Mutation screening of the 3q29 microdeletion syndrome candidate genes DLG1 and PAK2 in schizophrenia.

Deletion of chromosome 3q29, which is associated with mental retardation and autism, was recently identified as being present in excess or occurring de novo in schizophrenia cases, being present in approximately 1/1,000 cases and 1/40,000 unscreened controls. Of the ∼20 genes in the commonly deleted region two are prominent candidates for involvement in the behavioral features of the microdeletion syndrome: DLG1 and PAK2. We report the result of mutation screening of the entire protein coding sequence of both genes in a sample of 234 unrelated cases and 272 unrelated controls from the UK. We find no evidence for any amino acid changing genetic variants in PAK2. We observe several rare and singleton non-synonymous genetic variations at DLG1, however there is no excess of these variants in cases when compared to controls. Our sample was underpowered to detect very rare or low-penetrance disease relevant alleles in the studied genes. Therefore very rare, low-to-moderate penetrance protein coding mutations or non-coding mutations at DLG1 and/or PAK2, or a nearby gene, may reproduce the behavioral characteristics of the 3q29 microdeletion.

Evidence for rare and common genetic risk variants for schizophrenia at protein kinase C, alpha.

We earlier reported a genome-wide significant linkage to schizophrenia at chromosome 17 that was identified in a single pedigree (C702) consisting of six affected, male siblings with DSM-IV schizophrenia and prominent mood symptoms. In this study, we adopted several approaches in an attempt to map the putative disease locus. First, mapping the source of linkage to chromosome 17 in pedigree C702. We refined the linkage region in family C702 to a 21-marker segment spanning 11.7 Mb at 17q23-q24 by genotyping a total of 50 microsatellites across chromosome 17 in the pedigree. Analysis of data from 1028 single nucleotide polymorphisms (SNPs) across the refined linkage region identified a single region of homozygosity present in pedigree C702 but not in 2938 UK controls. This spanned ~432 kb of the gene encoding protein kinase C, alpha (PRKCA), the encoded protein of which has been implicated in the pathogenesis of psychiatric disorders. Analysis of pedigree C702 by oligonucleotide-array comparative genome hybridization excluded the possibility that this region of homozygosity was because of a deletion. Mutation screening of PRKCA identified a rare, four-marker haplotype (C-HAP) in the 3' untranslated region of the gene, which was present in the homozygous state in all six affected members of pedigree C702. No other homozygotes were observed in genotype data for a total of 6597 unrelated Europeans (case N=1755, control N=3580 and parents of probands N=1262). Second, association analysis of C702 alleles at PRKCA. The low-frequency haplotype (C-HAP) showed a trend for association in a study of unrelated schizophrenia cases and controls from the UK (661 cases, 2824 controls, P=0.078 and odd ratio (OR)=1.9) and significant evidence for association when the sample was expanded to include cases with bipolar (N=710) and schizoaffective disorder (N=50) (psychosis sample: 1421 cases, 2824 controls, P=0.037 and OR=1.9). Given that all the affected members of C702 are male, we also undertook sex-specific analyses. This revealed that the association was strongest in males for both schizophrenia (446 male cases, 1421 male controls, P=0.008 and OR=3.9) and in the broader psychosis group (730 male cases, 1421 male controls, P=0.008 and OR=3.6). Analysis of C-HAP in follow-up samples from Ireland and Bulgaria revealed no evidence for association in either the whole sample or in males alone, and meta-analysis of all male psychosis samples yielded no significant evidence of association (969 male cases, 1939 male controls, 311 male probands P=0.304 and OR=1.4). Third, association mapping of the pedigree C702 linkage region. Independent of pedigree C702, genotype data from the Affymetrix 500k GeneChip set were available for 476 patients with schizophrenia and 2938 controls from the United Kingdom. SNPs in PRKCA showed evidence for association with schizophrenia that achieved gene-wide significance (P=0.027). Moreover, the same SNP was the most significantly associated marker out of the 1028 SNPs genotyped across the linkage region (rs873417, allelic P=0.0004). Follow-up genotyping in samples from Ireland, Bulgaria and Germany did not show consistent replication, but meta-analysis of all samples (4116 cases and 6491 controls) remained nominally significant (meta-analysis P=0.026, OR=1.1). We conclude that, although we have obtained convergent lines of evidence implicating both rare and common schizophrenia risk variants at PRKCA, none of these is individually compelling. However, the evidence across all approaches suggests that further study of this locus is warranted.

Evidence that putative ADHD low risk alleles at SNAP25 may increase the risk of schizophrenia.

Synaptosomal Associated Protein 25 kDa (SNAP25) has been implicated in the pathogenesis of schizophrenia by numerous neuropathological studies and genetic variation at SNAP25 has been reported to be associated with ADHD. Expression levels of the putative schizophrenia susceptibility gene DTNBP1 has been shown to influence the levels of SNAP25 in vitro. We undertook directed mutation screening of SNAP25 in UK schizophrenic cases followed by direct association analysis of all variants identified and identified known exonic SNPs that showed evidence for association (rs3746544 P = 0.004 OR = 1.26, rs8636 P = 0.003 OR = 1.27), although these SNPs are highly correlated (r(2) > 0.99). We additionally genotyped a further 31 tag SNPs spanning the SNAP25 locus and identified several independent SNPs that were nominally associated with schizophrenia (strongest association at rs3787283, P = 0.006, OR = 1.25) however, due to the number of tests performed no SNP met experiment-wise significance (minimum permuted P-value = 0.1). Post hoc analysis revealed that the SNPs nominally associated with schizophrenia (rs3787283, rs3746544) were the same as those previously demonstrated to be associated with ADHD but with the opposite alleles, allowing the intriguing hypothesis that genetic variation at SNAP25 may be differentially associated with both schizophrenia and ADHD.

Accumulated background variation among H2 mutant congenic strains: elimination through PCR-based genotyping of F2 segregants.

Many commercially and privately available congenic strains of laboratory animals were founded decades ago and are likely to differ from one another by dozens of fixed mutational differences at background loci. This problem is often ignored despite growing evidence that such background variation exists. Eliminating this confounding variation can be largely accomplished by crossing congenic strains to produce F2 segregants that are homozygous (or heterozygous) for relevant genes. Discriminating F2 homozygotes can be difficult when strain differences are minor, as are mutant mouse strains differing at single major histocompatibility loci (H2 mutant congenics). Here, we describe a two-step polymerase chain reaction (PCR) method utilizing heteroduplex analysis and sequence specific primers (SSP-PCR) that efficiently discriminates the F2 progeny of two such H2 mutant congenic mice crosses (bm1xB6 and bm1xbm3). A third H2 mutant cross cannot be resolved by heteroduplexing, but is discriminated (albeit less efficiently) with SSP-PCR alone. This sensitive application can be extended to any congenic mutant strains.

Localization and release of FMRFamide-like immunoreactivity in the cerebral neuroendocrine system of Manduca sexta.

The distribution of FMRFamide-like immunoreactivity (FLI) in the cerebral neuroendocrine system of the moth, Manduca sexta, is described and evidence is provided for calcium-dependent release of FLI from the neurohaemal organs. FLI was detected by indirect immunofluorescence in approximately 25 bilaterally symmetrical pairs of somata in the pupal protocerebrum. In addition, FLI was observed in neurites in the brain, as well as in axons of the nervi corporis cardiaci and nervi corporis allati, and in terminals in the neurohaemal corpora cardiaca (CC) and corpora allata (CA). All immunocytochemical staining was blocked by preabsorption of the anti-FMRFamide antiserum with synthetic FMRFamide. We localized FLI to identified protocerebral neurosecretory cells (NSCs) by combining intracellular injection of Lucifer Yellow and indirect immunofluorescence. Among the NSCs in each hemisphere, FLI was observed in both group IIa (lateral) cells, in most group IIb (lateral) cells, and in two cells of group Ib (medial). FLI was extracted from the brain and neurohaemal organs and measured using radioimmunoassay (RIA). Calcium-dependent release of FLI was evoked from isolated CC-CA by high potassium depolarization in vitro and was quantified by RIA of the bathing medium. These results suggest that FLI may have a neurohormonal or neurotransmitter function in Manduca.