However, no PRS genes have been cloned, let alone studied for their biological function in rubber tree. In this study, we identify a novel protein (PRS4) that interacts in vivo with rubber tree anaphase promoting complex/cyclosome (APC/C) subunit 10 (HbAPC10) by yeast two-hybrid assays. PRS4 has been cloned from rubber tree and named as HbPRS4. Blastp search in the genome of Arabidopsis thaliana showed that HbPRS4 shared the highest similarity with AtPRS4, with 80.71% identity. qRT-PCR was used to determine the expression of HbPRS4 in different tissues and under various treatments. HbPRS4 was preferentially expressed in the bark. Moreover, the expression level of HbPRS4 was significantly induced by the proteasome inhibitor MG132 treatment, suggesting it might be regulated by the ubiquitin/26S proteasome pathway. The amount of HbPRS4 transcript was obviously decreased after mechanical wounding and abscisic acid (ABA) treatments, while a slight increase was observed at 24 h after ABA treatment. HbPRS4 transcript in the latex was significantly upregulated by ethephon (ET) and methyl jasmonate (MeJA) treatments. These results suggested that HbPRS4 may be a specific substrate of HbAPC10 indirectly regulating natural rubber biosynthesis in rubber tree.5-Phospho- PRPP is a precursor to synthesize pyrimidine, purine, pyridine nucleotides, tryptophan and histidine [2+ and phosphate (P) for activity, can be inhibited allosterically by ADP and prefer ATP or dATP as the diphosphoryl donor [Methanocaldococcus jannaschii, which was activated by P and used only ATP or dATP as diphosphoryl donors, but lacking allosteric inhibition [PRS gene has been identified in their genomes. In plant Arabidopsis thaliana and yeast Saccharomyces cerevisiae, five PRS genes are present in their genomes [PRS genes present in the mammals rat (Rattus norvegicus) and human (Homo sapiens), respectively [PRS (SoPRS1–4) are present in spinach (Spinacia oleracea) [Arabidopsis AtPRS1 and AtPRS2 belong to Class I and require P for activity, whereas SoPRS3, SoPRS4, AtPRS3 and AtPRS4 belong to Class II and do not require P for activity [AtPRS2 and AtPRS3 were differentially regulated under P starvation. The AtPRS2 transcript was increased in roots and shoots of P-starved plants, whereas AtPRS3 was constitutively expressed. AtPRS3 may play a novel role in providing PRPP to cellular metabolism under P starvation [5-phospho-istidine . Based oyl donor . Class Iyl donor ,5. Classhibition . PRS is  genomes ,8. Thereectively ,10,11. AS SoPRS1– are presactivity . Furtherarvation . These sHevea brasiliensis Müll. Arg.) is the most important source of natural rubber (NR). NR is extracted from latex of rubber tree, which is collected by cutting in the bark of the tree . NR molecules are produced, aggregated and packaged in the laticifers of rubber tree. The latex, a cytoplasmic component of the laticifers, expels from the laticifers upon tapping. Most of the latex volume (up to 50%) is made up of rubber particles  and lutoids (20%) [2+ and P for activity, and used only ATP as the diphosphate group donor [PRS genes have been cloned, let alone studied for their biological functions in rubber tree.Rubber tree (ds (20%) . Latex fds (20%) . Naturalds (20%) ,15. Jasmds (20%) . The conds (20%) . NR biosds (20%) ; while Pup donor . HoweverArabidopsis. The APC/C is constituted by 11–13 subunits [Arabidopsis [Oryza sativa L.), the interaction of OsTAD1 and OsMOC1, together with OsAPC10 forms a complex and regulates rice tillering [Hevea brasiliensis APC/C subunit 10 (HbAPC10) using the yeast two-hybrid system. PRS4 cloned from Hevea brasiliensis was named as HbPRS4. qRT-PCR  was used to determine the expression of HbPRS4 in different tissues and under different treatments. The results in this study suggested that HbPRS4 may serve as a specific substrate of HbAPC10 indirectly regulating NR biosynthesis in rubber tree.The anaphase promoting complex/cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that regulates cell-cycle progression by ubiquitinating and degrading mitotic regulatory proteins via ubiquitin/26S proteasome system ,20. Sincsubunits ,21. APC1subunits ,23. The subunits . AtAPC10bidopsis ,26. In rillering ,28. ThesArabidopsis thaliana showed that this putative protein shared the highest similarity with AtPRS4 (with 80.71% identity), it was designated as HbPRS4. Protein architecture analysis by NCBI conserved domain search and SMART online service demonstrated that HbPRS4 has a Pribosyltran_N domain (from the 16th–132nd amino acid) and a Pribosyltran domain (from the 154th–297th amino acid)  was screened using the yeast two-hybrid system, and a positive clone showed a strong interaction with the bait pGBKT7-HbAPC10. Then, this clone was isolated and sequenced. It did not contain a full-length of the target gene since it was shorter than its homologous genes. The sequence of this positive clone was used for blastn searching in the transcriptome shotgun assembly (TSA) database of rubber tree. A TSA sequence (GenBank accession: JT933457) obtained from rubber tree clone RRIM 600 contained the ORF of target gene, and then, the full length of the ORF was amplified by RT-PCR in CATAS 7-33-97 and sequenced. The amplified cDNA has a complete ORF that is predicted to encode a protein of 325 amino acids with a molecular mass of 36.3 kDa and a theoretical isoelectric point of 6.89. Since blastp search in the genome of no acid) A.HbPRS4 (GenBank accession: LVXX01000028). The genomic sequence of HbPRS4 was extracted and used for further analysis of the gene structure. Comparing the genomic sequence of HbPRS4 with its corresponding cDNA sequence, it was revealed that there were seven exons and six introns present in the genomic sequence of HbPRS4, with the total length of 4858 bp  nutrient mediums DDO (SD/-Leu/-Trp) and QDO (SD/-Ade/-His/-Leu/-Trp) for the interaction test. Yeast two-hybrid assays revealed that there was no interactions in a series of negative controls, i.e., pGBKT7-HbAPC10 with pGADT7, pGBKT7 with pGADT7-HbPRS4, pGBKT7 with pGADT7 and pGBKT7-Lam with pGADT7-T. However, pGADT7-HbPRS4 interacted strongly with pGBKT7-HbAPC10, and a strong interaction was also observed in the positive control (pGADT7-53 with pGBKT7-T)  . This reHbPRS4 was constitutively expressed in rubber tree, but its expression level was varied between different tissues. The maximum was present in the bark, flower and leaf with moderate expression, which was 26.5-, 5- and 3.1-fold over that in the latex. The preferential expression of HbPRS4 in the bark indicated its main function in this tissue. Yeast two-hybrid assays demonstrated that HbPRS4 can interact with HbAPC10, and APC10 is an E3 ubiquitin ligase regulating protein degradation via the ubiquitin/26S proteasome pathway. Therefore, MG132, an inhibitor of the 26S proteasome, was used to treat rubber tree seedlings of CATAS 7-33-97. The HbPRS4 transcript was rapidly induced by MG132. It reached the largest amount at 0.5 h and increased 6.7-fold compared with the control (0 h)  treatment, the amount of HbPRS4 transcript was obviously decreased and reduced by 70% at 10 h compared with 0 h, but there was a slight increase from 24 h  . This surom 24 h . Ethephopression . As for pression . HbPRS4  tapping . These rSaccharomyces cerevisiae. The ScPRS interacts with each other and exists in vivo as three minimal functional entities, ScPRS1/ScPRS3, ScPRS2/ScPRS5 and ScPRS4/ScPRS5. Each of the multimeric complexes can support yeast viability [Arabidopsis and tobacco plants, overexpression of the Ashbya gossypii AgPRS2,4 gene or its mutated variant could increases PRS activity and substantially enhances biomass accumulation along with significant changes in the content of sugars and other metabolites [2+ and P affected the activity of this latex PRS; and it used only ATP as the diphosphate group donor [Spinacia oleracea SoPRS4 and Arabidopsis thaliana AtPRS4  in this study was later than the increase of total adenine nucleotides in Amalou et al.’s study [HbPRS4. Together with the latex PRS purified by Gallois et al. [PRS gene present in the rubber tree genome. These PRSs may work together in response to ET induction. The effect of ET on PRS activity needs to be further studied, since it controls the synthesis of PRPP, which is a requirement for nucleotide and protein biosynthesis associated with latex regeneration.Comparing the expression of the bark . This reeatments . Howeverpression . ET is w’s study . Blastn s et al. , we specArabidopsis accelerates CYCLIN B1;1  degradation and cell division at the early stages of leaf development and leads to increasing final cell number and leaf size [APC10 is an E3 ubiquitin ligase and plays a direct and crucial role in ubiquitinating specific substrates, which will be degraded by the 26S proteasome ,22,23. Ieaf size ,26. APC1−1 MG132 (an inhibitor of proteasome) and 200-µmol·L−1 ABA treatments. These treatments were performed according to our previous report [v/v) ET and 1.0% (v/v) MeJA were spread over the tapping cut on the bark, and 3 trees were used for each treatment. The trees were tapped every three days after treatments, and latex was collected for total RNA extraction.Rubber tree clone CATAS 7-33-97 (bred from crossing RRIM 600 × PR 107) was planted at the experimental field of Institute of Tropical Agriculture and Forestry, Hainan University, Danzhou City, China. Grafted budding seedlings of CATAS 7-33-97 were used for wounding, 50-µmol·Ls report . ET and Arabidopsis AtAPC10 (GenBank accession: NP_565433) sequence was used for tblastn searching in the Hevea brasiliensis TSA database, and an accession (JT940912) from RRIM 600 encoding a completed ORF of rubber tree APC10 (HbAPC10) was obtained. In order to screen the interacting protein of HbAPC10, the ORF of HbAPC10 was amplified by RT-PCR in CATAS 7-33-97 and then cloned into the EcoRI/BamHI restriction sites of pGBKT7 to generate the bait vector pGBKT7-HbAPC10. This bait vector was transformed into the yeast strain Y2HGold to generate a bait clone. A normalized yeast two-hybrid cDNA library derived from the latex of CATAS 7-33-97 cloned in the pGADT7 vector was introduced into yeast strain Y187 using the Make Your Own “Mate & Plate™” Library System  [Escherichia coli strain DH5α and then sequenced with the T7 promoter primer to determine the sequence of the inserted cDNA.The CA, USA) . The baiHbPRS4 was amplified and cloned into pGADT7 to construct a prey plasmid pGADT7-HbPRS4, and pGBKT7-HbAPC10 was used as the bait plasmid. Primers used for generating constructs for yeast two-hybrid assays are listed in To analyze the interaction between HbAPC10 and HbPRS4, the ORF of HbPRS4 was obtained by sequencing the positive clone of the yeast two-hybrid screening HbAPC10 interacting proteins. The partial sequence of HbPRS4 was performed blastn searching in the Hevea brasiliensis TSA database. A sequence (GenBank accession: JT933457) generated from rubber tree RRIM 600 contained the whole ORF of HbPRS4. Primers PRS4-F1 and PRS4-R1  and sequenced.The partial sequence of  PRS4-R1  were usehttp://web.expasy.org/compute_pi/). NCBI conserved domain search (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi?) and SMART (http://smart.embl-heidelberg.de/) were used to identify the architecture of HbPRS4. Sequences of all the PRS proteins were aligned by the ClustalX 1.83 [The theoretical isoelectric point and molecular weight of HbPRS4 was calculated by the ProtParam tool  according to the recommended protocol. Specific primers designed for HbPRS4  algorithm was used to calculate the relative expression level of target gene [p < 0.05. Figures were drawn and viewed by OriginPro 9.0.Total RNA was extracted with the RNAprep Pure Plant Kit (Polysaccharides & Polyphenolics-rich)  and used for cDNA synthesis with the RevertAidget gene . For eacHbPRS4 gene was first cloned from rubber tree. Phylogenetic analysis supported HbPRS4 as a Class II PRS with a high degree of similarity with Spinacia oleracea SoPRS4 and Arabidopsis thaliana AtPRS4. HbPRS4 was preferentially expressed in the bark. Moreover, the expression level of HbPRS4 was significantly induced by the proteasome inhibitor MG132 treatment, suggesting that HbPRS4 was regulated by the ubiquitin/26S proteasome pathway. HbPRS4 transcripts in the latex were remarkably upregulated by ET and MeJA stimulus. These results suggested that HbPRS4 may be a specific substrate of HbAPC10 indirectly regulating natural rubber biosynthesis in rubber tree.Yeast two-hybrid assays demonstrated that HbPRS4 can interact in vivo with HbAPC10. This is the first report on the interaction of APC10 with PRS4, and


Elodea is used as a model species. Additionally, we use PCLake to estimate how much phosphorus is removed with the harvested biomass, and evaluate the long-term effect of harvesting. Our model indicates that mowing can temporarily reduce nuisance caused by submerged plants in the first weeks after cutting, particularly when external nutrient loading is fairly low. The risk of instigating a regime shift can be tempered by mowing halfway the growing season when the resilience of the system is highest, as our model showed. Up to half of the phosphorus entering the system can potentially be removed along with the harvested biomass. As a result, prolonged mowing can prevent an oligo—to mesotrophic lake from becoming eutrophic to a certain extent, as our model shows that the critical nutrient loading, where the lake shifts to the turbid phytoplankton-dominated state, can be slightly increased.Submerged macrophytes play an important role in maintaining good water quality in shallow lakes. Yet extensive stands easily interfere with various services provided by these lakes, and harvesting is increasingly applied as a management measure. Because shallow lakes may possess alternative stable states over a wide range of environmental conditions, designing a successful mowing strategy is challenging, given the important role of macrophytes in stabilizing the clear water state. In this study, the integrated ecosystem model PCLake is used to explore the consequences of mowing, in terms of reducing nuisance and ecosystem stability, for a wide range of external nutrient loadings, mowing intensities and timings.  Shallow lake ecosystems depend on the presence of submerged aquatic plants (macrophytes) for good water quality and high biodiversity , mowing intensity and timing of mowing during the growing season. We express the effect of mowing both in terms of remaining plant cover, and in terms of days without nuisance caused either by macrophytes or cyanobacteria. This exercise also allows us to evaluate under which conditions mechanical cutting of macrophytes results in a critical regime shift to the alternative turbid state. Secondly, we use the model to obtain quantitative estimations of the amount of P that can be removed from the system via harvesting of macrophytes. Removal of P may help to remediate eutrophication effects in the lake, and potentially can be recovered for sustainable reuse. Finally, we explore the long term impacts of mowing to analyze whether mowing is a measure that also can be applied to help prevent undesired eutrophication effects in shallow lakes.a, Secchi depth, vegetation cover and nutrient concentrations in the water column  is at a much higher value than the critical nutrient loading where the reverse switch takes place, back to clear conditions during re-oligotrophication (CNLoligo). Hence, at intermediate loading levels both the clear-water state and the turbid water state can exist as alternative stable states and the prevalent state depends on the foregoing conditions—a phenomenon known as hysteresis. Between the critical nutrient loading values, strong perturbations, such as discrete mowing events, may instigate a regime shift from one state to the other . Waterweed species are non-native yet widespread in NW Europe and they are often among the first macrophytes to return after restoration measures have been taken /p*V, where h is the harvested biomass (g m−2 day−1), f is the intensity (-), p is the duration (days) and V is the total aquatic plant biomass in the lake (g m−2). We applied a 'clean' mowing strategy throughout this study, whereby all biomass is removed from the lake. We did briefly consider potentially harmful side effects of mowing, including enhanced resuspension and incomplete removal of plant material from the water column, but present these findings as an appendix as they did not affect the conclusions of our main analyses (see Online Resource 2).A mowing function is available in PCLake, which requires defining a date when the mowing event takes place, the duration of the mowing event and a mowing intensity (i.e. fraction of the biomass that is removed). The mowing intensity is independent of the duration of the mowing event: a natural logarithm is used to calculate the amount of biomass that is removed per day: −1 (100 day residence time), a lightly clayish soil , no infiltration or seepage and no surrounding wetland area , and mowing intensity, ranging from 0 to 0.9 in steps of 0.1 (−). We did not consider P loadings above 1.7 mg m−2 day−1 as the modeled lake then resides in the turbid water state without macrophytes. Each simulation was started from a clear water state and we ran the model for 20 years before starting the mowing procedure to ensure the lake to be in  equilibrium. Note that internal nutrient loading in PCLake is not an independent variable, and by running the model 20 years we achieve that the internal loading in the system associates with the corresponding levels of external P loading. The initialization period was followed by three succeeding years where a mowing event took place. We considered 3 years to include the effect of mowing on the biomass in the next year (Kimbel and Carpenter p) was kept at the default value of 10 days in all of these and subsequent cases. Next, we repeated the foregoing simulations, but this time focusing on different combinations of mowing intensity and timing. Again the mowing intensity ranged from 0 to 0.9 in steps of 0.1, while the mowing dates ranged from June 1st to September 1st in steps of 7 days. We performed this analysis for three different nutrient loading settings .In this study on the impact of mowing on the lake we varied three independent variables of the model that can be controlled by lake managers: (1) external nutrient loading, (2) mowing intensity and (3) timing of the mowing. We first focused on the interplay between the first two. We simulated different combinations of external P loading, ranging from 0.7 to 1.7 in steps of 0.05  of submerged plants until 200 g DW m−2 is reached and the cover is 100%. Also, we calculated the days with nuisance during the peak of the holiday season (beginning of July until the end of August) caused by either submerged water plants or cyanobacteria. We presumed that water plants cause nuisance when they cover more than 40% of the area  vegetation cover and cyanobacterial chlorophyll-−2 day−1) and present the within-season dynamics of the vegetation cover and chlorophyll-a in response to several different mowing intensities, to also obtain a more detailed view on the dynamics of the lake.Additionally, we zoomed in on one intermediate nutrient loading  year of mowing, to evaluate the potential to impoverish the lake. The amount of P removed from the system via harvesting provides an indication of the P that can potentially be recovered for reuse. In addition, we calculated the relative removal of P, that is, the ratio of P in the harvested biomass to the total amount of P added to the system via external loading. The relative removal thus allows to assess the extent to which harvesting may contribute to the closing of the P cycle.eu of the lake, that is, the amount of external nutrient loading the lake can withstand without switching to a phytoplankton-dominated turbid state. Following Janse et al.  after 20 years was used to evaluate the state of the lake, to determine which P loading is the CNLeu. Previous analyses have shown that the ratio of Secchi depth to lake depth is a suitable response variable to determine the CNLeu . In the vicinity of the critical nutrient loading, a mowing intensity of >30% is sufficient to trigger a collapse when mowing is applied in three succeeding years.The model shows that the summer average plant cover can be reduced by mowing Fig. . When exZooming in on the seasonal dynamics clearly reveals the time window where plant cover is reduced due to mowing lasting for at least several weeks Fig. . It also−2 day−1), mowing can create conditions where hardly any nuisance is experienced during the peak of the summer holiday season .Our model exercises show that in the long run repeated mowing is able to enhance the resilience of the clear water state to nutrient loading for a wide range of mowing intensities and mowing dates, as it leads to an increase max. 7%) of the critical nutrient loading . This compensatory growth is not sufficient however to compensate for the entire loss of biomass, as plant cover does not recover to pre-harvesting levels  is in mid-summer, but also because the risk of inducing a regime shift increases when mowing is conducted later in the growing season. In our model, mowing late in the growing season provides the aquatic plants with little opportunity to regain biomass before the growing season ends. This is in line with Engel , who obsels Fig. .Elodea canadensis—produce overwintering organs  lake with a diameter of 1000 m, when receiving 1.2 mg P mFrom our analyses it appears that harvesting 10% of the standing crop has only a marginal effect on reducing nuisance. This situation may change however when it is possible and desirable to spatially divide functions over the lake area. By harvesting in such a way that only certain patches are cleared, it may become possible to reduce nuisance locally e.g. in a zone designated for swimming or a channel for navigation. The model we used (PCLake) is not spatially explicit and is therefore not suited to evaluate the effect of a local disturbance by harvesting, as it is intended to provide a general indication of the harvesting pressure the lake can withstand. There is only little known about the effect of spatial heterogeneity on alternative stable states in shallow lakes. Theoretical studies suggest that the potential of local disturbances to instigate an ecosystem-wide regime shift increases with interconnectedness (dispersion) within the system (Van Nes and Scheffer Elodea spp.) spread by vegetative fragmentation  can have a large impact on vegetation density . Thus, based on the long term mowing scenarios we argue two points. The first is that phytoremediation can be a worthwhile measure to prevent a lake from becoming eutrophic when it is still oligotrophic and its capacity to withstand perturbations is still high. The second is that it is much more difficult to use phytoremediation to impoverish a lake when it is already eutrophic, even though the absolute removal of nutrients is high, because the capacity to withstand perturbation is much reduced. As many vegetated lakes in NW Europe have only recently recovered from the turbid state, and their sediments are likely to be saturated with nutrients, mowing schemes should be designed with great care.Model analysis of the long-term effects of harvesting suggests that harvesting can potentially be used to prevent nutrient over-enrichment by increasing the resilience of the system to external loading, that is, by increasing the CNLeu Fig. . It is in = 1), but that there are general mechanisms that are key to the ecological functioning of every lake. PCLake has been developed to include the most important biotic and abiotic processes and lake characteristics  has a strong trophic interaction with aquatic plants (Hussner et al. Of course, it is conceivable that the ecology of certain lakes may differ fundamentally from the system currently portrayed by PCLake. For example, Blindow et al.  distinguOur integrated modeling analysis of a typical shallow lake in the temperate zone indicates that harvesting submerged macrophytes can be effective in temporarily reducing nuisance in lakes which are oligo—or mesotrophic, particularly when mowing is executed in mid-summer. Designing a successful mowing strategy becomes less easy with increasing nutrient loading. More eutrophic lakes are less resilient to perturbations, and when the external nutrient loading approaches the critical level, relatively small reductions in plant cover are sufficient to trigger an unwanted shift to the alternative phytoplankton dominated state. By extracting nutrients from the lake, negative effects of eutrophication may be partially remediated. Our modeling indicates that the largest amounts of P can be recovered close to the tipping point, although the highest removal of P relative to the input of P is realized when the external P loading is low. Particularly when a lake is still oligotrophic, phytoremediation can be an effective measure to counteract slowly increasing nutrient inputs, while it appears more difficult to use harvesting to impoverish a lake which is already eutrophic, as more eutrophic lakes are also more sensitive to perturbations. These insights provide a basis for more tailored studies on the effects of harvesting in specific lakes systems.Supplementary Information


Attention deficit hyperactivity disorder (ADHD) is a prevalent and multifactorial neuropsychiatric disorder in the human population worldwide. Complex etiology and clinical heterogeneity have challenged the research, diagnostics and treatment of the disease. Hyperactive and impulsive behaviour has also been observed in dogs, and they could offer a physiologically relevant model for human ADHD. As a part of our ongoing study to understand the molecular etiology of canine anxiety traits, this study was aimed to pilot an approach to identify metabolic biomarkers in canine ADHD-like behaviours for research, diagnostics and treatment purposes.We collected fresh plasma samples from 22 German Shepherds with varying ADHD-like behaviours. All dogs were on the same controlled diet for 2 weeks prior to sampling. A liquid chromatography combined with mass spectrometry (LC–MS)-based non-targeted metabolite profiling was performed to identify plasma metabolites correlating with the ADHD-like behaviour of the dogs.raw < 0.05), and three of them  had significant correlations also after FDR correction (pFDR < 0.05). Phospholipids were found to negatively correlate with ADHD-like behavioural scores, whereas tryptophan metabolites 3-indolepropionic acid (IPA) and kynurenic acid (KYNA) had negative and positive correlations with ADHD-like behavioural scores, respectively.649 molecular features correlated with ADHD-like behavioural scores  contains supplementary material, which is available to authorized users. Attention deficit hyperactivity disorder (ADHD) is a multifactorial neuropsychiatric disorder with a high prevalence 5–10 %) among children worldwide. It is also increasingly reported in adults 0 % among. In geneOne potential approach to unravel the biological pathways of ADHD is to utilise animal models, like dogs, which spontaneously show ADHD-like behaviours, such as hyperactivity, impulsivity and inattention. Many young dogs often show hyperactive and impulsive behaviour, but some hunting and working breeds, like the German Shepherd and the Belgian Shepherd, may continue to show these behavioural extremes later in life as well . Due to One of the challenges in ADHD research lies in the genetic complexity of the disorder. It is expected that multiple small effect genes contribute to ADHD susceptibility , 4. TherIn this study, we investigated the association between plasma metabolites and ADHD-like behavioural scores in diet-controlled dogs with varying ADHD-like behaviours in order to identify ADHD-related pathways and biomarker candidates using a LC-qTOF-MS –based approach. Our results reveal associations between ADHD-like behaviours and plasma phospholipids and tryptophan metabolites in dogs.Data on dog ADHD-like behaviour was collected using our validated owner-completed behavioural survey . The queThe owner-completed behavioural survey included both general questions concerning the details of their dogs’ background, daily routines, and everyday behaviour and 13 more specific questions concerning the activity, impulsivity and inattention behaviour of the dog , and they are previously presented in detail . BrieflyThe LC–MS data was collected using the vendor’s software MassHunter Qualitative Analysis B.05.00 (Agilent Technologies), where the ions were extracted to compounds utilising the “Find by molecular feature” algorithm. The data were output as compound exchange format (.cef) files into the Mass Profiler Professional software  for compound alignment and data pre-processing. In order to reduce noise and remove insignificant metabolite features, only the features found in at least 50 % of the samples were included in the analysis. This resulted in a dataset comprising 7058 features in four separate analytical runs .raw <0.05 were included in the identification analysis, and metabolites with pFDR <0.05 were considered statistically significant. Finally, the remaining features in the lists were inspected in the LC–MS chromatograms and spectra using the MassHunter software to locate chromatographic peaks with poor retention time accuracy and peak symmetry, which were removed from downstream analysis. Peak lists were also investigated to ensure that the molecular ion of a compound was included into automatic MS/MS fragmentation, and if not, targeted MS/MS analysis was performed.The four datasets were exported into Microsoft Excel (2013), and filtered according to peak area >20,000 to exclude small and insignificant features from further analysis. To investigate the associations between the peak areas of the metabolites and each of the ADHD-like behavioural scores , the Spearman correlation analysis was used. The results were adjusted for multiple comparisons by the Benjamini-Hochberg false discovery rate (FDR) correction  used in To investigate whether sex, age or fasting status had any effects on the associations between the metabolites and ADHD-like behavioural scores, a partial correlation analysis including sex, age and fasting status as covariates was performed. All the statistical analyses were performed using the R project for Statistical Computing version 3.0.1.m/z 184.07 for PCs and LysoPCs and m/z 196.03 for LysoPEs), as well as the deprotonated fatty acid fragments visible in the negative ionization mode. For LysoPCs, the product ion at m/z 104 was used to distinguish sn-1 and sn-2 isomers. The MS/MS fragmentation data for all of the identified metabolites is presented in Table The identification of metabolites was based on the accurate mass and isotope information; i.e., ratios, abundances and spacing, as well as product ion spectra (MS/MS) acquired either in the automatic MS/MS analysis, during the initial data acquisition, or via re-injection of the samples in targeted MS/MS mode. The spectra were compared with the METLIN Metabolite Database , the HumIn this study, we used non-targeted LC–MS-based metabolite profiling to create the plasma metabolite profiles of 22 German Shepherds with varying ADHD-like behavioural scores describing the hyperactive, impulsive and inattentive behaviour of the dog. The variation in the dietary profiles was controlled by feeding the same food to all dogs for a 2-week period prior to sampling.raw <0.05). These molecular features were subjected to manual inspection to identify metabolites, resulting in a set of 22 identified and five unidentified metabolites  5.77 min and m/z 190.050 and rt 3.23 min in RP ESI(+) analysis showed identical fragmentation with 3-indolepropionic acid (IPA) (CAS No. 830-96-6) and kynurenic acid (KYNA) (CAS No. 492-27-3), respectively, according to the METLIN database (Table m/z 176.071 and rt 5.01 in RP ESI(+) analysis was identified as indoleacetic acid (IAA) (CAS No. 87-51-4), based on its MS/MS fragmentation pattern. Higher ADHD-like behavioural scores were associated with lower plasma levels of IPA  and IAA  .Three molecules related to tryptophan metabolism correlated with the ADHD-like behavioural scores Table ; Fig. 1.s = −0.769, praw = 2.93E − 05, pFDR = 0.016; inattention: rs = −0.740, praw = 8.26E-05, pFDR = 0.030; impulsivity: rs = −0.765, praw = 3.32E-05, pFDR = 0.036), sn-1 LysoPE(18:2) and inattention score , and PC(18:3/18:2) and total and inattention scores  also remained significant after FDR correction. Significant correlations were not due to outliers  PC18:3/18:2), PC(20:5/18:3), PC(20:4/14:0), PC(18:2/16:1) and PC(15:0/18:2)), five lysophosphatidylcholines (LysoPC) (sn-1 LysoPC(18:3), sn-1 LysoPC(14:0), sn-1 LysoPC(15:0), sn-1 LysoPC(17:0) and sn-1 LysoPC(20:3)) and four lysophosphatidylethanolamines (LysoPE) (sn-1 LysoPE(18:2), sn-1 LysoPE(20:5), sn-1 LysoPE(18:1) and sn-1 LysoPE(18:0))  and C18:1 (m/z 283.26428)  and C18:1   analysis correlated positively with all three ADHD-like behavioural scores  and was identified as bilirubin (CAS No. 635-65-4) (Tables m/z 137.071 and rt 2.22 in the HILIC(+) analysis was identified as 1-methylnicotinamide (CAS No. 3106-60-3). It had a positive association with all three ADHD-like behavioural scores .The metabolite with m/z 796.516 correlated only with the inattention score. The impulsivity score correlated with 21 metabolites, and phenylalanine was found to specifically correlate only with the impulsivity score.Twenty out of the 27 reported metabolites correlated with all three ADHD-like behavioural scores  Table . Twenty-p value < 0.05). However, age, sex and fasting had significant effects on associations between IPA and the impulsivity score , sn-1 LysoPC(14:0) and the inattention score , sn-1 LysoPC(17:0) and the inattention score , unknown PC with m/z 796.516 and the inattention score , IAA and the inattention score , phenylalanine and the impulsivity score , and unknown metabolite with m/z 221.150 and the total ADHD score .Since there were differences in the fasting status of the dogs  in the intestines [Clostridium bartlettii) [Clostridium), negative correlations between the ADHD-like behavioural scores and these metabolites suggest differences in intestinal microbiota of dogs with different ADHD-like behaviours, since there were no differences in the diets (and intake of tryptophan) of the dogs. Gut microbiota may have a bidirectional effect on the nervous and the immune systems [We found three interesting metabolites in the tryptophan pathway, IPA, IAA and KYNA. Lower plasma IPA and IAA levels were associated with higher ADHD-like behavioural scores. IPA is a microbial deamination product of dietary tryptophan with antioxidant capacity , 32, protestines –35  , 38. Due systems –41. Sinc systems .Fig. 2SiN-methyl-d-aspartate receptor (NMDAR) in the CNS and as an agonist of a particular G-protein coupled receptor, GPR35, in immune cells and in the gastrointestinal (GI) tract [The third metabolite in the tryptophan pathway, KYNA, was positively correlated with the ADHD-like behavioural scores. KYNA is a neuroactive metabolite produced in the kynurenine pathway of tryptophan metabolism , 43. It I) tract . InteresI) tract , 46, depI) tract  and bipoI) tract . A recenI) tract . InteresI) tract , 50  tract . Thus, tI) tract . DecreasI) tract . Thus, tWe observed strong and negative correlations between ADHD-like behavioural scores and fifteen phospholipids, including five PCs, five LysoPCs, four LysoPEs and one unknown PC. The blood lipid composition is affected by nutrition and fasting , and we,Phospholipids are composed of hydrophobic and hydrophilic parts, which are joined together by glycerol . The hyd2) receptor binding in the frontal cortex [It has been suggested that the relationship between decreased omega-3 PUFAs and ADHD would lie in dopamine neurotransmission, as rodent studies have proposed that chronic omega-3 PUFA deficiency is associated with significantly decreased concentrations of endogenous dopamine and reduced dopamine-2 (Dl cortex , 61, accl cortex . In thisOur study also suggests the involvement of oxidative stress in canine ADHD-like behaviours. The ADHD-like behavioural scores showed a positive relationship with fatty acid C20:4, an oxidative stress marker, but a negative relationship with the antioxidant IPA. Impaired balance between the oxidant and antioxidant systems and increased levels of oxidative stress have already been demonstrated in ADHD, although precise mechanisms remain to be characterised –70.We have previously demonstrated the promise of the metabolomics approach in canine fear research , and nowOur metabolomics study suggests associations between canine ADHD-like behaviours and tryptophan and phospholipid metabolisms. A replication study with a larger sample size is needed to validate our findings and to confirm the overlap in the affected pathways between human ADHD and canine ADHD-like behaviours.


COMT, ANKK1) and the neurotrophic  system, associations with the Nogo-P3 as well as ADHD symptom severity were found suggesting that these systems are involved in response control deficits in ADHD. Methylation effects related to both functional aspects and ADHD behaviour were also observed for DPP10 and TPH2. Epigenetic mechanisms may play a role in ADHD-associated deficits but findings need to be replicated in larger samples and are limited by the fact that only peripheral methylation could be considered.In order to better understand the underpinnings of attention-deficit/hyperactivity disorder (ADHD), we targeted the relationship of attentional, cognitive control and motivational processes with DNA methylation patterns of 60 candidate genes in boys at early school age. Participants  were selected from a German longitudinal cohort (FRANCES). ADHD-related behaviour was assessed via maternal ratings. Performance and event-related potential measures , which were recorded in a motivational go/nogo task, indicated diminished attentional orienting, reduced inhibitory response control and a larger motivational effect on performance in ADHD already at this relatively young age. Methylation patterns were analysed in buccal cell DNA with the Illumina HumanMethylation 450K array. For CpG sites at genes of the dopaminergic ( ADHD is more common in boys than in girls with a ratio of about 4:14.Inattention, motor hyperactivity and impulsivity are the cardinal symptoms of attention-deficit/hyperactivity disorder (ADHD), one of the most prevalent child psychiatric disorders. Currently classified as a neurodevelopmental disorder in DSM-V, the symptoms need to be present early on (before the age of 12)6.ADHD is in many ways a heterogeneous disorder that has been associated particularly with cognitive and motivational deficits. According to the integrative view presented by Sonuga-Barke, multiple developmental pathways may lead on the background of person by environment interactions to ADHD: cognitive deficits related to executive functions  may be related to dysfunctions in the mesocortical dopaminergic system and associated brain regions , while impaired motivation (e.g. delay aversion) may be associated with dysfunctions in the mesolimbic dopaminergic system or ventral-striatal hyporesponsiveness to reward10. Reduced ERP components related to attentional orienting (Cue-P3), response preparation  and cognitive control during response inhibition (Nogo-P3) have repeatedly been reported. The Cue-P3 is linked to the posterior attention network mainly modulated by noradrenaline though processes triggering the Cue-P3 could also be under dopaminergic influence11. The subsequent Cue-CNV is a slow cortical potential with central topography reflecting resources allocated for response preparation. It is probably generated in the dorsal anterior cingulate, frontal cortex and midbrain dopaminergic nuclei, susceptible to dopaminergic modulation, and a lower CNV amplitude may reflect a persisting deficit in ADHD13. The Nogo-P3 with sources in the prefrontal cortex and the anterior cingulate cortex (ACC) may reflect inhibitory response control modulated by dopaminergic activity. The Nogo-P3 amplitude reduction is also considered a persistent deficit in ADHD13. Motivational effects, which can be assessed in the lab inter alia by contrasting task blocks with and without motivational contingencies, were at least partly found to be larger in children with ADHD at the performance and neuronal level15.Dysfunctions in neuronal networks associated with attentional processes and cognitive control were documented in a series of studies using event-related potentials (ERPs) with children usually being 8 years or older16. However, only relatively small effects between individual risk alleles and ADHD suggest that environmental factors  also play a significant role17. Environmental effects on brain development and behaviour may alter gene activity (without changing the order of their DNA sequence) via epigenetic modifications (like DNA methylation). So, epigenetic studies could be of particular value to unravel the underpinnings of ADHD. However, it has to be considered that epigenetic changes can be tissue specific18. So, effects found in peripheral tissue  may not reflect methylation patterns in the brain.Heritability estimates of more than 70% suggest a genetic component in ADHDDRD4) and the serotonin transporter (SLC6A4) gene, significant associations between methylation and ADHD behaviour were obtained in more than one study.So far, only a few epigenetic studies have been conducted in children showing ADHD symptoms. Some of the studies e.g., refs et al.23 and Wilmot et al.24. Using the ARIES (Accessible Resource for Integrated Epigenomics Studies) sample comprising more than 800 children, Walton et al.23 investigated if DNA methylation at birth was associated with trajectories of ADHD symptoms at the age of 7–15 years. They obtained 13 probes  that were associated with later ADHD trajectories applying FDR  correction. Gene network analysis revealed a complex interconnected network related to peroxisomal and neurodevelopmental processes. However, none of the probes turned out to be significant when considering methylation at age 7.Methylome-wide analyses were done by Walton et al.24 did their methylome-wide analysis in a smaller sample of boys with ADHD and typically developing boys aged 7–12 years. Several probes on the VIPR2 and MYT1L gene fulfilled their less stringent statistical threshold and enrichment analysis indicated pathways related to inflammatory processes and modulation of monoamine and cholinergic neurotransmission.Wilmot Summarizing the results, epigenetic studies in ADHD do not provide a congruent picture by now but indicate that methylation patterns may be associated with ADHD but need not to be stable in the course of development. The functional relevance of methylation variations regarding attentional and response control processes have not been considered yet.First, we investigated whether ERP markers and performance measures reflecting attention, cognitive control and motivational aspects already show differences in boys at early school age depending on their ADHD behaviour . As mentioned above, this age range has been underrepresented in previous studies though, according to DSM-IV and ICD-10, diagnostic criteria have to be fulfilled at this age.et al.25 and genes found in the methylome-wide analysis of Wilmot et al.24 for children in a comparable age range.Second, we studied associations between DNA methylation variations and ADHD behaviour and whether these variations are of functional relevance. In this respect, we tested if methylation levels are correlated with ADHD behaviour and associated performance and ERP measures, respectively. As sample size was too small to expect methylome-wide statistical significance (p < 1e-7), we considered candidate genes thought to play a role in the aetiology of ADHD  as reviewed by Banaschewski 26), we considered a sample of about 80 boys participating in a longitudinal study on child development. The associations of dimensional ADHD behaviour ratings, functional  measures and DNA methylation were assessed cross-sectional.Following a dimensional perspective of ADHD (interpreting ADHD as the extreme end of a continuous distribution of behaviour(s)2 = 0.13) indicating less impulsivity errors in blocks with motivational incentives .Results of the ANCOVAs  are summarized in Table 2 = 0.07) was obtained due to a larger influence of motivational contingencies with increasing FBB-ADHS total score. For the other performance parameters, no significant effects related to ADHD could be found  ≤ 2.96, p ≥ 0.089).Regarding reaction time variability, a significant ADHD × INCENTIVES interaction effect  = 5.69, p = 0.019, part. η2 = 0.07) indicating smaller amplitudes with increasing ADHD score. In Fig. 2 = 0.06).For Cue-P3 and Nogo-P3, significant ADHD effects could be revealed  ≥5.60, p = 0.020, part. η,80 ≥5.60Comparing children with FBB-ADHS scores in the normal range  and children with FBB-ADHS scores in the clinical range  regarding the difference in reaction time variability between blocks with incentives and blocks without incentives, the Cue-P3 (at electrode Pz) and the Nogo-P3 (at electrode CPz) revealed significant effects for all three measures (post-hoc t-tests: |t(51)| ≥ 2.29, p ≤ 0.026; see also Table These three functional parameters were considered in the subsequent methylation analysis.DNA methylation data of 67 (of the 82) boys could be included in the analysis. Data from non-Caucasian children (N = 2) were not considered. Four children had to be excluded due to errors concerning sample collection and, in 9 children, DNA sample failed quality control.COMT, ANKK1, BDNF and NGFR genes, i.e., genes related to the dopaminergic and neurotrophic system, respectively (see Table BDNF (cg11806762) and the FBB-ADHS total score was no longer significant  explaining about 34% of the variance  = 11.8, p = 3.69e-06) and in the model regarding the FBB-ADHS total score  explaining ca. 31% of the variance  = 10.2, p = 1.69e-05), while the CpG linked to NGFR (cg04613258) fell short of significance.Computing linear regressions (backward elimination) starting with those four CpG sites fulfilling our statistical criteria as predictor variables, three CpGs remained in the model for the Nogo-P3 (DPP10 gene) was associated with the Cue-P3 and ADHD behaviour.CpG site cg19651219  and a reduced Nogo-P3 (reflecting inhibitory response control) as well as a larger motivational influence on reaction time variability in a go/nogo task. ERP effects were of medium-to-large effect sizes. For several candidate genes, associations of CpGs sites with those functional aspects and with ADHD behaviour of at least medium size were obtained.7 as well as in adult patients with ADHD10 and, thus, may be considered as a robust finding in ADHD. Spronk et al.27 also investigated relatively young (5–7 years old) children and, as in our study, children did not have a clinical diagnosis of ADHD. They also obtained a reduced Cue-P3 and at least a tendency for a reduced Nogo-P3 in their small sample. So, it may be concluded that deficient attentional orienting and inhibitory response control characterize (at least) boys with ADHD from mid-childhood into adulthood. Findings from our present study also indicate that these deficits are not related to motivational issues.Reduced Cue-P3 and Nogo-P3 components have been found in older children and adolescents with ADHDet al.27 even observed a larger CNV at occipital sites in their 5–7 years old group of children with ADHD symptoms. So, due to developmental effects, the central CNV may be considered as a neurophysiological marker for ADHD starting in late childhood.For the CNV (reflecting cognition preparation), the major part of the studies reported a reduced amplitude at central electrodes in patients with ADHD though findings are not as consistent as for the Cue-P3 and the Nogo-P3. Spronk 28 revealed a large effect size for increased reaction time variability in children and adolescents with ADHD but this could not be found in our data, probably due to the character of the task (using a S1–S2 paradigm). However, a larger decrease of reaction time variability in the blocks with motivational contingencies in the group of children with a higher ADHD score may be seen in line with the notion of motivational processes as a pathway to ADHD5. The number of impulsivity errors was not significantly higher with increasing ADHD score, which is in line with other studies using a cued go/nogo task e.g., refs 7.A meta-analysisCOMT, ANKK1, BDNF, NGFR, DPP10, TPH2), have not been considered in the candidate-gene methylation studies in ADHD e.g., refs 24.The genes, for which our data showed associations of CpGs sites with functional aspects and ADHD behaviour fulfilling our statistical criteria  with the Nogo-P3 and ADHD behaviour further support the notion that the dopaminergic system is affected in ADHD. COMT (catechol-o-methyltransferase) is critical for monoamine signaling at the site of cortical synapses. Interestingly, in a study related to malnutrition, an association of a CpG site with attention (ADHD score) in adults was reported29 though it was a different CpG site than the CpG sites we found in our study.Associations of probes linked to genes of the dopaminergic system (ANKK1 (ankyrin repeat and kinase domain containing 1) gene was originally associated with the DRD2 gene. Due to its close proximity to DRD2, ANKK1 is assumed to regulate DRD2. It may also play a role in the development of alcohol dependence. In a recent methylation study related to maltreatment, children with early onset maltreatment had higher methylation values at ANKK1 indicating an increased risk for adverse mental health outcomes30. These effects may complement the association of Nogo-P3 and impulsive behaviour in a cued continuous performance test with a DAT1 haplotype11 but further research is needed to link developmental with genetic and epigenetic effects to actual phenotypes.The BDNF (brain derived neuroptrophic factor) gene and the NGFR (nerve growth factor receptor) gene belong to the neurotrophin family. They are involved in the development, plasticity and survival of dopaminergic and serotonergic neurons and may play an important role regarding learning and memory but also cognitive functions. Up to now, methylation studies related to BNDF have mainly been conducted in patients with depression, bipolar disorder and schizophrenia33.The DPP10 was detected. The DPP10 gene encodes dipeptyl peptidase 10, which binds to specific voltage-gated potassium channels and alters their expression. Potassium channel function can affect dopaminergic tone and may thus be involved in the development of neuropsychiatric disorders like schizophrenia, autism and ADHD34. As the Cue-P3 is linked to the posterior attention network mainly modulated by noradrenaline35, we would have expected associations with corresponding CpG sites. Applying less stringent criteria (see Table NET1/SLC6A2) gene. Moreover, in 3 of the 17 CpG sites linked to the DRD4 gene, associations with the Cue-P3 were nominally significant (p < 0.05) which can be seen in line with the findings of Albrecht et al. (2014) that processes triggering the Cue-P3 response may be influenced by DRD4 7 R polymorphisms  or the study by Gizer & Waldman36 reporting DRD4 7 R related to inattention.Regarding the Cue-P3, only an association with CpG sites in the TPH2 gene and the influence of motivational incentives on reaction time variability. TPH2 encodes tryptophan hydroxylase which is the rate-limiting enzyme in the production of serotonin. We are not aware of other studies considering DNA methylation in the TPH2 gene related to reward aspects. Guillemin et al.37 found that the promotor of TPH2 is differentially methylated in adults already showing aggressive behaviour in childhood. Interestingly, Neufang et al.38 reported that the TPH2 genotype  modulated the response to monetary rewards in the nucleus accumbens which could provide a link between TPH2, reward-related processes and impulsive behaviour.In our data, we obtained an association between CpG sites in the In summary, reflecting the literature may indicate that DNA methylation variations in those genes, for which we obtained significant findings, are involved in the development of neuropsychiatric disorders like ADHD. However, as only limited data are available by now, it does not allow to support our findings directly.39.ADHD-related behaviour was only assessed via maternal ratings which may be biased. On the other hand, ratings by teachers, who divide their attention over 20–30 children in a class and observe a child only for a limited period during the day, might be more prone to measurement erroret al.23) and to study to what extent findings are comparable or different for boys and girls. Generally, it has to be noted that findings obtained in the methylation studies in ADHD do not provide a converging picture. Differences in age ranges considered (methylation at birth vs. early or later childhood), sample composition  and methodological issues regarding methylation analysis may account for the non-congruent picture.Sample size was relatively small in our study, and a confirmation sample was not available. So, replication in a larger sample is urgently needed. Larger sample will also allow methylome-wide analysis , it is common practice in ERP studies not to correct for multiple testingIt remains open to what extent methylation variations in the identified CpG sites exert functional effects on gene expression and activity as no RNA was available. Moreover, genetic influences and further environmental factors (as well as their interactions) need to be considered for better understanding biological vulnerability and ADHD.COMT and BDNF, there are some hints for (strong) correlations between methylation variations in peripheral and brain tissue41 but more research is needed before the associations found in our current study can be integrated into functional pathways from genes, environment and epigenetic processes towards brain functions and mental disorders.Lastly, epigenetic changes can be tissue specific so that methylation patterns in the brain may be different than in peripheral tissue (and vice versa). However, at least for Diminished attentional orienting, reduced inhibitory response control and larger motivational performance effects appear to characterize boys with ADHD symptoms already at early school age.DNA methylation variations may be associated with functional deficits in ADHD. Particularly, the dopaminergic system and the neurotrophic system could contribute to the response control deficits in ADHD.COMT, BDNF and DPP10 are considered as neuropsychiatric risk genes.The associations between methylation and brain functions may, at least partly, not be specific to ADHD but may also occur in other disorders as genes like The following conclusions may be drawn from our study:Despite the limitations of our study, we expect that future studies targeting associations between DNA methylation, brain functions (resp. associated circuits) and behaviour with larger samples longitudinally will further enhance our understanding of ADHD. Using larger samples will allow to conduct methylome-wide analysis and to address the heterogeneity of the disorder.42), aims at investigating the effects of prenatal risk factors on child development at early school age43.82 boys (aged 6 to 8 years) were included in the present investigation. They were selected from the FRANCES (Franconian Cognition and Emotion Studies) cohort. FRANCES, a follow-up study of a prospective longitudinal study in a region located in the southern part of Germany  were excluded as well as children with insufficient comprehension of the go/nogo task or poor EEG data quality (N = 8). Sample characteristics are summarised in Table In FRANCES, 618 women who had participated in FRAMES were contacted and a total of 245 children (127 boys) agreed to participate. 215 children 113 boys) took part in the EEG lab session. Only those boys/families (N = 91), who responded to an invitation letter , were included here to prevent an overrepresentation of prenatal risk factors and to achieve a more representative sample. Children did not use psychotropic medication except for four boys taking methylphenidate. These four boys were at least drug-free for 24 hours before the assessments. Children with an IQ less than 75 . Each item is rated from 0  to 3 (‘notably’). The total score represents the mean value across all 20 items.ADHD-related behaviour was assessed via the German ADHD rating scale (FBB-ADHS)Recording of event-related potentials during a motivational go/nogo task and DNA collection took place at the same day.The study was approved by the Ethics Committee of the University Hospital of Erlangen and conducted in accordance to the declaration of Helsinki. Parents and children received written information and provided informed consent.The task , the same amount of money was subtracted. The participants received acoustic feedback for fast, correct responses and incorrect responses.In the second and third block of the task (ABBA design), a monetary reward  was given for fast responses in go trials to increase motivation. The reaction time threshold for receiving rewards was dynamically adjusted to the 75th percentile of reaction times in go trials of the previous block applying a tracking algorithmThe number of hits, impulsivity errors, the median reaction time as well as reaction time variability were determined and considered in the analysis.The task was implemented using Presentation .During the cued go/nogo task, EEG activity was recorded from 25 electrodes , CPz, Oz and mastoid electrodes TP9 and TP10; ground electrode: CP2). Electrode caps with sintered Ag/AgCl electrodes  were used. Impedances had to be below 20 kΩ. Raw data were recorded with a Brainproducts system  at a sampling rate of 500 Hz (filter bandwidth: 0.016–120 Hz).46 and re-referencing to linked mastoids. Amplitudes exceeding ± 150 μV were interpreted as artefact and corresponding segments as well as trials with performance issues (errors or responses faster than 200 ms or slower than 1500 ms after the S2 stimulus) were excluded from further ERP analyses.Preprocessing steps comprised downsampling to 250 Hz, filtering , correction of artefacts induced by blinks and eye movementsThe following segments were built (related to the S2 stimulus): cue segments ranging from −1850 ms to 100 ms and go and nogo segments lasting from −150 ms to 1150 ms. After averaging, ERP components were measured at the electrode(s) with highest amplitude: CNV , Cue-P3  and Go-P3  and Nogo-P3 .47. Buccal cells were collected from participants using buccal swabs  and stored at 4 °C. Subsequently, DNA was extracted using DNeasy Blood & Tissue Kit  according to the manufacturer’s recommendation. A total of 500 ng purified DNA was used for further methylation analysis.For our study, DNA was extracted from buccal cells and analysed with the Infinium Human Methylation 450K BeadChip  by the Helmholtz-Zentrum München (Germany)et al.48 and implemented in R (v. 3.3.1) using the minfi and wateRmelon packages. It was performed on all children (boys and girls) of the complete FRANCES sample with DNA methylation data of sufficient quality available (N = 174). Illumina Background correction was applied to the raw intensity values. Probes with a detection p-value ≥ 0.001, containing SNPs or located on gender chromosomes were removed as were samples with a call rate < 97%. Raw intensity values were normalized using quantile normalization and subsequently converted to beta values, as the proportion of DNA methylated at a single CpG site . Probes with mean values below 0.01 and above 0.99, respectively, were not considered further. Values four standard deviations above or below the mean value were considered as outliers and excluded from further analysis.Preprocessing of the methylation data was based on the pipeline of Lehne 48. In this regression-based approach, a principal component analysis (PCA) of the control probes is computed first to reduce multicollinearity as control probes are highly correlated. Besides the 22 PCA factors (explaining 95% of the variance), sex and age were included as predictors in a linear regression model . As shown in Fig. Control probe adjustment was used to reduce technical bias, which appears to be superior to using experimental factors like array number or position on arrayA second PCA was performed on the residuals  to account for further variance . The first two factors of this second PCA were included as additional predictors (besides sex and age and the 22 factors of the first PCA) in a second  regression model . The resulting residuals  were used for further analysis.et al.25. We considered CpG methylation in the following genes:DRD1, DRD2, DRD3, DRD4, DRD5), DAT1/SLC6A3, COMT, ANKK1, DDC, DBHgenes involved in the dopaminergic system: dopamine receptor genes  receptor genes.other candidate genes: Selection of candidate genes was primarily based on a review by Banaschewski VIPR2 and MYT1L, i.e., those genes identified in a methylome-wide study on ADHD in boys aged 7–1224.Additionally, we analysed CpGs linked to We studied all CpGs linked to those genes as defined in the HumanMethylation450K Manifest File (v.1.2) except probes containing SNPs and virtually showing no variance (as described above). In total, 60 genes  were included in the analysis  were subjected to ANCOVAs containing the within-subject factor INCENTIVES  and the covariate ADHD . Main and interaction effects were interpreted. For the Nogo-P3 amplitude, an additional factor ELECTRODE  was introduced to take a possible differential nogo-anteriorization effect into account.2) were also considered with part. η2 > 0.01 indicating small effects, part. η2 > 0.06 reflecting medium effects, and part. η2 > 0.14 representing large effects49.For these analyses, IBM SPSS v.21.0 was used. Statistical significance was assumed if p < 0.05. Effect sizes ; ADHD-low: (>0.5/≤1); ADHD-high (>1); see Table Associations between methylation and behavioural (ADHD) level and methylation and functional level , respectively, were tested using Pearson’s correlation coefficients (r). Only functional measures that showed differential effects regarding ADHD behaviour were considered. As these measures were not correlated with age in the narrow age range of our sample, the factor age was not controlled for when testing those associations.|r| ≥ 0.3 correlations of CpGs linked to a gene with the ADHD score and one of the functional measures FDR < 0.05 for an association between a certain CpG and either the behavioural or the functional level p < 0.05 correlation for that CpG with the other level|r| ≥ 0.3 correlation of a second CpG of this gene with the level involved in criterion (2).To reduce the probability of false positive effects, four criteria had to be fulfilled:To test whether methylation findings were affected by prenatal risk factors, we additionally computed regression models for the CpGs fulfilling our statistical criteria with maternal smoking, alcohol exposure and presence/absence of maternal depressive symptomatology during pregnancy as covariates. As the 24 h-washout may be considered too short to exclude a medication effect on DNA methylation, we repeated the correlational analyses without the four participants receiving methylphenidate.If a functional measure turned out to be associated with more than one probe fulfilling the above-mentioned criteria, we computed linear regression models for the functional measure and the FBB-ADHS total score considering these probes as predictor variables . No other covariates were included in the analysis.Statistical analyses related to the methylation data were conducted using R (v.3.3.1).Supplementary MaterialTable S-2


Objective: This study investigates whether impairments associated with persistent ADHD—impaired attention allocation (P3 amplitude), peripheral hypoarousal (skin conductance level [SCL]), and adjustment in preparatory state (contingent negative variation [CNV])—reflect enduring deficits unrelated to ADHD outcome or are markers of ADHD remission. Method: Young people with childhood ADHD (73 persisters and 18 remitters) and 144 controls were compared on neurophysiological measures during two conditions (baseline and fast-incentive) of a four-choice reaction time task. Results: ADHD remitters differed from persisters, and were indistinguishable from controls, on baseline P3 amplitude and fast-incentive CNV amplitude (p ≤ .05). ADHD remitters differed from controls (p ≤ .01), and were indistinguishable from persisters (p > .05), on baseline SCL. Conclusion: Preparation-vigilance measures were markers of ADHD remission, confirming previous findings with other measures. Yet, SCL-measured peripheral hypoarousal emerges as an enduring deficit unrelated to ADHD improvement. Future studies should explore potential compensatory mechanisms that enable efficient preparation-vigilance processes in ADHD remitters. In many individuals with childhood ADHD, the symptoms and impairments persist into adolescence and adulthood . Yet othIn our recent follow-up study from childhood to adolescence and early adulthood, ADHD persistence rate was 79% . We usedFurther candidates as markers of remission are other measures that show malleability in individuals with ADHD. Using the Fast Task, a four-choice reaction time task under two conditions , we have studied the extent to which individuals with persistent ADHD can improve their performance and associated neurophysiological functions between the two conditions. The baseline condition of the Fast Task induced impairments in RTV, attention allocation (P3 amplitude), and hypoarousal (skin conductance [SC] level [SCL]) in adolescents and young adults with persistent ADHD, but each of these improved significantly between conditions in the persistent ADHD group, indicating malleability of these measures in individuals with ADHD . In the Although our recent analyses indicated that RTV consistently emerges as a marker of remission across various tasks, the most robust effect was in the Fast Task . HoweverSD] = 1.1 years) after initial assessments: A total of 110 had a diagnosis of combined-type ADHD in childhood (10 sibling pairs and 90 singletons), and 169 were control participants (76 sibling pairs and 17 singletons). Full details on this sample can be found elsewhere (DSM-IV) levels of ADHD symptoms and impairment (ADHD “persisters”), whereas 23 (21%) were below the clinical cutoff ; siblings of ADHD probands were excluded unless they had an ADHD diagnosis themselves. The final sample consisted of 73 ADHD persisters , 18 ADHD remitters , and 144 controls . At follow-up, ADHD persisters, remitters, and controls differed in age and IQ, and there were significantly more males in the remitted group than in the other two groups .From the original follow-up sample, 252 participants  had SC measured . Due to SC equipment failure, 10 ADHD-persistent participants and eight control participants were excluded. For analyses, both members of control sibling pairs formed the control group  to 3 (very often). Participants were classified as “affected” at follow-up if they scored a “yes” on ≥six items in either the inattention or hyperactivity/impulsivity domains on the DIVA and if they scored ≥2 on two or more areas of impairments on the BFIS.The diagnostic interview for ADHD in adults DIVA;  was condThe slow-unrewarded (baseline) condition followed a standard warned four-choice reaction time (RT) task . A warniThe EEG was recorded from 62 channel DC (direct current)-coupled recording system (extended 10-20 montage), with a 500 Hz sampling rate, impedances kept below 10 kO, and FCz as the reference electrode. The electrooculograms (EOGs) were recorded from electrodes above and below the left eye and at the outer canthi. The EEG data were analyzed using Brain Vision Analyser Version 2.0 . After down-sampling the data to 256 Hz, the EEG data were rereferenced to the average and filtered offline with digitally band-pass  Butterworth filters. Ocular artifacts were identified from the data using independent component analysis (ICA). The extracted independent components were manually inspected, and ocular artifacts were removed by back-projection of all but those components. Data with other artifacts exceeding +100mV in any channel were rejected. All averages contained at least 20 sweeps. P3 amplitude was analyzed as the area amplitude measure (µV*ms) at Pz between 250 and 450 ms, to reduce bias due to the varying noise levels induced by the different task conditions . For theSC data were measured by attaching a pair of reusable 8 mm diameter silver-silver chloride electrodes on the palm of the hand (thenar eminence and hypothenar eminence) of participant’s nondominant hand at the start of the testing session. A nonsaline gel was used to increase impedance and help establish an electrical signal. A constant imperceptible voltage (0.5 V) was applied. SC was recorded using PSYCHLAB SC5 24 bit equipment system, which has an absolute accuracy of ±0.1 µS  removed; the pattern of results remained the same (results are available from first author upon request). RTV and SCL data were skewed and transformed using the optimized minimal skew (lnskew0) command in Stata Version 11.1 . As these were sibling data, the data were analyzed using random intercept models and regression in Stata. The random intercept model is a multilevel regression model that can be used as an alternative to ANCOVA to control for genetic relatedness  in a repeated-measures design, using a “robust cluster” command to estimate standard errors , where 0.2 is considered a small effect, 0.5 a medium effect, and 0.8 a large effect. By controlling for differences in the baseline condition, we were additionally able to investigate whether groups differed in the slope from the baseline to fast-incentive condition, indexing the degree of change. Pearson correlations were also conducted on these measures to examine their associations with DIVA ADHD symptom scores, and clinical impairment within those who had a childhood ADHD diagnosis, with age and gender included as covariates.Age was used as a covariate in all analyses. Analyses were initially performed without controlling for IQ, but we subsequently reran all analyses with IQ as a covariate to examine IQ effects. Gender was not included as a covariate in the group analyses to avoid controlling for ADHD status . Insteadd errors . We firsThe results for comparisons involving the ADHD-remittent group are new , main effect of group , but no main group-by-condition interaction , but did not differ from controls ; ADHD persisters had significantly increased RTV compared with controls . In the fast-incentive condition, ADHD remitters had significantly decreased RTV compared with ADHD persisters  but did not differ from controls . The within-group decrease from the baseline to fast-incentive condition was significant in ADHD remitters , ADHD persisters , and controls . The slope in RTV (indexing the degree of change from the baseline to the fast-incentive condition) in ADHD remitters was significantly less steep compared with ADHD persisters , but was not significantly different compared with controls . The slope in RTV was significantly greater in ADHD persisters compared with controls .For RTV data, a random intercept model indicated a significant main effect of condition , main effect of group , and a trend level significance of group-by-condition interaction  or controls . In the fast-incentive condition, ADHD remitters showed significantly increased CNV amplitude, compared with ADHD persisters , but were not significantly different compared with controls . There was a significant within-group increase in CNV amplitude from the baseline to fast-incentive condition in ADHD remitters , ADHD persisters , and controls . In ADHD remitters, the slope in CNV amplitude (indexing the degree of change from the baseline to the fast-incentive condition) was significantly steeper compared with ADHD persisters , but did not differ compared with controls ; the slope in CNV amplitude was significantly steeper in controls compared with ADHD persisters .For CNV amplitude, a random intercept model indicated a significant main effect of condition , but no main effect of group , or group-by-condition interaction , but were not different compared with controls . In the fast-incentive condition, ADHD remitters were not significantly different in P3 amplitude compared with ADHD persisters  or controls . There was a significant within-group increase in P3 amplitude from the baseline to fast-incentive condition in ADHD remitters , ADHD persisters , and controls . The slope in P3 amplitude between the baseline and fast-incentive condition in ADHD remitters was significantly less than in ADHD persisters , but did not differ from controls ; the slope in P3 amplitude was significantly greater in ADHD persisters, compared with controls .For P3 amplitude, a random intercept model indicated a significant main effect of condition , a significant group-by-condition interaction  but no significant main effect of group , but had decreased SCL compared with controls , or between ADHD remitters and controls . Analyses between ADHD persisters and controls in the identical sample have previously been reported: ADHD persisters had significantly decreased SCL compared with controls in the baseline condition , but the groups did not differ in the fast-incentive condition . Significant within-group increases in SCL, from the baseline to fast-incentive condition, in ADHD persisters and controls have previously been reported  or controls ; the slope in SCL was steeper in ADHD persisters, compared with controls .For SCL data, a random intercept model indicated a significant main effect of condition .n = 91), ADHD impairment at follow-up correlated significantly with baseline RTV and P3 amplitude, and with CNV amplitude in the fast-incentive condition  and peripheral hypoarousal (SCL) during baseline reaction time performance, as well as to an inability to adjust the preparatory state in a changed context (CNV amplitude in a fast condition with incentives; The finding of SCL-indexed hypoarousal reflecting an enduring impairment in the baseline condition is therefore not mirroring the remission pattern observed for RTV as expected, because we have previously found a link between SCL-indexed hypoarousal and RTV in individuals with persistent ADHD, under identical testing conditions . OverallAnalyses on continuous measures of ADHD outcome further confirmed the lack of an association between SC measures of arousal and either ADHD symptoms or impairment at follow-up. The ERP markers of remission in the group analyses—P3 in the baseline condition and CNV in the fast-incentive condition—were significantly associated with the continuous impairment scores, though only RTV was significantly associated with both ADHD symptoms and impairment .Although the Fast Task paradigm is not ideal for measuring CNVs due to the different interstimulus intervals, in the fast-incentive condition, where group differences between ADHD remitters and ADHD persisters emerged, we observed a typical CNV distribution in all groups , suggestOverall, our results indicate an enduring deficit in peripheral hypoarousal during baseline RT performance in ADHD remitters, whereas preparation-vigilance processes (P3 amplitude in the baseline condition and CNV amplitude in the fast-incentive condition, (as well as RTV


Autism spectrum disorder (ASD) symptoms frequently occur in individuals with attention-deficit/hyperactivity disorder (ADHD). While there is evidence that both ADHD and ASD have differential structural brain correlates, knowledge of the structural brain profile of individuals with ADHD with raised ASD symptoms is limited. The presence of ASD-like symptoms was measured by the Children's Social Behavior Questionnaire (CSBQ) in a sample of typically developing controls (n = 154), participants with ADHD (n = 239), and their unaffected siblings (n = 144) between the ages of 8 and 29. Structural magnetic resonance imaging (MRI) correlates of ASD ratings were analysed by studying the relationship between ASD ratings and grey matter volumes using mixed effects models which controlled for ADHD symptom count and total brain volume. ASD ratings were significantly elevated in participants with ADHD relative to controls and unaffected siblings. For the entire group , mixed effect models revealed that the left caudate nucleus volume was negatively correlated with ASD ratings . The current findings are consistent with the role of the caudate nucleus in executive function, including the selection of goals based on the evaluation of action outcomes and the use of social reward to update reward representations. There is a specific volumetric profile associated with subclinical ASD-like symptoms in participants with ADHD, unaffected siblings and controls with the caudate nucleus and globus pallidus being of critical importance in predicting the level of ASD-like symptoms in all three groups. Attention-deficit/hyperactivity disorder (ADHD) and autism spectrum disorder (ASD) are both severely impairing, highly heritable neurodevelopmental disorders ,2 3]. A. A3]. ASThe presence of milder, subclinical ASD symptoms has been shown to be continuously distributed in the general population ,8,9, altAbnormalities of regional volume have been found in ASD. Cerebellum , amygdalClinical symptoms have been associated with anatomic differences, for example, abnormalities in Broca's and Wernike's areas have been related to impaired language and social communication . AdditioNo study to date has aimed to identify the regional volumetric correlates of elevated ASD symptoms within ADHD. In previous work using the same cohort we established that ASD ratings in ADHD were predicted by the interaction between global white matter (WM) and global GM volumes, with increasing ASD ratings associated with greater GM volume . We exteStructural and functional MRI studies have found the caudate nucleus to be altered (with both increased volume and activation and decreased volume and activation relative to controls) in ASD and to be associated with dysfunctions in multiple domains related to ASD, such as repetitive and stereotyped behaviour , reward Previous research has also indicated that frontostriatal areas in general play an important role in modulating reward and motivation which in turn influence the expression of ASD symptom in ADHD , 47]. S. S47]. SParticipants were selected from a follow-up (2009–2012) of the Dutch part of the International Multicenter ADHD Genetics (IMAGE) study, performed between 2003–2006 (as described in detail in –62. WritSD = .72) in the NeuroIMAGE study (www.neuroimage.nl). In order to balance the distribution of gender and age between the ADHD and healthy control groups, additional girls with ADHD  and healthy control boys (N = 50) were recruited for NeuroIMAGE. Inclusion criteria were the same for all participants, and largely consistent with IMAGE: participants had to be between 5–30 years, of European Caucasian descent, have an IQ ≥ 70 and no diagnosis of autism, epilepsy, general learning difficulties, brain disorders and known genetic disorders (such as Fragile X syndrome or Down syndrome). Relating to the NeuroIMAGE MRI protocol, participants were excluded if they were younger than 8 years or had any contra indication to MRI scanning . 79% of participants from the IMAGE study also participated in the NeuroIMAGE follow-up study with no evidence for selective attrition [At first enrolment for IMAGE, 365 families with at least one child with combined type ADHD and at least one biological sibling (regardless of ADHD diagnosis) were recruited, in addition to 148 control families with at least one child, with no formal or suspected ADHD diagnosis in any of the first-degree family members. Recruitment of ADHD families was accomplished through probands with ADHD attending outpatient clinics in the regions Amsterdam, Groningen, and Nijmegen, as well as a Vrije Universiteit Amsterdam (VU University) affiliated ADHD research institute. Control families were recruited through primary and high schools in the same geographical regions as the participating ADHD families. All family members, also those who did not participate in IMAGE, were invited for follow-up measurement with a mean follow-up period of 5.9 years  , completTo determine ADHD diagnoses at the follow-up measurement, all participants in the study were assessed using a combination of Conners' ADHD questionnaires –67 and aThe parent-reported Children’s Social and Behavior Questionnaire (CSBQ) contains 49 items on a 3-point Likert scale. All items in the CSBQ are found in Similar to previous studies ,72, an aDuring the testing day, participants were motivated with short breaks, and at the end of the day, children received a reward of €50 and a copy of their anatomical MRI scan. Informed consent was signed by all participants (parents signed informed consent for participants under 12 years of age), and the study was approved by the local medical ethical committee .3, GRAPPA acceleration 2.Whole brain T1 weighted MPRAGE images were acquired at 1.5T using an 8 channel phased array headcoil on a Siemens Sonata scanner at the VU University in Amsterdam and a Siemens Avanto MR scanner at the Donders Institute for Brain, Cognition and Behaviour in Nijmegen. A breakdown of the distribution of subjects scanned at the two sites is included in The FIRST algorithm was applied to separately estimate the left and right volumes of eight regions; amygdala, hippocampus, nucleus accumbens, caudate nucleus, putamen, pallidum, thalamus and brain stem. FIRST is part of FMRIB's Software Library (FSL) and performs both registration and segmentation . Within During registration, the input data (3D T1 images) were transformed to the MNI  152 standard space, by means of affine transformations based on 12 degrees of freedom –77. ThisAfter subcortical registration, a sub-cortical mask was applied, to locate the different subcortical structures, followed by segmentation based on shape models and voxel intensities. Absolute volumes of structures were calculated, taking into account the transformations made in the first stage . The folAn average of two T1 scans was used for volume calculation In most instances, an average of two T1 scans, that were acquired on the same testing day, were used for volume calculation (n = 509). For 28 individuals only one T1 scan was available for volume calculation.Our approach was to begin by developing an initial behavioural factors or “external” model to determine which features, other than brain regions , would affect ASD ratings, calculated as the log-transformed aggregate of the four CSBQ subscales . We used a mixed-effects general linear model  with ageNext, we developed an initial brain volumes model to determine which of the subcortical brain regions under investigation significantly affected ASD ratings. We termed this an “internal” model as it contained only brain structures. Because of the previously found effects of laterality on ASD symptoms , we sepaRidge regression is a robust way of dealing with the problem of variance inflation and parameter mis-estimation associated with correlated explanatory variables , as wereP < 0.001). We removed this collinearity by using the residuals of caudate nucleus regressed on globus pallidus values. We also included the potential for interaction between brain regions in affecting ASD rating.For the left hemisphere ‘internal’ models, the globus pallidus and caudate nucleus were returned as significant. When including the left globus pallidus and left caudate nucleus into the external model to create the final comprehensive left hemisphere model, we followed Graham's  sequentiFor the right hemisphere ‘internal’ models, ridge analysis did not reveal any significant brain structures, and thus the final comprehensive right hemisphere model collapsed to the generic ‘external’ model.As a complementary test to assess whether one side of the brain would better explain ASD ratings, we fit separate linear models for each hemisphere (right and left) of each brain structure on the log-transformed ASD spectrum scores. We then directly compared the respective fits of right and left hemispheres for each structure using separate Cox likelihood ratio tests (with the lmtest package in R) for non-nested model comparisons ,86. For The demographic characteristics of the cohort are shown in t = 5.61, P < 0.001), but ASD scores of unaffected siblings and typically developing controls did not differ   .t = 0.036, P = 0.971), which was entirely due to the ADHD diagnosis group  from the control group with respect to the effect of age, the unaffected sibling and control groups did not differ with respect to the effect of age  and the globus pallidus , which was the same as the final right-sided comprehensive model .For the internal models of the effect of brain region on ASD rating, the final left-sided model included the volumes of the caudate nucleus   . On the To test for an effect of laterality of brain region on ASD spectrum scores, we fit separate linear models for each structure and hemisphere on the log-transformed ASD spectrum scores. The respective fits of right and left hemispheres for each structure were then directly compared using separate likelihood ratio tests for non-nested model comparisons using the lmtest package in R. For each test, two comparisons are made: one for the significant improvement of right-sided models when left-sided data are included, and the other for the improvement of left-sided models when right-sided data are included. Because the likelihood ratio tests were not independent (i.e. they tested the results of models on structures that were themselves correlated), we controlled for the inflated false-discovery rate by adjusting their resulting p-values according to Benjamini and Hochberg's approach for non-independent tests   noted a Contrary to the above finding, a study with a small number of subjects (n = 12) found a positive correlation between high-order repetitive behaviour and caudate volume . FurtherThe fact that only the left caudate predicts ASD-like symptoms is suggestive of a lateralized dysfunction in ASD, something that has been reported previously . In addiIn our statistical model, the globus pallidus was not a significant factor by itself, which is largely consistent with previous studies ,99. The The absence of a significant interaction between structural volume and diagnosis indicates that the caudate and globus pallidus volumes predict ASD ratings equally for typically developing controls and unaffected siblings as for ADHD participants. This was contrary to our expectation and leads to the conclusion that dysfunction in the anatomy of the striatum may influence the degree of ASD-like symptoms independent of clinical ADHD or ASD pathology.The hypothesis that these structures would be significant in our models in controls and unaffected siblings, as well as in patients with ADHD, stems from the fact that elevated levels of ASD at a subclinical level have been reported extensively 2,5–7,48), while subclinical levels of ASD have also been shown to be continuously distributed in the general population ,5–7,48, .The higher ASD ratings noted in male subjects are consistent with previous studies , and alsThere was a significant age-related decrease in ASD-like symptoms in the participants with ADHD, which is consistent with previous cross-sectional and longitudinal studies finding age-related improvements in ASD ,104 105105. NormThe current study should be viewed in the context of some strengths and limitations. Clear strengths of the work are the large sample size, the inclusion of unaffected siblings of ADHD participants, as well as the use of the CSBQ to probe subclinical ASD symptoms in ADHD participants–an area of study that has been significantly neglected. Limitations include the cross-sectional design of the study. Future longitudinal studies of MRI-measured developmental trajectories are needed for assessing the impact of age on developing brain structures.Overall, the current results highlight a specific volumetric profile that is associated with subclinical ASD symptoms in participants with ADHD, unaffected siblings and controls. The results point to the caudate nucleus and globus pallidus volumes as being of critical importance in predicting the level of ASD-like symptoms of participants with ADHD indicating that an interaction between these two structures was a significant predictor of ASD scores.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(XLS)Click here for additional data file.


GCH1, GTP-cyclohydrolase I), involved in maintaining nitric oxide synthetase (NOS) function and normal blood pressure, that harbors many potentially adaptive variants in Tibetans. We resequenced an 80.8 kb fragment covering the entire gene region of GCH1 in 50 unrelated Tibetans. Combined with previously published data, we demonstrated many GCH1 variants showing deep divergence between highlander Tibetans and lowlander Han Chinese. Neutrality tests confirmed a signal of positive Darwinian selection on GCH1 in Tibetans. Moreover, association analysis indicated that the Tibetan version of GCH1 was significantly associated with multiple physiological traits in Tibetans, including blood nitric oxide concentration, blood oxygen saturation, and hemoglobin concentration. Taken together, we propose that GCH1 plays a role in the genetic adaptation of Tibetans to high altitude hypoxia.Tibetans are well adapted to high-altitude hypoxia. Previous genome-wide scans have reported many candidate genes for this adaptation, but only a few have been studied. Here we report on a hypoxia gene ( Tibetans are a well-known example of successful adaptation to extreme environments at high-altitude. Compared to lowlanders moving to high altitude, Tibetans show greater lung capacity, function, diffusion, and ventilation, as well as lower hemoglobin (Hb) levels, better blood oxygen saturation, low hypoxic pulmonary vasoconstriction, high nitric oxide (NO) concentrations, and lower incidence of reduced birth weight . These tHIF2α, also called EPAS1, and EGLN1) in the hypoxic pathway showing deep between-population divergence (EPAS1 and EGLN1 protect Tibetans from the over production of red blood cells (polycythemia), a common deleterious physiological response when lowlanders migrate to high altitude areas  gene is a reported candidate that shows relatively deep allelic divergence between Tibetans and Han Chinese, a sign of positive Darwinian selection (GCH1 is located on human chromosome 14 (14q22.1-q22.2), spanning 60.8 kb with six exons. Furthermore, GCH1 is a rate-limiting enzyme in the de novo synthesis of tetrahydrobiopterin (BH4). It has been reported that under hypoxia, GCH1 can promote cancer growth, and its expression and that of endothelial nitric oxide synthetase (eNOS) is upregulated (GCH1 is considered as a major factor in maintaining nitric oxide synthetase (NOS) function and normal blood pressure, and its inhibition can increase blood pressure due to NOS uncoupling, which is found in many cardiovascular diseases such as hypertension and atherosclerosis  concentration, blood oxygen saturation level, and hemoglobin concentration. Hence, GCH1 might play an essential role in high-altitude adaptation in Tibetans.We resequenced an 80.8 kb fragment covering the entire gene region of GCH1 and its flanking sequences (10 kb up-and down-stream of GCH1). For association analysis, we collected blood samples and extracted DNA from 226 unrelated adult Tibetans, whose physiological data were also collected with written informed consent. The protocol of this study was evaluated and approved by the Internal Review Board of Kunming Institute of China, Chinese Academy of Sciences.The 50 unrelated Tibetan samples were obtained from previous research . We reseFST was calculated between 83 Tibetans and three reference populations  following D-test was also performed following the published procedure  showing a significant deviation from the Hardy-Weinberg Equilibrium (HWE < 0.000 1), and those with an excessive missing genotype rate (MGR > 0.05). Four methods were used for selection testing, including two allele-frequency-based and two haplotype-based tests. Locus specific rocedure .SD) over the genome.The two haplotype-based tests included the iHS and XP-EHH tests . The iHSFunctional enrichment analyses of the candidate variants were performed using the Combined Annotation Dependent Depletion (CADD) database , which incorporates data from ENCODE  and NIH Roadmap Epigenomics using ChromHMM  .Evolutionary constraint is an indication of functional importance. We used Genome Evolutionary Rate Profiling (GERP) to evaluate how conserved a test SNP was compared with SNP-containing sequences from different species . The GERP++ method was used to calculate site-specific RS scores . A positive GERP++ score indicates evolutionary constraint, and the greater the score, the greater the level of evolutionary constraint inferred to be acting on that site.P value indicates evidence for open chromatin. The transcription factor binding site (TFBS) is indicated by the number of different overlapping ChIP transcription factor binding sites. In addition, the splice site results indicate if the tested variant is within an ACCEPTOR or a DONOR (Supplementary Table S1).The H3K4Me1 value indicates the maximum ENCODE H3K4 methylation level , suggestive of an enhancer or other regulatory activities. The H3K4Me3 value indicates the maximum ENCODE H3K4 trimethylation level, and is an indication of a promoter. The DNase n=135, 37.41±3.8 years old) at an elevation of 4 700 m and Lhasa city  at an elevation of 3 600 m. Written informed consent from all participants was obtained. For physiological parameters, we collected hemoglobin (Hb) concentration, arterial oxygen saturation (SaO2) level, and blood nitric oxide concentration, representing key adaptive physiological traits in Tibetans . Blood NO was measured using a nitric oxide analyzer .The Hb level in blood was measured using a HemoCue Hb 201+ analyzer . The SaOGCH1  as well as the flanking sequences (10 kb from upstream and 10 kb from downstream regions). In total, we sequenced 50 unrelated Tibetan individuals, as reported previously  in the 83 unrelated Tibetans, among which 245 were shared between Tibetans and the three lowland reference populations from the 1000 Genomes Project  . The remaining 139 SNPs were rare in Tibetans ( < 1.0%), and therefore not included in our analysis. The 245 SNPs were all located in the non-coding regions of <italic>GCH1</italic>.GCH1 was under selection in Tibetans, we conducted four different tests of selection, including two allele-frequency-based tests (FST and Tajima's D) and two haplotype-based tests (iHS and XP-EHH). We identified 49 GCH1 SNPs with large between-population (Tibetan vs. Han) divergence (FST > 0.2), much larger than the genome average (FST=0.03). These high-FST variants also showed high iHS and XP-EHH values (iHS > 0.2 and/or XP-EHH > 0.2) To detect whether H > 0.2) . They weTo determine the potential function of the SNPs with selection signals, we performed functional predictions using evolutionary constraint (GERP), transcription factor binding sites (TFBS), splicing motif, H3K4Me1/H3K4Me3 sites, and DNase-I hypersensitive sites. Results showed that there might be multiple functional sites, reflected by the consistent signals of different functional predictions (Supplementary Table S1).GCH1 variants contribute to the adaptive physiological traits of Tibetans, we measured blood NO concentrations, SaO2 levels, and Hb concentrations in 226 unrelated adult Tibetans (refer to methods for details). For genotyping, we selected nine tag SNPs to represent the entire gene region of GCH1. Notably, four SNPs  showed significant association with blood NO levels, with the adaptive alleles exhibiting decreased NO. Each SNP accounted for 1.88%-3.76% of NO variance  showing significant association with blood NO levels, functional prediction analysis indicated that they were located in the EPAS1 and EGLN1 were reported to be responsible for these blunted physiological responses  as well as other deleterious physiological changes . Tibetanesponses . HoweverGCH1 in Tibetans. We identified more than 40 variants showing deep allelic divergence between highlander Tibetans and lowlander Han Chinese, with some having potential functional effects based on prediction using ENCODE. GCH1 is a rate-limiting enzyme, acting as a crucial factor for maintaining normal NO synthetase function and blood pressure. Inhibition of GCH1 activity is related to several cardiovascular diseases, with GCH1 found to prevent hypoxia-induced pulmonary hypertension , a vital regulator of eNOS, the endothelial-form enzyme producing NO, an important molecule for vasodilation, which is considered the main reason for the superior blood flow and pulmonary pressure in Tibetans (GCH1 in mice could prevent hypoxia-induced pulmonary hypertension due to the augmentation of BH4 (GCH1 regulates pulmonary vasoconstriction responses in Tibetans by influencing NO production in the blood. We observed four GCH1 variants showing significant association with blood NO levels. As these variants are located in the GCH1 intron regions with peak enhancer and/or promoter activity signals, they are likely involved in the regulation of GCH1 expression and eventually affect blood NO production, which needs further investigation. We also observed associations of GCH1 SNPs with oxygen saturation and hemoglobin; however, the underlying molecular mechanisms are yet to be studied.Hypoxic pulmonary vasoconstriction and pulmonary vascular structural remodeling are dominant pathophysiological characteristics of hypoxic pulmonary hypertension . When loTibetans . In hph-Tibetans . The oven of BH4 . Hence, GCH1 has been under positive selection in Tibetans. We identified many variants with deep allelic divergence between Tibetans and lowlanders. The association and known function results suggest the potential involvement of GCH1 in the regulation of multiple physiological traits in Tibetans.In summary, we demonstrated that We are grateful to all the volunteers participated in this study.