Partial replication of a DRD4 association in ADHD individuals using a statistically derived quantitative trait for ADHD in a family-based association test.

BACKGROUND: Previous research found an association between single nucleotide polymorphisms (SNPs) in the promoter region of DRD4 and statistically derived phenotypes generated from attention-deficit/hyperactivity disorder (ADHD) symptoms. We sought to replicate this finding by using the same methodology in an independent sample of ADHD individuals. METHODS: Four SNPs were genotyped in and around DRD4 in 2631 individuals in 642 families. We developed a quantitative phenotype at each SNP by weighting nine inattentive and nine hyperactive-impulsive symptoms. The weights were selected to maximize the heritability at each SNP. Once a quantitative phenotype was generated at each SNP, the screening procedure implemented in PBAT was used to select and test the five SNPs/genetic model combinations with the greatest power to detect an association for DRD4. RESULTS: One of the four SNPs was associated with the quantitative phenotypes generated from the ADHD symptoms (corrected p-values = .02). A rank ordering of the correlation between each of the ADHD symptoms and the quantitative phenotype suggested that hyperactive-impulsive symptoms were more strongly correlated with the phenotype; however, including inattentive symptoms was necessary to achieve a significant result. CONCLUSIONS: This study partially replicated a previous finding by identifying an association between rs7124601 and a quantitative trait generated from ADHD symptoms. The rs7124601 is in linkage disequilibrium (LD) with the SNPs identified previously. In contrast to the previous study, this finding suggests that both hyperactive-impulsive and inattentive symptoms are important in the association.     

Production of delayed hypersensitivity by antigen associated with peritoneal exudate cells and the effect of pretreatment with Freund's complete adjuvant

The immunogenicity of antigens associated with peritoneal exudate cells was compared with the immunogenicity of free antigens for the induction of delayed hypersensitivity. Oil induced peritoneal exudate cells were incubated with antigens labelled with radioactive iodine, washed and injected intraperitoneally into recipient guinea-pigs. Comparable amounts of free antigens were also injected. All sixteen inbred guinea-pigs given human or bovine gamma-globulin associated with peritoneal exudate cells gave positive reactions (greater than 5 mm) while the eighteen guinea-pigs given free antigens failed to show a reaction. Similar results were obtained with serum albumins in inbred guinea-pigs. The increased immunogenicity of antigens associated with peritoneal exudate cells was also seen in outbred guinea-pigs.

Pretreatment with Freund's complete adjuvant did not depress the immunogenicity of antigens associated with peritoneal exudate cells and actually increased the induration of the 24-hour reaction to picrylated human serum albumin and human serum albumin associated with peritoneal exudate cells.

     

Non-specific cytotoxicity by T cells activated with plant mitogens in vitro and the requirement for plant agents during the killing reaction

The ability of mouse lymphoid cells to show non-specific cytotoxicity to ⁵¹Cr-labelled mastocytoma cells in vitro was assessed by the release of ⁵¹Cr.

Little cytotoxicity was shown by normal lymphoid cells. However, incubation for several days with mitogens for T cells, such as PHA, Con A, P. graveolens and lentil mitogen, activated spleen cells for cytotoxic killing providing PHA was present during the killing reaction. Pokeweed mitogen, which is an inferior mitogen for T cells, was less active. Three agglutinins, which did not transform T cells, were ineffective. Con A and PWM also activated cortisone-resistant thymus cells.

Activated T cells only killed target cells when a plant agent was present during the killing reaction. The T cell mitogens, which are also strong agglutinins, were effective. PWM was less effective and agents which were not T cell mitogens, including leucoagglutinins, had a variable and usually small effect.

Cell division was not essential for the activation of cytotoxic cells as colcemid diminished but did not abolish activation. DNA synthesis was not required during the killing reaction as judged by the failure of hydroxyurea, an inhibitor of DNA synthesis, to block cytotoxicity when added 2 hr before the killing reaction.

The relation between the number of lymphocytes added and the number of target cells killed was compatible with the `one hit' hypothesis that an effective interaction between a single attacking cell and a target cell led to target cell death.

Non-specific cytotoxicity does not require a strong antigenic difference between attacking and target cells as syngeneic lymphocytes will kill mastocytoma cells. Killing of human cell lines also occurs. The data suggest that the present type of non-specific cytotoxicity is due to activated T cells (presumably blasts) and that plant mitogens are required both for the conversion of the resting T cells to activated cells and to approximate the activated T cells to the target cells and/or initiate a special terminal cytotoxic activation.

     

The serotonin transporter gene as a QTL for ADHD.

Molecular studies of attention deficit hyperactivity disorder (ADHD) have identified susceptibility genes for the categorically diagnosed disorder using operational diagnostic criteria. Here, we take a QTL approach to mapping genes for ADHD using a composite continuous index of ADHD behavior in a large epidemiological sample. Previous studies of clinical ADHD suggest that two functional polymorphisms in the serotonin transporter gene (SLC6A4), one in the 5'-regulatory region of the gene (5-HTTLPR) and the other a VNTR (5-HTTVNTR) in the second intron, as well as a single nucleotide polymorphism in the 3'-untranslated region (3'-UTR SNP), may be associated with the disorder. Here, we investigate these polymorphisms as well as an additional ten SNPs spread across the gene. We found significant association with the long (L) allele of the 5-HTTLPR; P = 0.019, but neither the 5-HTTVNTR nor the 3'-UTR SNP were significantly associated. Significant associations (P < 0.05) were found for a further 5 the 10 other markers tested. We found evidence for two haplotype blocks spanning the region. We found strong evidence for association with the first haplotype block (comprised of four markers), with the significance of a combined primary and secondary test of association reaching an empirical P value = 0.0054 for the global test and an empirical P value = 0.00081 for the largest local test. Thus, we show here that SLC6A4, which has a major influence on brain serotonin availability, may be a QTL for ADHD.     

No association between CHRNA7 microsatellite markers and attention-deficit hyperactivity disorder.

Attention-deficit hyperactivity disorder (ADHD) is a highly heritable, common psychiatric disorder of childhood that probably involves several genes. There are several lines of evidence suggesting that the nicotinic system may be functionally significant in ADHD. First, nicotine promotes the release of dopamine and has been shown to improve attention in adults with ADHD, smokers, and nonsmokers. Second, ADHD is a significant risk factor for early initiation of cigarette smoking in children and maternal cigarette smoking appears to be a risk factor for ADHD. Finally, animal studies in rats and monkeys also suggest that nicotine may be involved in attentional systems and locomotor activity. The nicotinic system has previously been studied in schizophrenia where the neuronal nicotinic acetylcholine receptor alpha 7 subunit gene (CHRNA7) has been implicated in decreased P50 inhibition and attentional disturbances in patients with schizophrenia and in many of their nonschizophrenic relatives. Three known microsatellite markers (D15S165, D15S1043, and D15S1360) near the nicotinic acetylcholine alpha 7 receptor gene, CHRNA7, were studied in 206 ADHD parent-proband trios of children aged 5-16 with ADHD according to DSM-IV criteria. Children with known major medical or psychiatric conditions or mental retardation (IQ < 70) were excluded from the study. Markers D15S165 and D15S1360 were in linkage disequilibrium. The extended Transmission Disequilibrium Test analyses demonstrated no evidence that variation at the microsatellite markers D15S1360, D15S1043, and D15S165 influences susceptibility to ADHD. However, it remains possible that the CHRNA7 gene and other nicotinic system genes may be involved in conferring susceptibility to ADHD.     

The passive transfer of delayed hypersensitivity in the guinea-pig: I. The synergic effect of immune cells and immune serum on the 24-hour skin reaction and a study of the histology

Donor guinea-pigs were immunized with antigen in Freund's complete adjuvant. Three weeks later these donor guinea-pigs showed strong delayed hypersensitivity to the antigen. Peritoneal exudate cells (macrophages) and serum were then transferred intravenously from the immunized donors to normal recipients. Good 24-hour skin reactions were obtained in the recipients which had received cells and serum from donors immunized with bovine serum albumin, γ-globulin, ovalbumin and haemocyanin. The reaction transferred by immune cells and serum was greater than the reaction transferred by either singly. This synergy was not found with PPD or blood group substance as antigen. Good transfers of 24-hour skin reactions to PPD were obtained with peritoneal exudate cells both alone and with serum. Good transfers could not be obtained with blood group substance.

The synergic action of cells and serum in the passive transfer of 24-hour skin reactions was specific. The ability of peritoneal exudate cells to transfer 24-hour skin reactions was present at 1 week and greater at 3 weeks. In contrast the ability of serum to enhance passive transfers was absent at 1 week and present at 2 and 3 weeks.

The histology of reactions passively transferred by spleen, lymph node and peritoneal exudate cells in the presence and absence of serum was studied. Serum alone produced a predominantly polymorphonuclear infiltrate which was maximal at 18–24 hours and almost absent at 48 hours. Cells alone produced only a very slight reaction. The combination of cells and serum produced lesions at 4 and 18 hours resembling those caused by serum alone. At 24 hours however the histiocytes and lymphocytes were more numerous than in the pure serum reaction and by 48 hours the infiltrate consisted of histiocytes and lymphocytes.

It was concluded that, with certain antigens, there is a synergic effect of immune cells and serum in the passive transfer of 24-hour skin reactions and reasons are given for considering that these passively transferred 24-hour skin reactions are indeed delayed hypersensitivity reactions.

     

Desensitization in vitro—The specific inhibition, by antigen, of the passive transfer of delayed hypersensitivity by peritoneal exudate cells

A preliminary experiment showed that the injection of bovine γ-globulin into guinea-pigs with delayed hypersensitivity to bovine γ-globulin reduced the 24-hour skin reactions to bovine γ-globulin and (to a lesser extent) PPD. The peritoneal exudate cells from the desensitized donors had a reduced ability to transfer delayed hypersensitivity to bovine γ-globulin but a normal ability to transfer delayed hypersensitivity to PPD.

Likewise, it was possible to diminish the passive transfer of delayed hypersensitivity to bovine γ-globulin by peritoneal exudate cells, by exposure of the cells to bovine γ-globulin in vitro. The recipients were tested immediately after cell transfer. This in vitro desensitization was specific, in that the transfer of delayed hypersensitivity to PPD was unaffected.

Exposure of cells in vitro to hypotonic conditions and antibody to guinea-pig γ-globulin did not prevent the passive transfer of delayed hypersensitivity.

     

Continuity and Change in Preschool ADHD Symptoms: Longitudinal Genetic Analysis with Contrast Effects

The genetic and environmental mediation of continuity and change in parent-reported ADHD symptoms were investigated in a cohort of over 6000 twin pairs at 2, 3 and 4 years of age. Genetic analyses of the cross-sectional data yielded heritability estimates of 0.78–0.81 at each age, with contrast effects. A common pathway model provided the best fit to the longitudinal data, indicating that genetic influences underlie 91% of the stable variance in ADHD symptomatology. In other words, what is stable for ADHD symptoms is largely genetic. Contrast effects acting in the same direction at different ages contributed to the observed continuity:longitudinal correlations were greater for dizygotic than monozygotic twins.The Twins Early Development Study is funded by the Medical Research Council.     

Hypescheme: an operational criteria checklist and minimum data set for molecular genetic studies of attention deficit and hyperactivity disorders

Investigators engaged in mapping the genetic basis of attention deficit hyperactivity disorder (ADHD) currently use a number of measures for the collection of clinical information. This gives rise to difficulties in comparing datasets and research communications between independent groups. This paper describes the development of Hypescheme, which is an operational criteria checklist for ADHD, oppositional defiant disorder (ODD), and conduct disorder (CD), and is proposed as a minimum dataset for those engaged in molecular genetic studies of ADHD. Hypescheme consists of a computerised data checklist system that includes all the operational criteria required for both DSM-IV and ICD-10 diagnostic criteria and a systematic record of information about comorbid psychiatric, developmental, and neurological disorders. Using this data, an algorithm applies both DSM-IV and ICD-10 criteria to generate operational diagnostics under both these systems. Hypescheme is not designed to replace current assessment protocols but to be a final common checklist that can be completed by experienced researchers using all available data.