identify associated variants, as well as damaging and likely causal rare mutations, will require very large sample sizes of ADHD that are not yet globally available.Although ADHD is highly heritable, complex and polygenic, rare mutation discovery has been slower than for many other similarly heritable and polygenic disorders that include DD/ID, schizophrenia, ASD and Tourette disorder. For example, de novo CNV data are currently only available for around a total of 500 trios (Lionel and colleaguesIn summary, we present findings which suggest that de novo CNVs likely contribute to ADHD risk and highlight the need for larger discovery studies for future biological insights.Supplementary Figure 1


Attention Deficit/Hyperactivity Disorder (ADHD) is a self-regulation disorder, with impairments in error monitoring associated with underactivation of the related brain network(s). Psychostimulant medication improves ADHD symptoms and can upregulate brain function, but has side effects, with limited evidence for longer-term effects. Real-time functional magnetic resonance neurofeedback (fMRI-NF) has potential longer-term neuroplastic effects. We previously reported the effects of 11 runs of 8.5 min of fMRI-NF of the right inferior frontal cortex (rIFC) in adolescents with ADHD. This resulted in improvement of clinical symptom and enhanced rIFC activation post-pre treatment during response inhibition, when compared to a control group receiving fMRI-NF of the left parahippocampal gyrus (lPHG).In the current study we applied a novel analysis to the existing data by investigating the effects of fMRI-NF of rIFC in 16 adolescents with ADHD compared to fMRI-NF of lPHG in 11 adolescents with ADHD on the neurofunctional correlates of error monitoring during the same fMRI tracking stop task and potential associations with cognitive and clinical measures. We found stronger performance adjustment to errors in the rIFC-NF compared to the control lPHG-NF group. At the brain function level, fMRI-NF of rIFC compared to that of lPHG was associated with increased activation in error monitoring regions of the left IFC, premotor cortex, insula and putamen. The increased activation in left IFC-insular-striatal error monitoring regions in the rIFC-NF relative to the lPHG-NF group was furthermore trend-wise correlated with NF-induced ADHD symptom improvements.The findings of this study show, that during error monitoring, fMRI-NF training of rIFC upregulation elicited improvement in post-error behavioural adjustments and concomitant increased activation in left hemispheric fronto-insular-striatal and premotor regions mediating self-control and self-monitoring functions. This suggests that the administration of fMRI-NF of the rIFC may have had an impact on wider networks of self-regulation and self-monitoring in adolescents with ADHD. ADHD haPatients with ADHD have been shown to have consistent impairments in inhibition and error monitoring . IndividPsychostimulant medication is the gold-standard treatment for ADHD. Stimulants are associated with improvement of clinical symptoms in about 70% of patients . StimulaMPH has also been shown to influence error monitoring networks in children with ADHD. Thus a single dose of MPH resulted in a complete normalisation of the brain underactivation in children with ADHD during placebo in error monitoring regions compared to healthy controls, i.e. in bilateral IFC, insula, putamen, right caudate, left medial frontal cortex, parietal and occipital regions .Although stimulants are effective in treating ADHD symptoms, they have important limitations such as the potential for side effects, and the fact that they are not indicated for all patients . In addiA recent randomised controlled trial from our lab tested fMRI-NF of the rIFC compared to fMRI-NF of the left parahippocampal gyrus (lPHG) in adolescents with ADHD . Thirty-failed stop trials, which measure error monitoring. The aim of the current study was therefore to investigate whether fMRI-NF of rIFC also improved brain activation related to error monitoring in the stop task, in the same group of adolescents with ADHD  compared to an active control group of 13 boys with ADHD (lPHG-NF group). For the purpose of this analysis, only the participants who completed the stop task were included, resulting in 16 in the rIFC-NF group and 11 in the lPHG-NF group. The experimental design is detailed in .2.1Twenty-seven boys with ADHD between 12 and 17 years of age (mean (SD) = 14 (1.5)) were included in this study. The diagnosis of ADHD was made by an experienced child psychiatrist and confirmed with Schedule of Affective Disorders and Schizophrenia for School-Age Children-Present and Lifetime version (K-SADS-PL) . Twenty-Exclusion criteria were IQ below 80 as measured on the Wechsler Abbreviated Scale of Intelligence WASI; , alcoholThe local research ethics committee approved the study, which was conducted in accordance with the Declaration of Helsinki . Each participant/legal guardian gave informed written consent/assent. All participants received up to £150 for their participation to the study: £20 for each fMRI-NF scan, up to £10 for best performance during the session, as well as £30 for the post-training visit. They were all reimbursed for their travel expenses.2.2Both primary and secondary outcome measures were parent-rated questionnaires, the ADHD rating scale (ADHD-RS) and the CPRS-R, respectively. The ADHD-RS assesses ADHD symptoms according to the DSM-5 and also monitors treatment effects . The CPR2.3The fMRI-NF protocol consisted of four 1 h to 1.15 hr sessions within a 2-week period, totalling a maximum of 14 NF runs of 8.5 min each. Each fMRI-NF run consisted of 6 activation blocks of 40 s, separated by a rest block of 30 s. The run always started with a rest block showing an image of a dolphin. In the activation blocks, the neurofeedback of the target region was represented by a video of a rocket, which the participants were asked to move toward space using any strategy they could. Since explicit instructions have been shown to be less effective  and instIn addition to the fMRI-NF sessions, participants were instructed to practice daily brain self-regulation using a picture of the rocket. After the last fMRI-NF run, the participants underwent a “transfer” run, where they tried to self-regulate their brain without any feedback being presented. This task measures retention of learning and transfer of training strategies to everyday life .2.4Before and after NF training, participants underwent an fMRI tracking stop task . The parsuccessful stop versus go trials has been published previously  were included in the analyses. Full details of the fMRI-NF data acquisition and processing are available in TAR) were defined in Talairach space and mapped to the individual participant’s fMRI scans via the T1-weighted structural image and a two-volume EPI localizer image collected at the start of each session. In addition, a mask of the white matter was also created for use as reference ROI (ROIREF) to compensate for non-specific global brain effect. The mean BOLD signal was extracted from each ROI by applying the masks to the pre-processed fMRI data. For each new brain volume (e.g. every TR/two seconds), AFNI calculated a new set of values for each ROIs. These values were transferred to an in-house program that generated the feedback by displaying a moving rocket. The neurofeedback signal was calculated as:A custom fMRI-NF interface system  and the TAR-ROIREF) – (ROITARPrevious – ROIREFPrevious)) to the averaged activity of the previous rest block. All values were being measured relative to the corresponding white matter signal, that represents global signal of no interest. Before each fMRI-NF run, participants were informed/reminded of the delay (at least 6 s) in the feedback they would receive, caused by both hemodynamic delay and data processing time.where ROIDetails of the acquisition of the fMRI Stop task can be found in 2.62.6.1Some parents did not complete all questionnaires. Two ADHD-RS and 3 CPRS-R parent ratings were missing across all participants and all visits. There was a limited amount of missing data (<5%) and missing points were assumed to be completely at random. Multiple  imputations were used for all missing data. The individual estimates from the multiply-imputed datasets were then used to calculate a combined estimate by applying Rubin’s Rules . RepeateThe fMRI-NF performance was averaged across all runs and a between-group ANOVA was conducted to assess group differences in this performance.2.6.2The individual and group-level analysis methods are described in detail elsewhere . VersionIndividual Analysis. The main experimental condition of error monitoring in the stop task, e.g. failed stop trials, against an implicit baseline, e.g. the go trials, was obtained using a standard GLM approach. Individual goodness-of-fit statistic (SSQ-ratio) maps were created and transformed into standard space  for each NF group were produced for the experimental condition. A repeated-measures ANOVA was then conducted to test the interaction of time  by group . The voxel-level threshold was first set to p < 0.05 to give maximum sensitivity and to minimize type II errors. After that, a cluster-level threshold was computed for the resulting 3D voxel clusters and set in such a way as to produce less than one false positive cluster per map, in this analysis achieved at cluster-level p < 0.02. The cluster p-value is adjusted to eliminate any potential false positive cluster from the map, i.e. we can be confident that all the clusters we see are not false positives . The necessary combination of voxel- and cluster-level thresholds was not assumed from theory but rather was determined by direct permutation, giving excellent type I error control  for the NF condition versus baseline, averaged across all 11 runs. For this purpose, a group brain activation map was produced for the fMRI-NF condition contrasted with the baseline condition. Hypothesis testing was carried out at the cluster level. A voxel-wise test at p < 0.05 was conducted to identify any voxels that might plausibly be activated, followed by a subsequent test at a cluster-level threshold of p < 0.011 to remove false positive clusters produced by the voxel level test. Combined voxel/cluster tests with permutation testing allow for excellent Type 1 error control . For the33.1The between-group ANOVA revealed no significant between-group differences at baseline in demographic or clinical measures, in medication type or medication dose . The chiWithin-group. Within-group ANOVA comparisons showed a significant decrease in ADHD symptoms in both groups after relative to before fMRI-NF, for most primary and secondary outcome measures  = 17.030, p = 0.0001; ADHD-RS Inattention subscale: F = 16.773, p = 0.0001; ADHD-RS Hyperactivity/Impulsivity subscale: F = 10.659, p = 0.003; CPRS-R ADHD Index: F = 20.573, p = 0.0001), but no group or group by time interaction effects.We also tested for group differences in clinical measures post-treatment. The between-group ANOVA revealed no significant between-group differences in clinical measures after fMRI-NF .3.2Between-group repeated measures ANOVA comparisons showed a significant effect of time, with a reduction of standard deviation of reaction time in both groups  = 8.143, p = 0.009), but there was no effect of group in any dependent variables. The only significant group by time interaction effect was for the post-error reaction time to go signals  = 5.669, p = 0.025), which was increased in the rIFC-NF group relative to the lPHG-NF group post compared to pre fMRI-NF , Fig. 1.3.3The cluster-level p-value was determined in such a way to produce <1 false positive cluster per map . This wa3.4To test whether the group differences in brain changes to failed stop trials post minus pre fMRI-NF were correlated with NF-induced cognitive or clinical changes in the rIFC-NF group, Pearson correlation analyses were performed in this group. Analyses were undertaken between the post- versus pre- NF brain changes and the corresponding post- versus pre- changes in the primary clinical outcome measures and in the key cognitive measure which is post-error reaction times. For this purpose, the average BOLD response was extracted for each subject in the activation clusters that resulted from the repeated between-group ANOVA analysis. These BOLD responses were then correlated with the changes post- versus pre- fMRI-NF in post-error reaction times to go signals, as well as with the primary clinical outcome measures of the ADHD-RS. There was no significant correlation between the NF-induced changes in brain activation and the NF-induced changes in post-error reaction times. There were two trend-level negative correlations in the left insula/IFC/putamen between the NF-induced changes in brain activation and the NF-induced changes in primary outcome measures  in the rIFC-NF group . Once we3.5We found increased brain activation, averaged across all runs, in both groups in the bilateral inferior frontal lobe/insula, basal ganglia, thalamus, anterior cingulate, SMA, premotor cortex, superior/middle/inferior temporal cortices, right middle frontal, right inferior/superior parietal/precuneus/occipital, the right ventrolateral prefrontal cortex/insula and the left ventrolateral prefrontal/superior temporal cortices , Fig. 4.4This study investigated the effects of fMRI-NF of rIFC relative to lPHG on error monitoring performance and underlying brain activation, in adolescents with ADHD. We found slower post-error reaction times to go signals after rIFC fMRI-NF compared to lPHG-NF, suggesting stronger performance adjustment to errors. At the neurofunctional level, fMRI-NF of rIFC compared to that of lPHG was associated with increased activation in error monitoring regions of the left IFC-insular-striatal areas and premotor cortex. This increased activation was trend-wise correlated with rIFC-NF-induced improvements in the primary outcome measures of total ADHD and ADHD hyperactivity/impulsiveness symptoms.Behaviourally, we found that adolescents with ADHD in the rIFC-fMRI-NF group compared to those in the lPHG-NF group slowed down more after committing errors. In healthy controls, there is typically a performance adjustment after mistakes, reflected by slowing down in the following trials  and is cInterestingly, both groups improved in intrasubject response variability which is one of the most consistently observed impairments in patients with ADHD  and assoThe improvement in post-error performance adjustment was paralleled at the brain level, where rIFC fMRI-NF relative to lPHG fMRI-NF resulted in increased activation in adolescents with ADHD in error monitoring regions of the left IFC, anterior insula, putamen and premotor cortex. Although there is some overlap between inhibition and error monitoring networks, the right IFC has most consistently been associated with motor response inhibition  while thBoth medial and lateral prefrontal cortices have been implicated in error monitoring, but their roles appear to be related to different components . The medThe premotor cortex, connected to the anterior insula via the pre-supplementary motor area, has also been associated with error detection  to help Taken together, our results show that fronto-insular-striatal and premotor activation related to the cognitive control aspects of error monitoring in ADHD, which are typically been found to be underactivated relative to healthy controls , are incWe have previously shown that, relative to placebo, a single dose of MPH upregulated and normalised activation in ADHD adolescents during error monitoring in the anterior insula, left IFC, premotor cortex and striatal regions, as well as other posterior attention regions . The finThe finding of increased left fronto-striatal activation during error monitoring with fMRI-NF suggest that self-regulation of an isolated prefrontal region has a more widespread effect on other fronto-striatal systems in ADHD. During inhibition, we previously found increased NF-induced activation in the right IFC as well as in parietal regions in the rIFC-NF compared to the lPHG-NF group . The incRegions that are crucial for performance monitoring may be the same regions that are important for self-regulation. Indeed, a meta-analysis of fMRI-NF studies , showed The changes in error-monitoring activation induced by fMRI-NF were negatively correlated with positive changes in ADHD symptoms (which became significant once we removed extreme outliers), suggesting that larger increases of brain activation in left fronto-insular-striatal error monitoring regions were associated with greater improvement in ADHD symptoms. It has been found previously that the neural correlates of error monitoring correlated with severity of hyperactivity and impulsivity symptoms in adult ADHD . In our Although the implications of these brain-behaviour correlations are very promising with respect to the potential clinical future use of fMRI-NF, these latter results must be considered with caution as they did not reach statistical significance. We furthermore found no correlation between the changes in post-error slowing and brain activation changes, which would have further strengthened the association. Our sample size was relatively small and our study was not powered to show such associations, so further studies will be required in order to determine whether these results are valid and replicable.Notably, both groups showed improvement of ADHD symptoms, while the brain activation and performance effects were specific to the rIFC-NF group. This is similar to what we found in our previously study where only the rIFC-NF group had increased rIFC and parietal activation during inhibition in the fMRI stop task and improved in sustained attention . Since tper se were stronger than the behavioural findings, in line with evidence that fMRI is more sensitive in detecting treatment effects in ADHD than behavioural or clinical data  to KR. Additional support was provided by the National Institute for Health Research (NIHR) Biomedical Research Centre at South London and the Maudsley NHS Foundation Trust and King’s College London and by the Medical Research Council (MRC) (MR/P012647/1) to KR which also supported MC. AA was supported by a Ph.D studentship from the Institute of Psychiatry, Psychology and Neuroscience, King’s College London. The funders had no involvement in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the article for publication.M. Criaud: Formal analysis, Writing - original draft, Writing - review & editing, Visualization. M. Wulff: Investigation. A.A. Alegria: Investigation. G.J. Barker: Methodology, Software, Validation, Resources. V. Giampietro: Methodology, Software, Validation, Resources. K. Rubia: Conceptualization, Formal analysis, Resources, Data curation, Writing - review & editing, Supervision, Project administration, Funding acquisition.