Association study of SNAP25 and schizophrenia in Irish family and case-control samples.

SNAP25 occurs on chromosome 20p12.2, which has been linked to schizophrenia in some samples, and recently linked to latent classes of psychotic illness in our sample. SNAP25 is crucial to synaptic functioning, may be involved in axonal growth and dendritic sprouting, and its expression may be decreased in schizophrenia. We genotyped 18 haplotype-tagging SNPs in SNAP25 in a sample of 270 Irish high-density families. Single marker and haplotype analyses were performed in FBAT and PDT. We adjusted for multiple testing by computing q values. Association was followed up in an independent sample of 657 cases and 411 controls. We tested for allelic effects on the clinical phenotype by using the method of sequential addition and 5 factor-derived scores of the OPCRIT. Nine of 18 SNPs had P values <0.05 in either FBAT or PDT for one or more definitions of illness. Several two-marker haplotypes were also associated. Subjects inheriting the risk alleles of the most significantly associated two-marker haplotype were likely to have higher levels of hallucinations and delusions. The most significantly associated marker, rs6039820, was observed to perturb 12 transcription-factor binding sites in in silico analyses. An attempt to replicate association findings in the case-control sample resulted in no SNPs being significantly associated. We observed robust association in both single marker and haplotype-based analyses between SNAP25 and schizophrenia in an Irish family sample. Although we failed to replicate this in an independent sample, this gene should be further tested in other samples.

Replication of association between schizophrenia and ZNF804A in the Irish Case-Control Study of Schizophrenia sample.

A recent genome-wide association study reported association between schizophrenia and the ZNF804A gene on chromosome 2q32.1. We attempted to replicate these findings in our Irish Case-Control Study of Schizophrenia (ICCSS) sample (N=1021 cases, 626 controls). Following consultation with the original investigators, we genotyped three of the most promising single-nucleotide polymorphisms (SNPs) from the Cardiff study. We replicate association with rs1344706 (trend test one-tailed P=0.0113 with the previously associated A allele) in ZNF804A. We detect no evidence of association with rs6490121 in NOS1 (one-tailed P=0.21), and only a trend with rs9922369 in RGRIP1L (one-tailed P=0.0515). On the basis of these results, we completed genotyping of 11 additional linkage disequilibrium-tagging SNPs in ZNF804A. Of 12 SNPs genotyped, 11 pass quality control criteria and 4 are nominally associated, with our most significant evidence of association at rs7597593 (P=0.0013) followed by rs1344706. We observe no evidence of differential association in ZNF804A on the basis of family history or sex of case. The associated SNP rs1344706 lies in approximately 30 bp of conserved mammalian sequence, and the associated A allele is predicted to maintain binding sites for the brain-expressed transcription factors MYT1l and POU3F1/OCT-6. In controls, expression is significantly increased from the A allele of rs1344706 compared with the C allele. Expression is increased in schizophrenic cases compared with controls, but this difference does not achieve statistical significance. This study replicates the original reported association of ZNF804A with schizophrenia and suggests that there is a consistent link between the A allele of rs1344706, increased expression of ZNF804A and risk for schizophrenia.

Meta-analysis of 32 genome-wide linkage studies of schizophrenia.

A genome scan meta-analysis (GSMA) was carried out on 32 independent genome-wide linkage scan analyses that included 3255 pedigrees with 7413 genotyped cases affected with schizophrenia (SCZ) or related disorders. The primary GSMA divided the autosomes into 120 bins, rank-ordered the bins within each study according to the most positive linkage result in each bin, summed these ranks (weighted for study size) for each bin across studies and determined the empirical probability of a given summed rank (P(SR)) by simulation. Suggestive evidence for linkage was observed in two single bins, on chromosomes 5q (142-168 Mb) and 2q (103-134 Mb). Genome-wide evidence for linkage was detected on chromosome 2q (119-152 Mb) when bin boundaries were shifted to the middle of the previous bins. The primary analysis met empirical criteria for 'aggregate' genome-wide significance, indicating that some or all of 10 bins are likely to contain loci linked to SCZ, including regions of chromosomes 1, 2q, 3q, 4q, 5q, 8p and 10q. In a secondary analysis of 22 studies of European-ancestry samples, suggestive evidence for linkage was observed on chromosome 8p (16-33 Mb). Although the newer genome-wide association methodology has greater power to detect weak associations to single common DNA sequence variants, linkage analysis can detect diverse genetic effects that segregate in families, including multiple rare variants within one locus or several weakly associated loci in the same region. Therefore, the regions supported by this meta-analysis deserve close attention in future studies.

Genomewide linkage scan of schizophrenia in a large multicenter pedigree sample using single nucleotide polymorphisms.

A genomewide linkage scan was carried out in eight clinical samples of informative schizophrenia families. After all quality control checks, the analysis of 707 European-ancestry families included 1615 affected and 1602 unaffected genotyped individuals, and the analysis of all 807 families included 1900 affected and 1839 unaffected individuals. Multipoint linkage analysis with correction for marker-marker linkage disequilibrium was carried out with 5861 single nucleotide polymorphisms (SNPs; Illumina version 4.0 linkage map). Suggestive evidence for linkage (European families) was observed on chromosomes 8p21, 8q24.1, 9q34 and 12q24.1 in nonparametric and/or parametric analyses. In a logistic regression allele-sharing analysis of linkage allowing for intersite heterogeneity, genomewide significant evidence for linkage was observed on chromosome 10p12. Significant heterogeneity was also observed on chromosome 22q11.1. Evidence for linkage across family sets and analyses was most consistent on chromosome 8p21, with a one-LOD support interval that does not include the candidate gene NRG1, suggesting that one or more other susceptibility loci might exist in the region. In this era of genomewide association and deep resequencing studies, consensus linkage regions deserve continued attention, given that linkage signals can be produced by many types of genomic variation, including any combination of multiple common or rare SNPs or copy number variants in a region.

Monoamine oxidase A and childhood adversity as risk factors for conduct disorder in females.

BACKGROUND: Recent studies among males have reported a genotype-environment interaction (GxE) in which low-activity alleles at the monoamine oxidase A (MAOA) locus conferred greater sensitivity to the effects of childhood adversity on risk for conduct disorder (CD). So far, few studies of females have controlled for gene-environment correlation or used females heterozygous for this X-linked gene. METHOD: Logistic regression analysis of a sample of 721 females ages 8-17 years from the longitudinal Virginia Twin Study of Adolescent Behavioral Development (VTSABD) assessed the additive effects of MAOA genotypes on risk for CD, together with the main effect of childhood adversity and parental antisocial personality disorder (ASP), as well as the interaction of MAOA with childhood adversity on risk for CD. RESULTS: A significant main effect of genotype on risk for CD was detected, where low-activity MAOA imparted the greatest risk to CD in girls while controlling for the significant effects of maternal ASP and childhood adversity. Significant GxE with weak effect was detected when environmental exposure was untransformed, indicating a higher sensitivity to childhood adversity in the presence of the high-activity MAOA allele. The interaction was no longer statistically significant after applying a ridit transformation to reflect the sample sizes exposed at each level of childhood adversity. CONCLUSIONS: The main effect of MAOA on risk for CD in females, its absence in males and directional difference of interaction is suggestive of genotype-sex interaction. As the effect of GxE on risk for CD was weak, its inclusion is not justified.

A region of 35 kb containing the trace amine associate receptor 6 (TAAR6) gene is associated with schizophrenia in the Irish study of high-density schizophrenia families.

The TAAR6 gene has been previously associated with schizophrenia in 192 pedigrees of European and African ancestry. To replicate these findings we performed an association study of TAAR6 in 265 pedigrees of the Irish Study of High-Density Schizophrenia Families (ISHDSF). Of the 24 genotyped single-nucleotide polymorphisms only rs12189813 and rs9389011 provided single-marker evidence for association (0.0094

Alcohol dependence is associated with the ZNF699 gene, a human locus related to Drosophila hangover, in the Irish Affected Sib Pair Study of Alcohol Dependence (IASPSAD) sample.

Because tolerance is an important aspect of alcohol dependence (AD) in humans, recent evidence showing that the Drosophila gene hang is critically involved in the development of alcohol tolerance in the fly suggests that variation in related human loci might be important in the etiology of alcohol-related disorders. The orthology of hang in mammals is complex, but a number of human gene products (including ZNF699) with similar levels of amino-acid identity (18-26%) and similarity (30-41%), are consistently identified as the best matches with the translated hang sequence. We tested for association between the dichotomous clinical phenotype of alcohol dependence and seven single nucleotide polymorphisms (SNPs) in ZNF699 in our sample of 565 genetically independent cases and 496 siblings diagnosed with AD, and 609 controls. In analyses of genetically independent cases and controls, four of the seven single markers show strong evidence for association with AD (0.00003

Multicenter linkage study of schizophrenia loci on chromosome 22q.

The hypothesis of the existence of one or more schizophrenia susceptibility loci on chromosome 22q is supported by reports of genetic linkage and association, meta-analyses of linkage, and the observation of elevated risk for psychosis in people with velocardiofacial syndrome, caused by 22q11 microdeletions. We tested this hypothesis by evaluating 10 microsatellite markers spanning 22q in a multicenter sample of 779 pedigrees. We also incorporated age at onset and sex into the analysis as covariates. No significant evidence for linkage to schizophrenia or for linkage associated with earlier age at onset, gender, or heterogeneity across sites was observed. We interpret these findings to mean that the population-wide effects of putative 22q schizophrenia susceptibility loci are too weak to detect with linkage analysis even in large samples.

Genome-wide scans of three independent sets of 90 Irish multiplex schizophrenia families and follow-up of selected regions in all families provides evidence for multiple susceptibility genes.

From our linkage study of Irish families with a high density of schizophrenia, we have previously reported evidence for susceptibility genes in regions 5q21-31, 6p24-21, 8p22-21, and 10p15-p11. In this report, we describe the cumulative results from independent genome scans of three a priori random subsets of 90 families each, and from multipoint analysis of all 270 families in ten regions. Of these ten regions, three (13q32, 18p11-q11, and 18q22-23) did not generate scores above the empirical baseline pairwise scan results, and one (6q13-26) generated a weak signal. Six other regions produced more positive pairwise and multipoint results. They showed the following maximum multipoint H-LOD (heterogeneity LOD) and NPL scores: 2p14-13: 0.89 (P = 0.06) and 2.08 (P = 0.02), 4q24-32: 1.84 (P = 0.007) and 1.67 (P = 0.03), 5q21-31: 2.88 (P= 0.0007), and 2.65 (P = 0.002), 6p25-24: 2.13 (P = 0.005) and 3.59 (P = 0.0005), 6p23: 2.42 (P = 0.001) and 3.07 (P = 0.001), 8p22-21: 1.57 (P = 0.01) and 2.56 (P = 0.005), 10p15-11: 2.04 (P = 0.005) and 1.78 (P = 0.03). The degree of 'internal replication' across subsets differed, with 5q, 6p, and 8p being most consistent and 2p and 10p being least consistent. On 6p, the data suggested the presence of two susceptibility genes, in 6p25-24 and 6p23-22. Very few families were positive on more than one region, and little correlation between regions was evident, suggesting substantial locus heterogeneity. The levels of statistical significance were modest, as expected from loci contributing to complex traits. However, our internal replications, when considered along with the positive results obtained in multiple other samples, suggests that most of these six regions are likely to contain genes that influence liability to schizophrenia.

An association study of DRD5 with smoking initiation and progression to nicotine dependence.

A large body of genetic epidemiological data strongly implicate genetic factors in the etiology of smoking behavior. Polymorphisms of genes in the dopaminergic system are plausible functional candidate genes and a linkage and an association study suggested that the type 5 dopamine receptor gene (DRD5) may be etiologically involved. We investigated the association of four DRD5 polymorphisms with smoking initiation and progression to nicotine dependence in a population-based sample of over 900 subjects. For smoking initiation, there was no significant association with the four DRD5 markers we studied; however, maximum likelihood analyses suggested the presence of a haplotype protective against smoking initiation. For progression to nicotine dependence, there were no strongly significant associations with the four DRD5 markers or for the estimated haplotypes. These data are not consistent with a strong etiological role for DRD5 in the etiology of these complex smoking behaviors.

Haplotypes of four novel single nucleotide polymorphisms in the nicotinic acetylcholine receptor beta2-subunit (CHRNB2) gene show no association with smoking initiation or nicotine dependence.

Several types of evidence, including experiments with mice that lack the nicotinic acetylcholine receptor beta2-subunit gene (CHRNB2), have suggested that a beta2-containing nicotinic receptor is necessary for at least some of the reinforcing properties of nicotine. However, sequence variations in CHRNB2 have not been reported, and its role in influencing human smoking behavior and nicotine dependence is not known. We screened most of the introns and exons and found five novel single nucleotide polymorphisms (SNPs). We tested four of these SNPs in three large, carefully selected samples: nonsmokers (n = 317) and regular smokers low levels of nicotine dependence (ND, n = 238), or smokers with high-ND (n = 317). None of the four polymorphisms we tested, nor their estimated haplotypes, were associated with smoking initiation or progression to nicotine dependence.

Susceptibility genes for nicotine dependence: a genome scan and followup in an independent sample suggest that regions on chromosomes 2, 4, 10, 16, 17 and 18 merit further study.

Cigarette smoking is associated with considerable morbidity, mortality, and public health costs. Genetic factors influence both smoking initiation and nicotine dependence, but none of the genes involved have been identified. A genome scan using 451 markers was conducted to identify chromosomal regions linked to nicotine dependence in a collection of 130 families containing 343 genotyped individuals (308 nicotine-dependent) from Christchurch, New Zealand. By pairwise analysis, the best result was with marker D2S1326 which gave a lod score under heterogeneity (H-LOD) of 2.63 (P=0.0012) and a nonparametric linkage (NPL, Zall) score of 2.65 (P=0.0011). To identify regions that warranted further study, rather than comparing the pairwise scores from the scan to theoretical thresholds, we compared them to an empirical baseline, found here to be H-LOD scores of 0.5 and Zall scores of 1.0. We also found a number of large (31-88 cM) regions where many (8-16) consecutive markers yielded small but positive Zall scores. Selected regions of chromosomes 2, 4, 10, 16, 17 and 18 were investigated further by additional genotyping of the Christchurch sample and an independent sample from Richmond, Virginia (91 families with 264 genotyped individuals, 211 nicotine-dependent). Multipoint nonparametric analysis showed the following maximums for the Christchurch sample: Chr. 2 (Zlr=2.61, P=0.005), Chr. 4 (Zlr=1.36, P=0.09), Chr. 10 (Zlr=2.43, P=0.008), Chr. 16 (Zlr=0.85, P=0.19), Chr. 17 (Zlr=1.64, P=0.05), Chr. 18 (Zlr=1.54, P=0.06). Analysis of the Richmond sample showed the following maximums: Chr. 2 (Zlr=1.00, P=0.15), Chr. 4 (Zlr=0.39, P=0.34), Chr. 10 (Zlr=1.21, P=0.11), Chr. 16 (Zlr=1.11, P=0.13), Chr. 17 (Zlr=1.60, P=0.05), Chr. 18 (Zlr=1.33, P=0.09). It is probable that the small samples used here provided only limited power to detect linkage. It may have been difficult therefore to detect genes of small effect, or those that are influencing risk in only a small proportion of the families. When simply judged against the usual standards of linkage significance, none of the individual regions yielded strong evidence in either sample. Some or all of the most positive results in the genome scan of the Christchurch sample, therefore, could be due to chance. However, the presence in the Christchurch scan of multiple large regions containing many consecutive positive markers, coupled with the relatively positive results in these same regions in the Richmond sample, suggests that some of these regions may contain genes influencing nicotine dependence and therefore deserve further study.

Role of platelet-endothelial cell adhesion molecule (PECAM) in platelet adhesion/aggregation over injured but not denuded endothelium in vivo and ex vivo.

BACKGROUND AND PURPOSE: We previously demonstrated that a monoclonal antibody (MoAb) with anti-CD31, anti-platelet-endothelial cell adhesion molecule (PECAM)-like properties delayed platelet adhesion/aggregation at a site of minor endothelial injury. To our knowledge, this was the first in vivo demonstration of an effect of anti-CD31. There was no exposure of collagen or basal lamina at the injured site, and the modulation of adhesion/aggregation at such sites has not received much study. The present investigation attempted to replicate the first with the use of a different MoAb, definitely characterized as anti-PECAM. In addition, an ex vivo investigation was performed to see whether the in vivo action of anti-PECAM could have been caused by an effect of the MoAb on the platelets rather than on the endothelium. METHODS: A helium-neon laser, in the presence of intravascular Evans blue, was used to injure the endothelium of arterioles on the surface of the mouse brain. Intravital microscopy was used to determine the number of seconds required for the light to initiate the first recognizable platelet aggregate forming at the injured site. Mice injected with vehicle were compared with mice injected with 2 mg/kg anti-PECAM through the tail vein. The injection was 10 minutes before challenge with the laser. Additional studies were performed of aggregation produced in vitro by arachidonate and by ADP added to platelet-rich plasma (PRP) prepared from blood taken from MoAb-treated and vehicle-treated mice. RESULTS: Aggregation latency was significantly prolonged (P<.02) by anti-PECAM (121+/-59 versus 65+/-26 seconds in controls; n=10 each). Aggregation ex vivo was not affected. CONCLUSIONS: PECAM is an important modulator of platelet adhesion/aggregation at sites of minor endothelial damage in brain arterioles. The data are consistent with the hypothesis that PECAM sites on the endothelium are involved and may be exposed by the injury to promote adhesion/aggregation. Since the endothelial cell layer is intact at these sites, mechanisms such as this offer important alternatives to the more commonly studied pathways of platelet activation, which require exposure of collagen and are not applicable in this model.

Selective depression of endothelium-dependent dilations during cerebral ischemia.

BACKGROUND AND PURPOSE: Pial arterioles have diverse mechanisms for endothelium-dependent dilations. In mice, different mechanisms or endothelium-derived mediators exist for each of the following dilators: acetylcholine, bradykinin, and calcium ionophore A-23187. This study tests the response to each of these dilators during profound ischemia. The response to sodium nitroprusside, an endothelium-independent dilator, was also tested. METHODS: In each mouse, ischemia was produced by bilateral carotid artery ligation that reduced cortical blood flow by approximately 90% as determined by laser-Doppler flowmetry. In separate studies of 10 mice each, dilations of pial arterioles to two doses of each dilator were compared before and after 10 minutes of occlusion, with the occlusion continuing during the second set of measurements. The dilator was applied in the suffusate bathing the pial surface exposed at a craniotomy site. Diameters were monitored by in vivo television microscopy and image splitting. RESULTS: The dose-dependent dilations to acetylcholine, bradykinin, and calcium ionophore A-23187 were each profoundly depressed during ischemia. The response to sodium nitroprusside was not depressed. In all cases, the ischemia was accompanied by arteriolar narrowing of approximately 25% and by obvious slowing of blood flow observed by intravital microscopy. Superoxide dismutase plus catalase failed to prevent the depressed response to acetylcholine. CONCLUSIONS: Endothelium-dependent dilations, mediated by diverse endothelium-derived relaxing factors, are depressed during ischemia of 10 to 15 minutes' duration. This cannot be a nonselective effect on vessel responsivity caused by constriction, reduced flow, or reduced intraluminal pressure during ischemia because under the same conditions dilation to endothelium-independent sodium nitroprusside is preserved. The selective endothelial dysfunction may play a role in exacerbating ischemia by precluding the ability of some dilators, released during ischemia, to dilate the resistance vessels.

Calcium ionophore and acetylcholine dilate arterioles on the mouse brain by different mechanisms.

Pial arterioles on the surface of the mouse brain were observed in vivo under a chamber with a glass window. When placed under the window, calcium ionophore, acetylcholine, and previously acidified sodium nitrite each dilated the arterioles. If the cyclooxygenase inhibitors indomethacin or acetylsalicylic acid were first placed in the chamber, subsequent dilation of the arterioles by calcium ionophore was reduced to essentially zero. Similar blockade of cyclooxygenase failed to significantly reduce dilation by acetylcholine or sodium nitrite. We have previously shown that dilations by calcium ionophore and acetylcholine were endothelium dependent. Our present experiments show that the endothelium-dependent mechanism for dilation by calcium ionophore is cyclooxygenase dependent, while that for acetylcholine is not. This implies that, in pial arterioles, the endothelium-derived relaxing factor for acetylcholine differs from that for calcium ionophore. This agrees with data from other microvascular beds.