Prevalence of attention deficit hyperactivity disorder symptoms in patients with schizophrenia.

OBJECTIVE: To investigate the frequency of childhood and adult attention deficit hyperactivity disorder (ADHD) symptoms in a cohort of patients with schizophrenia (SCZ). METHODS: A systematic review was conducted to evaluate existing evidence. Two self-report questionnaires were used to investigate adult ADHD and childhood ADHD symptoms in 126 patients with ICD-10 diagnoses of SCZ. RESULTS: Five studies were included in the systematic review, with the prevalence of childhood and adult ADHD in SCZ subjects ranging between 17-57% and 10-47% respectively. Within our cohort, 47% of patients reported positive screening for ADHD symptoms either in childhood or adulthood. 23% reported symptomatology consistent with both childhood and adult ADHD. CONCLUSIONS: We demonstrate a greater presence of ADHD symptomatology in SCZ compared to that reported for ADHD in the general population. Our findings highlight the importance of improved clinical assessment and treatment considerations in a subgroup of patients with SCZ.

Rare variant analysis in multiply affected families, association studies and functional analysis suggest a role for the ITGΒ4 gene in schizophrenia and bipolar disorder.

Recent results imply that rare variants contribute to the risk of schizophrenia. Exome sequence data from the UK10K project was used to identify three rare, amino acid changing variants in the ITGB4 gene which segregated with schizophrenia in two families: rs750367954, rs147480547 and rs145976111. Association analysis was carried out in the exome-sequenced Swedish schizophrenia study and in UCL schizophrenia and bipolar cases and controls genotyped for these variants. A gene-wise weighted burden test was performed on a trio sample of schizophrenia cases and their parents. rs750367954 was seen in two Swedish cases and in no controls. The other two variants were commoner in cases than controls in both Swedish and UCL cohort samples and an overall burden test was significant at p=0.0000031. The variants were not observed in the trio sample but ITGB4 was most highly ranked out of 14,960 autosomal genes in a gene-wise weighted burden test. The effect of rs147480547 and rs145976111 was studied in human neuroblastoma SH-SY5Y cells. Cells transfected with both variants had increased proliferation at both 24 and 48h (p=0.013 and p=0.05 respectively) compared to those with wild-type ITGB4. Taken together, these results suggest that rare variants in ITGB4 which affect function may contribute to the aetiology of schizophrenia and bipolar disorder.

Whole-exome sequencing of individuals from an isolated population implicates rare risk variants in bipolar disorder.

Bipolar disorder affects about 1% of the world's population, and its estimated heritability is about 75%. Only few whole genome or whole-exome sequencing studies in bipolar disorder have been reported, and no rare coding variants have yet been robustly identified. The use of isolated populations might help finding variants with a recent origin, more likely to have drifted to higher frequency by chance. Following this approach, we investigated 28 bipolar cases and 214 controls from the Faroe Islands by whole exome sequencing, and the results were followed-up in a British sample of 2025 cases and 1358 controls. Seventeen variants in 16 genes in the single-variant analysis, and 3 genes in the gene-based statistics surpassed exome-wide significance in the discovery phase. The discovery findings were supported by enrichment analysis of common variants from genome-wide association studies (GWAS) data and interrogation of protein-protein interaction networks. The replication in the British sample confirmed the association with NOS1 (missense variant rs79487279) and NCL (gene-based test). A number of variants from the discovery set were not present in the replication sample, including a novel PITPNM2 missense variant, which is located in a highly significant schizophrenia GWAS locus. Likewise, PIK3C2A identified in the gene-based analysis is located in a combined bipolar and schizophrenia GWAS locus. Our results show support both for existing findings in the literature, as well as for new risk genes, and identify rare variants that might provide additional information on the underlying biology of bipolar disorder.

CACNA1C hypermethylation is associated with bipolar disorder.

The CACNA1C gene, encoding a subunit of the L-type voltage-gated calcium channel is one of the best-supported susceptibility genes for bipolar disorder (BD). Genome-wide association studies have identified a cluster of non-coding single-nucleotide polymorphisms (SNPs) in intron 3 to be highly associated with BD and schizophrenia. The mechanism by which these SNPs confer risk of BD appears to be through an altered regulation of CACNA1C expression. The role of CACNA1C DNA methylation in BD has not yet been addressed. The aim of this study was to investigate if CACNA1C DNA methylation is altered in BD. First, the methylation status of five CpG islands (CGIs) across CACNA1C in blood from BD subjects (n=40) and healthy controls (n=38) was determined. Four islands were almost completely methylated or completely unmethylated, while one island (CGI 3) in intron 3 displayed intermediate methylation levels. In the main analysis, the methylation status of CGI 3 was analyzed in a larger sample of BD subjects (n=582) and control individuals (n=319). Out of six CpG sites that were investigated, five sites showed significant hypermethylation in cases (lowest P=1.16 × 10(-7) for CpG35). Nearby SNPs were found to influence the methylation level, and we identified rs2238056 in intron 3 as the strongest methylation quantitative trait locus (P=2.6 × 10(-7)) for CpG35. In addition, we found an increased methylation in females, and no difference between bipolar I and II. In conclusion, we find that CACNA1C methylation is associated with BD and suggest that the regulatory effect of the non-coding risk variants involves a shift in DNA methylation.

Genome-wide association study of Alzheimer's disease with psychotic symptoms.

Psychotic symptoms occur in ~40% of subjects with Alzheimer's disease (AD) and are associated with more rapid cognitive decline and increased functional deficits. They show heritability up to 61% and have been proposed as a marker for a disease subtype suitable for gene mapping efforts. We undertook a combined analysis of three genome-wide association studies (GWASs) to identify loci that (1) increase susceptibility to an AD and subsequent psychotic symptoms; or (2) modify risk of psychotic symptoms in the presence of neurodegeneration caused by AD. In all, 1299 AD cases with psychosis (AD+P), 735 AD cases without psychosis (AD-P) and 5659 controls were drawn from Genetic and Environmental Risk in AD Consortium 1 (GERAD1), the National Institute on Aging Late-Onset Alzheimer's Disease (NIA-LOAD) family study and the University of Pittsburgh Alzheimer Disease Research Center (ADRC) GWASs. Unobserved genotypes were imputed to provide data on >1.8 million single-nucleotide polymorphisms (SNPs). Analyses in each data set were completed comparing (1) AD+P to AD-P cases, and (2) AD+P cases with controls (GERAD1, ADRC only). Aside from the apolipoprotein E (APOE) locus, the strongest evidence for association was observed in an intergenic region on chromosome 4 (rs753129; 'AD+PvAD-P' P=2.85 × 10(-7); 'AD+PvControls' P=1.11 × 10(-4)). SNPs upstream of SLC2A9 (rs6834555, P=3.0 × 10(-7)) and within VSNL1 (rs4038131, P=5.9 × 10(-7)) showed strongest evidence for association with AD+P when compared with controls. These findings warrant further investigation in larger, appropriately powered samples in which the presence of psychotic symptoms in AD has been well characterized.

Polygenic dissection of the bipolar phenotype.

BACKGROUND: Recent data provide strong support for a substantial common polygenic contribution (i.e. many alleles each of small effect) to genetic susceptibility for schizophrenia and overlapping susceptibility for bipolar disorder. AIMS: To test hypotheses about the relationship between schizophrenia and psychotic types of bipolar disorder. METHOD: Using a polygenic score analysis to test whether schizophrenia polygenic risk alleles, en masse, significantly discriminate between individuals with bipolar disorder with and without psychotic features. The primary sample included 1829 participants with bipolar disorder and the replication sample comprised 506 people with bipolar disorder. RESULTS: The subset of participants with Research Diagnostic Criteria schizoaffective bipolar disorder (n = 277) were significantly discriminated from the remaining participants with bipolar disorder (n = 1552) in both the primary (P = 0.00059) and the replication data-sets (P = 0.0070). In contrast, those with psychotic bipolar disorder as a whole were not significantly different from those with non-psychotic bipolar disorder in either data-set. CONCLUSIONS: Genetic susceptibility influences at least two major domains of psychopathological variation in the schizophrenia-bipolar disorder clinical spectrum: one that relates to expression of a 'bipolar disorder-like' phenotype and one that is associated with expression of 'schizophrenia-like' psychotic symptoms.

Genetic association study of GABRA2 single nucleotide polymorphisms and electroencephalography in alcohol dependence.

The gamma aminobutyric acid (GABA) system has been implicated in the susceptibility to develop alcohol dependence and in determining electroencephalogram (EEG) beta activity. The role of the GABA receptor alpha-2 gene (GABRA2) in human alcohol dependence was determined in a genetic and electrophysiological study. The study population comprised 586 white UK individuals with alcohol dependence but a very low prevalence of co-morbid drug dependence, and 603 ancestrally matched healthy controls. Genotyping for seven GABRA2 single nucleotide polymorphisms (SNPs), identified from the literature as positively associated with alcohol dependence, was performed with success rates of 90% or greater. EEGs were available in 32 selected patients who had been abstinent from alcohol for a minimum of 24 months and in 138 ancestrally matched healthy controls. None of the SNPs showed allelic or haplotypic association with alcohol dependence. All markers were in Hardy Weinberg equilibrium (HWE) in the controls. HWE for marker rs279841 in the alcohol dependent sample was p=0.0199 and combined p=0.0166. Linkage disequilibrium patterns appear to be very similar to that observed in the HapMap CEU data. A significantly higher prevalence of excess EEG fast activity was found in the patients (31 vs. 14%, p=0.018). A significant relationship was found between the presence of excess EEG fast activity and GABRA2 SNPs rs548583, rs279871 and rs279841. This allelic association study provides no evidence for an association between GABRA2 polymorphisms and alcohol dependence. However, a significant relationship was identified between GABRA2 and excess EEG fast activity. This dissociation of effect may reflect the fact that the EEG is a more direct marker of phenotypic GABRA2 expression than the more heterogeneous alcohol dependence phenotype.

NK1 (TACR1) receptor gene 'knockout' mouse phenotype predicts genetic association with ADHD.

Mice with functional genetic ablation of the Tacr1 (substance P-preferring receptor) gene (NK1R-/-) are hyperactive. Here, we investigated whether this is mimicked by NK1R antagonism and whether dopaminergic transmission is disrupted in brain regions that govern motor performance. The locomotor activity of NK1R-/- and wild-type mice was compared after treatment with an NK1R antagonist and/or psychostimulant (d-amphetamine or methylphenidate). The inactivation of NK1R (by gene mutation or receptor antagonism) induced hyperactivity in mice, which was prevented by both psychostimulants. Using in vivo microdialysis, we then compared the regulation of extracellular dopamine in the prefrontal cortex (PFC) and striatum in the two genotypes. A lack of functional NK1R reduced (>50%) spontaneous dopamine efflux in the prefrontal cortex and abolished the striatal dopamine response to d-amphetamine. These behavioural and neurochemical abnormalities in NK1R-/- mice, together with their atypical response to psychostimulants, echo attention deficit hyperactivity disorder (ADHD) in humans. These findings prompted genetic studies on the TACR1 gene (the human equivalent of NK1R) in ADHD patients in a case-control study of 450 ADHD patients and 600 screened supernormal controls. Four single-nucleotide polymorphisms (rs3771829, rs3771833, rs3771856, and rs1701137) at the TACR1 gene, previously known to be associated with bipolar disorder or alcoholism, were strongly associated with ADHD. In conclusion, our proposal that NK1R-/- mice offer a mouse model of ADHD was borne out by our human studies, which suggest that DNA sequence changes in and around the TACR1 gene increase susceptibility to this disorder.

A threonine to isoleucine missense mutation in the pericentriolar material 1 gene is strongly associated with schizophrenia.

Markers at the pericentriolar material 1 gene (PCM1) have shown genetic association with schizophrenia in both a University College London (UCL) and a USA-based case-control sample. In this paper we report a statistically significant replication of the PCM1 association in a large Scottish case-control sample from Aberdeen. Resequencing of the genomic DNA from research volunteers who had inherited haplotypes associated with schizophrenia showed a threonine to isoleucine missense mutation in exon 24 which was likely to change the structure and function of PCM1 (rs370429). This mutation was found only as a heterozygote in 98 schizophrenic research subjects and controls out of 2246 case and control research subjects. Among the 98 carriers of rs370429, 67 were affected with schizophrenia. The same alleles and haplotypes were associated with schizophrenia in both the London and Aberdeen samples. Another potential aetiological base pair change in PCM1 was rs445422, which altered a splice site signal. A further mutation, rs208747, was shown by electrophoretic mobility shift assays to create or destroy a promoter transcription factor site. Five further non-synonymous changes in exons were also found. Genotyping of the new variants discovered in the UCL case-control sample strengthened the evidence for allelic and haplotypic association (P=0.02-0.0002). Given the number and identity of the haplotypes associated with schizophrenia, further aetiological base pair changes must exist within and around the PCM1 gene. PCM1 protein has been shown to interact directly with the disrupted-in-schizophrenia 1 (DISC1) protein, Bardet-Biedl syndrome 4, and Huntingtin-associated protein 1, and is important in neuronal cell growth. In a separate study we found that clozapine but not haloperidol downregulated PCM1 expression in the mouse brain. We hypothesize that mutant PCM1 may be responsible for causing a subtype of schizophrenia through abnormal cell division and abnormal regeneration in dividing cells in the central nervous system. This is supported by our previous finding of orbitofrontal volumetric deficits in PCM1-associated schizophrenia patients as opposed to temporal pole deficits in non-PCM1-associated schizophrenia patients. Caution needs to be exercised in interpreting the actual biological effects of the mutations we have found without further cell biology. However, the DNA changes we have found deserve widespread genotyping in multiple case-control populations.

DISC1 association, heterogeneity and interplay in schizophrenia and bipolar disorder.

Disrupted in schizophrenia 1 (DISC1) has been associated with risk of schizophrenia, schizoaffective disorder, bipolar disorder, major depression, autism and Asperger syndrome, but apart from in the original translocation family, true causal variants have yet to be confirmed. Here we report a harmonized association study for DISC1 in European cohorts of schizophrenia and bipolar disorder. We identify regions of significant association, demonstrate allele frequency heterogeneity and provide preliminary evidence for modifying interplay between variants. Whereas no associations survived permutation analysis in the combined data set, significant corrected associations were observed for bipolar disorder at rs1538979 in the Finnish cohorts (uncorrected P=0.00020; corrected P=0.016; odds ratio=2.73+/-95% confidence interval (CI) 1.42-5.27) and at rs821577 in the London cohort (uncorrected P=0.00070; corrected P=0.040; odds ratio=1.64+/-95% CI 1.23-2.19). The rs821577 single nucleotide polymorphism (SNP) showed evidence for increased risk within the combined European cohorts (odds ratio=1.27+/-95% CI 1.07-1.51), even though significant corrected association was not detected (uncorrected P=0.0058; corrected P=0.28). After conditioning the European data set on the two risk alleles, reanalysis revealed a third significant SNP association (uncorrected P=0.00050; corrected P=0.025). This SNP showed evidence for interplay, either increasing or decreasing risk, dependent upon the presence or absence of rs1538979 or rs821577. These findings provide further support for the role of DISC1 in psychiatric illness and demonstrate the presence of locus heterogeneity, with the effect that clinically relevant genetic variants may go undetected by standard analysis of combined cohorts.

Case-control studies show that a non-conservative amino-acid change from a glutamine to arginine in the P2RX7 purinergic receptor protein is associated with both bipolar- and unipolar-affective disorders.

Three linkage studies of bipolar disorder have implicated chromosome 12q24.3 with lod scores of over 3.0 and several other linkage studies have found lods between 2 and 3. Fine mapping within the original chromosomal linkage regions has identified several loci that show association with bipolar disorder. One of these is the P2RX7 gene encoding a central nervous system-expressed purinergic receptor. A non-synonymous single nucleotide polymorphism, rs2230912 (P2RX7-E13A, G allele) and a microsatellite marker NBG6 were both previously found to be associated with bipolar disorder (P=0.00071 and 0.008, respectively). rs2230912 has also been found to show association with unipolar depression. The effect of the polymorphism is non-conservative and results in a glutamine to arginine change (Gln460Arg), which is likely to affect P2RX7 dimerization and protein-protein interactions. We have confirmed the allelic associations between bipolar disorder and the markers rs2230912 (P2RX7-E13A, G allele, P=0.043) and NBG6 (P=0.010) in a London-based sample of 604 bipolar cases and 560 controls. When we combined these data with the published case-control studies of P2RX7 and mood disorder (3586 individuals) the association between rs2230912 (Gln460Arg) and affective disorders became more robust (P=0.002). The increase in Gln460Arg was confined to heterozygotes rather than homozygotes suggesting a dominant effect (odds ratio 1.302, CI=1.129-1.503). Although further research is needed to prove that the Gln460Arg change has an aetiological role, it is so far the most convincing mutation to have been found with a role for increasing susceptibility to bipolar and genetically related unipolar disorders.

Whole-genome association study of bipolar disorder.

We performed a genome-wide association scan in 1461 patients with bipolar (BP) 1 disorder, 2008 controls drawn from the Systematic Treatment Enhancement Program for Bipolar Disorder and the University College London sample collections with successful genotyping for 372,193 single nucleotide polymorphisms (SNPs). Our strongest single SNP results are found in myosin5B (MYO5B; P=1.66 x 10(-7)) and tetraspanin-8 (TSPAN8; P=6.11 x 10(-7)). Haplotype analysis further supported single SNP results highlighting MYO5B, TSPAN8 and the epidermal growth factor receptor (MYO5B; P=2.04 x 10(-8), TSPAN8; P=7.57 x 10(-7) and EGFR; P=8.36 x 10(-8)). For replication, we genotyped 304 SNPs in family-based NIMH samples (n=409 trios) and University of Edinburgh case-control samples (n=365 cases, 351 controls) that did not provide independent replication after correction for multiple testing. A comparison of our strongest associations with the genome-wide scan of 1868 patients with BP disorder and 2938 controls who completed the scan as part of the Wellcome Trust Case-Control Consortium indicates concordant signals for SNPs within the voltage-dependent calcium channel, L-type, alpha 1C subunit (CACNA1C) gene. Given the heritability of BP disorder, the lack of agreement between studies emphasizes that susceptibility alleles are likely to be modest in effect size and require even larger samples for detection.

Fine mapping of a susceptibility locus for bipolar and genetically related unipolar affective disorders, to a region containing the C21ORF29 and TRPM2 genes on chromosome 21q22.3.

Linkage analyses of bipolar families have confirmed that there is a susceptibility locus near the telomere on chromosome 21q. To fine map this locus we carried out tests of allelic association using 30 genetic markers near the telomere at 21q22.3 in 600 bipolar research subjects and 450 ancestrally matched supernormal control subjects. We found significant allelic association with the microsatellite markers D21S171 (P=0.016) and two closely linked single-nucleotide polymorphisms, rs1556314 (P=0.008) and rs1785467 (P=0.025). A test of association with a three locus haplotype across the susceptibility region was significant with a permutation test of P=0.011. A two SNP haplotype was also significantly associated with bipolar disorder (P=0.01). Only two brain expressed genes, TRPM2 and C21ORF29 (TSPEAR), are present in the associated region. TRPM2 encodes a calcium channel receptor and TSPEAR encodes a peptide with repeats associated with epilepsy in the mouse. DNA from subjects who had inherited the associated marker alleles was sequenced. A base pair change (rs1556314) in exon 11 of TRPM2, which caused a change from an aspartic acid to a glutamic acid at peptide position 543 was found. This SNP showed the strongest association with bipolar disorder (P=0.008). Deletion of exon 11 of TRPM2 is known to cause dysregulation of cellular calcium homeostasis in response to oxidative stress. A second nonconservative change from arginine to cysteine at position 755 in TRPM2 (ss48297761) was also detected. A third nonconservative change from histidine to glutamic acid was found in exon 8 of TSPEAR. These changes need further investigation to establish any aetiological role in bipolar disorder.

Genome scan of Tourette syndrome in a single large pedigree shows some support for linkage to regions of chromosomes 5, 10 and 13.

OBJECTIVES: To localize genes influencing the susceptibility to Gilles de la Tourette syndrome (GTS) and associated chronic multiple tics (CMT). METHOD: A single, large, multiple affected pedigree containing 35 subjects diagnosed with GTS and a further 14 with CMT was genotyped for markers spanning the autosomes. Linkage analysis was carried out using classical lod score analysis and model-free lod score analysis. All markers were subjected to two-point analysis, and markers producing a two-point result significant at P<0.005 were subjected to three-point analysis using adjacent markers. RESULTS: The following markers produced at least one result significant at 0.005 using two-point analysis: D5S1981, D5S2050, D10S591, D10S189, D13S217, and D14S288. Three-point analysis with D5S2050 and D5S400 produced a lod of 2.9 with CMT. Three-point analysis of D10S591 and D10S189 produced lods of 1.9 with GTS and CMT. Three-point analysis of D13S217 and D13S171 produced a lod of 2.7 with GTS. No single haplotype appeared to account for the majority of cases within the pedigree. CONCLUSIONS: It seems likely that more than one susceptibility allele is present in the pedigree. Although none of the three positive regions is conclusively implicated, it seems probable that at least one contains a susceptibility locus. We recommend that association-based studies be carried out in these three regions to produce further evidence for a localization and to carry out fine-mapping.

Variation in the DCP1 gene, encoding the angiotensin converting enzyme ACE, is not associated with increased susceptibility to Alzheimer's disease.

OBJECTIVES: To attempt to replicate previous reports that polymorphic variation in the DCP1 gene causes increased susceptibility to the development of Alzheimer's disease, either on its own or in interaction with the effects of the gene for apolipoprotein E (APOE). METHOD: Subjects older than 65 years of age consisting of 81 dementia patients diagnosed as having possible or probable Alzheimer's disease and 68 controls were obtained from Camden, Islington and Harlow psychiatric services. Subjects were genotyped for APOE alleles e2, e3 and e4, and the common insertion/deletion polymorphisms for DCP1* I/D were genotyped. RESULTS: There was no statistically significant difference in the frequency of the DCP1* insertion/deletion alleles between the cases and controls (X2 =0.04, 1 degree of freedom, not significant). When subjects were subdivided according to whether they possessed at least one copy of the APOE e4 allele, there were still no differences in DCP1 allele frequencies between cases and controls. CONCLUSIONS: Further research is needed to elucidate any role that the DCP1 polymorphism may play in relation to Alzheimer's disease. Previous studies may be false positive, or inconsistency in replication may be due to heterogeneity.

Genetic association studies of schizophrenia using the 8p21-22 genes: prepronociceptin (PNOC), neuronal nicotinic cholinergic receptor alpha polypeptide 2 (CHRNA2) and arylamine N-acetyltransferase 1 (NAT1).

Schizophrenia is a common, genetically heterogeneous disorder with a lifetime prevalence of approximately 1% in the general population. Linkage studies of affected families have now strongly implicated a susceptibility locus on chromosome 8p21-22. Tests of allelic association with markers on 8p21-22 should be able to localise any quantitative trait nucleotides (QTN's) or susceptibility mutations to within a few hundred kilobases. Three brain expressed candidate susceptibility genes, prepronociceptin (PNOC), neuronal cholinergic receptor, nicotinic, alpha polypeptide 2 (CHRNA2) and arylamine N-acetyltransferase 1 (NAT1) have been mapped to chromosome 8p21-22. A case-control, allelic association study was performed using a novel highly polymorphic dinucleotide repeat, D8S2611 near the PNOC gene, two previously characterised dinucleotide repeats, D8S131 and D8S131P at the CHRNA2 locus and an RFLP at the 3'UTR of the arylamine N-acetyltransferase 1 (NAT1) gene. No differences were found in allele frequencies between the patient and control groups. DNA variations or mutations at or near the three genes under study are unlikely to increase susceptibility to schizophrenia in our population sample.

Genomewide genetic linkage analysis confirms the presence of susceptibility loci for schizophrenia, on chromosomes 1q32.2, 5q33.2, and 8p21-22 and provides support for linkage to schizophrenia, on chromosomes 11q23.3-24 and 20q12.1-11.23.

We have performed genetic linkage analysis in 13 large multiply affected families, to test the hypothesis that there is extensive heterogeneity of linkage for genetic subtypes of schizophrenia. Our strategy consisted of selecting 13 kindreds containing multiple affected cases in three or more generations, an absence of bipolar affective disorder, and a single progenitor source of schizophrenia with unilineal transmission into the branch of the kindred sampled. DNA samples from these families were genotyped with 365 microsatellite markers spaced at approximately 10-cM intervals across the whole genome. We observed LOD scores >3.0 at five distinct loci, either in the sample as a whole or within single families, strongly suggesting etiological heterogeneity. Heterogeneity LOD scores >3.0 in the sample as a whole were found at 1q33.2 (LOD score 3.2; P=.0003), 5q33.2 (LOD score 3.6; P=.0001), 8p22.1-22 (LOD score 3.6; P=.0001), and 11q21 (LOD score 3.1; P=.0004). LOD scores >3.0 within single pedigrees were found at 4q13-31 (LOD score 3.2; P=.0003) and at 11q23.3-24 (LOD score 3.2; P=.0003). A LOD score of 2.9 was also found at 20q12.1-11.23 within in a single family. The fact that other studies have also detected LOD scores >3.0 at 1q33.2, 5q33.2, 8p21-22 and 11q21 suggests that these regions do indeed harbor schizophrenia-susceptibility loci. We believe that the weight of evidence for linkage to the chromosome 1q22, 5q33.2, and 8p21-22 loci is now sufficient to justify intensive investigation of these regions by methods based on linkage disequilibrium. Such studies will soon allow the identification of mutations having a direct effect on susceptibility to schizophrenia.

Cationic liposome-mediated DNA transfection in organotypic explant cultures of the ventral mesencephalon.

We have examined the potential of cationic liposomes as a tool for approaches to gene therapy in the CNS. Our previous work has shown that cationic liposomes formulated from 3 beta-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol (DC-Chol) and dioleoyl-L-alpha-phosphatidylethanolamine (DOPE) could achieve high transfection levels in a neuronal cell line (McQuillin et al. Neuroreport 1997; 8: 1481-1484). We therefore wished to assess transfection efficiencies in organotypic cultures from the brain with a reporter plasmid expressing E. coli beta-galactosidase in order to mimic an in vivo model. Explant cultures were generated according to the method of Stoppini et al (J Neurosci Meth 1991; 37: 173-182) with slight modifications. Brain slices were maintained on transparent porous membranes and were observed to maintain their intrinsic connectivity and cytoarchitecture to a large degree over periods of up to 6 weeks in culture. CNS tissue was obtained from rats at birth or 5 days after birth. After transfection beta-galactosidase expression was detected in cells of both neuronal and non-neuronal morphology. Control cultures were exposed to liposome alone and a plasmid that had the beta-galactosidase gene insert removed. Only low levels of endogenous beta-galactosidase reactivity were seen in these control cultures. DC-Chol/DOPE-mediated transfection was confirmed using a RT-PCR protocol capable of differentiating between untranscribed plasmid DNA and RNA generated from the transfected vector. These results suggest that cationic liposomes, particularly DC-Chol/DOPE liposomes, will be useful as delivery agents for gene transfer to CNS cells in vitro and possibly in vivo.