Haplotype study of three polymorphisms at the dopamine transporter locus confirm linkage to attention-deficit/hyperactivity disorder.

BACKGROUND: Attention-deficit/hyperactivity disorder (ADHD) is often treated using methylphenidate, a psychostimulant that inhibits the dopamine transporter. This led E.H. Cook and colleagues to consider the dopamine transporter locus (DAT1) as a primary candidate gene for ADHD. That group reported a significant association between ADHD and the 480-base pair (bp) allele of the variable number of tandem repeats (VNTR) polymorphism located in the 3' untranslated region of the DAT1 gene. This association was later replicated in additional studies. METHODS: The DAT1 gene has additional common polymorphisms in intron 9 and exon 9. We investigated the possibility of linkage of DAT1 and ADHD using the VNTR polymorphism and two additional common polymorphisms in 102 nuclear families with an ADHD proband. Using the transmission disequilibrium test, we examined the transmission of the alleles of each of these polymorphisms, as well as the haplotypes of the polymorphisms. RESULTS: We did not observe significant evidence for the biased transmission of the alleles of either the VNTR or the additional two polymorphisms when examined individually, although there was a trend for the biased transmission of the 480-bp allele of the VNTR. When we examined the haplotypes of the three polymorphisms we found significant evidence for biased transmission of one of the haplotypes containing the 480-bp VNTR allele. We also genotyped six additional DNA sequence variants of the DAT1 gene. However, these variants were not sufficiently polymorphic in our sample to be informative. Two of the DNA variants that result in an amino acid change, Ala559Val and Glu602Gly, were not observed in our sample. CONCLUSIONS: Our results support previous findings of an association between the DAT1 gene and ADHD.

Linkage of the dopamine D4 receptor gene and attention-deficit/hyperactivity disorder.

OBJECTIVE: There is considerable evidence supporting a genetic component in the etiology of attention-deficit/hyperactivity disorder (ADHD). Because stimulant medications act primarily on the dopaminergic system, dopamine system genes are prime candidates for genetic susceptibility factors for ADHD. Previous studies by several groups have observed a significant association of ADHD and an allele with 7 copies of the 48 base pair repeat in the third exon of the dopamine D4 receptor. METHOD: The authors sought to replicate these previous findings by collecting an independent sample of families from Toronto, Ontario, Canada, and confirming this finding in an expanded sample of ADHD families collected from Irvine, California. Using the transmission disequilibrium test (TDT), the authors tested for biased transmission of the 7-repeat allele at the exon III polymorphism of the dopamine D4 receptor locus in these samples of ADHD subjects. RESULTS: Biased transmission of the 7-repeat allele from parents to ADHD probands and their affected siblings was observed in the 2 new samples of families collected in Toronto and Irvine (TDT chi2 = 2.711, 1 df, one-sided p value = .050) and for these samples combined with the 52 families previously reported from Irvine (TDT chi2 = 6.426, 1 df, one-sided p value = .006). CONCLUSIONS: The results of this study further support the possibility of a role of the dopamine D4 receptor locus in ADHD.

Effect of methylphenidate on attention in children with attention deficit hyperactivity disorder (ADHD): ERP evidence.

Methylphenidate is the most common treatment for attention deficit hyperactivity disorder (ADHD) and has been shown to improve attention and behaviour. However, the precise nature of methylphenidate on specific aspects of attention at different dose levels remains unclear. We studied methylphenidate effects in ADHD from a neurophysiological perspective, recording event-related potentials (ERPs) during attention task performance in normal controls and children with ADHD under different dose conditions. Twenty children with ADHD and 20 age matched controls were assessed with a continuous performance task requiring subjects to identify repeating alphabetic characters. ERPs and behavioural measures were recorded and analyzed for trials where a correct response was made. The ADHD group was assessed off drug (baseline) and on placebo, low (0.28 mg/kg) and high (0.56 mg/kg) dose levels of methylphenidate. The results showed that the ADHD group at baseline was more impulsive and inattentive than controls and had shorter P2 and N2 latencies and longer P3 latencies. Low dose methylphenidate was associated with reduced impulsivity (fewer false alarms) and decreased P3 latencies, whereas the higher dose level was associated with reduced impulsivity and less inattention (more hits), as well as increased P2 and N2 latencies and decreased P3 latencies. Amplitudes were unaffected and there were no adverse effects of the higher dose for any of the children. These results suggest differential dosage effects and a dissociation between dose levels and aspects of processing.

Dopamine D4 receptor gene: novelty or nonsense?

Although the role of genetics in personality has been studied extensively at a phenomenological level, only lately has the investigation of specific genes been performed. Recent reports suggest that DNA variants of the dopamine D4 receptor gene (DRD4) are associated with the personality trait of novelty seeking; however, others fail to replicate this finding. Such conflicting results suggest either a weak effect, an association only in certain populations, or a false-positive resulting from population stratification. We provide a critical analysis of genetic studies of DRD4 variants with novelty seeking, alcoholism, drug abuse, and attention deficit hyperactivity disorder. Evidence for the role of DRD4 in novelty seeking is inconclusive, with a number of methodological concerns. Use of more conservative statistical criteria for significance, employing gene haplotypes, as well as linkage disequilibrium studies, are recommended. The molecular biology of the D4 gene is also reviewed.

Characterization of an AGAMOUS homologue from the conifer black spruce (Picea mariana) that produces floral homeotic conversions when expressed in Arabidopsis.

Advances in elucidating the molecular processes controlling flower initiation and development have provided unique opportunities to investigate the developmental genetics of non-flowering plants. In addition to providing insights into the evolutionary aspects of seed plants, identification of genes regulating reproductive organ development in gymnosperms could help determine the level of homology with current models of flower induction and floral organ identity. Based upon this, we have searched for putative developmental regulators in conifers with amino acid sequence homology to MADS-box genes. PCR cloning using degenerate primers targeted to the MADS-box domain revealed the presence of over 27 MADS-box genes within black spruce (Picea mariana), including several with extensive homology to either AP1 or AGAMOUS, both known to regulate flower development in Arabidopsis. This indicates that like angiosperms, conifers contain a large and diverse MADS-box gene family that probably includes regulators of reproductive organ development. Confirmation of this was provided by the characterization of an AGAMOUS-like cDNA clone called SAG1, whose conservation of intron position and tissue-specific expression within reproductive organs indicate that it is a homologue of AGAMOUS. Functional homology with AGAMOUS was demonstrated by the ability of SAG1 to produce homeotic conversions of sepals to carpels and petals to stamens when ectopically expressed in transgenic Arabidopsis. This suggests that some of the genetic pathways controlling flower and cone development are homologous, and antedate the 300-million-year-old divergence of angiosperms and gymnosperms.

Directions of aetiologic research on attention deficit hyperactivity disorder.

OBJECTIVE: The aim of this paper is to review and integrate recent literature on aetiological factors that have been postulated for attention deficit hyperactivity disorder (ADHD). METHOD: Recent studies relating to perinatal brain damage, intra-uterine toxic effects, neurochemical, brain imaging and genetic studies are reviewed, and those considered most significant are discussed. Where possible, recent findings are integrated and directions of future research are suggested. Clinical implications are briefly discussed. RESULTS: Perinatal studies indicate that children with a birth weight under 750 g may be disadvantaged for attentional skills. Magnetic resonance imaging (MRI) and steady state visually evoked potential studies show differences in prefrontal, caudate and parietal areas in ADHD children, suggesting right hemispheric dysfunction. Functional MRI studies hold promise in further elucidating attentional systems in the central nervous system that are involved in ADHD. Genetic studies suggest genes related to dopaminergic systems may be important. CONCLUSIONS: Recent research on ADHD has made considerable advances, particularly in the areas of brain imaging and genetic studies. Genetic studies should provide further aetiological understandings of ADHD, leading to more targeted treatments.

Association of the dopamine receptor D4 (DRD4) gene with a refined phenotype of attention deficit hyperactivity disorder (ADHD): a family-based approach.

Previously in this journal, we reported an association of the dopamine D4 receptor gene (DRD4) and attention deficit hyperactivity disorder (ADHD). In a population-association (case-control) study of 39 children with a refined phenotype of ADHD and 39 ethnically matched controls, we observed an increased percentage of the 7 repeat allele (29% vs 12%) and the 7+ genotype (49% vs 21%) in the ADHD group compared to the control group. In a replication and an extension of our initial study, we recruited another sample of ADHD subjects and found percentages of the 7 repeat allele (28%) and the 7+ genotype (48%) consistent with our previous findings. We used a family-based approach to evaluate a predicted association of DRD4 and ADHD based on a test of allele transmission focused on the 7 repeat allele. We identified 52 families based on the diagnosis of the refined phenotype of ADHD in the proband and the availability of DNA from both biological parents as well as the proband. Haplotype relative risk (HRR) analysis was performed to test our a priori hypothesis and produced significant results (chi-square = 4.65, P < 0.035). This provides additional evidence that the DRD4 gene is associated with a refined phenotype of ADHD.

Effects of methylphenidate in children with attention deficit hyperactivity disorder: a comparison of event-related potentials between medication responders and non-responders.

Event-related potentials (ERPs) were compared among three groups, each with 13 subjects: (1) ADHD non-responders to methylphenidate treatment; (2) ADHD responders to methylphenidate treatment; and (3) normal control children. Response to methylphenidate was determined through extensive psychoeducational and cognitive assessments during a 4-week double-blind medication assessment. ERPs were recorded each week from 13 active electrodes during a visual feature detection task and a semantic classification task. Significant group effects were found for N2 and P3b latencies due to longer latencies for the ADHD children. Off medication, there were no differences between responders and non-responders. However, on methylphenidate non-responders had significantly longer P3b latencies than responders. Cognitive testing also revealed differential performance on medication between non-responders and responders on the paired-associate learning (PAL) task. Thus, both cognitive and ERP measures were found to differentiate ADHD non-responders and responders to methylphenidate treatment.

An acetohydroxy acid synthase mutant reveals a single site involved in multiple herbicide resistance.

Acetohydroxy acid synthase (AHAS) is an essential enzyme for many organisms as it catalyzes the first step in the biosynthesis of the branched-chain amino acids valine, isoleucine, and leucine. The enzyme is under allosteric control by these amino acids. It is also inhibited by several classes of herbicides, such as the sulfonylureas, imidazolinones and triazolopyrimidines, that are believed to bind to a relic quinone-binding site. In this study, a mutant allele of AHAS3 responsible for sulfonylurea resistance in a Brassica napus cell line was isolated. Sequence analyses predicted a single amino acid change (557 Trp-->Leu) within a conserved region of AHAS. Expression in transgenic plants conferred strong resistance to the three classes of herbicides, revealing a single site essential for the binding of all the herbicide classes. The mutation did not appear to affect feedback inhibition by the branched-chain amino acids in plants.

T-DNA tagging of a seed coat-specific cryptic promoter in tobacco.

T-DNA tagging with a promoterless beta-glucuronidase (GUS) gene generated a transgenic Nicotiana tabacum plant that expressed GUS activity only in developing seed coats. Cloning and deletion analysis of the GUS fusion revealed that the promoter responsible for seed coat specificity was located in the plant DNA proximal to the GUS gene. A 3.3 kb fragment corresponding to the insertion site was isolated from untransformed plants. No long open reading frames were detected in this region. Northern blots and RNase protection assays failed to detect transcripts from this region in untransformed plants. Furthermore, the insertion site was situated within the N. tomentosiformis genome of the allotetraploid species N. tabacum, in a region which is not conserved within the genus Nicotiana. It is concluded that seed coat-specific GUS expression in this transgenic plant resulted from T-DNA insertion next to a cryptic promoter. These results suggest that at least some of the fusions generated to marker genes in promoter trapping studies are not associated with conventional gene promoters. The possibility that similar insertion events play a role in gene evolution is discussed.

Multiple resistance to sulfonylureas and imidazolinones conferred by an acetohydroxyacid synthase gene with separate mutations for selective resistance.

The acetohydroxyacid synthase (AHAS) gene from the Arabidopsis thaliana mutant line GH90 carrying the imidazolinone resistance allele imr1 was cloned. Expression of the AHAS gene under the control of the CaMV 35S promoter in transgenic tobacco resulted in selective imidazolinone resistance, confirming that the single base-pair change found near the 3' end of the coding region of this gene is responsible for imidazolinone resistance. A chimeric AHAS gene containing both the imr1 mutation and the csr1 mutation, responsible for selective resistance to sulfonylurea herbicides, was constructed. It conferred on transgenic tobacco plants resistance to both sulfonylurea and imidazolinone herbicides. The data illustrate that a multiple-resistance phenotype can be achieved in an AHAS gene through combinations of separate mutations, each of which individually confers resistance to only one class of herbicides.

Transformation of Brassica napus canola cultivars with Arabidopsis thaliana acetohydroxyacid synthase genes and analysis of herbicide resistance.

A survey of selected crop species and weeds was conducted to evaluate the inhibition of the enzyme acetohydroxyacid synthase (AHAS) and seedling growth in vitro by the sulfonylurea herbicides chlorsulfuron, DPX A7881, DPX L5300, DPX M6316 and the imidazolinone herbicides AC243,997, AC263,499, AC252,214. Particular attention was given to the Brassica species including canola cultivars and cruciferous weeds such as B. kaber (wild mustard) and Thlaspi arvense (stinkweed). Transgenic lines of B. napus cultivars Westar and Profit, which express the Arabidopsis thaliana wild-type AHAS gene or the mutant gene csr1-1 at levels similar to the resident AHAS genes, were generated and compared. The mutant gene was essential for resistance to the sulfonylurea chlorsulfuron but not to DPX A7881, which appeared to be tolerated by certain Brassica species. Cross-resistance to the imidazolinones did not occur. The level of resistance to chlorsulfuron in transgenic canola greatly exceeded the levels that were toxic to the Brassica species or cruciferous weeds. Direct selection of transgenic lines with chlorsulfuron sprayed at field levels under greenhouse conditions was achieved.