Testing for gene x environment interaction effects in attention deficit hyperactivity disorder and associated antisocial behavior.

Gene x environment (G x E) interactions are increasingly thought to have substantial influence on the aetiology and clinical manifestations of complex disorders. In ADHD, although main effects of specific genetic variants and pre- or peri-natal variables have been reported and replicated using pooled analyses, few studies have looked at possible interactions. In a clinical sample of 266 children with ADHD, we tested for interaction between gene variants (in DRD4, DAT1, DRD5, and 5HTT) found to be associated with ADHD in pooled analyses and maternal smoking, alcohol use during pregnancy and birth weight. First, G x E effects on a diagnosis of ADHD were tested using conditional logistic regression analyses. Second, possible modifying effects of G x E on symptoms of associated conduct disorder and oppositional defiant disorder (ODD) were investigated using linear regression analysis. The sample size associated with each of the analyses differed as not each variant had been genotyped for each individual. No effects of G x E on ADHD diagnosis were observed. The results suggest that lower birth weight and maternal smoking during pregnancy may interact with DRD5 and DAT1 (birth weight only) in influencing associated antisocial behavior symptoms (ODD and conduct disorder). These preliminary findings showed no evidence of interaction between previously implicated variants in ADHD and specific environmental risk factors, on diagnosis of the disorder. There may be evidence of G x E on associated antisocial behavior in ADHD, but further investigation is needed.

Association analysis of 528 intra-genic SNPs in a region of chromosome 10 linked to late onset Alzheimer's disease.

Late-onset Alzheimer's disease (LOAD) is a genetically complex neurodegenerative disorder. Currently, only the epsilon4 allele of the Apolipoprotein E gene has been identified unequivocally as a genetic susceptibility factor for LOAD. Others remain to be found. In 2002 we observed genome-wide significant evidence of linkage to a region on chromosome 10q11.23-q21.3 [Myers et al. (2002) Am J Med Genet 114:235-244]. Our objective in this study was to test every gene within the maximum LOD-1 linkage region, for association with LOAD. We obtained results for 528 SNPs from 67 genes, with an average density of 1 SNP every 10 kb within the genes. We demonstrated nominally significant association with LOAD for 4 SNPs: rs1881747 near DKK1 (P = 0.011, OR = 1.24), rs2279420 in ANK3 (P = 0.022, OR = 0.79), rs2306402 in CTNNA3 (P = 0.024, OR = 1.18), and rs5030882 in CXXC6 (P = 0.046, OR = 1.29) in 1,160 cases and 1,389 controls. These results would not survive correction for multiple testing but warrant attempts at confirmation in independent samples.

DNA pooling and dense marker maps: a systematic search for genes for cognitive ability.

Pooling DNA from subjects within a group and comparing the pooled DNA across groups for a dense map of DNA markers offers a solution to the conundrum that linkage is systematic but not powerful whereas allelic association is powerful but not systematic. We used DNA pooling to screen 66 markers on chromosome 22 in original and replication samples of children of high general cognitive ability (g) and controls of average g. Although none of these markers survived our three-stage screening design (original pooling, replication pooling, individual genotyping), the results of DNA pooling were largely confirmed by individual genotyping. We can therefore exclude associations of major effect size on chromosome 22 for g, a key variable for cognitive neuroscience research on learning and memory.

No association between apolipoprotein E polymorphisms and general cognitive ability in children.

In this work we explored the hypothesis that variation in the gene encoding apolipoprotein E (ApoE) is a factor modifying general cognitive ability (g). A case control sample of 101 high g and 101 average g children was scored for ApoE genotypes and two variants in the transcriptional regulatory region of the gene (Th1/E47cs and -491 AT). No evidence of association between these polymorphisms and g was found. We conclude that variation at these loci is not a factor with a measurable impact on general cognitive ability in the healthy population.

Interaction between the ADAM12 and SH3MD1 genes may confer susceptibility to late-onset Alzheimer's disease.

The neuropathology of Alzheimer's disease (AD) is characterized by intracellular neurofibrillary tangles and the extracellular deposition of beta-amyloid (Abeta) in senile plaques. Abeta has been shown to mediate neurodegenerative and inflammatory changes associated with amyloid plaques, although the pathological mechanism of Abeta remains largely unknown. Recent evidence suggests that the FISH adapter protein binds to, and potentially regulates, ADAM12 (a disintegrin and metalloprotease 12) to mediate a neurotoxic effect of Abeta. The ADAM12 gene lies on chromosome 10q26.3, and the gene encoding FISH, SH3MD1, lies within a region of linkage to late-onset AD (LOAD) on 10q25.1. This study investigates whether there is a relationship between variation in ADAM12 and SH3MD1 and susceptibility to LOAD in a sample of 1,051 AD cases and 1,269 matched controls. We observe significant interactions between variants in the two genes that may influence susceptibility to LOAD. The most significant statistical interaction is between rs3740473, a synonymous single nucleotide polymorphism (SNP) in SH3MD1 and rs11244787, an intronic SNP in ADAM12 (effect size = 2.1 for interaction term, P = 0.006).

Association studies of 23 positional/functional candidate genes on chromosome 10 in late-onset Alzheimer's disease.

Late-onset Alzheimer's disease (LOAD) is a common neurodegenerative disorder, with a complex etiology. APOE is the only confirmed susceptibility gene for LOAD. Others remain yet to be found. Evidence from linkage studies suggests that a gene (or genes) conferring susceptibility for LOAD resides on chromosome 10. We studied 23 positional/functional candidate genes from our linkage region on chromosome 10 (APBB1IP, ALOX5, AD037, SLC18A3, DKK1, ZWINT, ANK3, UBE2D1, CDC2, SIRT1, JDP1, NET7, SUPV3L1, NEN3, SAR1, SGPL1, SEC24C, CAMK2G, PP3CB, SNCG, CH25H, PLCE1, ANXV111) in the MRC genetic resource for LOAD. These candidates were screened for sequence polymorphisms in a sample of 14 LOAD subjects and detected polymorphisms tested for association with LOAD in a three-stage design involving two stages of genotyping pooled DNA samples followed by a third stage in which markers showing evidence for association in the first stages were subjected to individual genotyping. One hundred and twenty polymorphisms were identified and tested in stage 1 (4 case + 4 control pools totaling 366 case and 366 control individuals). Single nucleotide polymorphisms (SNPs) showing evidence of association with LOAD were then studied in stage 2 (8 case + 4 control pools totaling 1,001 case and 1,001 control individuals). Five SNPs, in four genes, showed evidence for association (P < 0.1) at stage 2 and were individually genotyped in the complete dataset, comprising 1,160 LOAD cases and 1,389 normal controls. Two SNPs in SGPL1 demonstrated marginal evidence of association, with uncorrected P values of 0.042 and 0.056, suggesting that variation in SGPL1 may confer susceptibility to LOAD.

A family based study of catechol-O-methyltransferase (COMT) and attention deficit hyperactivity disorder (ADHD).

Neurobiological studies have suggested that altered dopaminergic function may contribute to the etiology of attention deficit hyperactivity disorder (ADHD). The gene encoding catechol-O-methyltransferase (COMT) is an attractive candidate for ADHD susceptibility as it plays a major role in the degradation of dopamine. Moreover, a functional Val158Met polymorphism in COMT that alters the activity of the encoded protein has been strongly implicated in frontal lobe function, with the high activity Valine allele being associated with poorer performance, and ADHD is thought to involve fronto-striatal pathways. We have examined this functional variant for association with ADHD in a family based association sample comprising 279 probands and their parents. We have also examined two other markers in the COMT gene (rs737865, rs165599) which, together with the Val/Met variant, have recently been shown to be associated with altered COMT expression rather than enzyme activity. No evidence for association was observed with any single marker or haplotype in a sample of 279 affected children and their parents.

No support for association between the dopamine transporter (DAT1) gene and ADHD.

Several groups have reported an association between the 10-repeat allele of a dopamine transporter (DAT1) 3'UTR VNTR variant and ADHD but the finding has not been universally observed. An association between DAT1 genotype and stimulant medication response has also been reported although again there are conflicting data. We tested the DAT1 3'VNTR and three SNPs in the putative promoter region of DAT1 for association with ADHD in 263 parent-proband trios. Analyses of genotypes, alleles, and haplotypes were performed using family-based association methods. Case-control analysis of the VNTR in 263 cases and 287 controls was also conducted. In addition, we tested for association between the VNTR marker and stimulant medication response. Comparing allele 10 versus all other alleles combined, no significant association was found with ADHD, using FBAT analysis (chi2 = 0.1 (df 1), P = 0.9, (odds ratio (OR) = 1.0, 95% CI 0.8-1.2), and case-control analysis (chi2 = 0.12 (df 2), P = 0.91). No evidence of association with any of the SNPs in the promoter region was found. Haplotype analysis was also non-significant (chi2 = 3.93, (df 9) global P = 0.85). Finally, no association was found between the DAT 1 VNTR and response to stimulant medication (chi2 = 1.63 (df 3) P = 0.65). We conclude that the 3' VNTR and three additional promoter variants in DAT1 do not appear to be associated with ADHD, or response to stimulant mediation in our sample.

A functional polymorphism in the succinate-semialdehyde dehydrogenase (aldehyde dehydrogenase 5 family, member A1) gene is associated with cognitive ability.

Succinate-semialdehyde dehydrogenase (SSADH) deficiency is a rare cause of learning disability. We have investigated SSADH to assess its contribution to cognitive ability in the general population in both case-control- and family-based analyses. Sequence analysis of SSADH revealed four changes affecting the encoded protein, only one of which had a minor allele whose frequency is even moderately common. We genotyped this functional polymorphism in 197 high-IQ cases, 201 average-IQ controls and 196 parent high-IQ offspring trios. The minor allele was significantly less frequent in high-IQ cases and was significantly less frequently transmitted by parents to high-IQ subjects than chance expectation. A previous study has shown that the minor allele encodes a lower activity enzyme than the major allele. These data suggest that higher SSADH activity is associated with higher intelligence across the general population. The effect is small, with each allele having an effect size translating to about 1.5 IQ points.

Direct analysis of the genes encoding G proteins G alpha T2, G alpha o, G alpha Z in ADHD.

We have followed up the extensive replicated evidence that the dopamine DRD4 receptor is involved in the aetiology of ADHD by undertaking direct analysis of genes encoding other proteins in this effector system. We prioritised the genes encoding G protein alpha subunits G alpha(T2), G alpha(o), G alpha(Z) as these have been shown to transduce the effects of ligand binding at DRD4. We screened the exons of all three genes for sequence variation in 28 unrelated subjects with ADHD and identified 13 novel polymorphisms. All were tested for possible association with ADHD using a combination of pooled and individual genotyping. The results of our study do not suggest that polymorphisms in these genes contribute to susceptibility to ADHD.

A simple method for analyzing microsatellite allele image patterns generated from DNA pools and its application to allelic association studies.

Allelic association studies provide the most powerful method for locating genes of small effect contributing to complex diseases and traits. However, in outbred populations, allelic association is usually maintained only over distances of <=1 cM. Therefore, systematic searches over large regions are costly. Here we present a method involving DNA pooling that can be used as a rapid preliminary screen for allelic association with the most common class of polymorphic markers, single-sequence repeats. Patient and control samples are pooled separately, and markers are typed in the two pools. By use of primers with fluorescent 5' ends, PCR products can be analyzed on an automated sequencing apparatus. Allele image patterns (AIPs) produced for the two groups are overlaid and differences in pattern area between pools computed. From this, a DeltaAIP statistic is calculated from the difference in areas between the two AIPs expressed as a fraction of the total shared and nonshared area. AIPs of a range of different-sized pools were generated by computer simulation for markers with a range of allele sizes and frequencies. DeltaAIPs from pools and chi2 values for individual genotypings were compared, with both simulated and real data from microsatellite markers. The results demonstrated a high correlation between DeltaAIP and chi2 values. DeltaAIP analysis of real microsatellite data indicated the feasibility of using this method in systematic searches for allelic association and generated a small number of false positives but few false negatives. We conclude that DeltaAIP analysis of DNA pools can be used effectively and efficiently as a rapid screen for allelic association in case-control studies.

Follow-up of genetic linkage findings on chromosome 16p13: evidence of association of N-methyl-D aspartate glutamate receptor 2A gene polymorphism with ADHD.

Attention deficit hyperactivity disorder (ADHD) is a childhood onset disorder, for which there is good evidence that genetic factors contribute to the aetiology. Recently reported linkage findings suggested evidence of a susceptibility locus on chromosome 16p13 (maximum LOD score of 4.2, P=5 x 10(-6)). The GRIN2A (glutamate receptor, ionotropic, N-methyl D-aspartate 2A) gene that encodes the N-methyl D-aspartate receptor subunit 2A (NMDA2A) maps to this region of linkage. As this is also a good functional candidate gene for ADHD, we undertook family-based association analysis in a sample of 238 families. We found significant evidence of association with a GRIN2A exon 5 polymorphism (chi(2)=5.7, P=0.01). Our data suggest that genetic variation in GRIN2A may confer increased risk for ADHD and that this, at least in part, might be responsible for the linkage result on 16p reported by Smalley et al. We conclude that replication is required and that further work examining for association of GRIN2A polymorphisms with ADHD is warranted.

A genome-wide scan of 1842 DNA markers for allelic associations with general cognitive ability: a five-stage design using DNA pooling and extreme selected groups.

All measures of cognitive processes correlate moderately at the phenotypic level and correlate substantially at the genetic level. General cognitive ability (g) refers to what diverse cognitive processes have in common. Our goal is to identify quantitative trait loci (QTLs) associated with high g compared with average g. In order to detect QTLs of small effect size, we used extreme selected samples and a five-stage design with nominal alpha levels that permit false positive results in early stages but remove false positives in later stages. As a first step toward a systematic genome scan for allelic association, we used DNA pooling to screen 1842 simple sequence repeat (SSR) markers approximately evenly spaced at 2 cM throughout the genome in a five-stage design: (1) case-control DNA pooling (101 cases with mean IQ of 136 and 101 controls with mean IQ of 100), (2) case-control DNA pooling (96 cases with IQ > 160 and 100 controls with mean IQ of 102), (3) individual genotyping of Stage 1 sample, (4) individual genotyping of Stage 2 sample, (5) transmission disequilibrium test (TDT; 196 parent-child trios for offspring with IQ > 160). The over all Type I error rate is 0.000125, which robustly protects against false positive results. The numbers of markers surviving each stage using a conservative allele-specific directional test were 108, 6, 4, 2, and 0, respectively, for the five stages. A genomic control test using DNA pooling suggested that the failure to replicate the positive case-control results in the TDT analysis was not due to ethnic stratification. Several markers that were close to significance at all stages are being investigated further. Relying on indirect association based on linkage disequilibrium between markers and QTLs means that 100,000 markers may be needed to exclude QTL associations. Because power drops off precipitously for indirect association approaches when a marker is not close to the QTL, we are not planning to genotype additional SSR markers. Instead we are using the same design to screen markers such as cSNPs and SNPs in regulatory regions that are likely to include functional polymorphisms in which the marker can be presumed to be the QTL.

DNA pooling identifies QTLs on chromosome 4 for general cognitive ability in children.

General cognitive ability (g), which is related to many aspects of brain functioning, is one of the most heritable traits in neuroscience. Similarly to other heritable quantitatively distributed traits, genetic influence on g is likely to be due to the combined action of many genes of small effect [quantitative trait loci (QTLs)], perhaps several on each chromosome. We used DNA pooling for the first time to search a chromosome systematically with a dense map of DNA markers for allelic associations with g. We screened 147 markers on chromosome 4 such that 85% of the chromosome were estimated to be within 1 cM of a marker. Comparing pooled DNA from 51 children of high g and from 51 controls of average g, 11 significant QTL associations emerged. The association with three of these 11 markers ( D4S2943, MSX1 and D4S1607 ) replicated using DNA pooling in independent samples of 50 children of extremely high g and 50 controls. Furthermore, all three associations were confirmed when each individual was genotyped separately ( D4S2943, P = 0. 00045; MSX1, P = 0.011; D4S1607, P = 0.019). Identifying specific genes responsible for such QTL associations will open new windows in cognitive neuroscience through which to observe pathways between genes and learning and memory.

Linkage disequilibrium mapping provides further evidence of a gene for reading disability on chromosome 6p21.3-22.

Linkage disequilibrium (LD) mapping was used to follow up reports of linkage between reading disability (RD) and an 18 cM region of chromosome 6p21.3-22. Using a two-stage approach, we tested for association between RD and 22 microsatellite markers in two independent samples of 101 (Stage 1) and 77 (Stage 2) parent/proband trios in which RD was rigorously defined. The most significant replicated associations were observed between combinations of markers D6S109/422/1665 (Stage 1, P=0.002 (adjusted for multiple testing); Stage 2, P=0.0001) and D6S506/1029/1660 (Stage 1, P=0.02 (adjusted), Stage 2, P=0.0001). The only two-marker association observed in both samples was with D6S422/1665 (P=0.01, 0.04). No single marker showed replicated association but D6S506 produced values of P=0.01 and 0.08 which were significant when combined (P=0.02). We observed weaker and less consistent evidence of association in a region of confirmed linkage to RD in previous studies. The most consistently significant haplotypic association D6S109/422/1665, showed association with single-word reading, spelling, phonological awareness, phonological decoding, orthographic accuracy and random automised naming, but not with vocabulary or Attention Deficit Hyperactivity Disorder. Our findings strongly support the presence of a gene contributing to RD in a region of chromosome 6 between markers D6S109 and D6S1260, but do not rule out the presence of a gene between D6S1556 and MOG.

Substantial linkage disequilibrium across the insulin-degrading enzyme locus but no association with late-onset Alzheimer's disease.

Insulin-degrading enzyme (IDE; insulysin; EC 3.4.24.56) is a 110-kDa neutral metallopeptidase that can degrade a number of peptides including beta-amyloid. The gene encoding IDE is located on chromosome 10 close to a region of linkage for late-onset Alzheimer's disease (LOAD) and thus is a functional and positional candidate for this disorder. We analysed all of the coding exons, untranslated regions and 1000 bp of 5'-flanking sequence of IDE by using denaturing high-performance liquid chromatography and sequencing. We detected eight single nucleotide polymorphisms (SNPs), three in the 5' flanking sequence and five in the coding sequence, of which three were found at lower than 5% frequency. None of them changed the amino acid sequence. We genotyped the five SNPs with allele frequencies of more than 5% in 133 Caucasian LOAD cases and 135 controls collected in the UK and 95 cases and 117 controls collected at the Mayo Clinic, Rochester, USA. Two of the SNPs were analysed in a further independent case-control sample (Washington University, St. Louis: 86 cases, 94 controls). No significant association was found with any individual SNP in any of the samples or with any haplotypes. Analysis of the marker D10S583, which maps 36 kb upstream of IDE, also failed to show association in 134 cases and 111 matched controls from the UK ( P=0.63). Strong linkage disequilibrium was detected between the five SNPs that spanned the whole of the 120-kb genomic region of IDE and one major and a number of minor haplotypes were detected in the populations studied. We conclude that IDE does not make a substantial contribution to the aetiology of LOAD and therefore cannot account for the linkage between LOAD and 10q.

Comparative sequencing and association studies of aromatic L-amino acid decarboxylase in schizophrenia and bipolar disorder.

Aromatic L-amino acid decarboxylase (AADC) is a relatively non specific enzyme involved in the biosynthesis of several classical neurotransmitters including dopamine and 5-hydroxytryptamine (5HT; serotonin). AADC does not catalyse the rate limiting step in either pathway, but is rate limiting in the synthesis of 2-phenylethylamine (2PE) which is a positive modulator of dopaminergic transmission and a candidate natural psychotogenic compound.1 We and others have proposed that polymorphism in AADC resulting in altered 2PE activity might contribute to the pathogenesis of psychosis. In order to test this hypothesis, we have used denaturing high performance liquid chromatography (DHPLC)3 to screen 3943 bases of the AADC gene and its promoter regions for variants that might affect protein structure or expression in 15 unrelated people with schizophrenia, and 15 unrelated people with bipolar disorder. Three polymorphisms were identified by DHPLC: a insertion/deletion polymorphism in the 5' UTR of the neuronal specific mRNA (g.-33-30delAGAG, bases 586-589 of GenBank M77828), a T>A variant in the non-neuronal exon 1 (g. -67T>A, GenBank M88070), and a G>A polymorphism within intron 8 (g. IVS8 +75G>A, GenBank M84598). Case-control analysis did not suggest that genetic polymorphism in the AADC gene is associated with liability for developing schizophrenia or bipolar disorder.

Serotonergic system and attention deficit hyperactivity disorder (ADHD): a potential susceptibility locus at the 5-HT(1B) receptor gene in 273 nuclear families from a multi-centre sample.

Attention deficit hyperactivity disorder (ADHD) is a highly heritable and heterogeneous disorder, which usually becomes apparent during the first few years of childhood. Imbalance in dopamine neurotransmission has been suggested as a factor predisposing to ADHD. However, evidence has suggested an interaction between dopamine and serotonin systems in the pathophysiology of the disorder. Studies using selective agonists of the different 5-HT receptors microinjected into selected brain structures have shown a positive modulating effect on the functional activities of the mesotelencephalic dopaminergic system. This suggests that some of the genetic predisposition to ADHD might be due to DNA variation at serotonin system genes. In this study, we investigated polymorphisms in HTR(1B) and HTR(2A) (which encode the serotonin receptors 5-HT(1B) and 5-HT(2A) respectively) in a European ADHD sample. Using haplotype based haplotype relative risk (HHRR) and transmission disequilibrium test (TDT) analyses, we observed significant preferential transmission of the allele 861G of the HTR(1B) in the total sample (for HHRR; chi(2) = 7.4, P = 0.0065 and TDT; (chi(2) = 6.4, P = 0.014). Analysis of HTR(2A) failed to reveal evidence of association or linkage between the His452Tyr polymorphism and ADHD in the total sample. However, a significantly increased transmission of the allele 452His was observed in the Irish sample alone (chi(2) = 4.9, P = 0.026). These preliminary data suggest an important role for the serotonin system in the development of ADHD. Further studies, preferentially including different ethnic groups are required to substantiate these findings.

Family-based association mapping provides evidence for a gene for reading disability on chromosome 15q.

Family-based association mapping was used to follow up reports of linkage between reading disability (RD) and a genomic region on chromosome 15q. Using a two-stage approach, we ascertained 101 (stage 1) and 77 (stage 2) parent-proband trios, in which RD was characterized rigorously. In stage 1, a set of eight microsatellite markers spanning the region of putative linkage was used and a highly significant association was detected between RD and a three-marker haplotype (D15S994/D15S214/D15S146: P and empirical P < 0.001). A significant association with the same three-marker haplotype was also observed in the second-stage sample (P = 0.009, empirical P = 0.006). Our data therefore provide strong evidence for one or more genes contributing to RD being located in the vicinity of the region including D15S146 and D15S994. In addition, our results provide support for association analysis being a useful method to map susceptibility loci for complex disorders.