Background: The co-existence of tic disorders and attention-deficit/hyperactivity disorder (TD + ADHD) has proven to be highly important in daily clinical practice. The factor ADHD is not only associated with further comorbidities, but also has a long-term negative psychosocial effect, while the factor TD is usually less disturbing for the major part of the patients. It remains unclear how far this is related to a different neurobiological background of the associated disorders or whether TD + ADHD reflects a common one. Objective: This review provides an update on the neurobiological background of TD + ADHD in order to better understand and treat this clinical problem, while clarifying whether an additive model of TD + ADHD holds true and should be used as a basis for further clinical recommendations. Method: A comprehensive research of the literature was conducted and analyzed, including existing clinical guidelines for both TD and ADHD. Besides genetical and environmental risk factors, brain structure and functions, neurophysiological processes and neurotransmitter systems were reviewed. Results: Only a limited number of empirical studies on the neurobiological background of TD and ADHD have taken the peculiarity of co-existing TD + ADHD into consideration, and even less studies have used a 2 × 2 factorial design in order to disentangle the impact/effects of the factors of TD versus those of ADHD. Nevertheless, the assumption that TD + ADHD can best be seen as an additive model at all levels of investigation was strengthened, although some overlap of more general, disorder non-specific aspects seem to exist. Conclusion: Beyond stress-related transdiagnostic aspects, separate specific disturbances in certain neuronal circuits may lead to disorder-related symptoms inducing TD + ADHD in an additive way. Hence, within a classificatory categorical framework, the dimensional aspects of multilevel diagnostic-profiling seem to be a helpful precondition for personalized decisions on counselling and disorder-specific treatment in TD + ADHD. More than a decade ago, a group of experts presented a collection of articles with an overview concerning different aspects  of the clinically important co-existence of tic disorders and attention-deficit/hyperactivity disorder (TD + ADHD)  see als. For exan = 6768), indicating that co-occurring ADHD in TD cases is markedly lower in population-based samples .In the meantime, large-scale studies shed further light on the prevalence and impact of TD + ADHD. Scharf et al.  reportedn = 65,540, aged 6–17 years). After controlling for co-occurring neurobehavioral conditions  the before negative findings were no longer significant for the TD group, Ricketts et al. [n = 3014, age 4–17 years) and found that children with TD + ADHD had higher mean impairment across all domains  than children with either TD or ADHD. The authors concluded that disorder-specific contributions to impairment could inform targeted interventions for TD and ADHD within TD + ADHD.The negative psychosocial impact of TD plus co-occurring conditions was outlined by Bitsko et al.  among a Box 1The most comprehensive and updated textbook on Tourette Syndrome with a worldwide group of experts  includesChronic tic disorders  are basically characterized by waxing and waning motor/vocal tics, with an onset around the age of seven years, increasing in frequency, diversity, and severity up to the age of 12–14 years and declining thereafter. Around the age of 10 years, additional premonitory phenomena  may be recognized by the child. Tics can be suppressed for a certain amount of time. This natural compensatory neuronal process using prefrontal mechanisms  can be iADHD  is also a neurodevelopmental disorder that presents with symptoms of inattention and/or hyperactivity–impulsivity. According to DSM-5, three subtypes/presentations are defined, namely inattentive, hyperactive/impulsive, and combined. ADHD can be diagnosed from the age of four years onward, often persists into adulthood, and is usually accompanied by psychosocial impairment. Treatment with behavioral therapy and/or stimulants/noradrenergic drugs is recommended according to existing evidence-based conclusions .In both TD and ADHD, about 70–80% of individuals show at least one additional behavioral problem. In TD, the co-occurrence of ADHD can be found in about 50% of patients , while in ADHD about 20% of cases also present with TD. Both percentages are well above what would be expected by chance alone . BesidesComorbidity of TD in ADHD has low or no impact on impaired psychosocial functioning and learning, while comorbidity of ADHD in TD is highly associated with additional cognitive and psychosocial problems as well as with the appearance of further psychopathologies ,13,15. TAccording to the variety of the developmentally determined core symptoms, their spectrum of severity and associated psychopathologies TD, ADHD, and TD + ADHD may present with a great heterogeneity. This makes it highly challenging for diagnostics, treatment, and research.The long-term negative effect of ADHD can be found even beyond psychosocial aspects. This was underlined recently by Brander et al. . They inIt remains unclear how far this is related to a different or a common neurobiological background of the two associated disorders in TD + ADHD.This review will provide an update on the neurobiological background of TD + ADHD, having in mind that growing new empirical evidence within this area of research is needed to give rise to a better understanding and treatment of this clinical problem. Within this context, it should be investigated whether a specific role of each factor  can be stated or not. In addition, it should be discussed whether, within such a classificatory framework, the dimensional aspects of transdiagnostic neurobiological overlap may play a role. Specifically, in TD and ADHD, categorical as well as dimensional approaches are worth following in clinical practice and research: having or not having a tic is clearly categorical, while attention, motor activity, and impulsivity are dimensional. This review is phenotypically guided by the clinical categories as defined in DSM-5 while looking from this standpoint to a specific/non-specific neurobiological background.When modelling the co-occurrence of TD + ADHD, it has to be clarified whether this combination represents two independent pathologies (reflecting an additive model), a separate nosological entity manifested by both tics and ADHD symptoms (interactive model), or a phenotypical subgroup of one of the two major clinical forms (phenotype model), as outlined in Banaschewski et al.  and RothSo far, a stepwise model for children and adolescents has been proposed, i.e., the co-occurrence of TD + ADHD might follow an additive model at the neurobiological level  while with higher cognitive functions/performance  a merely interactive modelling might come into play . BecausTo answer this question methodologically, a 2 × 2 factorial design of studies would be best to disentangle the factors TD vs. ADHD, i.e., optimally, studies should include four groups of patients, namely TD-only, ADHD-only, TD + ADHD, and typically developing probands (TDP). Concerning clinically oriented examples see Roessner et al.  for psycn = 314 patients with TD (ages 5–19 years), and at follow-up 6 years later , changes in the expression of tics and comorbidities were examined. There was found a significant decline of both TD and ADHD, while there were still 63% of co-existing psychopathologies at follow-up (not only TD + ADHD). The strongest predictors for future symptoms of TD or ADHD were the respective diagnoses at baseline. This stability of symptoms over time in parallel may be a hint for separate lines of development for TD and ADHD, even within TD + ADHD [The assumption of behavioral additivity for TD and ADHD is also supported by a longitudinal study on the clinical course of TD. In a cohort of, at baseline Deriving from behavioral findings and earlier neurobiological reports in TD + ADHD , with thn = 262) and without TD  as well as their 1606 full siblings (n = 388 of the ADHD + TD index patients and n = 1248 of the ADHD-TD index patients). It was tested whether the risk for ADHD, TD, and ADHD + TD in siblings was associated with the related index patients’ diagnosis. In order to obtain an estimate for psychopathological specificity, the four groups were also compared for general psychopathological symptoms using the strengths and difficulties questionnaire (SDQ).As a precondition of a common genetic background, TD + ADHD should run in families. Earlier studies with small sample sizes suggested that there may exist an increased risk of ADHD in relatives of TD probands and also an increased risk of TD + ADHD in families affected with this comorbidity ,27. In aThe main finding was a significantly higher risk to present with ADHD + TD (and a tendency for TD without ADHD) among siblings of patients with ADHD + TD. This suggests that the familiality of ADHD + TD may exist, probably driven by the factor TD, which is part of both groups, namely ADHD + TD and TD without ADHD. There was no significant hint for a higher general psychopathological vulnerability in siblings of index patients with ADHD-TD, and thus the results may reflect some psychopathological specificity for TD as well as an additive model for TD + ADHD.If familiality of TD + ADHD is true, the question remains, what kind of genes are responsible for the genetic background?11q24 chromosomal region. Tested in n = 174 nuclear families with TD and n = 242 nuclear families with ADHD, the study indicated that the potassium inwardly-rectifying channel J5 (KCNJ5) was associated with TD and with ADHD, but functional variants remain to be identified and a TD + ADHD group was not investigated.Both TD and ADHD seem to be heterogeneous polygenic disorders  due to n = 162 patients with TD with n = 180 healthy probands according to the BTBD9 gene polymorphisms and came to the conclusion that these gene variants are not associated with TD but with ADHD comorbidity. Unfortunately, an ADHD-only group was not also investigated.Further, Janik et al.  comparedn = 412 children and adolescents aged 3–16 years with a chronic tic disorder (CTD) including TS and n = 131, 3–10 years old non-tic siblings of CTD individuals. Further, cross-disorder non-specific neurobiological dysfunctions exist  of ADRA2A were genotyped and analyzed in 936 normal controls and 1815 ADHD probands, including 1249 trios. Approximately 16% (n = 290) of the ADHD probands had TD + ADHD. While no significant association was found between ADRA2A and ADHD, the allelic/genotypic distribution and allelic transmission were different between TD + ADHD vs. TD-ADHD , especially in males. Unfortunately, there was no group with TD-only, and thus it can only be speculated that the ADRA2A gene effect might be related to the TD part of TD + ADHD, supporting an additive model of this comorbidity.Because noradrenergic drugs such as guanfacine/clonidine/atomoxetine may be helpful for ADHD as well as TD, these substances may be good candidates to treat TD + ADHD. Hence, noradrenergic genes might reflect neurobiological commonalities and thus elucidate the neurobiological background of TD + ADHD. Xu et al.  investigProbably, the genetics of TD + ADHD reflects two different neuronal mechanisms: one related to “non-additive” cross-disorder genes influencing more joint functions such as stress regulation, and another “additive” one that creates disorder-specific disturbances in certain neuronal circuits, leading to specific clinical symptoms.As in other neurodevelopmental disorders, pre- and perinatal complications are known in TD and ADHD. However, what the role is of pre- and perinatal factors in relation to the expression of single diseases and moreover a comorbidity such as TD + ADHD remains an unresolved issue. In a literature review and critical commentary on epidemiological works evaluating the association between pregnancy-related and birth-related adversities, Hoekstra et al.  concluden = 120 healthy adults (19–31 years old) and n = 60 healthy children (7–12 years old) by resting-state functional connectivity MRI (rs-fc MRI) and found a decrease in functional connectivity of BG with the cortical somatomotor face system, expressed by a partial correlation coefficient. The authors speculated  that the developmental trajectory of BG functional organization might be atypical in TS and that a refined rs-fc MRI coefficient might be used for diagnostic purposes.Basal ganglia (BG) dysfunction is known from several developmental disorders, but the development of its functional connectivity with other brain regions over time is not well understood. Greene et al.  investigLocalizing neuroanatomical deviations of the brain has increased our understanding of psychiatric disorders within the last decade, specifically when developmental aspects could be detected . Howeverbasal ganglia, they concluded that in TD there exist smaller caudate nuclei in both children and adults, which may suggest that the hypoplasia of the caudate nucleus represents a trait morphological abnormality in persons with TD, while the co-occurrence of ADHD did not statistically alter these findings.Plessen et al.  summarizcortical regions, they detected a higher proportion of white matter in the right frontal lobe of 11 boys with TD, but not in 14 boys with TD + ADHD, 12 with ADHD-only, or 26 healthy controls. Both individuals with TD + ADHD and ADHD-only had smaller overall frontal lobes, which might point to a driving effect of ADHD in these cases. However, one could not statistically distinguish morphometric differences of the brain that were specific to either TD or ADHD or TD + ADHD.Concerning corpus callosum (CC) was considered because of earlier findings of reduced brain asymmetry in the basal ganglia of TD patients, which has not been confirmed so far. However, DTI (Diffusion Tensor Imaging) findings seem to point to a reduced interhemispheric connectivity in children with TD. A comorbid diagnosis of ADHD did not influence the results [Finally, the  results .In conclusion, the authors suggest that comorbid ADHD does not significantly alter the primary findings derived from samples with TD patients, thus providing only a little support of a shared genetic vulnerability as expressed in deviations of brain morphology in TD + ADHD.n = 141 children between 8–12 years old. The analysis of basal ganglia volume or shape did not find any evidence that TD, ADHD, or TD + ADHD are associated with structural variations compared to healthy controls. There was also no association with ADHD severity and no interaction between TD and ADHD—the latter is also statistically essential for the assumption of an additive model in TD + ADHD.Both TD and ADHD have been associated mainly with the structural variation of frontal lobes and striatum/basal ganglia. Taking this in focus, Forde et al.  looked alimbic system  may als and Hof ,47, beca and Hof . This sh and Hof .amygdala as a key component of the limbic system. Amygdala and total brain volumes were measured with MRI in 17 male patients with TD, including eight with TD + ADHD  and 17 age-matched healthy controls . There was an amygdala volume reduction in TD that did not correlate with tic severity but with symptoms of ADHD . Hence, primarily ADHD might be related to vulnerability of the amygdala, giving input of neuronal dysregulation to the striatum and frontal cortex. The latter usually may be able to compensate this disturbance, at least partly, and may be trained by neurofeedback in order to better regulate behavior [The small study of Ludolph et al.  aimed tobehavior .n = 12) including patients with TD + OCD + ADHD (n = 8) and TD + ADHD (n = 4) was compared to n = 17 with TD + OCD and n = 24 healthy controls by voxel-based morphometry (VBM). They found decreased grey matter volume in the left inferior frontal gyrus (IFG). By comparing TD + ADHD with TD without ADHD they registered a left sided amygdala volume increase. Although this study had only a very small subgroup of four patients with TD + ADHD, the deviation of amygdala volume for TD + ADHD in both studies (children and adults) suggests the importance of investigating the role of emotional regulation related to this brain area in TD + ADHD further.A similar approach was reported in adults  by Wittforth et al. . A groupn = 11 with TD + ADHD). The multiple morphometric changes, i.e., grey matter volume, decrease of hippocampus, putamen, and parietal cortex, were modulated by the presence of ADHD assuming an additive effect.Within this context, it seems worthwhile to have a critical eye on some results on brain volume/cortical thickness that assume morphometric deviations in TD extend from the motor cortex to the limbic system depending on the severity/complexity of tics and comorbidities . For exaDopaminergic abnormalities in the striatum, although of a different kind, seem to play a key role in the pathophysiology of both TD and ADHD ,12. Whiln = 7), TD + ADHD (n = 12), and healthy controls (n = 19) was recently investigated by Capetian et al. [The dopaminergic neurotransmission and its metabolism in the central nervous system of non-medicated patients with TD  in the Nevertheless, the overwhelming influence of a hyper-dopaminergic state in TD was also documented by Gilbert et al. , who coun = 136 children with TD + ADHD. Either clonidine  or methylphenidate  or either their combination, CLON + MPH, or placebo were administered in a 2 × 2 factorial design over 16 weeks. CLON + MPH best reduced tics and ADHD symptoms, suggesting an additive effect of both neurotransmitters in TD + ADHD [Besides dopamine, noradrenaline seems to be an important player in TD + ADHD, as can be seen from a clinical trial in D + ADHD . The difn = 15, ADHD n = 39, TD + ADHD n = 29, and healthy controls n = 53) to address confounds of comorbidity. Glutamate concentrations were evaluated in the anterior cingulate cortex (ACC) and the left dorsal striatum (LDS). Unexpectedly, no group differences could be found. Furthermore, variation in glutamate concentration in ACC and LDS did not show any relation to age, sex, medication use, IQ, tic, or ADHD severity. Obviously, there exists no empirical evidence for glutamatergic neuropathology in TD and/or ADHD.Both TD and ADHD have also been related to abnormalities in glutamatergic neurochemistry in the fronto-striatal circuitry, because of the importance of glutamate in the excitatory aspects of neurons, assuming higher concentrations of glutamate at least in the striatum of TD patients . To furtn = 38 adult patients with TD, these reported in 55% an improvement of comorbidities, including ADHD symptoms [Within the last decade some small clinical studies in adults and single-case observations in adults and adolescents suggest that cannabis-based-medicine (CBM) such as delta-9-tetrahydrocannabinol (THC), dronabinol, or street-cannabis may improve tics, and thus the endocannabinoid system (ECS) might be involved in the pathophysiology of TD . So far,symptoms , suggestGABAergic alterations in different functional domains of the striatum might help to elucidate the co-occurrence of TD + ADHD. Examining the neuronal activity of freely behaving rats before and after domain-focused injections for neuronal disinhibition, Israelashvili et al.  found thFurther, indicators for a higher immune activation in TD may exist , but itn = 259 children, with a first-degree relative with CTD, but no tics at entry at the age of 3–10 years) [n = 5811 ADHD-only, n = 1816 TD-only, n = 349 TD + ADHD) that patients with TD + ADHD had a significantly higher prevalence of allergic diseases than (in decreasing order) ADHD-only, TD-only, and controls, suggesting that ADHD is the essential factor.In a recent review on immunology in TD and ADHD, Martino et al.  stated t0 years) . A posit0 years)  reportedn = 327; first degree relatives with tics, n = 31; first degree relatives without tics, n = 93). Lower levels of vitamin D were not associated with tics but only with the presence and severity of ADHD in children and adolescents with TD, pointing to separate effects of ADHD in TD + ADHD, supporting independence of the two factors. Finally, the role of vitamin D (25(OH)D) serum level in comorbid TD + ADHD is unclear, although observational studies and a meta-analysis revealed a lower level in a clinically significant proportion (about 50%) of children with ADHD ,77,78. FMoreover, vitamin D deficiency might modify dopaminergic pathways and thus influence ADHD symptoms . WhetherPatients with TD, ADHD, and TD + ADHD have problems with controlling their movements in line with the hypothesis of impaired motor inhibition in TD, ADHD, and TD + ADHD . Pure neWhen Rothenberger and Heinrich  reviewedn = 47; TS + ADHD, n = 32; ADHD-only, n = 22; matched typically developing controls, n = 35).In general, all the electrophysiological reports underline the good abilities of children with TD for neuronal/behavioral compensation of their motor hyper-excitability. The question arises as to whether this still holds when TD is associated with ADHD, because for ADHD-only inhibitory abilities are reduced  latencies during tasks involving inhibitory abilities ,88, redun = 7–14) the statistical power was low and differentiating factor-related results could not be reported.This is in line with the experimental approach of Eichele et al. ,93, who n = 44 to n = 53 children, included four groups . The authors revealed that, first, neurodynamic sufficiency in ADHD and TD + ADHD seems to be similarly impaired with ADHD as the responsible factor and, second, that if very high cognitive performance is requested, the two associated disorders seem to interact at the level of electrophysiology, while at lower levels of cognitive demand the two factors  may be separately and additively at work.Further, electrophysiological studies in the domain of cognitive control ,94,95,96n = 15) and 19 patients with primary tic disorders with a stop-signal-task and TMS applied to the motor cortex. They found that automatic but not volitional inhibition (proactive and reactive) was impaired in tic disorders. Corticospinal excitability while preparing for movement execution was not different in patients with tic disorders, indicating that they do not have an abnormally excitable motor output. Although comorbidity with OCD appeared to affect results, sample size  was too small to draw firm conclusions.Other neurophysiological studies in children with a 2 × 2 factorial design  also presented informative examples for the additivity of TD + ADHD, usually with the weakest results for ADHD and the comorbid group with TD + ADHD ,53,97. IThe brain’s functional organization during resting state or task processing can also be captured by functional magnetic resonance imaging (fMRI), with the advantage of referring the results topologically to certain cortical and subcortical brain regions.n = 109, 8–12 year old children with TD (n = 27), TD + ADHD (n = 19), ADHD (n = 23), and HC (n = 40). Local efficiency and clustering coefficient were significantly lower in children with TD in the default mode network compared with HC, and in the fronto-parietal network compared with ADHD. The findings for TD + ADHD were more similar to TD than to HC, and thus may represent a TD-related deficit. This gives support for a lower short-range network connectivity in the default mode network of TD, assuming the misguided synchronization of neuronal oscillations, as already outlined by Rothenberger [Openneer et al.  registerenberger .n = 28; TD + ADHD, n = 23; HC, n = 52) with fMRI during a stop-signal task. They observed an impaired response inhibition performance in the group with TD + ADHD, while showing that ADHD severity was the driving force and neural activation during failed inhibition was stronger in TD + ADHD at the inferior frontal gyrus and temporal and parietal areas. Because a group with ADHD-only was missing, a 2 × 2 factorial design could not be conducted. Nevertheless, these data may help to topographically localize the networks of known inhibitory deficits in TD + ADHD and ADHD ..n = 28; n = 18; TD + ADHD, n = 29; ADHD, n = 29; HC, n = 48). Neural activation during reward anticipation and receipt was not associated with TD but with ADHD and is in line with the view of nucleus accumbens hypoactivation, related to transdiagnostic ADHD symptom severity and an additive model of TD + ADHD.A third paper of this fMRI-group from the Netherlands  investign = 90 children). Kirov et al. (with n = 18 children in each of the four groups) concluded that in children with TD + ADHD, sleep problems seem to be higher, having both REM sleep increase (for ADHD) and lower sleep efficiency/elevated arousal (for TD) in an additive manner [n = 108), ADHD (n = 316), TD + ADHD (n = 79), and HC (n = 336). Compared to HC, TD and TD + ADHD groups had significantly lower sleep efficiency and higher sleep onset latency. TD + ADHD also had significantly increased time in bed and total sleep time. No significant differences were observed between the highly heterogeneous ADHD group and HC. Hence, TD + ADHD was associated with more pronounced differences. Unfortunately, most of the studies evaluated in this meta-analysis did not include all the four groups and thus it was not possible to create a 2 × 2 design in order to determine which factor  was responsible for the results, although adding ADHD to TD might have led to the pronounced differences, as also seen in a wrist-worn, seven days accelerometer study on physical activity, sleep, and neuropsychiatric symptom severity in n = 110 children with TD with/without different psychopathologies including ADHD [Polysomnographic sleep-laboratory registrations in children of the four groups in question  confirmed the earlier findings of short arousals and tics during sleep in TD. When ADHD symptoms accompanied TD, in addition, nightly general motor restlessness and disorganized behavior may take place  are available. Although not every problem is solved, existing data extend and strengthen the assumption that TD + ADHD may be considered basically as an additive model at the levels investigated . However, researchers are well aware that, while applying a clinical-categorical approach to this modelling, the ADHD core symptoms and associated psychopathology  are dimensional with certain clinical cut-offs but also sometimes showing subclinical/subcategorial values higher than in typically developing children. In addition, for example, attentional problems may hinder a child from suppressing his/her tics, or increased general motor restlessness might interfere with tics. Hence, some behavioral interdependence between the factors TD and ADHD have clinically to be taken into consideration but, overall, disorder-specific contributions to impairments shall guide targeted interventions for TD and ADHD within TD + ADHD .Thus, for practical reasons, this favors a more dimensional approach to child psychiatric disorders, finally leading to a multilevel/multisymptomatic diagnostic-profiling as a helpful precondition for decisions on counselling and specific treatment programs within the framework of “personalized medicine”; in the case of TD + ADHD, a treatment “in parallel” at different levels such as psychoeducation, counselling, psychotherapy, and psychopharmacotherapy.At the same time, the presented findings make clear that some overlapping (“non-additive”) deviations between TD and ADHD exist, which are of a more general neurobiological character, as can be seen for other early-onset neurodevelopmental disorders such as autism spectrum disorder. The parameters of these non-specific cross-disorder functional systems  may or may not be quantitatively different between TD, ADHD, and TD + ADHD , while disorder-specific disturbances in certain subcortical–cortical neuronal circuits may lead to disorder-related symptoms and can finally induce TD + ADHD in a more or less additive way, including interactive effects when higher cognitive functions are demanded, i.e., TD + ADHD represents a clinical picture appearing higher than chance, expressing as a special co-occurring psychopathology without representing a separate diagnostic entity. Such reasoning should be studied and discussed further, helping to develop a positive perspective for a fruitful joint neurobiological and clinical research on TD + ADHD. In this respect, a large-scale European Multicenter Tics in Children Study (EMTICS) that is currently underway in order to explore different aspects of TD  will probably shed further light on the associations of TD + ADHD ,105.


E. coli, has not been previously purified. Here we report the purification of the full-length form of viperin from HEK293T cells transfected with viperin. The purification method utilizes nanodiscs to maintain the protein in its membrane-bound state. Unexpectedly, the enzyme exhibits significantly lower catalytic activity once purified, suggesting that interactions with other ER-membrane components may be important to maintain viperin’s activity.Viperin is a radical S-adenosylmethionine enzyme that catalyzes the formation of the antiviral ribonucleotide, 3’-deoxy-3’,4’-didehydroCTP. The enzyme is conserved across all kingdoms of life, and in higher animals viperin is localized to the ER-membrane and lipid droplets through an N-terminal extension that forms an amphipathic helix. Evidence suggests that the N-terminal extension plays an important role in viperin’s interactions with other membrane proteins. These interactions serve to modulate the activity of various other enzymes that are important for viral replication and constitute another facet of viperin’s antiviral properties, distinct from its catalytic activity. However, the full-length form of the enzyme, which has proved refractory to expression in  Viperin-like enzymes appear conserved in all six kingdoms of life, pointing to an ancient role for this family enzymes in combating viral infections7.Viperin 9. The enzyme is integrated into the broader innate immune response to viral infection and has been shown to regulate both the activity and expression levels of numerous cellular and viral proteins. This network of protein–protein interactions results in the enzyme exerting a wide array of antiviral properties beyond just the synthesis of ddhCTP. Cellular enzymes shown to interact with viperin include the mitochondrial trifunctional protein11, which is involved in fatty acid β-oxidation; squalene monooxygenase and lanosterol synthase13, which catalyze key steps in sterol biosynthesis; and interleukin receptor-associated kinase 1 (IRAK1) and the E3 ubiquitin ligase, TRAF616, which are components of the TLR7/9 innate immune signaling pathway. Viperin also binds to a wide range of viral proteins from viruses such as hepatitis C, Dengue, tick-borne encephalitis and Zika viruses9. Depending on the virus, viperin interacts with both non-structural proteins that are responsible for viral replication and assembly and with structural proteins that form the viral capsid19.In higher animals, viperin expression is highly up regulated by type I interferons and viruses20. Although the membrane-localizing sequence is not required for enzymatic activity, the N-terminal extension likely plays an important role in mediating viperin’s interactions with other proteins—either by making direct contacts with other proteins or, indirectly, by localizing viperin to the ER membrane and lipid droplets. For example, we recently showed by co-immunoprecipitation that viperin forms a complex with the hepatitis-C viral protein, NS5B, and the cellular protein, VAP33, both of which also bind to the ER membrane19. However, these interactions were lost when the membrane-localizing sequences were removed to facilitate expression and purification of these proteins from E. coli.In animals, viperin is localized to the cytosolic face of the ER membrane and lipid droplets through an N-terminal extension that is unstructured in solution but adopts an amphipathic α-helical conformation upon binding the lipid bilayerE. coli. Therefore, most mechanistic studies and all structural studies on the enzyme have been conducted on truncated constructs lacking this sequence, which can be over-expressed and purified from E. coli7. Here, we report the purification of the full-length enzyme from transfected HEK293T cells in which we have used nanodiscs to retain the interaction between the N-terminal sequence and the lipid bilayer. Nanodiscs are discoidal lipid bilayers, ranging in diameter between 8 and 16 nm22. The discoidal structure is stabilized by alpha-helical proteins known as membrane scaffold proteins (MSPs) that are derived from apolipoprotein A1 and form a ‘belt’ around the disc that stabilizes lipid bilayers in solution23. This work represents the first purification of functional full-length viperin from a mammalian system. The ability to purify the full-length, membrane associated form of viperin should facilitate efforts to elucidate the molecular mechanisms by which viperin interacts with other membrane-associated protein and the effect of these interactions on viperin’s antiviral activity.So far, it has not proved possible to express full-length viperin  as recombinant protein in a bacterial expression system such as The HEK293T cell line was purchased from ATCC.A synthetic gene encoding human viperin (GenBank accession no. AAL50053.1) was synthesized and subcloned into pcDNA3.1(+) vector (GenScript). A 3X FLAG tag (DYKDHDGDYKDHDIDYKDDDDK) was introduced at the N-terminus of the protein to facilitate purification. A Kozak consensus sequence (5′-GCCAAC-3′) was introduced ahead of the gene to facilitate expression in eukaryotic cells. The gene encoding the membrane scaffold protein with an E3 amphipathic helical region insertion (MSP1E3D1) was cloned into pET28a(+) vector and equipped with an N-terminal His tag to facilitate purification.S-(5′-Adenosyl)-L-methionine p-toluenesulfonate salt and cytidine 5′-triphosphate disodium salt hydrate were purchased from Sigma Aldrich. POPC  and POPE  were purchased from Avanti Polar Lipids. Amberlite XAD-2 hydrophobic beads, Anti-FLAG M2 magnetic beads, and 3X FLAG peptide were purchased from Millipore Sigma. Pierce silver stain kit was purchased from ThermoFisher Scientific. Rabbit polyclonal viperin antibody (11833–1-AP) was purchased from ProteinTech. Goat anti-rabbit Ig secondary antibody (170-6515) was purchased from BioRad.For DNA transfection in HEK293T cells, transfection grade linear polyethylenimine (PEI) hydrochloride was purchased from Polysciences, Inc. 24. Typically, 50 µg of DNA was mixed with 100 µg PEI in 1:2 ratio (w/w) and incubated for 10 min at room temperature before adding it to HEK293T cells at 50–60% confluence in a 150 mm culture dish. The cells were grown at 37 °C under CO2 for 36–40 h, gently harvested, and cell pellets were stored at − 80 °C.The gene encoding viperin housed in the pcDNA3.1 shuttle vector was overexpressed in HEK293T cells (cultivated in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin) using standard transient transfection protocols with polyethylenimine transfecting agent, as described previouslyE. coli BL21 (DE3) and purified by Ni–NTA metal affinity chromatography as described previously25. Further purification by ion exchange chromatography26 was found to be necessary to remove protease contamination remaining in the MSP solution. A prepacked Q-Sepharose  column was equilibrated in 40 mM Tris–HCl pH 7.4, 100 mM NaCl buffer. Ni-purified MSP was loaded onto the column and proteins were eluted with an increasing concentration of NaCl. MSP eluted at ~ 200 mM NaCl. The purified protein was buffer exchanged into 40 mM Tris–HCl pH 7.4, 100 mM NaCl, concentrated, and stored at − 80 °C.MSP1E3D1 (MSP) containing an N-terminal His tag was expressed in The nanodisc lipid composition for viperin incorporation was optimized at 80% POPC and 20% POPE. POPC and POPE were dissolved in chloroform and mixed in the desired ratio and dried under nitrogen. The dried lipids were stored in a desiccator under vacuum overnight. For nanodisc assembly, a stock solution (25 mM) was prepared by dissolving the dried lipid mixture in buffer A  with 100 mM Na-cholate by sonication in a water bath for 30 min until the solution turned clear. The lipid solution was mixed with MSP1E3D1 in 20 mM Na-cholate and HEPES buffer in a final molar ratio of 130:1 (phospholipid:MSP).All steps were performed inside a Coy anaerobic chamber unless mentioned otherwise. All buffers and stock solutions were thoroughly degassed before introducing them into the anaerobic chamber. Incubations at 4 °C were performed outside the chamber; the tubes were tightly wrapped with parafilm to exclude air before being removed from the chamber.7 cells. The cell lysate was cleared by centrifugation at 12,500 rpm for 20 min at 4 °C. A portion of lysate was saved for assay of viperin activity. The total protein concentration in the cell lysate was then measured using the BCA assay. In an Eppendorf tube, the cell lysate was then incubated with the phospholipid/MSP solution at 4 °C for 2 h with end-to-end mixing. The amount of phospholipid/MSP was adjusted so that the concentration of MSP was approximately double that of the proteins in the cell lysate. In a typical purification the cell lysate protein concentration was ~ 16 µM and the concentrations of phospholipid and MSP were 4.2 mM and 32 µM respectively in a total volume of 2.5 mL. To remove the detergent and facilitate nanodisc assembly, Amberlite XAD-2 beads were then added in equal volume (2.5 mL) to the lysate, MSP, and lipid mixture and incubated at 4 °C overnight.HEK293T cells transfected with viperin were harvested from four 150 mm culture dishes and resuspended in 500 µL  of 1X TBS  with 5 mM DTT, 0.1% (w/v) Tween-20 and protease inhibitor cocktail and sonicated for 10 pulses. Each plate yielded ~ 2 × 10The nanodisc-lysate mixture was filtered through 0.22 µm syringe filter to remove the Amberlite beads. Ni–NTA beads (~ 1 mL) were equilibrated with buffer B  and incubated with nanodisc lysate mixture at 4 °C for 1 h. The resin was then washed with 4 column volumes of buffer B twice before eluting protein with 2 column volumes of buffer C . The eluted protein was concentrated by ultrafiltration against a 10 kDa cutoff membrane. A portion of concentrated Nickel-NTA-purified nanodisc sample was saved for assay of viperin activity.The concentrated, nickel-affinity purified nanodisc sample (~ 900 µL) was incubated with anti-FLAG magnetic beads (~ 40 µL) equilibrated with buffer A  at 4 °C for 16 h. The resin was washed with 4 column volumes of buffer A three times. The viperin incorporated nanodiscs were finally eluted by incubating anti-FLAG magnetic beads with 3X FLAG peptide solution (2.5 µg/µL) prepared in buffer A at 4 °C for 2 h with end-to-end mixing. The purity of the protein was analyzed by SDS-PAGE (4–20% gradient gel), with proteins visualized by silver staining. To confirm the presence of viperin, the duplicate SDS-PAGE gels were immunoblotted against anti-viperin antibody using standard protocols.3 (100 µM) and Na2S (100 µM) were then added slowly in small additions  to fully reconstitute the [4Fe-4S] cluster of viperin, as described previously24.The NTA- and FLAG-purified viperin nanodiscs samples were incubated with 5 mM DTT at 4 °C for 20 min. Stock solutions of FeCl6 for the N-terminal truncated forms of the enzyme that have been expressed and purified from E. coli.Viperin activity was assayed under anaerobic conditions in a Coy anaerobic chamber as follows: Samples of HEK293T cell lysate, nickel-affinity purified nanodiscs, or FLAG-affinity purified viperin nanodiscs (100 µL each) were initially incubated with 5 mM dithionite and 0.3 mM CTP  at 25 °C for 20 min. The reaction was initiated by the addition of 0.3 mM SAM  to the sample and incubated at 25 °C for 1 h with end-to-end mixing. The concentrations of CTP and SAM were assumed to be saturating based on the previously reported kinetic parameters12.The reaction mixtures were then removed from the Coy chamber and quenched by heating at 95 °C for 10 min followed by incubation on ice for 5 min. The precipitated proteins were removed by centrifugation at 14,000 rpm for 20 min. 5′-dA was extracted from the supernatant (~ 90 µL) with a fivefold excess of acetonitrile (450 µL). Samples were analyzed in triplicate by UPLC-tandem mass spectrometry as described previously15. The activity of the various samples was normalized with respect to the amount of viperin in the sample.The amount of viperin in the various samples was determined by immunoblotting with reference to a standard curve constructed from purified, truncated enzyme as described previouslySilver stained polyacrylamide gels and immuno-stained western blots were imaged using a BioRad ChemiDoc™ Touch system. Imaging of immuno-stained blots was performed using the instrument’s standard chemiluminescent application with autoexposure; silver stained gels were imaged using white light transillumination with autoexposure.Protein samples were negatively stained using uranyl formate on FCF100-Cu grids (Electron Microscopy Sciences) for imaging. Grids were imaged on a Morgagni electron microscope with a high tension of 100 kV, at 22,000 × magnification, and a pixel size of 2.1 Å/pixel at this magnification.E. coli. This is because, lacking the requisite membrane structure to bind and chaperones that may be needed for them to fold, they are prone to misfolding and are rapidly degraded by the host cell. Thus, despite multiple attempts using various E. coli strains developed to facilitate the expression of “difficult to express” proteins we were unable to detect expression of full-length viperin. In contrast, we have found that the N-terminal truncated form of the enzyme expresses well in E. coli19. However, previous studies in our laboratory had established that viperin is expressed well in HEK293T cells transfected with the pcDNA3.1 (+) expression vector housing a gene for the full-length enzyme24. Therefore, we sought to develop a method to purify the full-length enzyme from transfected HEK293T cells.Mammalian membrane-associated proteins such as viperin have proven to be especially challenging to recombinantly express and purify from bacteria such as 20, we employed lipid nanodiscs to provide a native-like environment that would substitute for the ER-membrane21. The purification strategy is illustrated in Fig. Initial attempts to purify viperin directly from HEK293T cells utilizing a 3X FLAG-tag introduced to at the N-terminus of viperin (either with or without non-ionic detergents present) were unsuccessful and resulted in very low yields of inactive enzyme. Therefore, to facilitate purification of viperin, which localizes to the ER-membrane through its N-terminal amphipathic helix6 cells at confluency. Cells were either used immediately after harvesting or flash frozen and stored at – 80 °C. Because viperin, like all radical SAM enzymes contains an oxygen-sensitive [4Fe-4S] cluster27, all lysis and purification steps were performed in an anaerobic glove box.To obtain sufficient material to purify viperin, 3 or 4 150-mm culture dishes were used to culture viperin-transfected HEK293T cells, with each dish yielding approximately 20 × 1025 which is appropriate to incorporate one molecule of viperin per nanodisc. To maximize the incorporation of viperin, the final concentration of MSP1E3D1 was adjusted to be approximately double the nominal concentration of cellular proteins in the lysate, assuming an average molecular weight of 350 kDa for a hypothetical human cellular protein. Based on this number, the molar ratio of lipids (POPC + POPE) to MSP1E3D1 was then adjusted to be 135:1. In a typical experiment, the final concentrations of each of the components was 16.2 µM nominal concentration of cell lysate proteins , 32.4 µM MSP1E3D1, 4.2 mM POPC + POPE, and 20 mM Na cholate. The mixture was incubated in buffer A  at 4 °C for 2 h with gentle mixing, after which Amberlite XAD-2 beads were added to remove the detergent and incubation was continued at 4 °C overnight. Removing the detergent resulted in spontaneous assembly of the nanodiscs.After lysis by sonication and centrifugation to remove cellular debris, the clarified lysate was incubated with the scaffold protein, MSP1E3D1, a mixture of 80% POPC and 20% POPE lipids (preliminary experiments established that this ratio appeared to give the highest incorporation of viperin into nanodiscs) and sodium cholate to facilitate extraction of membrane proteins into nanodiscs. MSP1E3D1 is designed to facilitate the formation of nanodiscs of ~ 12.9 nm diameterAfter incorporating proteins into the nanodiscs, the nanodiscs were purified from the cell lysate by Ni–NTA affinity chromatography using the His-tag appended to MSP1E3D1. This step yielded nanodiscs containing viperin and other membrane-bound proteins along with free MSP1E3D1 Fig. A. To purThe purity of the viperin nanodiscs was examined using SDS-PAGE Fig. A. The si15, by using a standard curve constructed from known amounts of N-terminal-truncated viperin, which was expressed and purified from E. coli. After reconstituting the [4Fe-4S] cluster, the activity of viperin was assayed by measuring the formation of 5’-dA when the dithionite-reduced enzyme was incubated with SAM and CTP, as described previously19. These data are summarized in Table We next examined how the specific activity of viperin varied at each step of the purification. The amount of viperin present at each step was determined by immunoblotting, as described previouslykobs = 3.8 ± 0.6 h−1 which is comparable to previously reported values15. The specific activity of viperin was essentially unchanged after extraction into nanodiscs and purification by NTA-affinity chromatography. However, somewhat surprisingly, the final purification step significantly reduced the specific activity of viperin by ~ 8-fold to kobs = 0.5 ± 0.09 h−1. This reduction in activity was observed over several independent purifications.In the crude HEK293T cell lysate, the specific activity of viperin, calculated as an apparent turnover number, was 15. Therefore it seems reasonable that interactions with other endogenous proteins or other components of the lipid membrane may help stabilize the enzyme.It is unclear why the activity of the purified enzyme is reduced so dramatically. Incorporating the enzyme into nanodiscs per se does not seem to affect the activity. One possibility is that nonspecific interactions between viperin and the anti-FLAG beads used in the purification result in partial misfolding of the protein. This explanation may be in accord with the poor recovery of enzyme eluted from the anti-FLAG beads, discussed below, which is a problem often encountered using this technique. A more interesting possibility, however, is that other protein(s) or small molecules present in the cell lysate are involved in stabilizing or activating viperin. Such a protein or small molecule would presumably be associated with the ER membrane or liposomes because it would have to co-purify with the nanodiscs isolated by NTA-affinity chromatography. In support of this idea, we note that we have observed in other studies that the activity of viperin can be altered quite significantly by its interactions with other proteins. For example, co-expression of viperin with IRAK1 and TRAF6 in HEK293T cells results in these enzymes forming a complex, with the activity of viperin increasing ~ tenfold as a result28.The yield of viperin after the final purification step was calculated to be ~ 15% . Notably, a large loss of protein accompanied the final step in the purification. This is due in part to difficulties encountered in eluting the enzyme from the anti-FLAG beads using the 3x-FLAG peptide. Even after prolonged incubation only about 50% of the protein bound to the beads could be eluted. After taking these losses into account, the amount of purified full-length viperin obtained is comparable to other membrane proteins purified with the aid of nanodiscs: for example, ~ 0.25 µg/100 mm culture dish of the family B GPCR (GLP1R) receptor protein was obtained when purified from a mammalian cell line using a similar approachHere we describe, for the first time, the purification of full-length human viperin, rather than its truncated form, from a transfected mammalian cell line. Purification has been facilitated by the use of nanodiscs that allow the interaction between the amphipathic N-terminal extension of viperin and the lipid bilayer to be preserved. Surprisingly, the purified enzyme has rather low activity. It is currently unclear whether the loss of activity results from the purification conditions employed, which might result in some misfolded protein, or whether it reflects the removal of an unknown activating factor that is presumably retained in the nanodiscs. We consider the latter explanation more likely, given that viperin is known to interact with multiple cellular proteins that alter its specific activity. If correct, our observations imply that the activating factor(s) is incorporated into a different nanodisc from viperin and that the interactions between them are sufficiently weak that the factor does not co-purify with viperin. However, further experiments are needed to better understand what additional factors contribute to viperin’s enzymatic activity.Although the amount of protein that can be purified from HEK293T cells by this method is relatively small, the amounts are nevertheless sufficient to undertake further studies on the factors affecting viperin’s catalytic activity and to undertake structural studies using single-molecule techniques such as cryo-EM. Such studies could reveal the details of the interaction between viperin and the membrane that appear to be important for its biological function. Furthermore, the ability to purify viperin bound to nanodiscs should facilitate structural characterization of the various complexes that viperin forms with other proteins that make important contributions to its multifaceted antiviral activity.Supplementary Information.


COMT) and dopamine receptor D2 (DRD2) gene polymorphisms, and if genetic predisposition affects response and safety of MPH treatment. The polymorphisms of COMT (rs4680) and DRD2  were analyzed in DNA samples. A borderline significance was found for the correlation between MPH side effects and the G allele of COMT (rs4680) (p = 0.04994) in all ADHD children. No effect of COMT (rs4680) and DRD2  polymorphisms and the treatment efficacy was observed. The analyzed DRD2 and COMT gene polymorphisms seem to play no role in MPH efficacy in ADHD children with hPAE, while low-activity COMT (Met158) variant carriers may be more intolerant to MPH. The MPH treatment is effective in ADHD independent of FASD, although the ADHD-FASD variant requires higher doses to be successful. These results may help in optimization and individualization in child psychiatry.Fetal alcohol spectrum disorders (FASD) in a course of high prenatal alcohol exposure (hPAE) are among the most common causes of developmental disorders. The main reason for pharmacological treatment of FASD children is attention deficit hyperactivity disorder (ADHD), and methylphenidate (MPH) is the drug of choice. The aim of the study was to assess whether children born of hPAE with ADHD, with or without morphological FASD, differ in terms of catechol-O-methyltransferase ( Alcohol consumption during pregnancy may lead to permanent damage in the offspring, resulting in, for example, Fetal Alcohol Spectrum Disorders (FASD). FASD include Fetal Alcohol Syndrome (FAS), partial Fetal Alcohol Syndrome (pFAS), alcohol-related neurodevelopmental disorders (ARND), and alcohol-related birth defects (ARBD) . FAS is According to worldwide studies, alcohol consumption among women of childbearing age is increasing. At the same time, a large proportion of pregnancies are unplanned, which leads to more frequent cases of prenatal alcohol exposure (PAE) which may be explained by harmful and sustained alcohol intake during any trimester of pregnancy which, in consequence, may produce morphological, biochemical, and epigenetic (among others) consequences in the fetus. Alcohol has a prolonged effect in pregnancy in terms of abundance and efficiency caused by the differences in maternal and fetal enzymes, oxidative stress/reactive oxidative species (ROS) and, additionally, reduced elimination. In 120 min after the mother’s alcohol intoxication, its concentration in her blood increases to maximum . Owing to placenta transport of alcohol, its concentration after 120 min gets maximal BAC which is nearly the same as maternal. During pregnancy there is no safe dose of alcohol and most of PAE state in the first trimester of pregnancy . The strThe multiple clinical consequences of the prefrontal cortex area damage in FASD children involve cognitive domains , motor functions and activity levels, language skills and adaptive functioning ,12,13,14In addition to psychological and behavioral therapies, NICE guidelines  allow a pharmacological treatment of individuals older than 5 years with a severe presentation of ADHD. Stimulants, such as methylphenidate (MPH), are considered to be the most effective medications for the improvement in ADHD symptoms and MPH is often the first-choice substance owing to its effectiveness and safety, as demonstrated in several studies ,27.DRD2), e.g., Taq1A (rs1800497), C957T (rs6277), and −141Cins/del (rs1799732), in neuropsychiatric disorders such as schizophrenia, alcoholism, Parkinson’s disease, substance addiction, and ADHD . The transition resulting in the substitution of methionine for valine has been linked to low COMT enzyme activity and is designated as the L (low-activity) allele, in contrast to the H (high-activity) allele   . The stuer (ASD) . The lower (ASD) . An impoSo far, several studies have tried to understand the genetic variants underlying interindividual variability in pharmacological parameters, as well as adverse effects and efficacy, however, with inconsistent results. Possible reasons may be small group sizes, different methodologies, or selection of other polymorphisms. Importantly, only a few publications have directly addressed the association of genetic variation with efficacy and safety of MPH therapy for ADHD in children with FASD. Animals with PAE have an exaggerated response to psychostimulants; similarly, children with FASD who have structural or neurochemical changes in the central nervous system are often hypersensitive to side effects of the treatment, which may be further enhanced by a genetic factor .COMT allele implies that in the case of an early onset of MPH adverse effects without achieving a therapeutic effect, especially in children without features of FASD, an attempt should be made to reduce the dose before discontinuation [The presented data on the impact of the A (Met158) inuation .DRD2 and COMT gene polymorphisms do not play a role in methylphenidate efficacy in children with high PAE and ADHD. Low-activity variants of COMT (Met158) carriers may be more intolerant to MPH. This result may be of potential importance in the treatment individualization of ADHD children with FASD syndrome.Our findings suggest that the analyzed DRD2 and COMT gene polymorphisms seem to play no role in MPH efficacy in ADHD children with hPAE, while low-activity COMT (Met158) variant carriers may be more intolerant to MPH. The MPH treatment is effective in ADHD independent of FASD, although the ADHD-FASD variant requires higher doses to be successful. These results may help in optimization and individualization in child psychiatry.As was mentioned above, the multiple structural and neurobehavioral consequences in a course of teratogenic hPAE effects are well documented. Some of the brain lesions are clinically common to FASD and ADHD children, especially in an area of cognitive domains, motor hyperactivity, immoderate impulsivity, and social functioning in general. All these symptoms are also typical of the so-called morphological organic changes which are clinically expressed in children by means of emotional and behavioral disturbances coexisting with ADHD symptoms. It is rather difficult to differentiate if they are of an organic or functional background and at the moment of choosing the most proper medicament. The experiment conducted by the authors could have shown rather positive observations that only a portion (about one third in our study) of children who had been exposed early to alcohol poison effects (hPAE) in their gravy manifested ADHD symptoms. Moreover, most of these children were methylphenidate treatment successful (over 90%), especially those with FASD and hyperactivity/impulsivity  compared to those with non-FASD who showed a better response in the cognition/impulsivity skills. We might achieve a rather optimistic prognosis for children with a diagnosis of ADHD and probable hPAE interview ; unless there are many alarming data in the beginning , the prognosis of their proper development seems to be good, especially when the symptoms tend to be reduced during methylphenidate treatment. Organic factors seem not to be so severe and functional disturbances dominate, which are sensitive to MTH therapy. It is worth pointing out that such children with probable hPAE factors are in greater demand for MTH doses . Such good and successful MPH therapy for ADHD–FASD coexisting syndrome might give a better prognosis of its course because of dominant dopaminergic immaturity than endogenous (genetic or morphologic/organic) changes. Further observations in a bigger group of children (divided into sex subgroups) with hPAE are going to be continued. The analyzed ADHD features should be further investigated.The epigenetic role of alcohol on the clinical intercourse and possible therapeutic CNS response to catecholamine-involved drugs in children with FASD and The limitations of the present study mostly concern the small sample size, a potential sex bias FASD diagnosis, and exclusivity on morphology, because of other scanty precise methodological tools . The authors are worried that using valid tests such as kiddie–SADS and CGI  is surely more appropriate and precise to compare gathered results with other published data. It is worth mentioning that the time for therapeutic effect observation was too short and often ceased because of added agents, for example the necessity to change the model of conducted therapy . Such remarks required verification of the pharmacological form of therapy, and methylphenidate therapy was given up or completed by means of another drug . These activities were outside the range of the subject of this paper.


Attention-deficit/hyperactivity (ADHD) and autism spectrum (ASD) disorders are commonly co-occurring conditions characterized by neurocognitive impairments. Few studies have directly compared neurocognitive profiles in ADHD and ASD and fewer still have controlled for comorbidity of ADHD and ASD. All direct comparisons have been in clinic samples, leaving the question of generalizability of results unaddressed. We compared neurocognitive performance in clinically ascertained ASD (n = 261) and ADHD (n = 423) cases and controls (n = 162), 6.0–17.9 years of age. We also compared ASD (n = 190) and ADHD (n = 926) cases ascertained in the community with controls  of similar age. Using the stop-signal task (SST), we measured response inhibition , sustained attention (defined as reaction time variability–RTV), and reaction time (RT). We controlled for comorbidity using ADHD and ASD trait scores and categorically-defined ADHD. Compared with controls, both clinic ADHD and ASD had significantly longer SSRT and RTV than controls and did not differ from each other. ADHD traits accounted for neurocognitive impairment in ASD, but not vice versa. There were no group differences for RT. Similar patterns of neurocognitive impairment were observed in the community sample. In the largest direct comparison of ADHD and ASD to date, we found impaired response inhibition and sustained attention in both disorders. However, neurocognitive impairment in ASD was almost completely accounted for by comorbid ADHD. Results generalized in the community sample indicating that referral bias alone did not drive results. Response inhibition and sustained attention likely play a role in ADHD and ASD.The online version contains supplementary material available at 10.1007/s10802-022-00958-6. Attention-deficit/hyperactivity disorder (ADHD) and autism spectrum disorder (ASD) are impairing, persistent and heritable neurodevelopmental disorders APA,  that shaMore compelling evidence for the uniqueness of the neurocognitive profiles of ASD and ADHD would come from direct comparisons that would allow control over variations in task conditions, performance metrics, testing environments  and demographic variables including age, sex, and medication status during testing  identified 60 direct comparisons of neurocognition  in ADHD and ASD. There was considerable variation in the measures used, the indices of performance derived from these measures and the neurocognitive domains covered in these studies. Only five studies reported performance on the stop-signal task (SST), a widely used measure of response inhibition  were recruited from five different hospital- or university-based child and adolescent clinics. For ASD participants, recruitment into the study followed assessment and for ADHD participants, recruitment was concurrent with the initial assessment. Most participants received treatment in the community prior to and following research participation. Treatment involved a mix of medication, psychosocial intervention and school consultation. Each participant was assessed by a psychiatrist and/or a psychologist to confirm their primary diagnosis using the Autism Diagnostic Observation Schedule–2 (ADOS), Autism Diagnostic Interview–Revised (ADI-R) for ASD  to a public science museum  scale. For this reason, all effects are represented as % difference. Because the effect is expressed as % difference, the corresponding effect in ms will be greater where predictors are associated with larger predicted values  and smaller where predictors are associated with smaller predicted values. For example, a 10.2% greater SSRT in Community ADHD compared to Controls will correspond to a predicted difference of 36 ms at age 6 when the predicted SSRT in Controls is 355 ms but only 21 ms at age 16 when the predicted SSRT in Controls is 207 ms. Raw SSRT and RTV scores values for each group at ages 6, 12 and 16 and effect sizes estimates using the pooled error estimate from the linear regression model are presented in We compared each group  to Controls and to each other. We predicted a main effect of group on SSRT and RTV and tested for differences in group trajectories across age, for a total of 8 primary hypotheses: 2 SST outcomes × 2 settings × 2 effects (age x group interaction and group main effect). We use a conservative adjustment for multiple comparisons and α of 0.05/8 = 0.0063. Although this is a very conservative adjustment, results were unchanged even when the more stringent criterion was applied for significance. Where there were no group differences in trajectories across age, the group x age interaction was dropped for a more parsimonious comparison of the main effect of group across all ages.We did not include differences in RT among our primary hypotheses based on previous findings  using a score of 2 or 3 on the SWAN as a symptom  = 13.56, p < 0.0001). SSRT was 18.8% longer in the Clinic ADHD group than in the Clinic Control group and 23.1% longer in the Clinic ASD group than in the Clinic Control group . Controlling for SWAN t-scores reduced the Clinic ASD effect by 52% (Clinic ASD effect p = 0.048). Given that ADHD is essentially “defined” by the presence of ADHD symptoms it was not surprising that controlling for SWAN t-scores reduced the Clinic ADHD group effect by 72% confirming that the effect of ADHD on SSRT was largely attributable to ADHD trait severity. SCQ symptom count was not significant when added to the models indicating that ASD traits did not account for poorer response inhibition in the Clinic ADHD group (data not shown).F = 2.89, p = 0.09). A 10-point difference in IQ was associated with a 2.0% lower SSRT in the clinic sample in a model that does not control for SWAN . Controlling for IQ did not have any impact on the ASD and ADHD differences in SSRT from controls.There was a clear age effect but no significant group difference by age effect  on SSRT Table  = 14.12, p < 0.0001). As shown in Table There was a significant main effect of group on SSRT in the community sample  = 8.77, p = 0.0002) with no group differences in the age effect  indicating that ASD traits did not impact the performance of the ADHD group. The Clinic ASD + ADHD group had greater RTV than controls (14.9% greater), but the difference between Clinic ASD-ADHD and Control groups (7.7%) was not significant , but did not change the results of the group comparisons. A 10-point difference in IQ was associated with 2.0% lower RTV in the clinic sample . Controlling for IQ reduced but did not eliminate the ASD and ADHD differences from controls.There was a significant main effect of group on RTV in the Clinic sample  = 18.24, p < 0.0001). RTV was 7.2% greater in the Community ADHD group and 11.4% greater in the Community ASD group than in the Community Controls  in the Community but there was no gender effect in the Clinic sample. There were no significant gender x diagnosis interactions in any of the models , p = 0.007] in the Clinic sample but was not significant in the Community sample. Figure Females had 4.4% longer SSRT than males [2.7,6.1), e models . There we models  which me, e modelWe compared ADHD and ASD cases with controls on two indices of neurocognition (SSRT and RTV) with and without control for comorbid ADHD and ASD to determine whether ADHD and ASD share neurocognitive profiles. We conducted these comparisons in specialty clinics where ADHD and ASD diagnosis was established after rigorous assessment and in a community sample where ADHD and ASD was defined by parent and self-report in order to assess the generalizability of findings. This study was motivated by the fact that there have been few direct comparisons of neurocognitive function, in particular response inhibition, in large samples of individuals with ADHD and ASD  response inhibition (SSRT) and greater reaction time variability (RTV) than age-matched controls, but did not differ in response time (RT). The differences in SSRT and RTV between ASD and ADHD were not significant. Because it can be difficult to estimate the clinical significance of differences in reaction time measures, we estimated the magnitude of the impairments using “age equivalents''–the age at which statistical models predicted that a control would perform at the same level as a case. The difference between ADHD, ASD and controls was substantial. Clinic ADHD and Clinic ASD showed a 2-year delay in SSRT. For RTV, the delay was 1–2 years. The delay for Community cases was somewhat less for SSRT but similar for RTV. The functional implications of this effect needs further study as measures of impairment were not collected, but we note that a 2-year delay is the typical criterion for learning disability. We caution that while the observed impairments were statistically significant and may be clinically or aetiologically relevant, the associations were not sufficiently strong or sufficiently specific to use as a proxy for diagnosis  that we did not measure. The greater number of invalid SST administrations in the ADHD and ASD sample means those with the worst performers were excluded at a greater rate in those groups than in the control group, resulting in underestimation of the true magnitude of the difference between the groups.These results support the conclusion that ADHD and ASD share a neurocognitive profile characterized by deficient response inhibition and sustained attention reflected in greater reaction time variability but not RT. ADHD traits and/or comorbidity accounted for the observed impairment in ASD.Supplementary file1 (DOCX 120 KB)Below is the link to the electronic supplementary material.


Effective diagnostic and treatment pathways for ADHD are needed in prison settings due to the high prevalence of ADHD and comorbidities in the prison population.In this presentation, we will describe two studies conducted in seperate London prisons in England. In the first study, the aim was to identify prisoners with ADHD with a focus on describing comorbidity. In the second study, using QI  methodology, the aim was to measure the practicability and effectiveness of a specialist ADHD diagnostic and treatment pathway for prisoners.Two studies were carried out in two separate prisons in London. Firstly, data were collected to understand the prevalence of ADHD and the comorbidities. The second study used quality improvement (QI) methodology to assess the impact of a diagnostic and treatment pathway for prisoners with ADHD.Of the prisoners, 22.5% met the diagnostic criteria for ADHD. Nearly half of them were screened positive for autistic traits, with a higher prevalence of mental disorders among prisoners with ADHD compared to those without. The QI project led to a significant increase in the number of prisoners identified as requiring ADHD assessment but a modest increase in the number of prisoners diagnosed or treated for ADHD.Despite various challenges, an ADHD diagnostic and treatment pathway was set up in a prison using adapted QI methodology. Further research is needed to explore the feasibility of routine screening for ADHD in prison and examine at a national level the effectiveness of current ADHD prison pathways.No significant relationships.


Existing research demonstrates that children with attention-deficit/hyperactivity disorder (ADHD) underperform in cognitive tasks involving working memory (WM) due to hypo-arousal, which has led to the development of arousal regulation models to determine proper levels of arousal and optimal cognitive outcomes. The present study focuses on investigating the effects of external auditory stimuli on verbal WM in children with ADHD. Thirteen children with ADHD (aged 6–10 years old) and thirteen age- and gender-matched children with typical development (TD) completed the verbal WM task when listening to no sound, white noise, or pleasant music. A two-way repeated-measures analysis of variance was used to compare the verbal WM performance between groups in the three auditory conditions. Children with ADHD showed the best verbal WM performance when listening to white noise and the worst performance when listening to no sound. Yet, children with TD performed the best in the no-sound condition and the worst in the white noise condition. Our findings suggest auditory white noise is beneficial for ideal arousal regulation and cognitive performance involving verbal WM for children with ADHD and support the moderate brain arousal model. Providing external white noise is a non-invasive and cost-effective approach to improving verbal WM in children with ADHD in real-world contexts. Effective strategies to improve the working memory (WM) performance of children with attention-deficit/hyperactivity disorder (ADHD) are urgently needed. Characterized by a high level of inattention and hyperactivity-impulsivity , childreOne viable solution is to improve the problem of hypo-arousal, a less-than-normal level of physiological alertness  that is Researchers have attempted to validate the three theories, and the results indicate that particular extra stimuli may facilitate cognitive outcomes. Following the optimal stimulation theory, children with ADHD perform better on arithmetic tasks when listening to music than when listening to conversations irrelevant to the cognitive task but that are still meaningful, or when listening to no sound. However, children with typical development (TD) perform equally well under all the three auditory conditions . VerifyiSince providing proper auditory stimuli is a feasible and cost-effective approach to improving cognitive outcomes for children with ADHD in real-world contexts, in the current study, we sought to identify what external stimulation is the most ideal and appropriate to improve cognitive performance. We compared the performance of verbal WM in the presence of three types of exogenous auditory stimuli  between children with ADHD and TD to answer the following questions:Are there differences in the performance of verbal WM between children with ADHD and TD under different auditory conditions?Do exogenous auditory stimuli improve the performance of verbal WM for children with ADHD?Which external auditory stimulation is the best one for children with ADHD to enhance verbal WM?We hypothesized that children with ADHD and TD would demonstrate differences in task performance involving verbal WM in the three auditory conditions; that both white noise and pleasant music would ameliorate the performance of verbal WM for children with ADHD; and finally, that white noise would be the best auditory external input for children with ADHD for an improved verbal WM.F = 0.28, p = 0.76; η2 = 0.01).A 2 × 3 mixed design was used in this study. The between-subjects factor was ADHD diagnosis (ADHD vs. TD), and the within-subjects factor was the auditory condition . We adopted an interval of one week between each auditory condition to avoid carry-over effects. A counterbalancing procedure was applied to eliminate the order effects for repeated measures in the three conditions; the order effect was examined, and no order effect was found in this study  and symptoms of ADHD were assessed to ensure their eligibility for this study.We recruited 13 boys (6 to 10 years old) diagnosed with ADHD by psychiatrists (mean age = 8.25 years) and 13 age- and gender-matched children with TD . One child with ADHD was taking medication but was asked to stop taking it 24 h before conducting the auditory WM tasks. Nine children in the ADHD group were undergoing rehabilitation therapy, and two children with ADHD were enrolled in special education classes at school. The TD group was recruited from after-school programs based on their scores in the Chinese version of the Swanson, Nolan, and Pelham IV (SNAP-IV) Parent Rating Scale (<95th percentile) . All parr = 0.90), test-retest reliability (r = 0.90), and concurrent validity with the Wechsler Intelligence Scale for Children-Third Edition . The Peabody Picture Vocabulary Test-Revised Edition (PPVT-R) is a norm-referenced instrument for measuring the receptive vocabulary of 3- to 12-year-old children with an established norm based on the Taiwanese population . It contr = 0.83), and concurrent validity with Raven’s Progressive Matrices (r = 0.78).The Test of Nonverbal Intelligence, Third Edition (TONI-3) is a norm-referenced, language-free test for measuring the aptitude, intelligence, abstract reasoning, and problem-solving abilities of 4- to 16.5-year-old children . It consr = 0.73), and concurrent validity with Conners’ Parent Rating Scales and Children Behavioral Checklist .The SNAP-IV Parent Rating Scale is a screening tool for ADHD symptoms. The norms of the parent’s scale for 6- to 13-year-old children (first to eighth grade) in Taiwan have been developed, and the 95th percentile has been recommended as the cutoff score for ADHD symptoms . The scar = 0.96), test-retest reliability (r = 0.94), and concurrent validity with the WISC-III (r = 0.58). However, according to the definition of WM [The Digit Span Test was pulled from the Chinese version of the Wechsler Intelligence Scale for Children, Fourth Edition to assess participants’ verbal WM. It is a standardized, norm-referenced assessment of the verbal memory of 6- to 16-year-old children with an established norm based on the Taiwanese population . The exaon of WM ,8,9, onlGiven the verbal tasks and different auditory conditions used in this study, we conducted the Weber test with a 512-Hz tuning fork to evaluate the hearing loss of all participants before tasks, and no participants showed hearing impairments. All participants completed the auditory WM tasks in three auditory conditions: no sound, white noise, and music (the no-sound condition was used as the control/baseline condition). The stimuli were broadcasted from two 60-Hz speakers placed 60 cm in front of the participants, whereas the auditory WM tasks were presented at 86 dB in all three auditory conditions and lasted about 15–20 min for each auditory condition.In each condition, after a 5 min exposure to no sound, white noise, or music, the participants were asked to complete the auditory WM tasks. In the no-sound condition, there was no exogenous auditory stimulus; the average sound level was 61 dB. In the white noise condition, the white noise was presented at 78 dB. In the music condition, each participant selected one of the 10 pieces of music that they liked the most , and the chosen music was played repeatedly. The average sound level ranged from 76 to 79 dB. After a 5 min exposure to the chosen music, the participants also rated how much they liked the music selection on a 10-point Likert-type scale . The 10 pieces of the music library comprised music nominated by twenty 6- to 10-year-old children who did not participate in this study as their favorite music.t-tests, χ2 tests, and descriptive analyses to describe and compare the characteristics of participants with ADHD and TD. Second, we conducted a 3 × 2 two-way repeated-measures analysis of variance (ANOVA) with one within-subjects factor  and one between-subjects factor (group: ADHD or TD) to compare the verbal WM performance of the two groups in the three auditory conditions. Given the statistically significant interaction between the condition and group (see below), we estimated the simple effects of condition and group in addition to the post hoc tests. The significance level was set at p < 0.05 (two-tailed), and the corresponding effect sizes were calculated. We analyzed the data with IBM SPSS Statistics for Windows, Version 26.0 .First, we used independent-samples t(24) = 0.67–1.81; p = 0.08–0.53; d = 0.27–0.71). Given the relationship between IQ and cognitive performance, we included verbal IQ and nonverbal IQ as covariates in the following analyses of verbal WM. As expected, the score of inattention (t(24) = 4.90, p < 0.001; d = 1.92) and impulsivity (t(24) = 4.12, p < 0.001; d = 1.62) of SNAP-IV in the ADHD group was significantly higher than those in the TD group. There were no significant differences between groups in having experiences of learning musical instruments or habitually listening to music when doing homework  = 4.19; p = 0.02; η2 = 0.16; F = 0.54; p = 0.58; η2 = 0.02) and group  = 1.02; p = 0.32; η2 = 0.04; see In the repeated-measures ANOVA model, we observed a statistically significant interaction effect between group and auditory condition  = 4.01; p = 0.06), but there were no differences between two groups when listening to white noise  = 0.58; p = 0.45) or music  = 1.58; p = 0.22).Simple effect analysis showed that, probably due to the small sample size, the ADHD group performed marginally worse in verbal WM than the TD group when listening to no sound , the positive affect did not have beneficial impacts on the verbal WM of either group. Our findings were not aligned with the dopaminergic theory of positive affect  and did There are some limitations to this study. As a pilot study with 26 participants, the preliminary data ought to be replicated with a larger sample size to avoid the disadvantage of low statistical power. Second, although there were neither order nor carry-over effects, we used the same verbal WM task in only one form, without parallel forms, three times for each participant. Future researchers should adopt parallel forms of the digit span task to tackle learning effect. Third, each participant possessed different levels of arousal and levels of tonic DA. However, in order to standardize the auditory conditions , individualized levels of auditory input for each participant could not be provided in this study. To optimize children’s cognitive performance, the individualized level of white noise should be evaluated for each child when applying white noise in practical and clinical settings in the future. Finally, as our findings support the moderate brain arousal model, researchers in the future should investigate the cross-modal effect of auditory white noise on different sensory modalities and cognitive functions .Our findings support the moderate brain arousal model that suggest that the presence of auditory white noise may enhance the moderate arousal regulation and cognitive processing of children with ADHD. When exogenous auditory white noise is provided via speakers or headphones, this non-invasive and cost-effective approach can improve the verbal WM performance of children with ADHD in daily contexts.


These participants were enrolled at birth and followed-up prospectively  at the Boston Medical Center. Higher levels of cord 5-MTX was associated with a lower risk of ASD  and ADHD  per Z-score increase, after adjusting for potential confounders. Similarly, children with cord 5-MTX ≥ 25th percentile (vs. <25th percentile) had a reduction in ASD  and ADHD risks . In contrast, higher levels of cord tryptophan, 5-HTP and N-acetyltryptophan were associated with higher risk of ADHD, with aOR: 1.25, 95% CI: 1.03, 1.51; aOR: 1.32, 95% CI: 1.08, 1.61; and aOR: 1.27, 95% CI: 1.05, 1.53, respectively, but not with ASD and other DD. Cord serotonin was not associated with ASD, ADHD, and other DD. Most findings remained statistically significant in the sensitivity and subgroup analyses.Alterations in tryptophan and serotonin have been implicated in various mental disorders; but studies are limited on child neurodevelopmental disabilities such as autism spectrum disorder (ASD) and attention-deficit hyperactivity disorder (ADHD). This prospective cohort study examined the associations between levels of tryptophan and select metabolites (5-methoxytryptophol (5-MTX), 5-hydroxytryptophan (5-HTP), serotonin, N-acetyltrytophan) in cord plasma (collected at birth) and physician-diagnosed ASD, ADHD and other developmental disabilities (DD) in childhood. The study sample ( Autism spectrum disorder (ASD) is characterized by social and communication disabilities as well as restrictive and repetitive behaviors and interests  with 1 iTryptophan, an essential amino acid, serves as a precursor to neurotransmitters through multiple metabolic pathways Fig. . Some ofBeyond tryptophan, serotonin, and melatonin, very few studies to date have assessed other less known tryptophan metabolites, especially in the context of neurodevelopmental conditions. One such metabolite is 5-methoxytryptophol (5-MTX)  which isBuilt on the previous observations and to address the major knowledge gaps, we set out to assess the association of tryptophan and select metabolites  measured in the cord blood plasma collected at birth and subsequent risk of physician diagnosis of ASD and ADHD. We will leverage a well-defined prospective birth cohort, which allows us to examine the temporal and dose-response associations of cord tryptophan metabolites and the risk of ASD, ADHD, and other developmental disabilities (DD), considering the extent of comorbidity between these conditions possibly suggesting a common underlying etiology .Detailed information on the participation and enrollment in the Boston Birth Cohort (BBC) has been discussed previously , 31. BriA sub-set of the children originally enrolled continued to receive pediatric care at the Boston Medical Center (BMC), were followed up from birth to 21 years of age and were included in this study. These mother-infant dyads recruited at birth remained in the follow-up study from October 1, 1998 to June 30, 2018. As reported earlier, there were no major differences in the baseline demographic and clinical characteristics of those that continued to be part of postnatal follow-up and those that did not . Of the Written informed consent was obtained from the mothers and depending on the age of the child, verbal or written consent was also obtained from the participating children. The database for research did not contain personal identifier information and is accessible only to authorized investigators. The Institutional Review Boards of Boston University Medical Center and the Johns Hopkins Bloomberg School of Public Health approved the study protocol. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline for cohort studies.Umbilical cord blood samples were collected at the time of delivery and were processed and fractioned into cells and plasma shortly after collection by the field team at BMC. Quantitative profiling of analytes including tryptophan and its select metabolites  from cord was assessed in a random sub-set of children as part of a metabolome panel and was measured using a liquid chromatography tandem mass spectrometry technique at the Broad Institute Metabolite Profiling Laboratory at the Massachusetts Institute of Technology. The distribution of most of the cord analytes was skewed and hence inverse normal transformation was used to render the distribution approximately normal. Details on laboratory methods, quality control, and data processing have been described elsewhere , 34.International Classification of Diseases, Ninth Revision (ICD-9) or International Classification of Diseases, Tenth Revision (ICD-10) codes. We defined four mutually exclusive groups based on the physician diagnoses, as documented in the Electronic Medical Records (EMR): ASD, ADHD only, other DDs only and neurotypical. Children in the ASD group included those ever diagnosed with autism ICD-9 code 299.00, 299.01; ICD-10 code 84.0), Asperger syndrome , and/or pervasive developmental disorder not otherwise specified . When children had concurrent diagnoses, such as ASD and ADHD, or ASD and other DDs, they were still categorized as having ASD. Children in the ADHD only group included those with ADHD-related codes (ICD-9 codes 314.0-314.9 or ICD-10 codes F90.0-F90.9) but excluded those with ASD diagnosis. Children in the other DD only group included those who were diagnosed with mental, behavioral, and neurodevelopmental disorders (ICD-9 codes 290-319 or ICD-10 codes F01-F99) but excluded children with ASD and ADHD diagnosis. Neurotypical children were those that were never diagnosed with ASD, ADHD, and other DDs.For every postnatal clinical visit since 2003, we obtained primary and secondary diagnoses using Based on the existing literature and our previous work , 35, we Maternal and child characteristics of the study children in the four outcome groups  were compared using chi-square tests for categorical variables and analysis of variance test for continuous variables. Cord tryptophan metabolites were normalized using rank-based inverse normal transformation (similar to Z Scores) and were used for all subsequent analyses. The distribution of concentrations of cord tryptophan metabolites was compared across all the four groups. Pre-determined logistic regression models were applied to estimate the crude and adjusted associations between cord tryptophan metabolites and outcomes. After categorizing cord tryptophan metabolites into quartiles, adjusted logistic regression was used to examine the associations with the risk of ASD, ADHD, other DD with the neurotypical considered as the referent group. Our final model adjusted for maternal age at delivery, parity, maternal education, smoking, maternal race/ethnicity, maternal BMI, diabetes status, child sex, preterm birth and year of baby’s birth. We conducted stratified analyses using each stratum of covariates, including child’s sex, maternal race ethnicity and preterm birth to assess possible effect modification. We also conducted supplemental analysis to assess if any of the covariates  affWe performed the following sensitivity analyses: 1) using a stringent criterion of ASD diagnosis on two occasions, one of them being a visit to specialists such as developmental-behavioral pediatrician, pediatric neurologist, or child psychologist; 2) using a stringent criterion for neurotypical by further excluding children with Congenital Anomalies (740–759.9). All results are presented as odds ratio. Two-sided tests were used with a 0.05 significance level. Given the five metabolites that were considered in the analyses, we have also presented an adjusted P-value using Bonferroni correction (cutoff: 0.05/5 = 0.01). Data were analyzed using STATA version 13.0 . Data described in the manuscript, code book, and analytic code is available upon request pending IRB review and approval.P = 0.004), smoked 3 months prior or during pregnancy  and had diabetes mellitus . Children in the ASD group were more likely to be male , born preterm  and born during or after year 2007 . Similarly, mothers of children in the ADHD-only group, were more likely to have smoked 3 months prior or during pregnancy , compared to the mothers of children in the neurotypical group. Children in the ADHD only group were more likely to be male , born preterm  and born prior to year 2007 , compared to their neurotypical counterparts.Of 996 participants, the final sample included 326 neurotypical children, and 87 any ASD, 269 ADHD only and 314 other DD only group children , evident by the 5-MTX distribution shifted slightly left  , 5-HTP (P < 0.001) and N-acetyltryptophan (P < 0.001) were higher in those who were subsequently diagnosed with ADHD, as seen in the distribution of these metabolites shifted slightly right , with the distribution slightly shifted to the left  Fig. . That isght Fig. . On the P < 0.01). When stratified, those with cord 5-MTX above the cutoff (≥25th percentile), compared to those in the lowest quartile (<25th percentile), had a 73% reduction in the odds of ASD . Further stratifying by quartiles showed consistent associations, with cord 5-MTX in higher quartiles having lower odds of subsequent ASD, when compared to the referent group (quartile 1). Cord tryptophan and other metabolites  were not associated with the subsequent risk of ASD.Table Next, we assessed the association between cord tryptophan and its metabolites with the subsequent risk of ADHD. When considered as a continuous variable, one unit increase in tryptophan , 5-HTP  and N-acetyltryptophan  were associated with an increased risk of subsequent ADHD, after accounting for potential confounders. These findings were mostly consistent when the metabolites were further stratified by quartiles, as well as when the metabolites in the lowest quartile (<25th percentile) were compared against those above this cutoff (≥25th percentile). Cord blood serotonin, however, was not associated with the risk of ADHD. In contrast to the other tryptophan metabolites, 5-MTX was inversely associated with ADHD risk. That is, when 5-MTX was considered as a continuous variable, higher cord 5-MTX levels were associated with a lower odds of ADHD , after adjusting for potential confounders. Further stratifying cord 5-MTX ( < 25th percentile vs. ≥25th percentile) showed a consistent inverse association with ADHD . The only exception was that the children that had highest 5-MTX levels (quartile 4), whose odds of ADHD was not significantly different compared to the referent group (quartile 1). There was no association between 5-MTX and other DDs, when 5-MTX was considered as a continuous or categorical variable.We repeated the main analysis by restricting the sample to high-risk sub-groups for each of the metabolites and risk of ASD Fig. , ADHD F and otheP = 0.04) and maternal diabetes or obesity (P = 0.04) was significantly associated with lower cord 5-MTX levels. Next, we sequentially added covariates to the model, but none of these affected the association between 5-MTX and ASD . Sixth, because of our observational study design, we cannot exclude the possibility of residual confounding. Finally, considering that our study was conducted in a high-risk population, a predominantly urban, low-income, racially diverse, with a high proportion of preterm births, caution should be exercised when extrapolating our findings to other populations with different characteristics.Our findings should be considered in the context of the following limitations. First, this study included one-time measurement of cord tryptophan and its metabolites at birth and did not measure it subsequently to understand how they varied with age. The fact that some of the metabolites showed an association with incident ASD and ADHD in childhood is an important finding and worth additional investigation. Our study only examined certain metabolites in the pathway; future studies should consider melatonin, another important chronobiotic in the pathway, to simultaneously assess their inter-relationships and joint association with ASD and ADHD. Further, current metabolomics platform generates relative intensity rather than actual concentration of the metabolites. Second, the study children were characterized as neurotypical vs. having neurodevelopmental disability  based on physician diagnosis, as documented in the EMR and this could have led to some potential misclassification. However, this misclassification may not be differential given the prospective study design and objective lab measurements of cord tryptophan and its metabolites, which were conducted by laboratory personnel who were unaware of the case status. As such, any misclassification would have biased the results towards the null. Third, other DD group in our analysis included a number of conditions due to disabilities in physical, learning, language, or behavioral areas. While it would have been insightful to assess the association between cord 5-MTX and each of these specific conditions, summarized under the broad other DD category, our study was not powered to do so. Fourth, this study was limited to those who continued to seek pediatric care at the BMC. While selection bias is a concern, this is assuaged by that the baseline characteristics of the participants initially enrolled in the study and remaining BBC sample is comparable. Fifth, sunlight is known to strongly influence circadian rhythm. As shown in supplemental eFig. In summary, this is the first prospective birth cohort study that linked multiple metabolites of tryptophan as measured at birth with the risk of ASD, ADHD, and other DD. We found that 5-MTX in cord blood at birth was associated with subsequent ASD and ADHD diagnosis. Multiple tryptophan metabolites considered in this study including tryptophan, 5-hydroxytryptophan, and N-acetyltryptophan were associated with the risk of ADHD, possibly suggesting a global disruption in the tryptophan pathway. This study should be regarded as hypothesis-generating, since very little is known about the role of 5-MTX in humans. Additional research is needed to study the association of 5-MTX at different time points with the evolution of clinical features of ASD and ADHD to better understand the temporal relation and pathophysiological significance of 5-MTX. Future studies can also explore the combination of metabolites on ASD and ADHD, since some research suggest this may lead to good discrimination between individuals with vs. without ADHD , 54.Supplemental Tables and Figures