Secale cereale L.) has been at the basis of agriculture for centuries in most mountainous and northern areas of Eurasia, because it is more resistant than other cereals to low temperatures and poor soils. Rye deserves to be re-evaluated as a source of “environmentally resilient” genes in the future as well, and particularly in a perspective to grow cereals able to withstand global warming. According to recent studies, modern rye varieties have a relatively narrow genetic pool, a condition that is worsening in the most recent breeding processes. The preservation of local landraces as unique sources of genetic diversity has therefore become important, in order to preserve the genetic heritage of rye. In this study, genetic diversity of rye landraces collected in a sector of the Italian Alps particularly suited to traditional agriculture was investigated using the ddRADseq technique. A few landraces still managed with family farming turned out to be genetically distant from the commercial varieties currently in use, highlighting that the phenomenon of homogenization of the local genetic pool can be still circumvented. Ex situ conservation of genetically divergent landraces is a valid tool to avoid the dissipation of an as yet unexplored genetic potential.Rye ( Globally, there are approximately 382,000 species of vascular plants on the Earth  and moreSecale cereale L.), as for other crops, the replacement of local landraces with modern varieties is one of the main reason for the loss of genetic variation.Even if the genetic diversity within a crop can be broad, genetic erosion due to loss of variation and genetic vulnerability at the species level are growing matters of concern in several crops. In common rye  and barley  [Secale are open pollinated and therefore much more difficult to maintain via regeneration of seed accessions.According to a 2020 world survey genetic resources of rye include 26,100 seed accessions encompassing most of the species in the genus essions) . Rye ex Landraces are genetically heterogeneous populations generated in subsistence agriculture and, in this regard, they share with wild rye ancestors much more variation than modern varieties . Thanks In recent years, several initiatives have started to re-evaluate the importance of crop local varieties and to preserve their diversity by ex-situ and on-farm conservation, also in the perspective of feeding 50 billion people in 2050 . Rye lanClaviceps purpurea (Fr.) Tul.), rye presents several disease resistance genes reducing the need for phytochemicals [pers. comm.).In Europe, for its use as food, feed and energy in biogas plants, rye is widely cultivated in Eastern, Central and Northern Europe, particularly in Poland, Germany, Ukraine and Russia. Even if subject to the ergot disease  to Pennine Alps (Verbano-Cusio-Ossola Province) in NW-Italy. In this area, rye was traditionally the most cultivated cereal for the production of flour [Secale strictum (Presl.) Presl. .In this study, ddRAD-seq has been applied to examine the genetic diversity and structure within different of flour ,37 and oof flour . Less thThe aim of this study was to explore the genetic diversity in still-viable Italian NW-Alps rye landraces, in order to understand which of them deserve conservation efforts, due to their “authentic” adaptation to local environmental conditions. The most promising candidates will be kept as repositories of potential genetic diversity.We obtained 482,236,822 demultiplexed sequences representing 30,835 polymorphic loci among 31,595 genotyped loci. Globally we recorded 173,451 polymorphic sites. After clone-correction and informative loci filtering we obtained 17,705 loci and 119,814 polymorphic sites.p > 0.05). This is confirmed by the high differentiation  between landraces/varieties. The overall variation within individuals is significant , revealing the presence of distinctive samples significantly divergent from the other individuals, and thus the presence of an inner genetic structure ; nevertheless, a group of four landraces grouped together at an intermediate distance between S. strictum samples and all the other landraces and varieties  . The larEstimated nucleotide diversity values (π) between populations rounded around the average value of 0.00231 (±0.00031 SD) . We obseResults of the DAPC analysis helped to better understand genetic relationships between varieties and alpine landraces included in the large group of rye accessions  and an important source of livelihood, and nowadays, it has become a relict crop ,38, histSeed accessions were obtained from local farmers, institutions (EAPAM) and from two seed banks (IPK and IAR). Landraces were genetically compared with two commercial ryes, largely marketed throughout Europe. The ddRAD sequencing allowed the analysis of 17,705 loci, an unprecedented deep analysis in rye, where several recent studies do not exceed the hundreds of loci analyzed ,35.Traditional rye varieties are panmictic populations, characterized by high levels of heterozygosity and heterogeneity . PrevioHangenlad et al.  have expSelf-incompatibility in rye is not strict and, with the exception of early landraces and ancestors, mutations at two multiallelic loci, S and Z, mapped on chromosomes 1R and 2R, respectively, has led to self-fertility in the modern species. It has been proposed that self-fertility mutations have been captured in current hybrid ryes during breeding programs after introgression of a dominant self-fertility gene  from IraIn DID, STR, MIN, MUS, CAR, PEV, and SUN, conversely, F values were negative, pointing to a condition of excess of observed heterozygosity. For SUN, this result was expected in consideration of its breeding history; current hybrids were obtained starting from genetically divergent inbreeds. In open-pollinated populations, experiencing a reduction of the effective size, an heterozygosis excess occurs, i.e., recently bottlenecked populations .Under different circumstances, this can be the case in some of our rye accessions. For example, DID is repeatedly propagated at IPK to renew the ex situ accession, probably starting from a small number of seeds. In MIN, which is employed for the production of straw for packsaddles and only marginally for the grains, a condition of heterozygosity exceeding what is expected in a population at mutation drift equilibrium has most probably occurred.Breeding lines in rye are thought to be exposed to severe inbreeding depression when repetitively self-fertilized due to the high load of recessive deleterious mutations accumulated in highly heterozygous species . This seSurprisingly, EST counterpoised the highest F value with the highest nucleotide diversity (π) recorded between accessions a, meaninS. strictum Presl., which grows in southern Italy as a feral plant [S. strictum together with S. vavilovii are in fact wild ryes related to the cultivated winter rye. However, while many authors have questioned the validity of S. vavilovii as a separate species [S. strictum [S. vavilovii, it occurs throughout the Mediterranean Basin [We included in our analysis an Italian accession of al plant  and thatal plant ,54. S. s species , others an Basin ,56 in pean Basin . HoweverThe structure of alpine rye populations turned out to be composed of three main genotypes, as observed in previous analyses using microsatellites and SNP array over a very large number of accessions , and of Since the two commercial varieties were included in cluster I, we further inspected the genetic structure of this group  together with MIN and MAR , where genetic diversities are typical of many “traditional” landraces ,57.Divergent landraces of cluster II are similarly managed with family farming, despite coming from four rather distant sites. MIN was grown to be used as a filling for packsaddles and saddles. This rye has particularly short and thin stems. EST and MAR landraces showed high and thin stems, suitable for making thatched roofs, the traditional roofs from the SW-Alps villages. ARN comes from Arnad  in the NW-Alps, a peculiar area in the Italian landscape influenced by French culture, where thatched roofs were less common, but where rye stems were traditionally used for stable and barn roofs. An AFLPs analysis conducted by IAR and the University of Torino  on rye vGenetic distances between cluster I and cluster II were found to be well statistically explained by 39 loci, and eight of them were functionally assigned in previous studies . Given tNo apparent geographic structure was found among the alpine landraces of this study; a lack of correspondence between genotypes and the place of origin in rye has been documented ,28,31. RUniformity of the genetic pool of cluster I could be interpreted as a form of genetic contamination induced by random cross-pollination between different crops or depending on the farmers which deliberately mixed the different seeds. Alternatively, these landraces have originated from a common modern variety as Antoninskie itself or a very similar commercial variety.The presence of two clear genetic clusters within the W-Alps was likely to depend on the main local uses of these rye landraces; not only as foodstuffs, but also selected for roof building and the production of other manufacts. Landraces belonging to cluster II are rare, difficult to be obtained, and exposed to genetic erosion. The poor shelf life of rye seeds increases the vulnerability of this germplasm. On farm and ex situ preservation of the divergent landraces is encouraged, given their tendency to hybridize and the great wealth of genes they conceal.It could be useful to remember that “authentic” local landraces are worthy of conservation, not only as part of a relict biodiversity, but also because landraces host genetic traits to respond to specific environmental stresses: traits which can be used in plant breeding and for the development of new resilient varieties.As far as landraces of cluster I are concerned, a gene flow from commercial varieties towards them seems probable. Only four up to 16 landraces were completely separated, thus further investigation into their origin would be necessary. Morphological and agronomic analyses will clarify the relationship between some of these landraces and modern varieties. Most interesting landraces selected in this study are valuable under several agronomic aspects and they can be proposed in programs of crop enrichment and enforcement.S. strictum seed accession was provided by IPK, supplying seeds from Italian Apennines, where the species grows wild.In spring 2018, we started a survey to recover local rye varieties in NW-Italy. We recovered seeds of 10 rye varieties declared as “local” by the interviewed farmers. The Institut Agricole Régional (IAR) , Ente Aree Protette Alpi Marittime (EAPAM) , and Liebniz Institute of Plant Genetics (IPK)  supplied seven landraces from Maritime, Graian, and Pennine Alps . A comme® Plant II from Macherey-Nagel  as described in the product manual, using the PW2 buffer protocol. Samples’ quality was assessed by means of a NanoDrop® ND-1000 spectrophotometer from Thermo Scientific  respecting the ranges 260/280 ≥ 1.7 and 1.8 ≤ 260/230 ≤ 2.2. We quantified samples DNA concentration between 20 and 50 ng/μL, using a Qubit® 2.0 fluorometer by Invitrogen  using the DNA BR Assay Kit. Samples were shipped to IGA Technology Services  to be prepared and sequenced for ddRAD sequencing.Globally we analyzed 19 different seed accessions. Forty rye seeds (caryopsis) for each accession were sterilized for 60 s in 70% ethanol and then gently shaken twice in a sodium hypochlorite solution  for 5 min. Seeds were placed on wet sterile filter paper in petri dishes and incubated for two days at 4 °C and then moved to 24 °C with a 16/8 h light/dark cycle. After a few days (from one to seven), all mature seeds germinated. About 200 mg of fresh material (the first two leaves) for each sample were freeze-dried and stored at −20 °C. DNA extraction was performed with the NucleoSpinddRAD (double digest Restriction Associated DNA) libraries were produced following the IGATech  custom protocol, with minor modifications with respect to Peterson et al. . Genomicr = 0.75 in order to retain only loci that are represented in at least the 75% of the population.We analysed ddRAD-seq raw sequences using Stacks v2.0  followinThe populations.structure file produced by the populations program was used as input file in the following analysis performed in the R v3.6.2 environment  with theS. strictum samples (STR). We applied the clonecorrect function to identify duplicated multilocus genotypes into the original dataset and the informloci function to remove uninformative loci, setting the MAF threshold to 0.01. Expected and observed heterozygosity were calculated with the function poppr; the inbreeding index was calculated as F = (He − Ho)/He [5 iterations, followed by 106 Markov chain Monte Carlo steps. Following Evanno et al. [http://www.ncbi.nlm.nih.gov/genbank/, accessed on 10 August 2021), then functionally annotated for the locus function (when available).The following steps were done to compare rye varieties’ genetic differentiation, excluding − Ho)/He ; and the− Ho)/He ,70. Anal− Ho)/He  was perf− Ho)/He , was useo et al. , we defio et al. . A DAPC o et al.  was perfCalibration of threshold levels for molecular characteristics against the minimum distance in traditional characteristics” [We further inspected genetic distances within rye accessions calculating Prevosti’s distances among populations  with theristics” ; thus we


Attention deficit/hyperactivity disorder (ADHD) is one of the most common mental disorders in childhood and adolescence and is associated with functional, psychosocial and cognitive impairment. As part of the second wave of the German Health Interview and Examination Survey for Children and Adolescents (2014-2017), parents of children and adolescents aged between 3 and 17 years reported whether their child was diagnosed with ADHD by a physician or psychologist. Overall, 4.4% of children and adolescents have been diagnosed with ADHD in Germany. In comparison, the KiGGS baseline study (2003-2006) showed a reduction of lifetime ADHD diagnoses of almost one percentage point over a period of ten years. The reduction of parent-reported ADHD diagnoses primarily occurred among 3- to 8-year old children and boys. The results are discussed in terms of health promotion and the introduction of health care measures. Attention deficit/hyperactivity disorder (ADHD), with its three core symptoms of inattentiveness, hyperactivity (motor unrest) and impulsivity, is one of the most common mental disorders in childhood and adolescence . ADHD isAccording to the diagnostic criteria used by the classification systems  and diagnostic guidelines (e.g. the ADHD working group of physicians in child and adolescent medicine ), a diagComorbidities are highly prevalent among children and adolescents with ADHD and vary between 60% and 80% depending on the study , 12. TheSecond follow-up to the German Health Interview and Examination Survey for Children and AdolescentsData owner: Robert Koch InstituteAim: Providing reliable information on health status, health-related behaviour, living conditions, protective and risk factors, and health care among children, adolescents and young adults living in Germany, with the possibility of trend and longitudinal analysesStudy design: Combined cross-sectional and cohort studyCross-sectional study in KiGGS Wave 2Age range: 0-17 yearsPopulation: Children and adolescents with permanent residence in GermanySampling: Samples from official residency registries - randomly selected children and adolescents from the 167 cities and municipalities covered by the KiGGS baseline studySample size: 15,023 participantsKiGGS cohort study in KiGGS Wave 2Age range: 10-31 yearsSampling: Re-invitation of everyone who took part in the KiGGS baseline study and who was willing to participate in a follow-upSample size: 10,853 participantsKiGGS survey waves► KiGGS baseline study (2003-2006), examination and interview survey► KiGGS Wave 1 (2009-2012), interview survey► KiGGS Wave 2 (2014-2017), examination and interview surveywww.kiggs-studie.de/englishMore information is available at This study presents lifetime prevalences of parent-reported ADHD diagnoses from the second wave of the German Health Interview and Examination Survey for Children and Adolescents . Furthermore, it also describes time trends for a period of ten years by comparing data with the KiGGS baseline study (2003-2006).New data for action. Data collection for KiGGS Wave 2 has been completed in issue S3/2017 as well as KiGGS Wave 2 cross-sectional study – participant acquisition, response rates and representativeness in issue 1/2018 of the Journal of Health Monitoring [The German Health Interview and Examination Survey for Children and Adolescents (KiGGS) is part of the health monitoring system established at the Robert Koch Institute. KiGGS includes repeated cross-sectional surveys of children and adolescents aged between 0 and 17 years . Both the KiGGS baseline study (2003-2006) and KiGGS Wave 2 (2014-2017) were conducted as a combined examination and interview survey. A detailed description of the methodology used in KiGGS Wave 2 can be found in nitoring , 22.The lifetime prevalence of ADHD was assessed for children and adolescents aged 3 to 17 years using parent-reported ADHD diagnoses given by a physician or a psychologist (see ). The reThe analyses were carried out using a weighting factor that corrected for deviations within the sample from the population structure with regard to age in years, gender, federal state, German citizenship and the parents’ level of education . ResultsOverall, 4.4% of children and adolescents between 3 and 17 years of age showed a parent-reported lifetime diagnosis of ADHD given by a physician or psychologist for KiGGS Wave 2 (2014-2017) . For theIn comparison to the KiGGS baseline study, the results suggest that the diagnostic gap between girls and boys may be closing, however, the data show that boys are still diagnosed with ADHD twice as often as girls. Moreover, the time trend demonstrates that lifetime ADHD diagnoses significantly dropped among 3- to 5-year-olds and 6- to 8-year-olds. No valid findings about gender specific differences within age groups can be made due to the small sample size within each group.Children and adolescents living in socioeconomically disadvantaged families are more frequently diagnosed with ADHD compared to their peers from high SES families . PreviouOver the last decade, a continuous increase in media reports and research, as well as the reported rise in the diagnostic prevalence of ADHD, have driven a broad debate involving society, health policy, medical and psychological health care professionals, and service providers in the health system (see e.g. ).This debate has resulted in the initiation of several measures related to health care policy and provision. One example is the directive issued by the Federal Joint Committee (G-BA) for the modification of drug policies aimed at a more restrictive prescription of psychostimulants (i.e. methylphenidate) in the case of children and adolescents with ADHD . The dirIt cannot be ruled out that these changes may have led to the introduction of a more restrictive diagnostic practice in the case of ADHD. This assumption is supported by the recent reduction in the lifetime prevalence of ADHD among the youngest age groups, particularly as these were at the focus of the debate and have continued to be so. Other initiatives to promote children’s health at the federal level have also been introduced. These include the German government’s strategy for the promotion of children’s health, the promotion of the national centre for early support, which began in 2007, and the medical check-up (U10) put in place in 2006. However, the impact that they might have had on the prevalence of ADHD diagnoses remains open.The results show that boys are still diagnosed twice as often with ADHD as girls. Boys are referred for treatment for ADHD more often and earlier as their behaviour tends to be more disruptive (i.e. more hyperactive or impulsive) than girls . A previConclusions about the accuracy of ADHD diagnoses, whether these follow the guidelines, or the severity of the disorder cannot be drawn from the parent-reported ADHD diagnoses collected for the KiGGS study. Additionally, the question as to whether the parents’ response behaviour towards ADHD diagnoses might have been influenced by the changing public and professional perception over the last ten years remains open.Overall, the study found a significant reduction in the diagnostic prevalence of about one percentage point over a period of ten years. Hence, it cannot be ruled out that this reduction might be a consequence of a more restrictive diagnostic practice for ADHD. This explanation is currently supported by routine data gained from statutory health insurers that report a small reduction in ADHD diagnoses .4.4% of children and adolescents between 3 and 17 years of age had received a parent-reported lifetime diagnosis of ADHD from a physician or psychologist at KiGGS Wave 2 (2014-2017).In contrast to the KiGGS baseline study (2003-2006), KiGGS Wave 2 showed a significant reduction in the prevalence of ADHD diagnoses of about one percentage point.A reduction of parent-reported ADHD diagnoses was identified among boys and 3- to 8-year old children.


Bartonella spp. are emerging pathogens transmitted by arthropod vectors, possibly including ticks. We have investigated signs of bartonellosis in Swedish patients with presumed tick-bite exposure and symptom duration of at least 6 months.Bartonella henselae and Bartonella quintana was performed in 224 patients. Symptoms, tick exposure, evidence of co-infection and previous treatments were evaluated. Seropositive patients were compared to a matched group (twofold larger and negative serology) from the same study cohort.Serological testing for B. henselae and 1% for B. quintana, with one patient testing positive to both agents. Tick bites were reported by 63% of the patients in the seropositive group and 88% in the seronegative group and presumed tick exposure was more common in the seronegative group. Animal contact was equally common in both groups, along with reported symptoms. The most common symptoms were fatigue, muscular symptoms, arthralgia and cognitive symptoms. Exposure to co-infections was evenly distributed in the seropositive and seronegative groups.Seroprevalence was 7% for Bartonella were more common in this cohort of patients than in cohorts of healthy Swedish blood donors in previous studies but lower than those in blood donors from southern Europe. Positive Bartonella serology was not linked to any specific symptom, nor to (suspected) tick-bite exposure.Antibodies to  Bartonella spp. are slow-growing facultative intracellular bacteria that may cause various diseases, both in humans and animals. These zoonotic bacteria can be transmitted directly from animals to humans (e.g. Bartonella henselae) or by different blood-sucking vectors, including lice and flies. More than 40 Bartonella spp. are known at the present time, among which at least 14 are considered to be human pathogens [Bartonella quintana, B. henselae and Bartonella bacilliformis [B. bacilliformis being endemic to the Andes mountains of South America.athogens . Most inliformis , with B.Bartonella bacterium may invade and persist in red blood cells and endothelial cells and then be transferred by different arthropod vectors [ Bartonella transmission [Bartonella spp. have been demonstrated in Ixodes ricinus ticks [Borrelia burgdorferi [Bartonella henselae and other Bartonella spp. have been demonstrated in the blood of patients exposed to ticks [The  vectors . Ticks hsmission –6. Diffeus ticks , 8, whicus ticks . This tigdorferi , 10, andgdorferi , 11, 12.to ticks .B. quintana is the cause of trench fever with recurring fever, headache and bone pain [B. henselae may cause cat-scratch disease with lymphadenopathy, fever and myalgia [B. quintana and B. henselae may cause endocarditis. Other Bartonella spp. have also been reported as the cause of endocarditis in a few patients [Bartonella spp. has been demonstrated in humans [Human bartonellosis can present with a wide range of symptoms and diseases –17. For one pain , and B.  myalgia . Atypica myalgia –21. Bothpatients . Moreoven humans  and animBartonella spp. are transmitted by ticks, an investigation for Bartonella infection may be relevant in this patient population. Therefore, we examined a cohort of Swedish patients with suspected previous tick exposure for the prevalence of antibodies against B. henselae and B. quintana and analyzed their epidemiological, clinical and baseline demographic data compared to a group of seronegative patients attending the same clinic.Patients suffering from persistent unexplained symptoms sometimes attribute these to a previous tick bite . If BartWe purposely selected participants from an exploratory study of human tick-borne infections conducted at the Center for Vector-borne Infections (CVI), Uppsala University Hospital, Uppsala, Sweden between October 2015 and December 2018 . A totalB. henselae and B. quintana by indirect immunofluorescence assay (IFA) using the Anti-Bartonella henselae/quintana IIFT Mosaic kit from Euroimmun AG  according to the manufacturer’s instructions. Titers at 1:64 were considered to be the limit value for B. henselae and B. quintana and titers at 1:128 or higher were considered to indicate seropositivity. Positive samples were titrated to end titer.Patient sera were analyzed as part of routine diagnostics at the Uppsala University Hospital, Uppsala, Sweden for IgG antibodies against Borrelia burgdorferi, Borrelia afzelii and Borrelia garinii  and Anaplasma phagocytophilum . Serological tests (IFA) for antibodies against Babesia microti and Babesia divergens were performed at the Public Health Agency of Sweden, Solna, Stockholm and the National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands. Serological testing (IFA) and PCR assays for Rickettsia spp. and tick-borne encephalitis virus  and the PCR assay for Candidatus Neoehrlichia mikurensis in blood were performed at Sahlgrenska University Hospital, Gothenburg, Sweden as previously described [All patients were also examined for other tick-borne infections known to be present in Sweden, such as serological testing for escribed .The Chi-square test and Fisher’s exact test were conducted using IBM SPSS Statistics .B. quintana or B. henselae, or both. An anti-Bartonella spp. IgG-seronegative group of 32 patients was matched for age, sex and region of residence from the same cohort of patients investigated at the CVI. The seropositive group included 10 women and six men with a mean age of 57 (range 23–77) years; the seronegative group comprised 20 women and 12 men with a mean age of 56 (range 20–80) years. Most patients in both groups (88%) were from central Sweden, with the majority  from the region of Uppsala and Stockholm. The remaining patients were from the southern part of Sweden; none came from the northern region of Sweden.The study included 224 patients, of whom 16 (7%) were seropositive for antibodies against B. henselae, one against B. quintana and one against both B. henselae and B. quintana. Anti-Bartonella IgG titers against B. quintana were between 1:64 and 1:512. The patient with the highest titer was also seropositive for B. henselae (titer: 1:64). For B. henselae, nine patients had a titer of 1:64; four, 1:128; one, 1:256; and one, 1:512. Seroprevalence was 7% for B. henselae and 1% for B. quintana in the investigated cohort.Sixteen patients (7%) were seropositive: 14 against Candidatus Neoehrlichia mikurensis in one of the Bartonella-seropositive patients. Previous exposure to, but no ongoing infection of B. burgdorferi, A. phagocytophilum and Rickettsia spp. was demonstrated. The number of patients in each group is listed in Table B. divergens or B. microti. There was no difference between the groups in the number of patients with previous exposure to tick-borne pathogens.The search for other tick-borne pathogens revealed a possible infection with Bartonella reported tick bites approximately once per year. Although the difference was not statistically significant, the frequency of tick bites tended to be higher in seronegative patients. Furthermore, when the prevalence of tick bites was investigated in all anti-Bartonella spp. IgG-negative patients in the entire cohort investigated at the CVI clinic (n = 208), 79% reported tick bites. All participants in the present study reported that they walked in woods and fields over the years. Patients in the seronegative group reported berry picking (P = 0.012) and being on a boat or in the archipelago (P = 0.005) more frequently than patients in the seropositive group. When we looked at tick exposure only in the group of patients with a titer > 1:64 (i.e. 7 patients) and compared these patients to the seronegative group, we found was no difference in terms of the factors related to tick exposure. Travel history was not assessed due to incomplete or missing information on this variable from the patients.Epidemiological factors related to tick exposure are shown in Table Bartonella-seropositive and -seronegative patients are listed in Table Borrelia, which was interpreted as previous neuroborreliosis. There was no significant statistical difference between the two groups in terms of symptoms. Comparison of the symptoms in the group of patients with a positive anti-Bartonella spp. IgG titer > 1:64 (i.e. 7 patients) to those of the seronegative group revealed that cognitive symptoms were more common (P = 0.022) in the seronegative group. However, this finding should be considered uncertain due to the small size of the groups.Reported symptoms in Bartonella-seropositive and Bartonella-seronegative patients is shown in Table Bartonella-seropositive group were recommended doxycycline treatment; one patient was positive for Candidatus Neoehrlichia mikurensis in the blood and had a titer against B. henselae of 1:128, and the other patient had a titer of 1:256 against B. henselae that persisted during follow-up 2 months later. These findings together with long-lasting diffuse symptoms and no previous treatment with antibiotics effective against Bartonella infection resulted in the recommended treatment.Previous antibiotic treatment in Bartonella infection should be included in this investigation has yet to be determined. In the present study of Swedish patients with presumed tick exposure and persistent unexplained symptoms, we demonstrated a slightly higher seroprevalence against B. henselae and B. quintana than reported in previous studies of healthy blood donors [Bartonella serology could not be linked to any specific symptom or to a higher prevalence of any epidemiological risk factor related to tick exposure.Many patients suffer from persistent medically unexplained symptoms, and if these patients have been exposed to ticks, an investigation into possible tick-borne infection may be advisable. Whether d donors , 26. HowBartonella spp. in ticks (including I. ricinus), both in Europe and the USA [Bartonella spp. in ticks varies greatly from only 0.6% in Denmark [Bartonella spp. in ticks has been investigated in two studies: one on 167 ticks [Bartonella spp. in the examined ticks.Ticks are considered to be one of the most important vectors in Europe, but infected patients may not always have noted a tick bite . In seve the USA , 6, 12.  Denmark  to 11.8% Denmark . In Swed67 ticks  collecte67 ticks . Both stBartonella spp. differs between countries. In Europe, the seroprevalence against B. henselae in healthy populations is higher in the central and southern countries, ranging from 19% in Germany to 23% in Austria to 57% in Croatia [B. henselae is 3% in orienteers [Bartonella spp. in populations of homeless and addicts is also known from other studies [B. henselae, which is higher than that reported previously in Sweden (except for homeless persons), even in persons at high risk of exposure to tick bites (such as orienteers). The prevalence of antibodies against B. quintana was 1% in the current study, which is somewhat higher than previously demonstrated in healthy blood donors (0.2–0.3%) [B. henselae and B. quintana, which may represent cross-reactivity; the cross-reactivity between these species has been shown in other studies to be high [Bartonella spp.The seroprevalence of antibodies against  Croatia –33. In Sienteers , 14% in ienteers  and 29% ienteers , and ranienteers , 26. A h studies , 36, 37..2–0.3%) , 26 but .2–0.3%) , 35. One be high , 39. UnfBartonella serology. Rather, there was a tendency to a higher prevalence of some factors in the group with negative serology; these included, in particular, a higher frequency of gardening, berry picking in forested areas and being on a boat in the archipelago. Also, previous tick bites tended to be more common in the seronegative group, as did possession of a cat. This latter finding was against expectation, as it was expected that the seropositive group would have more contact with cats given that cat scratches are the primary mode of transmission of B. henselae to humans [B. henselae [B. henselae with culture or PCR assays the prevalence was 2% [The patients included in the present study were all being investigated for possible tick-borne infections. Hence, all had most likely been exposed to ticks to some extent. However, our investigation of the different epidemiological factors associated with tick exposure did not show a higher prevalence of any factor in patients with positive o humans . Howeverhenselae , and whee was 2% .Bartonella spp. [B. burgdorferi being the most common. No difference between the two groups was noted for co-infections. As a result of the study design, a high prevalence of exposure to various tick-borne pathogens is not surprising.Co-infections are known to occur both in ticks and humans, including lla spp. , 42. MosB. henselae and usually presents with fever and enlarged lymph nodes. However, atypical symptoms occur in up to 20% of patients, mostly in adults [Bartonella seroprevalence and a high rate of positive Bartonella PCR in the blood [Cat-scratch disease is caused by n adults . Neurolon adults , and musn adults . An invehe blood . The symB. henselae and B. quintana in presumed tick-exposed patients than reported previously in blood donors in Sweden. However, Bartonella-seropositive patients did not have a higher prevalence of epidemiological risk factors associated with tick exposure, nor did they have more tick bites, when compared to Bartonella-seronegative patients from the same study cohort. Symptoms were also the same in both seropositive and seronegative patients. Accordingly, these findings do not add support to ticks being vectors for Bartonella spp. transmission. Why the prevalence of Bartonella antibodies is higher in this population remains to be determined.We have demonstrated a somewhat higher seroprevalence of antibodies against


Attention-deficit hyperactivity disorder (ADHD) is a neurological and neurodevelopmental childhood-onset disorder characterized by a persistent pattern of inattentiveness, impulsiveness, restlessness, and hyperactivity. These symptoms may continue in 55–66% of cases from childhood into adulthood. Even though the precise etiology of ADHD is not fully understood, it is considered as a multifactorial and heterogeneous disorder with several contributing factors such as heritability, auxiliary to neurodevelopmental issues, severe brain injuries, neuroinflammation, consanguineous marriages, premature birth, and exposure to environmental toxins. Neuroimaging and neurodevelopmental assessments may help to explore the possible role of genetic variations on ADHD neuropsychobiology. Multiple genetic studies have observed a strong genetic association with various aspects of neuropsychobiological functions, including neural abnormalities and delayed neurodevelopment in ADHD. The advancement in neuroimaging and molecular genomics offers the opportunity to analyze the impact of genetic variations alongside its dysregulated pathways on structural and functional derived brain imaging phenotypes in various neurological and psychiatric disorders, including ADHD. Recently, neuroimaging genomic studies observed a significant association of brain imaging phenotypes with genetic susceptibility in ADHD. Integrating the neuroimaging-derived phenotypes with genomics deciphers various neurobiological pathways that can be leveraged for the development of novel clinical biomarkers, new treatment modalities as well as therapeutic interventions for ADHD patients. In this review, we discuss the neurobiology of ADHD with particular emphasis on structural and functional changes in the ADHD brain and their interactions with complex genomic variations utilizing imaging genetics methodologies. We also highlight the genetic variants supposedly allied with the development of ADHD and how these, in turn, may affect the brain circuit function and related behaviors. In addition to reviewing imaging genetic studies, we also examine the need for complementary approaches at various levels of biological complexity and emphasize the importance of combining and integrating results to explore biological pathways involved in ADHD disorder. These approaches include animal models, computational biology, bioinformatics analyses, and multimodal imaging genetics studies. Severity status and symptoms of ADHD vary throughout a person’s lifespan; however, adult individuals with ADHD show less noticeable signs of hyperactivity and impulsivity than pediatric patients with ADHD6. ADHD may occur either as an isolated condition or as comorbidity with other neurological, psychiatric, and neurodevelopmental disorders8. The adverse impact of ADHD on society is profound and multifaceted as it affects not only all aspects of a child’s life but also those of siblings and parents, causing significant disturbances to routine family functioning10. Furthermore, there are financial burdens related to treatment costs and reduced employment prospects11. Based on the severity of the ADHD disorder, it may affect the child’s performance at school, and if not treated or left undiagnosed, it may persist into adulthood, affecting both personal and professional life11.Attention-deficit hyperactivity disorder (ADHD) is a clinically heterogeneous neurobiological disorder of inattention, impulsivity, and hyperactivity, affecting 5–7% of children worldwide12 , the current diagnostic criteria for ADHD include subjective measurements of inattentiveness (six or more symptoms of inattention) and hyperactivity and impulsivity (six or more symptoms of hyperactivity–impulsivity)13. Several neuroimaging studies have been performed to evaluate the anatomical, microstructural, functional, biochemical, and molecular changes in the brains of ADHD16. Numerous neural networks implicating attention, executive and reward functions, and molecular pathways such as dopaminergic, adrenergic, serotonergic, and cholinergic have been identified that play a critical role in the pathophysiology of ADHD, and these pathways can be studied in great details because of the advances in the field of neuroimaging18. Molecular genomic studies, on the other hand, have observed a strong genetic influence on ADHD with a heritability estimate rate between 70 and 90%21. Several candidate genes, variants, and chromosomes associated with ADHD symptoms have been found in multiple studies focussing on correlation, linkage, and meta-analysis investigating the genetic susceptibility of ADHD. Genome-wide association studies (GWAS) have found genomic DNA copy number variants and rare or large deletion/duplications in ADHD23. Studies using fine-mapping linkage analysis found variation in the gene encoding for neuronal signaling as a potential risk factor for ADHD26. Multiple gene polymorphisms showed an association with changes in the neuropsychobiological functions in ADHD28. Most of the individual candidate gene association studies in ADHD have been conducted on a small sample size and the reported variants were not strongly associated with the ADHD pathology. Also, a recent genome-wide association (GWAS) based meta-analysis study, performed in a significantly large sample size, did not observe any association between previously reported candidate genes and ADHD29.Clinical symptoms of ADHD are associated with aberrant structural and functional changes in the brainStructural and functional imaging-derived phenotypes can be used to understand neuropsychobiology of the disorder caused by genetic changes, and this can lead to a better understanding of the clinical presentation of ADHD phenotypes. Intermediate phenotypes are those characteristics of a disorder that are correlated explicitly with its neurobiology than clinical features and contribute to the disorder’s genetic susceptibility itself. Phenotypes derived from neuroimaging, such as structural, functional, and molecular characteristics considered to be of high inheritance, are shown to be significantly altered in ADHD and found to be vital intermediate phenotypes for ADHD. This review aims to provide an overview of the structural and functional changes in the ADHD brain and their interactions with complex genomic variations. We also discuss the requirement for combinatorial methodologies that can be achieved by combining neuroimaging-derived phenotypes with genomic components to better understand ADHD neuropsychobiology.37. Changes in the ADHD brain can be classified into various categories, as described in more detail in the following sections.The behavioral/cognitive profiles in ADHD are likely to be controlled in early childhood by the structural, functional, and molecular brain changes. Many characteristic attributes, such as gene mutations/polymorphisms, neural development, neuronal maturation, neuronal functions, cortical and subcortical structures, metabolite levels, brain blood flow, and connectivity patterns, are abnormal in the brain of ADHD patients40. Several meta-analyses studies using automated voxel-based morphometric analysis have been performed to explore the global and regional gray matter volume changes in ADHD44. A meta-analysis, based on 14 studies, observed that patients with ADHD demonstrated a reduced overall volume of gray matter, particularly in the right caudate and lentiform nuclei43.Magnetic resonance imaging (MRI) is a widely used noninvasive modality for mapping in vivo brain changes in various neurological disorders. Whole-brain volumes, mainly gray and white matter volumes, have been used to differentiate patients with ADHD from healthy controls. T1-weighted high-resolution brain imaging provides superior contrast between the brain tissues and is frequently used to measure brain tissue changes, including gray matter and subcortical regions in different neurological disorders, including ADHD. MRI studies in patients with ADHD have shown lower overall brain gray matter volume (3–5%) than controls44. A cross-sectional MRI study showed a reduction in global brain volume by 2.5% and total gray matter volume by 3% in ADHD patients than control. These patients have shown significantly lower caudate nuclei and putamen volumes on regional brain analysis31. Another study relatively conducted on a large number of subjects including 307 subjects with ADHD, 169 siblings without ADHD, and 196 healthy controls reported lower gray matter volume in 5 brain sites, including the medial and orbitofrontal cortex, precentral gyrus, and para cingulate cortices in the ADHD group. In comparison, siblings without ADHD exhibited a pattern of lower volumes of gray matter in all brain sites except the precentral gyrus compared to healthy controls45.Furthermore, a meta-analysis involving 931 patients with ADHD and 822 control subjects found a reduction in the volume of gray matter in bilateral basal ganglia and insular regions in ADHD patients compared to controls47. A higher degree of gyrification is necessary to maximize the cortical surface area while preserving the compact brain size. The gyral and sulcal folding in the brain relates to the efficient corticocortical connections and neuronal fibers compaction. The abnormal cortical folding signifies deficits in structural and functional connectivity. Several studies have reported changes in cortical thickness, showing both increased and decreased cortical thickness in ADHD55. A longitudinal study found a lower global cortical volume in ADHD patients, driven mainly by a reduction in the volume of the front lobe associated with a smaller surface area and gyrification, and almost all changes remained significant throughout the development56. Another study showed a synchronized delay in cortical thickness growth32 and surface area in children with ADHD, suggesting that there could be an overall slow cortical maturation in ADHD that could lead to abnormal neural functions33. Normal brain development in early childhood tends to increase grey matter volume, and throughout neuronal development, populations are pruned to provide optimal functional efficiency. Alterations in gray matter or cortical thickness reflect abnormal neuronal maturation. Shaw et al. observed a delay of 2–3 years in cortical thickness development in both motor and sensory cortices in children with ADHD compared to age-matched controls38. The surface area, another important parameter to assess brain maturation, has been found to be associated with developmental delay in ADHD, predominantly in the right prefrontal cortex33. A cross-sectional meta-analysis study on children and adults with ADHD (age range: 4–63 years) using enhancing neuroimaging genetics through meta-analysis observed significant smaller subcortical volumes from multiple brain regions such as caudate, putamen, accumbens, amygdala, hippocampus, and reduced total intracranial volume in ADHD relative to healthy controls37. Collectively, these studies suggest that the brain is altered in a more widespread manner in ADHD than has been previously hypothesized.Multiple studies were conducted to identify neurodevelopmental changes by analyzing cortical thickness, surface area, subcortical volume, and gyrification index in ADHD59. DTI is an MRI-based technique used to characterize the microstructural tissue integrity  and microfiber pathways by using diffusion properties of water molecules. Fractional anisotropy (FA) measures the directionality of water diffusion of the underlying tissue structures, while mean diffusivity (MD) estimates the magnitude of water diffusion in tissue. Both FA and MD are the most frequently used DTI metrics for quantitative estimation of the white matter integrity in various neurological and neurodegenerative disorders. Greater FA values are associated with higher directionality of diffusion, especially in white matter regions, and may suggest intact axonal integrity, while higher MD values are related to loss of myelin and tissue’s integrity. Other DTI-derived metrics such as axial diffusivity and radial diffusivity provide valuable information about the degree of axonal integrity and myelination62. A meta-analysis based on DTI findings observed significant microstructural tissue abnormality mainly in the white matter areas of the frontostriatal-cerebellar neurocircuitry in ADHD compared to healthy participants34. It has been reported that decreased FA in the corpus callosum of adult patients with ADHD was attributed to deviations in radial diffusion instead of axial diffusion, indicating abnormal myelination process may be a dominant factor for poor neurobiological performance in adult patients with ADHD35. A study on an adolescent patient with ADHD showed lower FA and higher radial diffusivity in multiple brain sites, including corpus callosum and major fiber tracts in the left hemisphere, and the lower FA values were correlated with the inhibition performance in ADHD57. Another tract-based analysis performed in relatively bigger sample size on adult patients with ADHD showed reduced FA and increased MD and radial diffusivity in various brain regions35. A meta-analysis of DTI-derived tract-based spatial statistics observed reductions in FA from corpus callosum regions that extended to the right cingulum region, the left tapetum, and the right sagittal stratum. This study also reported that the reduced FA in splenium was negatively correlated with the age of ADHD patients58. Using whole-brain voxel-based morphometry analysis, a recent meta-analysis documented both decreased and increased FA from multiple brain regions in patients with ADHD compared to typically developing (TD) children59. Integrating the structural imaging-based morphometric and diffusion tensor imaging-based tractography, it has shown that the altered fronto-accumbal circuit was associated with a higher frequency of aggression in ADHD children. These findings implicate an important role of the fronto-accumbal circuit in the pathophysiology of ADHD, which can be further explored to treat aggressive behaviors in ADHD children63.White matter tissue makes deep brain regions and consists of bundles of myelinated axons. It is organized into tracts and primarily involved in coordinating communication between different brain areas. Intact white matter is responsible for improved learning and brain functions. Various neurological and psychiatric disorders, including ADHD, are associated with white matter abnormalities, as revealed by several diffusion tensor imaging (DTI) studies68. The fMRI quantifies the cerebral activity based on oxygen consumption by active neuronal cells, resulting in a shift in the blood oxygenation level. The fMRI can be used to measure changes in the neuronal activity against a specific task and at the resting-state fMRI (rs-fMRI). fMRI studies have reported altered neuronal signals in multiple brain sites, especially in the prefrontal and cerebellum region in patients with ADHD76. Task-based fMRI is performed mainly to explore brain activity against a specific task. In response to working memory, inhibitory control, and attentional tasks, ADHD patients showed lower activation in frontostriatal, parietal, and attentional networks than healthy controls69. A meta-analysis fMRI study against attention and inhibition tasks showed functional abnormalities in two different domains connected to fronto-basal-ganglion networks in ADHD patients70. The fMRI study based on reward task demonstrated decreased activation in the striatum brain region in ADHD patients compared to control71. Another reward task-based fMRI study observed increased activation in the anterior cingulate, anterior frontal cortex, cerebellum, orbitofrontal, occipital cortex, and ventral striatum in children with ADHD compared to control72. The meta-analysis study related to time-based task showed decreased activation in the brain area responsible for the timing, including the insular, cerebellum, and the left parietal lobe, in patients with ADHD compared to healthy controls70. Recently, a stop-signal task-based fMRI study showed a significant hypoactivation in the left superior frontal, inferior frontal, medial frontal, and bilateral temporal and parietal areas in children with ADHD compared to siblings without ADHD and healthy participants, suggesting hereditary patterns in the activation.Functional MRI (fMRI) is a neuroimaging technique widely used to measure brain activity in vivo77. Multiple metrics are quantified for rs-fMRI, including regional homogeneity (ReHo) and amplitude of low-frequency fluctuation (ALFF). The ReHo quantifies the regional similarity of the brain activity, while ALFF measures the brain signal variability of a given voxel. The rs-fMRI showed brain abnormalities in various domains, including sensorimotor, default mode network, cerebellum, cortex, anterior cingulated cortex, and other related brain sites in ADHD patients78. Abnormal global and local brain neural activity has emerged as discerning parameters to distinguish ADHD from healthy subjects80. It has been reported that atypical resting-state functional connectivity belongs to the cortical–striatal–thalamic circuitry74, and this connectivity is mostly associated with the neuropsychological status of ADHD76. Another study demonstrated that changes in the default mode network correlated with the behavioral changes in ADHD81. Emotion regulation is a common issue in children with ADHD and is shown to be associated with altered amygdala–cortical resting-state functional connectivity82.The rs-fMRI is considered a powerful technique and has recently had considerable interest in ADHD neuroimaging studies. It has been shown that at resting-state spontaneous fluctuations in blood oxygenation level were observed in the human brain without any functional task83. It was further suggested that the possible cause of hypoperfusion and abnormal vascular response in patients with ADHD could be attributed to impaired dopamine and nitric oxide systems84. Second, a lateralization trend was observed, as hypoperfusion areas were located primarily in the left cerebral hemisphere. Some earlier studies86 have also reported atypical lateralization  in ADHD patients with altered CBF and brain connectivity observed from the left hemisphere. Taken together, these findings indicate that one potential component of ADHD is the over-aroused right cerebral hemisphère.In a recent study, Tan et al. combined pseudocontinuous arterial spin labeling and rs-fMRI techniques simultaneously to study alterations in cerebral perfusion and functional connectivity in a cohort of medication naïve male adults with ADHD. The study observed several interesting findings that may help to comprehensively understand the neuropathogenesis of ADHD. First, a significant reduction in cerebral blood flow (CBF) from subcortical regions and other regions involved in functional networks such as somatomotor network, ventral attention network, the limbic network was observed compared to those of age-matched healthy controls87, the disrupted connectivity between these two regions support a notion of emotional dysregulation that is generally observed in patients with ADHD. Collectively, these findings provide valuable insights into the pathophysiological mechanisms of neurovascular coupling in ADHD.Another notable finding was that significant negative associations with severity of the disorder were found between the CBF in the left amygdala, hippocampus and the left putamen, global pallidum. In addition, the left amygdala showed significantly decreased functional connectivity with the prefrontal cortex (PFC). As the amygdala is involved in “bottom‐up” (support or influence emotion regulation) emotional processing and PFC is vital for the emotional processing in “top‐down”  regulation1H MRS) is the most commonly used MRS technique to quantify the different metabolites such as N-acetyl aspartate (NAA), choline (Cho), glutamate/glutamine (Glx), creatine (Cr), and myoinositol (mI) by targeting their aliphatic protons (−CH2 or −CH3). Collectively, these metabolites play an essential role in maintaining the brain’s structures and regulating various physiological processes; for example, NAA is considered as a marker of neuronal integrity and viability, and reduced NAA levels indicate neuronal loss. Cho is a cell membrane marker, and its elevation indicates increased turnover of cell membranes and the lower choline levels are associated with demyelinating processes. Glutamate/glutamine (Glx) are neurotransmitters of the glutamatergic system and play a key role in neuronal signaling, neuronal maturation, proliferation, and migration. Changes in the levels of Glx are associated with abnormal neurophysiology of the brain. Cr is mainly involved in phosphate metabolism and responsible for energy consumption and storage. Several 1H MRS studies have reported significant alterations in brain metabolism in ADHD patients. For instance, children with ADHD had higher levels of Glx from the frontalstriatal and right dorsolateral frontal region than healthy controls88. Also, adults with the combined type (both inattention and hyperactivity/impulsivity) of ADHD were found to have significantly reduced NAA levels in the dorsolateral prefrontal cortex relative to the inattentive type of ADHD patients and control subjects89. In addition, higher levels of Glx in the anterior cingulate cortex were observed in children with ADHD compared to children with bipolar disorder and healthy controls90. However, 1H MRS study from a large population of adult patients with ADHD found significantly lower Glx levels from the right anterior cingulate than healthy controls91. The difference in the Glx level between pediatric and adult patients with ADHD indicates that age is a key factor that regulates excitatory/inhibitory neurobiological activities in ADHD. A meta-analysis study reported increased levels of Cho from the prefrontal cortex, striatum, and anterior cingulate cortex in ADHD patients compared to healthy controls92.Magnetic resonance spectroscopy (MRS), a noninvasive imaging technique, provides a quantitative assessment of in vivo brain metabolites. Proton MRS  knock-out mouse model observed that CDH13 modulates the synaptic activity of hippocampal interneurons and cognitive domains and it was suggested to be a risk gene for ADHD108. A linkage analysis-based study on multigenerational families identified the adhesion G protein-coupled receptor L3 (ADGRL3) gene (previously known as LPHN3) variants susceptibility in developing ADHD109. Another GWAS found enrichment of rare copy number variants110, such as CHRNA7, NPY genes, and genes encoding for glutamate receptors, suggesting that rare variant involvement in ADHD neuropsychobiology is very mixed and is similar to a common variant contribution111. ADHD-related genes are spread across the entire genome but, as found on clustering analysis, they tend to be clustered into specific functional groups. A study using group comparison enrichment analyses observed that enriched functions for the ADHD-GWAS association were linked to neuronal projections, synaptic structures, nervous system structures, neuronal morphogenesis, cell–cell interaction, glutamatergic signaling113. Another GWAS on five common psychiatric disorders, including ADHD, from Psychiatric Genomics Consortium, found the association of calcium channel signaling genes with multiple psychiatric and neurological disorders, suggesting that gene variants in calcium channel activity may have pleiotropic effects in the evolution of ADHD neuropsychobiology.An International Multi-site ADHD Genetics (IMAGE) project performed an in-depth analysis of more than 50 candidate genes from European multicenter samples of around 674 families showed significant associations of several candidate genes with ADHD114. Such genes are involved in many neurobiological processes, including neurotransmitter biosynthesis, modulation of synaptic membrane dynamics, glutaminergic signaling, and various transcriptional mechanisms.Due to their strong effect on ADHD genetics, the risk genes identified in ADHD are associated with multiple processes, and some of them are known as hot genes. Hot genes are defined as candidate genes reported by at least five studies. Currently, 24 hot genes represent the top 7% of ADHD candidate geneshttps://string-db.org) visualizes the results in their genomic context , adrenoceptor alpha 2A (ADRA2A), dopa decarboxylase (DDC), dopamine receptor D2 (DRD2), 5-hydroxytryptamine receptor 1B (HTR1B), adrenoceptor alpha 2C (ADRA2C), dopamine receptor D4 (DRD4), monoamine oxidase A (MAOA), monoamine oxidase B (MAOB), catechol-O-methyltransferase (COMT), dopamine beta-hydroxylase (DBH), tryptophan hydroxylase 1 (TPH1), and tryptophan hydroxylase 2 (TPH2) out of 24 hot genes showing five or more than five connections, considered as hub genes. These clusters of genes are more connected to one another than they are to other groups of genes and thus can help identify functional modules. These genes are involved in various biological processes, molecular functions, cellular components displaying polymorphisms, and maybe the potential risk factors for ADHD  webserver. By an iterative approach, this server retrieves the genes that are indirectly (via other genes) associated with the query gene. The web interface  (http://fuma.ctglab.nl/), was employed. In the case of ADHD, a set of 24 genes was used as input. Gene enrichment analysis revealed that in the biological processes, the significant enrichment with maximum threshold was observed in neurotransmitter signaling (P = 10−28) with 19 overlapping genes (19/24); in cellular components, the significant enrichment with maximum threshold was observed in neuron projections (P = 10−16) and neuron part (P = 10−16) with 18 overlapping genes (18/24) in both the components. In contrast, in molecular functions, the significant enrichment with maximum threshold was observed in ammonium ion binding (P = 10−22) with 12 overlapping genes (12/24)  datasets grouped according to gene ontology (GO) categories related to biological processes, cellular components, and molecular functions. The MSigDB is one of the most widely used and comprehensive databases of gene sets for performing gene set enrichment analysis. For the study, a The presence of highly significant enrichment genes belongs to neurotransmitters, neuron projections, part of neurons, and ammonium ion binding, which all play a vital role in synaptic transmission, suggesting that the risk factor for ADHD is polymorphism in enriched genes.t test was performed against the remaining bands (types of tissue) per gene per tissue, and a P value < 0.05 was considered significant after Bonferroni correction. ADHD gene expression heatmap revealed higher relative expression levels across several brain tissues for the following genes; ADRA2C, COMT, dopamine receptor D1 (DRD1), DRD2, MAOA, MAOB, synaptosome associated protein 25 (SNAP25), and tyrosine hydroxylase (TH), suggesting that these genes may be considered as a risk factor for ADHD. We also performed the differently expressed gene (DEG) analysis for ADHD hot genes using GTEX v8 (54 tissue types). The DEG analyses showed significant upregulation of ADHD hot genes in the brain sites, including hypothalamus and substantia nigra suggesting the tissue-specific gene expression pattern could be associated with ADHD . Heatmap shows standardized expression value  with dark-red displaying maximum relative expression of that gene in each band, compared to a dark blue color Fig. . A two-sDHD Fig. .Fig. 6Ge29. The FOXP2 gene is located on chromosome 7, which encodes a forkhead/winged-helix transcription factor, characterized by a 100-amino acid monomeric DNA-binding domain and plays a critical role in the synapsis formation and neuronal mechanisms related to speech and learning116. The SORCS3 gene, located on chromosome 10, encodes for transmembrane receptors and is high expresses in the brain. This gene is important for neuronal development and plasticity117. On the other hand, the DUSP6 gene is located on chromosome 12 and encodes for the dual-specificity phosphatase 6 enzyme and regulates neurotransmitter homeostasis by affecting the dopamine level in the synapses. Though DUSP6 expresses at a low level in the brain but plays a critical role in brain development120.A recent GWAS study by Demontis et al. in a large sample size observed 12 independent loci surpassing the genome-wide significance threshold. The study identified three candidate genes  in the association regions125. A review article published by Gallo et al., with an intensive focus on neural circuits and genetic variants implicated in developing ADHD symptoms, discusses how circuitry abnormalities relate to symptom presentation and treatment. Authors suggested that for unraveling, ADHD’s causality can be pinpointed by exploring the endophenotype fine-tuning in both basic and clinical environments by advanced studies in animal models, performing neuromodulation, and discoveries based on pharmaco-imaging126.Advancement in imaging and molecular genomic technologies offers the opportunity to examine the impact of the genetic variations on the structural, functional, and neuronal connectivity of the brain along with the study of dysregulated biological pathways in various neurological and psychiatric disorders. Using neuroimaging genomics studies, a large body of work has demonstrated a significant association of structural and functional brain changes with genetic variations in ADHD Tables  and 2. RSLC6A3); previously known as DAT1 gene which encodes the transmembrane proteins involved in reuptake of dopamine from the synapse , which acts by inhibiting reuptake of dopamine and norepinephrine or Atomoxetine helps to increase gray matter in the prefrontal and occipital areas of ADHD children having 10/10-repeat allele with a variable number tandem repeat of 40 bp of the SLC6A3 genotype associated with ADHD compared to TD children128.A study performed on children with ADHD observed that SLC6A3 gene with two copies of the 10R allele showed lower cortical thickness in the right lateral prefrontal cortex compared to one or the absence of the 10R allele129. While investigating the effect of three SLC6A3 alleles  on striatum volume in ADHD patients, another volumetric-based study reported that carriers of the SLC6A3 haplotype 9–6 had around 6% bigger striatum volume than non-carriers130. In another seminal study, Fernandez-Jaen A et al. found that ADHD children homozygous for SLC6A3 with a 10-repeat allele had significantly higher cortical thicknesses in the right ventral and cingulate gyrus relative to 9-repeat carriers. The authors also suggested that the presence of the 10-repeat allele in ADHD influences the cortical thickness of the cingulate gyrus131.A study based on cortical thickness measurements in patients with ADHD observed that ADHD patients carrying the DRD4 7-repeat allele with lower cortical thickness in the right orbitofrontal/inferior prefrontal and posterior parietal brain sites in children with ADHD132. Another study observed that patients with ADHD having a 7-repeat allele of the DRD4 gene showed a lower volume of the superior frontal cortex and cerebellum cortex than ADHD patients without the DRD4 7-repeat allele. It was further suggested that volume changes in the brain of ADHD might indicate an intermediate morphological phenotype between the DRD4 genotype and the expression of the clinical characteristics of ADHD133. A structural MRI study has been performed to examine the effect of MPH treatment on brain structures in ADHD patients carrying the DRD4 7R allele. After treatment, increased volume of the frontal cortex and left hippocampal was observed in younger patients with ADHD, suggesting that younger patients with ADHD carrying DRD4 genotype are more sensitive to cortical remodeling after stimulant treatment134.An age-dependent study observed an association of COMT Val158Met polymorphism on grey matter in children with ADHD, and it was found that the presence of Met158-allele is associated with lower gray matter volume in the inferior frontal gyrus. However, children with ADHD homozygotes for the Val158-allele showed higher gray matter in the caudate nucleus when compared with TD children135. The COMT gene is involved in the deactivation of neurotransmitters such as dopamine, epinephrine, and norepinephrine and regulates the function of the enzymes involved in their synthesis. A study performed in ADHD to look for the differential effect of COMT on the brain of different ethnic groups observed that COMT polymorphism resulted in lower gray matter volume in the left striatum in ADHD children compared to healthy children. The COMT Met carrier ADHD children showed lower striatal gray matter volume than COMT Val/Val-genotype ADHD children. Among Caucasian children with ADHD, striatal gray matter volume alterations are correlated with the COMT Val-homozygotes. In contrast, in Japanese children with ADHD, striatal gray matter volume alterations are associated with the COMT met allele. Such findings suggest ethnic differences in the genetic effects of COMT on brain changes in ADHD patients136.A voxel-based morphometry study was performed to explore the impact of SLC6A4) gene encodes the integral membrane protein that regulates the serotonin transport from synaptic spaces into presynaptic neurons and polymorphism in this gene is associated with a higher risk of ADHD. It has been shown that SLC6A4 methylation is associated with lower cortical thickness in the right occipitotemporal region in children with ADHD137.The solute carrier family 6 member 4 (SNAP25) was associated with altered gray matter volume in ADHD patients138. The DBH gene involved in the synthesis of an enzyme dopamine beta-hydroxylase, responsible for the conversion of dopamine to norepinephrine, is presumed to play an essential role in the autonomic nervous system. A surface measurement-based study showed that DBH gene polymorphism is associated with larger left insula surface area in ADHD children with G carriers than AA homozygotes139.Polymorphism of gene coding for a synaptosomal associated protein  etc. have been shown to influence the functional brain activity in patients with ADHD  gene polymorphism on ReHo and functional connectivity in ADHD patients found that the presence of the DRD4 2R allele had both increased and decreased ReHo bilaterally in the cerebellum and the left angular gyrus, respectively. Patients with the DRD4 2R allele also showed lower functional connectivity to the left angular gyrus in the left striatum, right inferior frontal gyrus, bilateral lobes of the cerebellum, and increased functional connectivity in the left superior frontal gyrus, medial frontal gyrus, and rectus gyrus. Based on these findings, the authors suggested that DRD4 polymorphisms are associated with localized brain activity and specific functional connections150. Another study based on rs-fMRI observed decreased ReHo in the left superior occipital gyrus, cuneus, and precuneus in ADHD patients having SLC6A3 polymorphism SLC6A3 rs27048 (C)/rs429699 (T) haplotype and without the CT haplotype). Significant interactions of the ADHD disorder status (diagnosis) and CT haplotype with decreased ReHo were observed in the right postcentral gyrus127.Genetic variations, especially gene polymorphisms of the various genes such as HD Table 143–148. N-methyl-d-aspartate (NMDA) and dopamine receptor genes are found to have significant effects on functional connectivity in ADHD. A recent study investigated the effects of NMDA receptor gene glutamate ionotropic receptor NMDA type subunit 2B (GRIN2B) and dopamine receptor gene (DRD4) variants on ReHo in the ADHD group and healthy controls by using rs-fMRI. They found that the ADHD group with GRIN2B TC/TT genotype showed lower static and dynamic ReHo in the left superior parietal surface than the healthy controls. In contrast, the ADHD group with DRD4 variable number tandem repeat (VNTR)2 polymorphism showed lower dynamic ReHo in the right superior parietal surface. Considering the role of the superior parietal region in the selective attention process, decreased static and dynamic ReHo in the ADHD group in the superior parietal region may lead to worse performance outcomes during active states and may reduce the ability to respond to attention-based tasks in ADHD. Based on these findings, this study concluded that alterations in dopaminergic and glutamatergic systems contribute to impaired local functional connectivity leading to attention deficits in ADHD patients151.The DRD4 and SLC6A3 genes. The authors found that carriers of DRD4 7-repeat allele showed reduced activation in the superior frontal and middle gyrus during successful response inhibition and reduced activation in the supramarginal gyrus and parietal lobule during failed response inhibition. In contrast, SLC6A3 risk variants showed lower cerebellar activity during failed trials of successful response inhibition152. Another study explored to find interactions between variants in candidate plasticity genes  and social environments . The study showed that exposure to high positive peer affiliation was associated with the least reward speeding in serotonin-transporter-linked polymorphic region (HTTLPR) short allele carriers, while exposure to low positive peer affiliation or low maternal warmth was associated with the most reward speeding in HTTLPR short allele carriers. On the other hand, SLC6A3 10-repeat homozygotes carriers displayed the most extended reaction times when exposed to low maternal warmth. In contrast, DRD4 7-repeat carriers showed high neural activation when exposed to little maternal warmth and vice versa. Taken together, these findings emphasize the relevance of supportive social environments in sensitivity rewards and task performance with specific genotypes has differential environmental impacts153.A stop-signal task-based fMRI study was performed to measure response inhibition task in individuals carrying ADHD risk alleles of the SLC6A3 and DRD4) and by performing fMRI (go/no task). The study found that children with ADHD-RI showed reduced psychomotor speeds and higher activation of temporo-occipital regions during the Go/No-Go task as compared to TD individuals. In addition, ADHD-RI children had a higher presence of DRD4-7-repeat allele154. Another study employed DTI and N-back fMRI paradigms to investigate the effect of the DRD4-5-repeat allele on microstructural properties and functional connectivity in the brain in a healthy Asian population, mostly comprising of adolescent individuals. The study found that the presence of a 5-repeat allele was associated with poor processing speed performance, increased impulsivity, and reduced tendency to maintain attentional focus, suggesting that the presence of the 5-repeat allele of the DRD4 gene might contribute to the risk of developing ADHD142. A study conducted by Gilsbach S et al. investigated the effect of DRD4-7-repeat allele in a healthy population of children and adolescents using combined stimulus-response incompatibility task (IC) and time-discrimination task (TT). The study showed that the DRD4-7-repeat carriers demonstrated reduced neural activation of the middle and frontal gyrus in IC and reduced cerebellum activation in TT. Also, the DRD4-7 carriers showed reduced coupling between frontal brain regions compared to 7-repeat non-carriers155. An fMRI study investigated the effect of SLC6A3 VNTR polymorphism on the brain’s activity in a working memory task in ADHD and TD children. Authors observed that working memory-related activation was more significant in 9R carriers in ADHD subjects and only 10R homozygotes showed higher working memory-related activation than 9R carriers in multiple brain sites, including the parietal, temporal lobes, ventral visual cortex, orbitofrontal gyrus, and the head of the caudate nucleus in ADHD children. The findings suggest that the presence of SLC6A3 polymorphism can significantly influence the working memory in ADHD children156. A study performed a verbal n-back task in two fMRI runs to see the effect of one (9/10) copy of the 10-repeat allele of the SLC6A3 genotype in TD children. The study observed that 9/10 carriers showed more activation in frontal–striatal–parietal regions than 10/10 carriers in high load run. On the contrary, subthalamic nuclei tended to be more activated in 10/10 carriers under low load, which showed that SLC6A3 10R homozygosity is associated with reduced performance in higher demanding working memory tasks157. Another task-based study performed Go and No-Go paradigm to assess the impact of SLC6A3 3’ UTR genotype polymorphisms on brain activation in unmedicated ADHD youth and children. Youth with the SLC6A3 3’ UTR 10R/10R genotype showed higher activation in the left striatum, right dorsal premotor cortex, and bilaterally in the temporoparietal cortical junction as compared to the ADHD individuals who were heterozygous for the SLC6A3 3′ UTR 9R allele. These findings provide preliminary evidence that neural activity related to inhibitory control may differ as a function of SLC6A3 3′ UTR genotype in youth with ADHD144.ADHD heterogeneity is one of the major issues to be found in a study evaluating the neurobiological pinning of a new ADHD phenotype known as ADHD restrictive inattentive (ADHD-RI) and comparing ADHD-RI, ADHD inattentive, combined ADHD and TD individuals using genetic data involving dopamine transporters and receptor gene polymorphisms , previously known as 5HTT genes in ADHD patients159.In patients with ADHD, dopamine and serotonin-related genes play an essential role in the neurobiological response of inhibition and large-scale neural activation changes. A study observed large-scale changes in the response inhibition networks’ neural activation in prefrontal, parietal, and subcortical regions in relation to COMT polymorphism is associated with the alteration of cortico-cerebellar executive function in ADHD children using rs-fMRI. The study showed that ADHD COMT Met carriers exhibited decreased functional connectivity of right Crus I/II with the left dorsolateral prefrontal cortex compared to the ADHD children with Val/Val genotype160. Another study by Brown AB et al. showed that ADHD subjects homozygous for the SLC6A3 10R allele showed significant hypoactivation in the left dorsal anterior cingulate cortex, lateral prefrontal cortex, and cerebellar vermis as compared to SLC6A3 9R carriers. The SLC6A3 9R carriers showed greater activation in the left dorsal anterior cingulate cortex and cerebellar vermis and right lateral prefrontal cortex compared to the SLC6A3 10R carriers161. A study investigating the association between synaptosomal associated protein (SNAP25) rs3746544 polymorphism and functional connectivity density (FCD) in male children with ADHD found that rs3746544 TT homozygous carriers showed decreased local functional connectivity hubs in the anterior cingulate cortex and dorsal lateral prefrontal cortex as compared to rs3746544 G-allele carriers, suggesting that the SNAP25 polymorphism is linked with ADHD162. An additional study investigating the effect of MAOA genotype using stop signal fMRI task in adolescent boys and girls found that MAOA was correlated with ADHD symptoms and subsequently, single-nucleotide polymorphism (SNP) rs12843268 “A” hemizygotes lowered MAOA levels and reduced ventral striatal BOLD response during monetary incentive delay task. Whereas in “G” hemizygotes of SNP rs12843268 associated with higher MAOA levels and frontal gyrus and ventral striatal hyperactivation during monetary incentive delay task, and frontal gyrus hypoactivation during the stop signal task163. Another fMRI study conducted working memory tasks in adults with ADHD to investigate the effect of MAOA polymorphisms on working memory, distraction, and dual-tasking. The authors found increased activation for working memory in the lower bilateral frontal lobe and pars opercularis, and increased activation in the lingual gyrus in response to dual-tasking147.A study examined whether 1H MRS, a study investigated the relationship between SLC6A3 gene polymorphisms and brain metabolite responses in patients with ADHD following administration of MPH drug. Interestingly, no significant differences in NAA, Cr, and Cho levels were observed before and after drug administration. In ADHD individuals with SLC6A3 10R genotype, only high levels of Cr were identified in the cerebellum after MPH administration164. The increase in Cr levels (hypermetabolic state) after MPH administration might have been due to the normalization of CBF and glucose metabolism following psychostimulant therapy in ADHD. Similarly, in ADHD adults with SNAP25 gene polymorphism, another MRS study evaluated brain metabolite responses to MPH treatment. The study found high levels of NAA in adults with SNAP25 DdeI (rs1051312) and SNAP-25 MnlI (rs3746544) polymorphisms in the anterior cingulate cortex region after MPH treatment, suggesting that NAA levels in ADHD may be influenced by changes related to MPH165. Another MRS study examined changes in neurometabolite levels in ADHD adults with synapsin III (SYN3) gene polymorphisms in response to MPH. The study found higher levels of Cho in the striatum of ADHD subjects with synapsin III rs133945 polymorphism and higher NAA level in ADHD subjects with synapsin III rs133945 polymorphism166. A similar MRS study was conducted to determine the effect of treatment with MPH on brain metabolite levels in ADHD subjects with COMT gene polymorphism. Increased levels of NAA were reported in the anterior cingulate cortex and prefrontal dorsolateral cortex regions in the Val/Val and Val/Met genotype (rs4680) carriers and elevated levels of Cho were reported in the Val/Val and Val/Met genotype carriers striatum after treatment with MPH suggesting that MPH had a positive impact on impaired neuronal function and activity167. We believe that the integration of pharmacogenomics and metabolomics in future studies may open up new horizons in the diagnosis and treatment of ADHD.The brain metabolites or neurotransmitters’ levels are strictly controlled and regulated by both genetic and epigenetic factors. Gene abnormalities or polymorphisms can lead to changes in brain metabolite levels in ADHD via alterations of the cortico–striato–thalamic–cortical networks. Using 168. Notably, it is difficult to distinguish whether the symptoms are signs of ADHD or merely a sign of a young active child or symptoms of other neurodevelopmental or neuropsychiatric disorders171. The fundamental challenge in diagnosing ADHD is that the symptoms of ADHD are distributed in the general population, with a wide range of factors contributing to the etiology172. Since ADHD comprises different subtypes  and comorbidity with psychiatric conditions, a joint research strategy on ADHD and other neuropsychiatric disorders is required to define the basis of these phenotypes. The use of targeted next-generation sequencing for coding and noncoding regions can identify different genes and pathways involved in ADHD, paving the way for the development of enhanced diagnostic tools with improved treatment outcomes173. The advancement in neuroimaging and molecular genomics offers the opportunity to examine genetic variations on imaging-derived phenotypes in ADHD and the development of polygenic risk scores to predict the risk of developing ADHD and improve the diagnosis and tailored treatments. The epigenetic pathways involved in ADHD must be investigated and how they interact with genetic factors or risks involved in ADHD. Because of the broad-ranging consequences of the heterogenic nature of ADHD, it may be challenging to unravel the entire genetic profile of ADHD. However, it still appears to be achieved through genomic technology progressions and advanced neuroimaging. There is also a need to link the increasing evidence of genetic anomalies in ADHD with measures of brain dysfunction in longitudinal studies to determine whether the brain abnormalities change throughout the life cycle. It would be also helpful to conduct studies that use identical measures to assess neurobiological continuity in children and adults with ADHD. Besides clinical interventions, pharmaceutical medications that include psychostimulants are the most widely recognized medicines used to treat ADHD symptoms but have been found to be of limited utility due to the heterogeneous nature of ADHD disorder174.Accurate and specific diagnosis or profiling of ADHD symptoms remains a significant challenge in the pediatric cohortThe ADHD neurobiology is intricate and involves multiple neural pathways, but dopamine, noradrenaline, and serotonin are the critical neurotransmitters highlighted in ADHD pathogenesis. The combined efforts from psychologists, psychiatrists, geneticists, and neuroscientists have resulted in an improved understanding of ADHD etiology. Given the multifaceted symptomatology and multifactorial origin of ADHD, significant research efforts have been made over the past years to explore ADHD-related genetic and neural modifications. Notwithstanding the involvement of candidate genes and neurotransmitter systems in ADHD, genome-wide correlations between ADHD symptoms and individual genetic variants have yet to be established. Therefore, their contributions to our understanding of the etiology of ADHD are limited. Larger-scale, multicenter neuroimaging genetic approaches are now in progress, however, representing one hopeful avenue to translate this polygenic disorder’s genetic architecture. Besides, there has been considerable progress in finding vital brain circuits and regions whose structure, function, and connectivity are impaired in ADHD. One of the biggest challenges yet to be faced is developing causal relationships between these neural fluctuations and the disorder. Improved neuroimaging methods combined with experimental manipulations such as advanced neuromodulation and pharmacological approaches would probably require a deeper understanding of neural circuits and their functions. Also, relying on well-characterized animal models and the latest technologies, such as in vivo optogenetics, could allow selective manipulation of the neural circuitry involved during ADHD-related tasks. A critical future direction for ADHD research is to couple human and animal neuroimaging genetic studies to explore how the risk genes associated with ADHD neurobiology affect the brain changes in knock-out rodent models. This will help to identify the abnormal biological pathways involved in ADHD pathophysiology.The intermediate or endophenotype approach allows mapping the effects of individual risk genes on neurobiological parameters, such as brain structure, chemistry, and ultimately function. In addition, the combination of neuroimaging-related endophenotypes with genetic networks is now seen as an explanatory combinatorial model for understanding detailed ADHD pathogenesis including the generation of polygenic risk scores. The combination of neuroimaging, psychiatric genetics, and behavioral genetics will not only contribute to the diagnosis of ADHD but can also be a useful tool for personalized medicine. In the years to come, we will gain a more comprehensive understanding of ADHD, thereby allowing for new medications that are more successful than those currently in use.