It has been hypothesized that genetic and environmental factors relate to psychiatric disorders through the effect of intermediating, vulnerability traits called endophenotypes. The study had a threefold aim: to examine the predictive validity of an endophenotypic construct for the ADHD diagnosis, to test whether the magnitude of group differences at the endophenotypic and phenotypic level is comparable, and to investigate whether four factors  have an effect (moderation or mediation) on the relation between endophenotype and phenotype.Ten neurocognitive tasks were administered to 143 children with ADHD, 68 non-affected siblings, and 120 control children (first-borns) and 132 children with ADHD, 78 non-affected siblings, and 113 controls (second-borns) (5 – 19 years). The task measures have been investigated previously for their endophenotypic viability and were combined to one component which was labeled 'the endophenotypic construct': one measure representative of endophenotypic functioning across several domains of functioning.The endophenotypic construct classified children with moderate accuracy (about 50% for each of the three groups). Non-affected children differed as much from controls at the endophenotypic as at the phenotypic level, but affected children displayed a more severe phenotype than endophenotype. Although a potentially moderating effect (age) and several mediating effects  were found affecting the relation between endophenotypic construct and phenotype, none of the effects studied could account for the finding that affected children had a more severe phenotype than endophenotype.Endophenotypic functioning is moderately predictive of the ADHD diagnosis, though findings suggest substantial overlap exists between endophenotypic functioning in the groups of affected children, non-affected siblings, and controls. Results suggest other factors may be crucial and aggravate the ADHD symptoms in affected children. Psychiatric disorders as defined by the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV-TR)  have beeStudying endophenotypes may have certain advantages over studying phenotypes. It has been proposed that endophenotypes may be more suitable for detecting risk genes, because endophenotypes are genetically less complex than phenotypes (i.e. related to fewer genes than phenotypes) and, therefore, probably stronger linked to these disease genes than phenotypes -9. EndopIn the last two decades, substantial attention has been given to studying endophenotypes of psychiatric disorders. This has led to the discussion of what exactly constitutes an endophenotype and what criteria must be met for a neurocognitive function to be useful as candidate endophenotype -9,11-14.Thus far, several psychiatric conditions have been targeted for candidate endophenotypes and considerable knowledge has been gathered on the usefulness of different endophenotypes in for example, bipolar disorder, schizophrenia, substance abuse, and depression -19. ThisTwo studies have failed to find neurocognitive impairments in parents of children with ADHD ,21 and tTaken together, the majority of studies have found support for cognitive and motor endophenotypes for ADHD -31,33-35Practically all studies of ADHD endophenotypes conducted thus far have reported the same type of results: The group of non-affected siblings performs in between the affected siblings group and the normal control group. At a phenotypic level, non-affected siblings do not (or apparently to a lesser extent) deviate from the controls. The reverse appears to be true for affected children: Their phenotypic deficits are more pronounced than one would expect based on their cognitive and motor dysfunctioning. Therefore, the second aim of the paper was to confirm apparent observations that children with ADHD show more severe ADHD symptoms than one would expect based on their endophenotypic dysfunctioning, whereas non-affected siblings show less ADHD-like behaviour than one would expect based on their endophenotypic vulnerabilities.The question that automatically arises if these observations can be confirmed, is why endophenotypic vulnerabilities are not proportionally related to deviations at a phenotypic level? It might be that certain factors moderate and/or mediate the relation between endophenotype and phenotype. Moderation would imply that the relation between endophenotype and phenotype is not comparable across different levels of the moderating factor . MediatiWith respect to the first factor gender: ADHD is more frequently diagnosed in boys than girls , probablAn additional factor that may influence the relation between endophenotype and phenotype may be rater bias: Parents may underestimate the severity of ADHD symptoms in their undiagnosed/non-affected child and overestimate the severity of ADHD symptoms in their diagnosed/affected child. Teachers, however, may be less likely to be affected by this contrast effect, since they often do not have siblings of the same family in their class due to age differences between the siblings. Rater bias explaining the non-comparable magnitude of group differences at the endophenotypic and phenotypic level may thus be investigated by comparing the relation between endophenotype and phenotype as observed by parents and teachers.The first aim was to investigate if an endophenotypic construct, encompassing a broad battery of both cognitive and motor endophenotypes, is predictive of the ADHD diagnosis. The second aim was to confirm apparent observations that children with ADHD show more severe ADHD symptoms than one would expect based on their endophenotypic dysfunctioning, whereas non-affected siblings show less ADHD-like behaviour than one would expect based on their endophenotypic vulnerabilities. The third aim of this study was to explore the (moderating and mediating) effects of four factors on the relationship between endophenotype and phenotype: gender, age, IQ, and rater bias.Families with at least one child with the combined subtype of ADHD (proband) and at least one additional sibling (regardless of possible ADHD-status) were recruited in order to participate in the Dutch part of the International Multicenter ADHD Genes study (IMAGE). The IMAGE project is an international collaborative study that aims to identify genes that increase the risk for ADHD using QTL linkage and association strategies . AdditioT-scores were ≥ 63 on the Conners' ADHD-subscales  and > 90th percentile on the SDQ-Hyperactivity scale. A score of ≥ 15 on the SCQ was considered indicative of autistic like behaviours. Parents and teachers were asked to rate the behavior of the child when off medication. Concerning all children rated clinically on any of the questionnaires completed either by parents or teachers, the Parental Account of Children's Symptoms (PACS) was administered [The screening procedures and measures for phenotyping have been described elsewhere . Brieflynistered . Data frnistered . With reFull-scale IQ was estimated by four subtests of the Wechsler Intelligence Scale for Children III (WISC-III) or Wechsler Adult Intelligence Scale III (WAIS-III) (depending on the child's age): Vocabulary, Similarities, Block design and Picture completion ,56. ThesTesting of children with ADHD and their siblings took place at the VU University Amsterdam or at the Radboud University Nijmegen Medical Centre and was conducted simultaneously for all children in a family. Psychostimulants were discontinued for at least 48 hours before testing took place . ChildreThe ten experimental tasks described in this study have been fully described elsewhere -31,33,34SD 125 ms). Inter-trial intervals were 3000 ms. The delay between the go- and stop-signal was dynamically varied so that it could be estimated when the child successfully inhibited 50% of the stop-trials, and unsuccessfully inhibited the other 50%. At this point, the go-process and stop-process were of equal duration, which made it possible to estimate the latency of the stop-process: the Stop signal reaction time (SSRT) [The Stop task was used to measure speed of inhibition of an ongoing response ,60. Subje (SSRT) . A totale (SSRT) , which sShifting attentional set was designed to measure accuracy of motor inhibition and cognitive flexibility . The tasThe Timetest application version 1.0  was usedThe Visuo-spatial sequencing task was used to measure accuracy of visuo-spatial working memory . StimuliThe Digit span backwards of the WISC-III and WAIS-III ,56 was uThis task was designed to measure precision of motor control under continuous adaptation . The stiThis task aimed to measure precision of motor control without continuous adaptation required . The stiThis task measured variability of self-generated motor output . This taThis task was designed to measure variability on a simple reaction time task . StimuliSD of productions in ms). Previous results have shown that this measure to be a viable endophenotypic candidate [This task was designed to measure variability of motor timing . In thisandidate .z-scores by applying a Van der Waerden transformation (SPSS version 14). Some of the z-scores were multiplied by -1, so that the z-scores of all task variables would have the same meaning: A higher z-score was indicative of poor performance or underestimation. Correction for multiple comparisons according to the false discovery rate (FDR) controlling procedure was applied to the analyses with a q-value setting of 0.05 [Missing data for all variables for the sample described in this study was less than 5% and were replaced by means of expectation maximization . The tas of 0.05 .N = 331), thereafter repeated on the second-born children (N = 323). In this way, not only was the dependency of data handled, but this also gave the opportunity to study replicability of the results. Since most families consisted of two children, the samples of third- and fourth-borns were substantially smaller  than the samples of first- and second-borns. These sample ratio differences were considered too large (> 4) and therefore, the third- and fourth-borns were excluded from the analyses.In order to rule out dependency of the data (more than one child per family participated in the study), analyses were performed with the children split by birth order. First, analyses were run on the data from the first-borns  using a multiple discriminant analysis. Age was also entered in the model, because age differences had arisen between the first- and second-borns by splitting up the sample. There was assessed what percentage of children was correctly classified by the endophenotypic construct.The second aim of this paper was to test whether children with ADHD showed a more severe phenotype than one would expect based on their endophenotypical dysfunctioning and whether the reverse was true for the non-affected siblings. Therefore, the interaction of group  by functioning (2 within subject levels: endophenotypic construct and phenotype) was tested in a repeated measures ANOVA. Age was again implemented as covariate.a × b/√(b2sa2 + a2sb2), in which a represents the unstandardized path coefficient of the endophenotypic construct on the mediating factor, b represents the unstandardized path coefficient of the mediating factor on the phenotype, sa2 and sb2 represent the square of the standard error of the path coefficients a and b. It was examined whether the direct effect between the endophenotypic construct and phenotype was still significant after controlling for the indirect effect.The third aim of this paper was to investigate whether the factors gender, age, and IQ may moderate and/or mediate the relation between the endophenotypic construct and the phenotype. Using regression analyses, significant moderation for a factor would be demonstrated if the predictive effect of the endophenotypic construct was not constant for that factor and was tested by examining the interaction between the endophenotypic construct and the factor in predicting the phenotype . Mediatit-tests were used to compare parental and teacher ratings of ADHD to assess whether the phenotype differed when rated by parents or teachers. Additionally, in order to investigate whether the predictive validity of the endophenotypic construct was different for the phenotype as rated by the parents or by the teachers, the correlation coefficients between the endophenotypic construct – phenotype rated by parents on the one hand and the endophenotypic construct – phenotype rated by teachers on the other hand were compared using two-sided dependent samples t-tests [Rater bias could not be examined for moderation and mediation, since the phenotype was confounded with the moderating or mediating factor. Therefore, a different approach was taken. Paired sample  t-tests .2 = 1.24, df = 2, p = .54). The characteristics of both samples are presented in Table The percentage of affected, non-affected and controls was equally distributed across the first- and second-borns  = 33.24, p < .001; second-borns F  = 29.73, p < .001). In all groups, correct classification percentages were roughly around 50%. In the first-borns, respectively 55%, 52%, and 48% of the affected children, non-affected siblings, and controls were correctly classified. In the second-borns, respectively 67%, 41%, and 45% of the affected children, non-affected siblings, and controls were correctly classified.A multiple discriminant analysis was performed with age and the endophenotypic construct as predictors and diagnosis as the grouping variable. To correct for the unequal group sizes, prior probabilities for all groups were set to 1/3. The endophenotypic construct significantly predicted diagnostic status  = 45.46, p < .001, ηp2 = .22; second-borns F  = 56.89, p < .001, ηp2 = .26), suggesting group contrasts to be different at the endophenotypical and phenotypical level. When the analysis was repeated with affected children and controls, the interaction remained significant  = 67.68, p < .001, ηp2 = .21; second-borns F  = 92.13, p < .001, ηp2 = .28). As is visible in Figure F  = 0.24, p = .63, ηp2 < .01; second-borns F  = 0.02, p = .89, ηp2 < .01).The group by level interaction was analyzed to assess whether group differences were comparable at the endophenotypic and phenotypic level. This interaction was significant , which suggested that the relation between the endophenotypic construct and phenotype was comparable for boys and girls. There was evidence, however, for a partially mediating effect of gender in the first-borns , though this effect was completely non-significant in the first-borns . The significant interaction between the endophenotypic construct and age in predicting ADHD in second-borns appeared to be related to the non-affected siblings: Younger non-affected siblings performed in between their affected siblings and controls (non-affected versus affected p < .001 and non-affected versus control p = .002), but older non-affected siblings performed more like controls (non-affected versus affected p < .001 and non-affected versus control p = .54). Strong evidence was found for a partially mediating effect of age: The endophenotypic construct related to age, and age related to ADHD severity , indicating that the relation between the endophenotypic construct and phenotype to be constant across the IQ range studied here. However, there was a partial mediating effect of IQ , which was in line with our expectations. However, in contrast to our expectations, parents and teachers ratings agreed on the degree of ADHD-like behaviour in non-affected children , which is similar to the findings in control children . It appeared that parents were indeed possibly 'overestimating' the degree of ADHD in their affected child, but they did not seem to 'underestimate' the degree of ADHD in their non-affected child.Paired samples t-values between 0.47 – 2.47 and p-values between .36 – .98), when calculated separately for affected, non-affected, and control children or when calculated across groups. These findings suggest that the predictive validity of the endophenotypic construct was comparable for the phenotype as rated by the parents or by the teachers.None of the correlation coefficients between the endophenotypic construct and ADHD rated by parents, on the one hand, and the endophenotypic construct and ADHD rated by teachers, on the other hand, differed significantly from each other , which is in line with the aetiology of psychiatric disorders, in which endophenotypic vulnerabilities lead to phenotypic symptoms. The status of all groups was predicted with roughly similar percentages (around 50%). However, these percentages were only moderate and not high, indicating a substantial overlap in endophenotypic functioning of children with different diagnostic status. Thus, even an aggregated component encompassing multiple endophenotypic measures is not a firm predictor of diagnostic status and illustrates the causal heterogeneity of ADHD .In the second aim of our study, it was confirmed that affected children indeed portrayed a more severe ADHD phenotype than one would expect based on their endophenotypic dysfunctioning. This is in line with previous studies on cognitive dysfunctioning in patients with ADHD, in which not all patients portray neurocognitive dysfunctioning and effect sizes are generally modest . Based oIn line with the idea that some factors may influence the relation between endophenotype and phenotype, we analyzed the (moderating and mediating) effects of gender, age, IQ, and rater bias. No evidence was found for a moderating effect of gender, which suggests that the relation between the endophenotypic construct and phenotype in ADHD is comparable for boys and girls. There was evidence, though, for a partially mediating effect of gender in the first-borns. However, since this partial mediation was completely non-significant in the second-borns, and the direct effect between the endophenotypic construct and phenotype was still present after correcting for this partially mediating effect of gender, it appears that gender did not have a large impact on the relation between the endophenotypic construct and phenotype in ADHD. It could not account for the apparent disproportionately severe phenotype in affected children. Similar non-influential effects of gender in relation to ADHD have been documented previously ,73.Another factor possibly influencing the endophenotypic construct – phenotype relation is age. Age moderated the relation between the endophenotypic construct and phenotype in the second-borns, though not in the first-borns. This significant interaction between the endophenotypic construct and age in predicting the ADHD phenotype appeared to be related to the non-affected siblings: Younger non-affected siblings performed in between their affected siblings and controls, but older non-affected siblings performed more like controls, possibly suggesting endophenotypic vulnerabilities ease somewhat with increasing age in non-affected siblings. Strong evidence was found for a partial mediating relation of age, but correcting for this partially mediating effect of age did not result in a non-significant direct effect between the endophenotypic construct and phenotype. The findings of non-moderation of age in affected children are in line with studies showing symptoms of ADHD persist into adolescence and adulthood for the majority of patients ,74 and sLike gender and age, IQ also did not appear to account for these non-comparable group differences, since IQ did not have a moderating influence on the relationship between the endophenotypic construct and phenotype. IQ did have a mediating effect, but correcting for this mediating effect did not result a non-significant direct route between the endophenotypic construct and phenotype, suggesting the influence of IQ on this relation is not substantial. These findings give support to the idea that endophenotypic dysfunctioning and a lower IQ are both related to each other and to ADHD -49, thouAnother factor we studied was the effect of rater bias on the relation between the endophenotypic construct and phenotype. We hypothesized that parents, compared to teachers, may possibly overrate the ADHD severity in their affected children, whereas they may underrate symptoms in their non-affected children. This, in turn, might explain the disproportionate severe phenotype of the affected group compared to their endophenotypic construct. Parents indeed rated the degree of ADHD in their affected children more severely than teachers. This may be because parents wanted to secure their participation in the study  and/or bSeveral possible limitations of this study warrant consideration. Dividing the sample in first- and second-borns doubled the number of statistical tests that were performed. However, we corrected for multiple testing and this approach gave the opportunity to investigate replicability of the results and showed that almost all findings were comparable in first- and second-borns. Another possible limitation was the interpretability of the endophenotypic component measure. Since it was a composition of various cognitive and motor task variables, its exact representation remains unclear. It may be hypothesized that the endophenotypic construct taps into an underlying factor, which may represent general cognitive functioning ('g') possibly in combination with variability of reacting. We feel though, that combining individual task measures to one more robust component will facilitate heritability research in ADHD, since a component probably entails less error variance and may be a more reliable measure than individual task measures.An endophenotypic construct encompassing multiple endophenotypic measures is moderately predictive of diagnostic status, but substantial overlap exists between endophenotypic functioning in the groups of affected children, non-affected siblings and controls. Group differences at an endophenotypic and phenotypic level are not comparable for affected children, displaying a more severe phenotype than one would expect based on their endophenotype when compared to controls. Group differences were comparable for non-affected siblings compared to controls, suggesting subtle endophenotypic vulnerabilities translate proportionally into phenotypic deviations. Even though a potentially moderating effect (age) and several mediating effects  have been found affecting the relation between the endophenotypic construct and phenotype, none of the effects studied  could account for the finding that affected children deviated more from controls at the phenotypic than endophenotypic level. These findings suggest other factors come into play and aggravate the phenotype in affected children.th edition; IMAGE = International Multicenter ADHD Genes study; IQ = Intelligence Quotient; PACS = Parental Account of Children's Symptoms; SCQ = Social Communication Questionnaire; SDQ = Strengths and Difficulties Questionnaires; SSRT = Stop Signal Reaction Time.ADHD = Attention-Deficit/Hyperactivity Disorder; DSM-IV = Diagnostic and Statistical Manual for Mental Disorders 4NR, MA, NM, CB, and JO have no competing interests. SF has received grants from Eli Lilly, McNeil Consumer & Specialty Pharmaceuticals, and Shire; has been a consultant for Eli Lilly, McNeil Consumer & Specialty Pharmaceuticals, Shire, Noven Pharmaceuticals, and Cephalon; has been on the speakers' bureaus of Eli Lilly, McNeil Consumer & Specialty Pharmaceuticals, Shire, and Cephalon. JB has been a consultant to/member of advisory board of/and/or speaker for Janssen Cilag BV, Eli Lilly, Bristol-Myer Squibb, UBC, Shire, Medice. JS has been a member of advisory board of Eli Lilly, Shire, Janssen Cilag. This study was partly funded by a grant assigned to SF by the National Institute of Mental Health (NIH grant # R01 MH62873-01A1).NR contributed to the data ascertainment, study design, literature searches, analyses, and writing. MA and CB contributed to the data ascertainment and writing. NM contributed to the analyses and writing. SF, JB, JS and JO have written the study protocol and have contributed to the writing. All authors have read and approved the final manuscript.


The authors sought to map gray matter changes in Attention Deficit Hyperactivity Disorder (ADHD) using a novel technique incorporating neuro-imaging and genetic meta-analysis methods.A systematic search was conducted for voxel-based structural magnetic resonance imaging studies of patients with ADHD (or with related disorders) in relation to comparison groups. The authors carried out meta-analyses of the co-ordinates of gray matter differences. For the meta-analyses they hybridised the standard method of Activation Likelihood Estimation (ALE) with the rank approach used in Genome Scan Meta-Analysis (GSMA). This system detects three-dimensional conjunctions of co-ordinates from multiple studies and permits the weighting of studies in relation to sample size.For gray matter decreases, there were 7 studies including a total of 114 patients with ADHD (or related disorders) and 143 comparison subjects. Meta-analysis of these studies identified a significant regional gray matter reduction in ADHD in the right putamen/globus pallidus region. Four studies reported gray matter increases in ADHD but no regional increase was identified by meta-analysis.In ADHD there is gray matter reduction in the right putamen/globus pallidus region. This may be an anatomical marker for dysfunction in frontostriatal circuits mediating cognitive control. Right putamen lesions have been specifically associated with ADHD symptoms after closed head injuries in children. The core symptoms of Attention Deficit Hyperactivity Disorder (ADHD) are inattention, impulsivity and hyperactivity . The disAccording to one model of the neurobiology of ADHD, the aetiology involves a major genetic component. Gene variants, controlling proteins in catecholamine pathways, cause aberrant neurodevelopment of frontal brain networks supporting executive processes such as attention, memory and behavioural inhibition , in partIf ADHD involves abnormal neurodevelopment of brain circuits then this may be associated with structural brain changes in nodes within these circuits. There is evidence that genes controlling dopamine pathway proteins are associated with regional brain volume changes in the frontal lobe and caudate nucleus .There have been many studies investigating structural brain changes in ADHD. A meta-analysis of region of interest studies of ADHD found that regional volume reductions were present in the right caudate and frontal regions . HoweverOne problem with region of interest studies is that they usually only measure a small number of selected brain regions because the measurement process is labour-intensive. Furthermore, because of the difficulties in defining structurally complex regions of cortex, region of interest studies focus on structures that are easily definable, such as the hippocampus, at the expense of neocortical morphology. This may lead to a distorted picture of the pattern of brain changes in ADHD, with an undue concentration on relatively few cerebral structures, at the expense of a more comprehensive assessment of brain structure.The measurement problems associated with region of interest analyses have led to the development of automated 'whole-brain' techniques for analysing structural MR images. These methods have been called 'voxel-based morphometry' because scans are analysed for structural change at the level of 'voxels' – the individual elements within a three-dimensional digital image . HoweverIn this study we conduct a meta-analysis of voxel-based morphometry imaging studies of ADHD in order to localise regions where there are structural gray matter changes and avoiding the biases of region selection which may be present in region of interest studies. On the basis of the previous meta-analyses of region of interest studies  and funcStudies were considered for inclusion if they were published before February 2008 as an article (rather than a letter or an abstract), if they compared a group of subjects with ADHD (or Hyperkinetic disorder or subjects with high ADHD symptom scores) and a comparison group (either related or unrelated to the subjects), if they utilized voxel-based morphometry analysis of MRI datasets to investigate differences in whole-brain structure (gray matter density), and if they reported the three-dimensional co-ordinates of brain changes in stereotactic space.Study data were excluded if insufficient data were reported to extract the number of subjects in each group, if there were fewer than six subjects in either the ADHD group or the comparison group, or if the data contributed to another publication, in which case the publication with the largest group size was selected.Journal of Child Psychology and Psychiatry, the American Journal of Psychiatry, Archives of General Psychiatry and Biological Psychiatry. Finally, we searched the reference lists of the studies identified for inclusion. Table A systematic search strategy was used to identify relevant studies. First, we carried out a MEDLINE search using the following keywords: ADHD, magnetic resonance imaging, voxel, SPM , Talairach; the search was conducted in March 2008, and no time span was specified for date of publication. Second, a manual search was also conducted of the titles of published papers in four psychiatric journals for the period February 2007 to February 2008: Co-ordinates that were reported in the stereotactic space of the Montreal Neurological Institute (MNI) were converted to Talairach coordinates using the Lancaster transform  in GingerALE . TalairaMeta-analyses were performed using the Talairach stereotactic coordinates derived from the studies listed in Table Meta-analyses were carried out using a C++ program which modified the technique of Activation Likelihood Estimation (ALE) -10 by inFor each study, the reported loci of maximal anatomical difference were modeled as the peaks of three-dimensional Gaussian probability density functions with full-width half-maximum of 7 mm, within a brain mask of 210 069 voxels of linear dimension 2 mm. The voxels in this probability image were then ranked from 210 069 (highest probability) to 1 (lowest probability), giving voxels of equal probability a mean rank. This created a rank image for each study which was smoothed with a 7 mm Gaussian filter. These study images were then summated to create a sum-rank image.A null distribution for the sum-rank image result was derived by 10 000 permutations of the same process, but using an equal number of co-ordinates for each study derived from a random uniform distribution of coordinates within the brain mask. The voxel-wise probability of a sum-rank under the null hypothesis was calculated as the proportion of permutations giving a value equal or greater then the actual value. The data set being tested was included in the ranking of all known outcomes . The proA total of 6 articles -20 were The 6 articles provided 7 studies with data on gray matter decreases in ADHD. One article  includedThese studies included a total of 114 patients with ADHD (or related disorders) and 143 comparison subjects and provided 59 co-ordinates for gray matter decreases.3). By comparison, analysis of the co-ordinate data using an ALE approach identified 30% fewer voxels with voxelwise p = 0.0001 and this ALE result was not significant over the whole image with the False Discovery Rate set at P < 0.05.Meta-analysis of the co-ordinates from these studies (unweighted by sample size) identified a single cluster of gray matter decrease in ADHD subjects compared with controls on sum-rank analysis. This was located in the right putamen/globus pallidus region .A similar result to the unweighted meta-analysis was obtained when the meta-analysis was repeated, weighting studies by sample size, using the square root of the study participant numbers as the weighting value . In functional terms, corticostriatal circuits are thought to involve projections from cortex to caudate/putamen and back to the cortex via the globus pallidus and thalamus. The activity of these circuits is modulated by dopaminergic projections to the striatum from the substantia nigra.Evidence from a number of sources has implicated the striatum in the pathology of ADHD  includinA study of 76 children who suffered closed head injury with no prior history of ADHD identified 15 who developed ADHD symptoms. The children who developed ADHD had more lesions localized in the right putamen . AnotherThe meta-analysis of region of interest studies of ADHD found a significant regional volume reduction in the right caudate . On anatThere is evidence for white matter abnormality in ADHD in the region of the right basal ganglia. An investigation of white matter structure in familial ADHD found an association between a measure of myelination  in right frontostriatal fibre tracts and both functional activity in the frontal gyrus and caudate and performance of a go/nogo task . DisruptFunctional imaging studies have also detected abnormalities in the striatum in ADHD. In adults with ADHD, positron emission tomography studies have found lower dopamine D2/D3 receptor availability in the left caudate in ADHD . ChildreWhile activation patterns in functional MRI studies depend on the nature of the task and performance, a number of studies have identified hypoactivation of striatal networks in ADHD. Children with ADHD were found to show less activation in a right striatal-parietal network while performing a mental rotation task , less frWhen adolescents with ADHD performed a task involving a monetary incentive delay task, fMRI revealed reduced striatal activation during reward anticipation . ConversFinally, the right putamen is part of a circuit whose dysfunction has been linked to human spatial neglect . There iThere are some important limitations of this meta-analysis. Firstly, only a small number of studies have examined brain structure in ADHD using voxel-based morphometry. The gray matter reduction in the right putamen/globus pallidus region was identified as significant because of the conjunction of co-ordinates from four studies ,17,18,20a priori bias in region selection (which may occur with region of interest studies).A second limitation is that given the small number of studies in the meta-analysis, the power to detect changes was small. Heterogeneity among the studies  may also have reduced the power of the meta-analysis to detect differences. Therefore, although we did not confirm our original hypothesis of gray matter decreases in the right caudate and frontal lobes, there may be structural changes in ADHD which are present but were not identified. Therefore, we can not rule out the possibility that the gray matter decrease in the putamen/globus pallidus was bilateral (but only detected on the right), or that other striatal decreases were present (e.g. in the caudate), or that other brain regions were affected . However, one advantage of voxel-based studies is that all regions of the brain are analysed and so there is no A third limitation is that the meta-analysis does not have the spatial resolution to differentiate between a gray matter reduction in the putamen and globus pallidus. Although they are both components of the lentiform nucleus ,39 thereA fourth limitation is that there may be publication bias in the voxel-based morphometry literature on ADHD. Studies giving negative results may be less likely to be published than studies giving positive results . Negative studies should not affect the results of this meta-analysis  but a bias in favour of publishing studies with co-ordinates which the researchers considered to be in interesting locations could bias the meta-analysis to detect changes in these locations. Although the meta-analysis did not incorporate the significance level of the results returned in different component studies, one advantage of the Genome Scan Meta-Analysis approach is that it provides a systematic method for integrating studies with different analysis methods and statistical thresholds .Finally, the gray matter reduction in the subjects with ADHD may be modulated by medication. Most of the studies in this meta-analysis included patients receiving stimulant treatment. It is possible that methylphenidate (an indirect dopamine agonist) may contribute to basal ganglia volume decreases in broadly the same way that antipsychotics (dopamine antagonists) may cause basal ganglia volume increases in schizophrenia ,41. TherIn summary, this study demonstrates the application of a novel meta-analysis technique incorporating elements of Activation Likelihood Estimation and Genome Scan Meta-Analysis. The combination of these methods has theoretical advantages over the standard Activation Likelihood Estimation system, because extra significance is given to the spatial conjunction of co-ordinates when they derive from different studies (rather than a single study) and results can be weighted by sample size.The meta-analysis identified a right putamen/globus pallidus regional gray matter reduction in structural MRI studies of ADHD. The role of the right putamen in ADHD is supported by clinical studies of brain-injured children which have found a high incidence of right putamen lesions associated with the development of ADHD symptoms. Current neurobiological models of ADHD postulate frontostriatal dysfunction as a key component of the disorder and this may account for structural change in the right putamen/globus pallidus region during neurodevelopment.The presence of a structural brain change in this disorder provides an impetus to further research to establish whether the change is pathological or iatrogenic and whether it may be of diagnostic utility.IEW and ZEW disclose that they have no competing interests. EB is employed half-time by GlaxoSmithKline (GSK) and half-time by the University of Cambridge and is a stockholder in GSK.IEW developed the meta-analysis method, wrote the software and drafted the manuscript. ZEW participated in the ascertainment of studies, extraction of data and provided clinical expertise on Attention Deficit Hyperactivity Disorder. EB participated in the design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Bartonella infections are emerging in the Zhejiang Province of China. However, there has been no effort to date to explore the epidemiology of these infections in this region, nor to identify risk factors associated with exposure to Bartonella. The aim of this study was to investigate the seroprevalence of Bartonella in both patients bitten by dogs and blood donors (for control) in Eastern China, and to identify risk factors associated with exposure to Bartonella. As no previous data for this region have been published, this study will provide baseline data useful for Bartonella infection surveillance, control, and prevention.Bartonella in these samples. Risk factors associated with Bartonella exposure were explored using Chi-square tests and logistic regression analysis of epidemiological data relating to the study's participants.Blood samples were collected from industrial rabies clinic attendees and blood donors living in eight areas of the Zhejiang Province of China, between December 2005 and November 2006. An indirect immunofluorescent antibody test was used to determine the presence of Bartonella antibodies were detected in 19.60% (109/556) of blood samples. Seroprevalence varied among the eight areas surveys, ranging from over 32% in Hangzhou to only 2% in Jiangshan . We detected a significantly higher prevalence of Bartonella antibodies in people who had been bitten by dogs than in blood donors . Seroprevalence of Bartonella was similar among males  and females .Bartonella antibodies were encountered in people living across Zhejiang Province and the seropositivity rate among those exposed to dog bites was significantly higher than that among blood donors, indicating that dog bites may be a risk factor for Bartonella infection. Bartonella comprises of fastidious, Gram-negative hemotropic bacteria that infect blood erythrocytes and endothelial cells of their hosts [Bartonella have been described, of which at least 12 are recognized as human pathogens [The genus ir hosts . More thBartonella infections in Zhejiang Province is the second largest in China. However, to date, there have been no efforts to explore the epidemiology of these infections in this region, nor to identify risk factors associated with exposure to Bartonella. Directly detecting Bartonella in humans is problematic, but detection of antibodies as an indication of exposure to the agent has been widely used in previous studies. In this study, we adopted the latter approach for the first time to investigate human exposure to Bartonella in the Zhejiang Province of China.According to clinical reports, the number of patients with Patient blood samples  were collected from eight locations  of the Zhejiang Province of China from December 2005 to November 2006. The aims of our study were explained to all participants upon enrollment, and their consent was obtained prior to inclusion in this study. All enrolled participants provided information upon inclusion in the study, with regard to their age, gender, place of residence, and whether they had been exposed to dog bites.Experimental research reported in this study has been performed with the approval of the ethics committee of Zhejiang Provincial Center for Disease Control and Prevention. Human research was carried out in compliance with the Helsinki Declaration.Bartonella henselae (B. henselae)-based indirect immunofluorescence antibody (IFA) test kit  was used to assess the presence of IgG antibodies against B. henselae in human samples. After the samples were diluted 1:100, 1:320, 1:1000, 1:3200, and 1:10000 in phosphate-buffered saline PBS-Tween buffer (provided in the test kit), the IFA assay was conducted following the manufacturer's protocol. Positive and negative controls (provided in the test kit) were also used. Immunofluorescence was observed using an epifluorescence microscope at magnifications of 40× and 200×. According to the manufacturer's guidelines, a titer of 1:320 was considered indicative of an infection, and IgG antibody titers of 1:1000 or higher were judged as an indicator for acute infection.A commercially available Bartonella seroprevalence among sites, gender, age groups, and exposure to dog bites were conducted using the SPPS, version 11.0 statistical package . The dependent variable in the logistic regression was assigned as the serological status of patients and the independent variables were site, gender, age, exposure, and site × exposure. The method of logistic regression used was forward-conditional. The stepwise probability was set to 0.05 for entry and 0.10 for removal. the classification cut-off was 0.5 and the maximum number of iterations was 20. Omnibus tests of model coefficients were also conducted.Chi-square tests and logistic regression analysis of Table Bartonella based on antibody immunofluorescence results. Of note, seroprevalence of Bartonella varied significantly among sites within the Zhejiang Province , and between those patients bitten by dogs versus those not bitten . Seroprevalence of Bartonella was found to be similar among males  and females . Furthermore, participants were divided into four groups according to age and the seroprevalence in each was determined. In summary, we found that 17.78% (5/18) of those patients less than 15 years in age had Bartonella antibody, 18.27% (59/323) of patients between 15 and 44 years in age had antibodies, 18.42% (28/152) of patients between 45 and 59 years old had antibodies, and 26.98% (17/63) of those patients over 59 years in age had antibodies. These results suggest that age was an insignificant factor in Bartonella antibody expression .Blood samples were collected from 556 people living in eight locations across Zhejiang Province Table . Of thesThe Chi-square value in omnibus tests of model coefficients was determined to be 60.326 (P < 0.05). Furthermore, the overall correct percentage was found to be 81.5%. Variables in the equation included site, exposure, and site×exposure. The wald of site×exposure, site, and exposure were determined to be 25.477 (P = 0.001), 20.090 (P = 0.005), and 16.174 (P = 0.024), respectively.Bartonella species, including B. henselae, have previously been detected in domestic cats [Haemaphysalis longicornis and Ixodes sinensis ticks [Bartonella-related infection from any of these sources to humans is feasible. Therefore, it is necessary to study the prevalence of Bartonella in people throughout the Zhejiang Province and to analyze relevant risk factors of infection. In our study, the seroprevalence of Bartonella was found to be 14.81% among healthy patients, a percentage similar to the recently reported percentage (14.28%) recorded in the Yunnan Province in Southwestern China [et al. [B. henselae specific IgG seroprevalence of 3.1% among adult patients with cardiovascular disease and 10.9% in a high-risk population of healthy veterinary students in Japan, of whom only 0.8% had positive results of serologic testing for B. henselae specific IgM. The bases for these differences are not clear, but are likely to be multi-factorial.Various tic cats , small mis ticks  in the Zrn China , but mucrn China . Further [et al.  previousBartonella species in IFA assays [B. henselae antigen may actually represent a previous or current infection with another Bartonella species. According to our analysis (carried out according to the manufacturer's instruction), cross reactivity with other Bartonella species cannot be ruled out; however, this kit has more than 84% specificity and 88% sensitivity for B. henselae IgG. As a result, our data suggest that detected antibodies were against all Bartonella species, while the majority of them were against B. henselae.Due to the inherent serological cross-reactivity among A assays ,9, the oBartonella antibodies were detected across all eight study sites of the Zhejiang Province indicates that the general population in this region is at risk of exposure to Bartonella. Of note, the prevalence varied between sites within the province, although Hangzhou, Tiantai, and Longyou had the highest prevalence of positive reactions. Furthermore, the exposure rate due to dog bites, which was identified as a significant determinant of seroprevalence, also varied between sites, and was significantly higher in Hangzhou than other sites, and significantly lower in Jiangshan than other sites. Moreover, site×exposure fit the equation in logistic regression analysis, indicating that site and exposure results correlated to each other.The fact that patients with Bartonella in individuals bitten by dogs was significantly higher than that among unbitten blood donors. However, the explanation of this finding is not altogether obvious, as domestic cats, rather than dogs, are considered a primary reservoir host for Bartonella. It is established that dogs serve as hosts for Ctenocephalides felis, the suspected vector of Bartonella. Therefore, dogs may acquire and maintain infected and infectious fleas, thereby, indirectly acting as a source of infection. Alternatively, people living in proximity to dogs may also be more likely to live in proximity to cats. However, it remains a possibility that dog bites themselves may transmit Bartonella infection, as Bartonella DNA has been detected in dog saliva [We also found that seroprevalence of g saliva .B. henselae infections are more common in children than adults. However, we did not see a significantly higher seropositivity among children than adults in our studies. In contrast, in our study the highest seropositivity was observed in the oldest (>59 years) patient age group. Others have reported similar findings [Previously published studies have indicated that findings .Bartonella infections can be difficult to diagnose clinically, particularly as many of the clinical presentations associated with them have nonspecific symptoms, such as fever. Thus, reliance on clinical reports from physicians to determine the public health burden of infection is not feasible. In contrast, serological surveys of individuals offer a means of quantifying the level of exposure to Bartonella among the population. In this study, we used this approach to demonstrate that exposure to this pathogen is both common and widespread across the Zhejiang Province of China, although prevalence was higher in some parts of the province than others. Furthermore, it appeared that people bitten by dogs were significantly more likely to have Bartonella antibodies than blood donors without exposure to bites. This information should serve to help those in relevant settings to consider Bartonella infection in the differential diagnosis of their patients.The authors declare that they have no competing interests.JS designed the study and drafted the manuscript. GF carried out the patient blood sample collection. JL performed analysis and interpretation of data. XS carried out serological testing. LL participated in the design of the study and helped draft the manuscript. QL conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/121/prepub


Here we report that oto is the mouse ortholog of the gpi deacylase gene pgap1, and that the endoplasmic reticulum (ER)-resident Oto protein has a novel and deacylase-independent function during Wnt maturation. Oto increases the hydrophobicities of Wnt3a and Wnt1 by promoting the addition of glycophosphatidylinositol (gpi)-like anchors to these Wnts, which results in their retention in the ER. We also report that oto-deficient embryos exhibit prematurely robust Wnt activity in the Wnt1 domain of the early neural plate. We examine the effect of low oto expression on Wnt1 in vitro by knocking down endogenous oto expression in 293 and M14 melanoma cells using shRNA. Knockdown of oto results in increased Wnt1 secretion which is correlated with greatly enhanced canonical Wnt activity. These data indicate that oto deficiency increases Wnt signaling in vivo and in vitro. Finally, we address the mechanism of Oto-mediated Wnt retention under oto-abundant conditions, by cotransfecting Wnt1 with gpi-specific phospholipase D (GPI-PLD). The presence of GPI-PLD in the secretory pathway results in increased secretion of soluble Wnt1, suggesting that the gpi-like anchor lipids on Wnt1 mediate its retention in the ER. These data now provide a mechanistic framework for understanding the forebrain defects in oto mice, and support a role for Oto-mediated Wnt regulation during early brain development. Our work highlights a critical role for ER retention in regulating Wnt signaling in the mouse embryo, and gives insight into the notoriously inefficient secretion of Wnts.Precise regulation of Wnt signaling is important in many contexts, as in development of the vertebrate forebrain, where excessive or ectopic Wnt signaling leads to severe brain defects. Mutation of the widely expressed  Palmitic and palmitoleic acids are covalently linked to Wg, Wnt1 and Wnt3a during their maturation, which serve to stimulate their secretion and signaling activities Wnts are a family of secreted, cysteine-rich signaling proteins that affect cell fate and proliferation in a wide variety of contexts C. elegans, Xenopus, zebrafish, chicken, and mouse has indicated that Wnts act as morphogens in the developing embryo, forming concentration gradients across cell fields which generate pattern Evidence from Drosophila, xrayoto mutation causes forebrain truncations in mice that closely resemble those in dkk1, ICAT, six3 and hesx1 mutants six3 and hesx1 transcription factors are expressed in prechordal neurectoderm from early neural plate stages, and both are repressed by Wnt signaling six3 and hesx1-deficient embryos are reported as the molecular bases of the failure of forebrain development in these mice The recessive, lethal oto mouse oto, we now reveal the identity of the oto gene, and present an investigation into the molecular function of the Oto protein. Phenotypic parallels with Wnt-inhibitor mutant mice initially suggested that oto might act as a Wnt antagonist during early brain development. Instead, we find that Oto regulates Wnt secretion via a novel mechanism. We show that Oto is a widely expressed, ER-resident glycoprotein involved in adding atypical glycophosphatidylinositol (gpi) anchors to Wnts 1 and 3a, which results in their retention in the ER. We further show that Oto is required for the correct initiation of Wnt signaling in the Wnt1 domain of the early neural plate. In oto-deficient embryos, neurectodermal Wnt signaling in the Wnt1 domain begins prematurely and robustly, which is consistent with the loss of Oto-mediated Wnt retention. The early neural plate in oto mutants thus exhibits an abnormally large medial domain of Wnt activity, and the oto embryo subsequently develops with a truncated forebrain.We have used a forward genetics approach in our study of the oto, we show that active Wnt flows out of the cell in a relatively uninhibited manner. We thus describe a new mechanism involved in the production of Wnt ligands, that has an important role during mammalian embryogeneis. Given the widespread expression of oto throughout mammalian life, this novel mode of Wnt regulation likely extends to other Wnt-dependent processes in development and disease.Our results reveal the existence of a novel Oto-dependent mechanism that retains Wnts in the ER of Wnt producing cells. We also present evidence that intracellular cleavage of gpi anchor lipids stimulates Wnt secretion. We propose that gpi-anchoring of Wnts provides a means of accumulating, and then releasing, a regulated burst of Wnt ligands. Conversely, in the absence of xrayoto mutation was mapped to a 284 kilobase interval on chromosome 1 which contains two genes, TFIIIc and oto/pgap1  transgenic rescue approach. Three BACs, each containing the complete oto/pgap1 gene, individually rescued the developmental defects and restored viability to oto homozygotes  phases . Three bHydrophobicity of Wnt3a can be increased by palmitoylation on a conserved cysteine, C77 To measure the apparently novel hydrophobicity change in Wnt3a caused by Oto co-expression, we performed hydrophobic interaction chromatography (HIC) using butyl sepharose. Proteins bind to butyl sepharose via their hydrophobic surfaces and moieties Although Oto-modified Wnt3a migrates more slowly than naked Wnt3a, it is not glycosylated ; its sig35S-labeled Oto+Wnt3a insoluble extracts using the same method. The major protein that co-immunoprecipitates with modified Wnt3a is correctly sized to be Oto  To see if Oto modifies other Wnts, we also looked at Wnt1. Like Wnt3a, Wnt1 signals via the canonical pathway and is lipidated by porcupine in vivo conditions, their association could be sufficient to explain the observed ER retention of Wnts. It is also possible that the dramatic increase in Wnt hydrophobicity independently reduces the Wnt secretion. To explore this question further, we sought additional insight into the nature of the Oto modification. The analysis was continued using Wnt3a, since Oto apparently modifies and retains both Wnts similarly. Because Oto encodes the gpi deacylase PGAP1 3H-ethanolamine (3H-EthN), 3H-GlcN, and 3H-MN. When aliquots of all proteins from 3H-EthN-labeled insoluble extracts are resolved and blotted, a 42 kDa band consistent with the size of Oto-modified Wnt3a is visible in the autoradiograph .If Oto and Wnt bind directly in the ER under diograph . Probingdiograph . This laed Wnt3a . The sam, we examined oto expression in sections (oto is ubiquitously expressed (six3 (oto continues to be widely expressed in embryos throughout gestation (not shown). The expression results in Wnt1 is expressed in the medial neural plate in mouse, and is required for the development of the midbrain and anterior hindbrain  midline . During al folds . To looksections . In an asections , oto is xpressed . In a seed . However, some variation in staining was seen in embryos from E8.0–E8.25. It should be noted that only the anteriormost portions of embryos could be examined from E8.0 to E8.5, because canonical Wnt activity in the posterior portions of embryos is completely stained from E8.0 , no significant Wnt activity is apparent in the anterior neural plate , Wnt activity is first detected in the medial neural plate at 2–3 somites . We used two cell lines in oto knockdown experiments, 293 cells and the human melanoma M14 cell line oto shRNA (shoto), and for M14 melanoma cells, only shoto was transfected, since these cells express Wnt1 endogenously (data not shown and GNF SymAtlas) oto in 293 and M14 melanoma cells dramatically increases the amount of soluble Wnt1 recovered from the media  from shoto and control M14 cells, and applied these to NIH3T3 cells that stably express a canonical Wnt luciferase reporter. NIH3T3 cells are capable of responding to Wnt signals via the canonical Wnt pathway In al plate . Consistal plate . Howeveroto-deficient cells, and/or due to the fact that these are knockdown rather than knockout cells. Taken together with the overexpression studies, these activity experiments indicate an inverse dose-response relationship between oto expression levels and Wnt activity, with lower oto levels resulting in production of more active Wnt signal.In the CM activity experiments, there was variability in the intensity of the Wnt response . VariabiA lingering question remains; what is the role of the Wnt gpi anchor itself in Wnt secretion? We addressed this by co-transfecting mammalian gpi-specific phospholipase D (GPI-PLD) with Wnt1 alone or with Oto+Wnt1, and then examined the consequences on Wnt secretion. GPI-PLD is an enzyme that specifically cleaves lipids from gpi anchored proteins, and it can do so within the ER Ci-PGAP1 as an essential component of the canonical Wnt pathway during embryogeneis of the chordate Ciona intestinalisoto in embryos and adults. Our preliminary results indicate that Wnt5a is also Oto-modifiable (not shown). Gpi-anchoring by the widely expressed Oto protein likely contributes to explaining why Wnts are poorly secreted by mammalian cells We present evidence that Oto/PGAP1 functions as a novel component of the Wnt pathway during mouse forebrain development. Interestingly, another study has recently identified The characterizations reported here show that the hydrophobic modifications on Wnt3a and Wnt1 have the hallmarks of gpi anchors. While the appended moiety itself is clearly a chemical relative of the conventional gpi anchor, the process by which the protein is modified is apparently unrelated. For instance, a conventional gpi anchored protein is produced by a transamidation reaction in which the carboxy terminus of the protein is cleaved and replaced with the gpi anchor Others have shown that porcupine is responsible for adding palmitic acid and palmitoleic acid to Wnt3a at C77 and S209 respectively in vitro . The variety of Wnt-related phenotypes in the oto mutant, together with evidence that at least two distinct Wnts can be Oto-regulated, indicate that Oto plays a role in determining the timing or level of Wnt activity in multiple contexts.While it is possible that dysregulation of other secreted proteins may contribute to oto forebrain phenotype is the indicated underlying molecular mechanism. In other mutant mouse lines referenced here In conclusion, the most novel and exciting aspect of the All mice were treated in accordance with the established rules and recommendations of our IACUC protocols and UCSF's animal care committee, and with the welfare of the animals carefully considered.xrayoto mutation was successively backcrossed onto C57BL6/J and mapped by standard meiotic mapping. Animals were genotyped using PCR-based, polymorphic microsatellite markers (www.broad.mit.edu/mouse/) that vary by size between C57BL6/J and DBA2/J  were prepared using the Qiagen Large Construct kit, sizes verified on FIGE gels, and transgenic mice were made. Mice doubly heterozygous for a BAC and the xrayoto mutation were crossed to xrayoto heterozygotes. oto/oto; BAC+/− mice were born at normal Mendelian frequency and were viable and fertile . Mouse Wnt3a and Wnt1 DNA expression constructs encoding Wnts tagged with HA at their carboxy termini were purchased from Upstate. Wnt3aΔ31–107 was made by removing the ScaI/SmaI fragment from mouse Wnt3a. Candidate genes for oto were evaluated by RT-PCR, whole mount in situoto (AAGAAGCCAAACCATACAAAGTT) was cloned into the pSiRPG vector. Antibodies were purchased from the following sources: mouse anti-flag M5 (Sigma); rat anti-HA high affinity (Roche); goat and rabbit anti-Wnt1 ; rabbit anti-HDAC1 ; secondaries . Reagents: protein G agarose (Roche), Egfp and dsRedER vectors (Clontech), EndoH and PNGaseF (NEB).A sequence verified, wildtype 2. Transient transfections were done using Cytofectene  or the equivalent Metafectene . Transfected cells were cultured for 42–48 h, scrape harvested, and lysed in Hepes-buffered RIPA . Lysate viscosity was reduced with benzonase nuclease (Novagen). Lysates were spun at maximum speed for 10 min at 4°C, and supers collected in fresh tubes to yield soluble extracts. Insoluble extracts for immunoprecipitation (IP) were prepared by resuspending pelleted material in a small volume of 1% SDS, 20 mM Hepes pH 7, 150 mM NaCl, 10 mM DTT, heating for 10 min at 80°C, diluting 10× with SDS-free RIPA, then spinning at max speed for 10 min to pellet debris, and collecting supers in fresh tubes. Wnts were immunoprecipitated for at least 3 nights. Insoluble extracts for gels only were prepared by solubilizing pelleted material in 0.5% SDS, 1% β-mercaptoethanol. Samples were analyzed by reducing SDS-PAGE on 4–12% or 10% Bis-Tris gels in MOPS (Invitrogen). Proteins were transferred onto PVDF (Amersham and Millipore) using the X-Cell II system (Invitrogen) at 30V for 1.5–2 h on ice. Proteins on blots were detected with ECL Plus (Amersham) and Kodak Biomax Light film. Cells were labeled by adding 140 µCi/ml of 2∶1 35S-methionine∶35S-cysteine (ARC) for 30 min, or 0.5 mCi/mL 3H-labeled reagent (ARC) for 6–24 h. To visualize 35S, gels were dried and exposed to film. For 3H, gels were blotted and exposed to film for one month at −80°C using Kodak Transcreen LE intensifying screens. For butyl sepharose (Amersham) chromatography, insoluble extracts were prepared as for IP except they were diluted with RIPA lacking detergents, then incubated with butyl beads for 90 min at room temperature. Beads were washed, then eluted with Hepes-buffered solutions. For PI-PLC digests, 5 µL of immunoprecipitated modified Wnt3a on HA beads was sonicated in 15 µL PBS+0.1% Triton X-100 for one hour, then PI-PLC (Glyko) was added or not, and samples incubated overnight. In oto knockdown assays , and M14 cells in RPMI-1640 (ATCC), each plus 10% fetal calf serum (Hyclone), in 5–6% COn assays , shRNAs oto expression in stable shoto cells, mRNA was extracted from one million cells per line in triplicate, and RT-PCR products quantitated using a real-time assay.For activity experiments, stable shoto lines in M14 cells were generated by selecting shoto-transfected cells with 1 µg/mL puromycin. Wnt reporter cells were made by stably transfecting NIH3T3 cells with a canonical Wnt luciferase reporter, constructed by cloning the enhancer element of Super8xTOPFlash containing 8 TCF/LEF binding sites into the pGL4.28 vector (Promega) upstream of the minimal promoter element. Luciferase-mediated luminescence was measured using the Dual-Glo kit (Promega). In conditioned media (CM) experiments, cells were plated and CM was collected after 4 days. CM or purified Wnt3a at 50 ng/mL was applied to reporter cells for 6 hours. In co-culture experiments, on day 1 NIH3T3 reporter cells were plated at 20,000 cells per well (24-well plate) and transfected with the Renilla luciferase normalization control vector RL-SV40 (Promega). On day 2, 40,000 M14 or shoto cells were plated on top of the reporter cells. Control reporter cells (no co-culture) were stimulated with 50 ng/mL Wnt3a. On day 3, cells were lysed and luciferase activity measured. In all experiments, each condition was repeated 6–10 times, and the mean luminescence intensity was calculated and plotted with one standard deviation. For measuring Table S1oto. otoxray carriers were intercrossed with animals heterozygous for both otoxray and a BAC encoding the genomic oto gene. Representative data from one rescuing bac, RPCI23-34H22, is shown. Viability was scored. The otoxray mutation is perinatal lethal; only 0.4% of oto homozygotes have lived to adulthood , and these were infertile. The observed column lists the number of viable adult animals recovered with the indicated genotypes. Predictions are based on the total number of animals scored. Bac transgenic mutant animals are born at normal Mendelian frequency (yellow), have normal head morphology, and are fertile, revealing that BAC-mediated restoration of oto expression eliminates the oto phenotype.BAC transgenic rescue of (0.35 MB TIF)Click here for additional data file.Figure S1oto. Homologous recombination of MICER clone MHPN237e12 with the endogenous oto locus in the vicinity of exons 5 and 6 results in a duplication and frameshift after exon 6. 0, +1, and +2 show the reading frame between indicated exons. The otoins allele is a likely null.Derivation of the MICER insertional allele of (0.31 MB TIF)Click here for additional data file.


To the Editor:Bartonella and Rickettsia species are pathogens of humans and domestic mammals that may be transmitted by fleas and other arthropods. Rickettsia felis causes flea-borne spotted fever in humans who come into contact with flea-infested domestic and peridomestic animals; worldwide distribution of this pathogen in ectoparasites and mammals makes it an emerging threat to human health  has not been previously documented.Pulex irritans, Tunga penetrans, Ctenocephalides felis strongylus, Echidnophaga gallinacea, and Xenopsylla brasiliensis were collected in the Ituri district of northeastern DRC from March through April 2006, during an investigation of a plague outbreak. Our investigation area was limited to 4 villages: Djalusene and Kpandruma, which had confirmed plague patients, and Wanyale and Zaa, which had several suspect cases.Off-host We collected fleas by using a kerosene lamp hung above a 45-cm diameter tray containing water (Bartonella DNA was detected by PCR amplifying a 379-bp fragment of the citrate synthase gene (gltA) . All assays were run in duplicate, and positive and negative controls were included in all assays. Amplicons were purified with the QIAquick PCR purification kit (QIAGEN) and sequenced in both directions by using a BigDye sequencing kit  with the same primers used for PCR amplification. Resultant sequences of Bartonella spp. were aligned with MegAlign by using the Clustal algorithm , and compared with reference sequences obtained from GenBank.Yersinia DNA and R. typhi were not detected, 89 of the 193 pools were PCR positive for either Bartonella spp. or R. felis (www.cdc.gov/ncidod/dvbid/westnile/software.htm), we calculated an estimated infection rate of 10.72%  for R. felis, 3.66% for Bartonella species, and 0.91% (95% CI 0.40–1.78) for both Bartonella spp. and R. felis . Moreover, Bartonella from 3 pools of P. irritans demonstrated only 1.8% to 2.4% divergence to B. vinsonii subspecies arupensis isolated from a human patient in Wyoming, USA. Likewise, sequences of Bartonella from 1 pool of T. penetrans and 1 pool of P. irritans were 100% identical to Bartonella isolated from a Neotoma mexicana wood rat (GenBank accession no. AF110312); a sequence obtained from 1 flea pool of T. penetrans was 100% identical to the gltA Bartonella sequence found in Orchopeas sexdentatus, collected from Neotoma micropus in New Mexico, USA (data not shown). This finding indicates a new Bartonella species with multiple rodent origins and a more ubiquitous global dissemination than previously determined. Our results also demonstrate the previously unreported detection of R. felis in P. irritans, E. gallinacea, X. brasiliensis, and T. penetrans flea species.Phylogenetic analysis indicated several Bartonella spp. and R. felis exists in fleas within the DRC. In addition, we report Bartonella spp. and R. felis DNA in T. penetrans fleas and R. felis DNA in E. gallinacea fleas, vectors not previously associated with these pathogens. Co-infections were also observed in T. penetrans, P. irritans, and C. felis fleas, suggesting a common vector or mammalian host shared by R. felis and Bartonella spp. Flea feedings occur intermittently and on potentially different hosts, thus the vectors described here may acquire multiple bacterial strains for transmission to humans (This report suggests that Bartonella spp. and R. felis have been detected previously in fleas within northern and sub-Saharan Africa (Bartonella spp. and R. felis in the fleas is important because they were collected in close contact with humans at risk for multiple exposures within households. Our results suggest that both R. felis and Bartonella spp. are prevalent in this region of the DRC and should be included in the differential diagnosis of potential flea-borne infections in this region of sub-Saharan Africa.


The purpose of this study was to investigate whether paediatricians have appropriate knowledge, attitudes, and behaviours regarding vaccinations for infants in Italy.A random sample of 500 paediatricians received a self-administered anonymous questionnaire covering demographic and professional characteristics; knowledge about the mandatory, recommended, and not indicated vaccinations for infants; attitudes about vaccinations for infants; behaviour regarding current administration or willingness to administer mandatory or recommended vaccinations for infants and immunization education programs of the parents.Only 42.3% paediatricians knew all recommended vaccinations for infants and this knowledge was significantly higher in females, in those who worked a higher number of hours for week, and in those who use guidelines for immunization practice. Only 10.3% had a very favourable attitude towards the utility of the recommended vaccinations for infants and this was significantly higher in those who administered recommended vaccinations for infants. A large proportion (82.7%) of paediatricians routinely informed the parents about the recommended vaccinations for infants and this appropriate behaviour was significantly higher among younger, in those with a higher number of years in practice, and in those who administered the recommended vaccinations for infants.Training and educational interventions are needed in order to improve knowledge, attitudes, and behaviours regarding vaccinations for infants among paediatricians. It is very well established that the vaccinations of infants and adolescents have an extraordinary beneficial public health impact  by reducHaemophilus influenzae type b, measles, meningococcal, mumps, pertussis, pneumococcal meningitis, rubella, varicella) and they are administered by public health physicians and paediatricians working in a public network. All mandatory, Haemophilus influenzae type b, pertussis, and pneumococcal meningitis vaccines are administered according to a three-dose schedule at 3, 5, and 11-12 months of age, whereas the others with a single-dose in the second year of life. Those mandatory are actively offered free of charge whereas for the recommended the offer is independently planned by each region [In Italy, vaccinations for infants are mandatory  and recommended  of Italy. Study protocol and final questionnaire were approved by the ethical committee.The questionnaire was pilot-tested on a sample of 20 paediatricians to refine the wording of items and to ensure clarity of the text. The self-administered anonymous questionnaire, revised on the basis of the comments from the pilot-study, was mailed to the random sample. Each paediatrician was approached via an introductory telephone call and was invited to participate by receiving a pack which included a letter explaining the purpose, the importance of the study, and asking them to participate, an informed consent form, a questionnaire, and a self-addressed and pre-stamped envelope for returning the questionnaire. Participants were assured that response provided would be treated in confidence. The return envelopes were coded to allow contact with non-responders, and the coding file was stored separately to preserve confidentiality and destroyed at the conclusion of the study. Non-responders received a telephone call and a further questionnaire was sent approximately two months after the initial mailing.The questionnaire was designed to collect the following information: a) demographic and professional characteristics; b) knowledge about the mandatory, recommended or not indicated vaccinations for infants. Responses about the vaccine schedule were in an open format, whereas in the other questions respondents were asked to indicate their agreement with true or false statements; c) attitudes about vaccines were based on items measuring the perceived utility of the mandatory and recommended vaccinations for infants and beliefs regarding the severity of infectious diseases. Response to all questions on the utility of the vaccinations were on a 10-point Likert-type scale , whereas the other questions were with true or false statements; d) behaviour regarding current administration or willingness to administer mandatory or recommended vaccinations and immunization education programs of the parents. Responses about current or willingness administration were in the yes/no format, whereas all other responses were measured by 5-point Likert-type scale, ranging from "never" to "always"; e) source of information about vaccination.Participants were advised that the return of a complete questionnaire signified written consent to the study.Multivariable logistic regression models were constructed in order to determine significant independent predictors of the following outcomes of interest: knowledge of the recommended vaccinations for infants (Model 1); positive attitude towards the utility of recommended vaccinations for infants (Model 2); routinely inform parents regarding recommended vaccinations for infants (Model 3). In all models, the following independent variables were included: gender , age , primary practice type , number of year in practice (continuous), number of hours worked for week (continuous), number of patients seen in a workday (continuous), perception that the recommended vaccinations for infants are dangerous , use of guidelines for immunization practice , guidelines as source of information about vaccinations , and need of additional information about vaccinations . The variables knowledge of the recommended vaccinations for infants  and administration of recommended vaccinations for infants  were also included in Models 2 and 3.p value ≤ 0.25 were included into the multivariate analysis. The variables were added into the logistic regression models in a forward stepwise manner with inclusion criteria of p < 0.4 to enter the model and of p < 0.2 to remain in the final model. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. Statistical significance was assessed using two-sided tests with p-values of ≤ 0.05. All data were analyzed using the Stata version 10 statistical software [Univariate analysis was conducted using appropriate test statistics and those characteristics associated with the outcome variables with a Of the 500 questionnaires, 15 were excluded because the respondent was no longer in practice and 81 because were returned due to incorrect addresses. Of the 404 eligible, 156 paediatricians completed the questionnaire for an overall response rate of 38.6%. Table Table For the perception of severity of infectious diseases, the values were, in descending order, pneumococcal meningitis (71.8%), pertussis (68.6%), diphtheria (60.9%), polio 58.3%), and hepatitis B (56.4%). The mean total values of respondents' attitude regarding the utility of mandatory vaccinations were always higher than 9, on a scale 1 to 10, indicating a very favourable attitude. In contrast, only 10.3% had a very favourable attitude by responding 9 or 10 towards the utility of those recommended and the results of the multiple logistic regression analysis indicated that this positive attitude was significantly higher in those who administered recommended vaccinations for infants  , in those with a higher number of years in practice , and in those who administered the recommended vaccinations for infants  , followed by guidelines (70.5%), and educational courses (69.2%). More than half (59.6%) said that they were interested in learning more.As far as we know, this is the first observational survey specifically designed to collect detailed data on knowledge, attitudes, and behaviours regarding vaccinations for infants and to identify their determinants among a random national sample of paediatricians. The survey provides useful baseline information for further research and for policy makers.The results of this study show that the respondents' knowledge about recommended vaccinations for infants was surprisingly low, since only 42.3% know all of them. Concern should be expressed particularly because well-informed paediatricians are essential for educating patients and for an effective health promotion of infectious diseases. Therefore, it is important that parents receive clear and consistent messages about vaccinations as a routine aspect of health care. Furthermore, a primary goal should be the development of tools in order to assist the paediatricians to provide consistent, accurate, and appropriate advice, to promote vaccinations, and to raise their knowledge about related issues. Of interest was that the multivariable analysis demonstrated that female paediatricians, those who worked a higher number of hours per week, and those who used guidelines for immunization practice were more likely to know all recommended vaccinations for infants. Some of these findings are in accordance with a previous study .Surprisingly, paediatricians' attitude about the utility of recommended vaccinations for infants was not very positive because only 10.3% believed that they were extremely useful. Moreover, this positive attitude was significantly higher in those who administered these vaccinations. In this scenario, where poor recommended vaccinations related knowledge coexists with low positive attitude, one may argue that educational programs are not only needed but also welcomed. This is supported by the fact that 59.6% of respondents indicated that they desire more information about vaccinations.Another notable finding was regarding the behaviours, with a very low proportion of the sample that routinely administered to their patients the recommended vaccinations 25.6%). Such data deserve particular attention since it can significantly increase the risk for infectious diseases. Although this data may also be partly explained by the fact that the offer of the recommended vaccinations is independently planned by each region, considering the significance of paediatrician influence and support about the health promotion of their patients, paediatricians as well as patients may play a much more powerful role in increasing infants vaccination compliance, particularly the recommended. Although 100% compliance is probably not yet a realistic goal, any increase in adherence rates will hopefully reduce morbidity and mortality in this vulnerable population. The values observed in this study were considerably lower compared with those found in paediatricians in the United States. Indeed, for children at 12 months 78% adhere to standard immunization practices %. Such d, respectThis study may have a number of methodological limitations that are worthy of emphasis and that should be considered when interpreting the results. First, the cross-sectional nature carries with it the disadvantage of not being able to prospectively identify predictors of the outcomes. Although the findings are relevant to the magnitude and direction of the relationship between the different variables and the outcomes. Rather, we can only speak of factors that are significantly associated with the outcomes. Second, the study suffers from the same problem as does any survey using results based on self-reporting instrument. Although self-report is one of the few ways to assess attitudes, there is concern about the accuracy and desirability bias with respondents that may provide responses they think the researcher wants or expects. Moreover, it is possible the tendency of the respondents for overestimation their compliance with administration/recommendation the vaccinations and, therefore, actual rates may be lower. However, we attempted to minimize these potential bias by ensuring complete respondent anonymity and confidentiality. Third, we were unable to achieve a high response rate as desired, though typical for a mailed questionnaire, even though multiple follow-up attempts were conducted, and this introduces several obvious biases that are a common limitation in mailed surveys. The information displayed by participants may be not representative of the broader population. However, no significant differences have been observed between participants and non-participants according to some demographic characteristics, so we are confident that the findings, which are based on a random sample of the population of interest, may be representative of the paediatricians in Italy.In conclusion, the findings from this study extend the understanding of the knowledge, attitudes, and behaviours regarding vaccinations for infants among paediatricians and they point out the need for designing and implementing educational programs.The authors declare that they have no competing interests.Preliminary results were presented at the Research Meeting of the Second University of Naples, July 2-7, 2007, Naples, Italy.DA participated in the design of the study, collected the data, and contributed to the data analysis; GDG contributed to the data analysis and interpretation; PM participated in the design of the study and contributed to the interpretation of the data; IFA, the principal investigator, designed the study, was responsible for the data collection, statistical analysis and interpretation, and wrote the article. All authors have read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/9/463/prepub


A clinically validated attention deficit hyperactivity disorder (ADHD) scale in Arabic for evaluating children in Saudi Arabia who might be suspected of having ADHD is lacking. Thus, we studied the validity of an Arabic version of the ADHD Rating Scale in discriminating children with an ADHD diagnosis from normal children or from those with non-ADHD psychiatric diagnoses, including mental retardation.The guardians of 119 children provided demographic data and completed the standardized Arabic version of the ADHD Rating Scale on their children, who were either normal, had a diagnosis of ADHD, or had a non-ADHD psychiatric diagnosis. The mean rating scores of the groups were compared, and the cutoff points were calculated for both sexes.The scores discriminated children with ADHD diagnosis  from normal children (10.93 [8.009]), and those with a non-ADHD psychiatric diagnosis (16.63 [8.865]). ADHD cutoff points were obtained for male (23.5) and female (22.5) children. Psychosocial characteristics associated with children having ADHD were not associated with the diagnosis of ADHD.The ADHD Rating Scale (Arabic version), in terms of either the grand total score or the total score of each of its two subscales, demonstrated concurrent and discriminant validity by discriminating children with ADHD from other clinical and non-clinical children groups. The study obtained cutoff points for both sexes based only on the grand total score of the scale because of the relatively small sample size. Replication of the study, utilizing a larger sample and eliciting ratings from both parents and teachers, is recommended. There is a general consensus that well standardized behavior rating scales are essential in the evaluation of children with attention deficit hyperactivity disorder (ADHD). However, such rating scales have their limitations. They are subject to biases in opinion, such that various characteristics of the informants, including their education and emotional state at the time of conducting the ratings, may influence the ratings. Also, rating scales may fail to assess factors influencing a child's behavior.The ADHD Rating Scale (Arabic version)  is the tThe Department of Translation, King Saud University was consulted for the processes of translation and back-translation, while the technical adaptation of the scale was performed by the researchers who, after a pilot study (n=56), examined and adapted the Arabic version to ensure that it preserved the format, consistency and content of the English version of the scale. Personal and demographic data about the participants in the study, including information about the child, his or her parents, siblings, and the principal guardian if not one of the parents were collected. These include family structure and size, educational level of the child and his parent/guardian, status of parental marriage , the other socioeconomic data.The ADHD Rating Scale6In 1995, the original ADHD Rating ScaleThis study sample included 119 children, of which 90 (89.1%) had various psychiatric diagnoses confirmed by a psychiatrist, and 29 (10.9%) were clinically screened by a psychiatrist to be normal children. The normal children as well as those with psychiatric diagnoses were subsequently designated to the following diagnostic groups: normal, ADHD diagnosis only (ADHD Dx), ADHD diagnosis combined with another psychiatric (Axis I) diagnosis (ADHD+Psy Dx), Psychiatric (Axis I) diagnosis only (Psy Dx) and mental retardation only (MR).The data were tabulated and analyzed using the Statistical Package for Social Sciences, version 10 (SPSS 10). The chi-square test was used for categorical variables, the t test for numerical variables, and one-way analyses of variance (ANOVA) and Scheffé test for more than two-group analysis. For determining the cutoff points, including sensitivity and specificity measures, based on the grand total score of the scale, the receiver operating characteristics (ROC) curve was plotted.P =.0001) in terms of the children's years of formal schooling, with the children in the normal group having a higher mean school grade than that of the ADHD plus and the mental retardation groups.The study included 119 children   with a mP <.001) or in either the mental retardation (P <.05) or the psychiatric diagnosis (P <.001) groups (P <.001) from that of the children in either the normal (P <.001), the psychiatric diagnosis (P <.001), or the mental retardation (P <.01) groups (P <.001) (P <.001), the psychiatric diagnosis (P <.01), or the mental retardation (P <.05) groups.Children in the ADHD diagnosis only and the ADHD plus psychiatric diagnosis groups had significantly higher grand total scores compared to children in the normal group  groups . The mea) groups . The Sch) groups . The meaP <.001) . HoweverA valid and reliable behavioral rating scale for screening of children suspected of having ADHD is an essential step toward accurate diagnosis and optimal management. The need for such a scale in Saudi Arabia can hardly be over-emphasized. Our results show that the grand total scores obtained by the children on the ADHD Rating Scale (Arabic version) differentiated between the children with ADHD, whether as a pure clinical condition or as a co-morbidity, and the children who were either normal or had a psychiatric diagnosis other than ADHD, including the mental retardation diagnosis.Two prevalence studies on ADHD in Saudi Arabia reported rates of 12% and 16.5%.The original scale was developed and standardized for obtaining a direct rating of the essential symptoms of the disorder from both parents and teachers.The results of this study indicate that ADHD is not restricted to a particular social strata, family type, family size, or to parents with a certain educational level. For example, it was found that parents who rated the symptoms of their children on the ADHD Rating Scale (Arabic version) above the cutoff for ADHD diagnosis were of various educational levels, ranging from illiteracy to post-graduate education. This finding also indicates that parents' recognition of the behavioral manifestations of the disorder is irrelevant to their educational level.Not surprisingly, the study groups differed significantly in terms of the school grade that a child had achieved . Children who were mentally retarded and children with ADHD combined with another Axis I psychiatric diagnosis  had a significantly lower grade level. An examination of the data has shown that the mentally retarded children either did not attend any school, or they attended what is locally termed “special schools” where the mentally retarded children attend ‘school time’ during daytime with very little to do except playing games, and they usually do not progress from one grade to a higher one.Although the parents' educational level did not show a significant group difference in this study, a trend was observed for some parents with high educational level to rate their children relatively higher on ADHD symptoms, compared to those with low or no education.In this study, the psychiatrist who reached the diagnosis for a child is different from the one who supervised the completion of the data collection form by the parent of the same child. Some of the guardians who rated their children on the ADHD Rating Scale (Arabic version) preferred to complete the scale at home due to time constraints. This arrangement did not seem to have influenced the quality of their rating in any inappropriate way. However, a future similar study would be expected to apply additional controls on the study, specifically by blinding the process of diagnosing children from the process of rating the same children, preferably with inter-rater protocols.The sample of this study is largely a clinical sample. Future studies on the prevalence of ADHD in the Saudi community, involving children recruited from their community settings, are highly recommended. However, unless a solid screening tool with strong validity indices is established, community screening for prevalence of ADHD may be misleading. Therefore, it is recommended that a future similar study administer the validated ADHD rating scale to a larger number of participants. Such an exercise may re-confirm or modify the cutoff points established in this study.In summary, the ADHD Rating Scale (Arabic version) successfully differentiated children with a clinically proven ADHD diagnosis from both normal children and children with non-ADHD psychiatric diagnoses that include mental retardation. The “inattention” and “impulsivity-hyperactivity” subscales were equally successful in such a differentiation. The study re-emphasized the importance of developing and validating ADHD rating scales against some solid gold standards before administering them for obtaining prevalence rates of the disorder in communal settings. The acknowledged limitations, such as small sample size and utilization of a rating scale that is probably out-dated, may not overshadow the fact that the study probably provided the clinical and investigative grounds for an improved research work on ADHD in Saudi Arabia in the recent future.


Numerous studies have shown the pathological influence anti-phospholipid antibodies (APLA) have on the physiology of the single neuron as well as the function of the entire human nervous system. The influence is well demonstrated in the antiphospholipid syndrome (APS). This syndrome is characterized by a triad of arterial or venous thrombotic events, recurrent fetal loss and thrombocytopenic purpura. The syndrome exhibits different neurological pathologies such as: chorea, seizures, transverse myelopathy, migraine, cerebral ataxia, hemiballismus and transient global amnesia, which are not fully explained by the procoagulopathic trait of APLA. A study on mice induced with APS demonstrated hyperactive behavior when compared to the control group. The information gathered from these different studies raised the question whether APLA has any part in the etiology of Attention Deficit/Hyperactive Disorder (ADHD) in children.            We compared 41 children diagnosed with ADHD to a control of 28 healthy children. Blood drawnfrom the two groups was screened using ELISA for the presence of anti-cardiolipin antibodies, antiβ2GPantibodies, anti-phosphatidyleserine antibodies and anti-ethanolamine antibodies. The resultsshow no significant difference in the level of antiphospholipid antibodies (APLA) measured betweenthe children diagnosed with ADHD and the control group.


The current study investigated the personality characteristics of mothers of children with attention deficit hyperactivity disorder (ADHD) using the Minnesota Multiphasic Personality Inventory (MMPI).Fifty mothers (average age of 38.1±4.2 years) of children with ADHD not having comorbidity  and 59 mothers (average age of 38.1±2.7 years) of comparison children  completed the Korean version of the MMPI. Only mothers whose psychiatric health was verified by the Structured Clinical Interview for axis-I DSM-IV disorders (SCID-IV) were included in current study.After controlling for maternal age, maternal education level, children's gender, age, and total and verbal intelligence quotient (IQ), the MMPI scores of the mothers of children with ADHD were significantly higher on the depression (D), hysteria (Hy) and psychasthenia (Pt) scales than those of the mothers of children in the comparison group.These results suggested that even psychologically healthy mothers of children with ADHD alone might be depressed, histrionic and anxious. A number of previous studies have noted an increased incidence of psychopathology and personality disorder in parents of children with attention deficit hyperactivity disorder (ADHD).Fifty-six biological mothers of outpatients in the Seoul Metropolitan Eunpyeong Hospital with Diagnostic and Statistical Manual-fourth edition (DSM-IV)Finally, fifty mothers (average age of 38.1±4.2 years) of children with ADHD  and 59 mothers (average age of 38.1±2.7 years) of comparison children  completed the Korean version of the MMPI. The MMPI is one of the most frequently used tests for personality assessment. It was developed in 1943 as a screening tool for psychopathology and assessment of personality.The mean scores on all of the MMPI scales were within the normal limits for both groups of participants. There were no significant differences in maternal age, maternal educational level, or children's age between the ADHD and comparison groups (Independent t-test; p=0.933, p=0.773 and p=0.201 respectively, To the best of our knowledge, this is the first study to assess the personality characteristics of mothers whose psychiatric health was verified using the SCID-IV. We also did not enroll children with ADHD who had other psychiatric comorbidities in the current study based on previous findings suggesting that there may be a strong association between coexisting conduct disorder and parental personality disorder.7In the current study, the mothers of children with ADHD scored higher on the following MMPI scales: D, which measures depression; Hy, which measures hysterical reactions and denial of stressful situations; and Pt, which measures anxiety, than those of the comparison children. These findings support those of previous studies reporting that mothers of children with ADHD tended to be more depressed, self-blaming and socially isolated.In the current study, the mothers of the children with ADHD did not score significantly high on the Pd scale of the MMPI. A higher Pd score was a consistent finding in two previous family studies that measured the parental MMPIs of children with ADHD.Many aspects of personality are known to be under partial genetic influence. In a prior study, the heritability estimates of MMPI scores were from 0.26 to 0.61.16However, a non-genetic understanding of these findings is possible. The MMPI scores could be more state-dependent than trait-dependent in the individual's lifetime.Parents play an important role in delivering the proper treatment to children with ADHD. It would not be surprising that maternal depression is related to their poor parenting behavior.The present study has several limitations, including its relatively small sample size and the lack of a parametric assessment investigating the severity of psychopathology in the mothers or children. As conduct behaviors in ADHD could become an issue in later in life, the exclusion of children with ADHD and behavioral problems might not be perfect. In addition, the total and verbal IQ scores of children with ADHD were significantly lower than those of the comparison children. This finding could support the previous findings that the mean IQ scores of children with ADHD were lower than those of normal controls and that the IQ scores correlated negatively with ADHD symptoms.In conclusion, these results suggested that even psychologically healthy mothers of children with ADHD alone might have a characteristic personality, which was observed to be more depressed, more histrionic and more anxious.