The impact of clinical heterogeneity in schizophrenia on genomic analyses.

Though clinically useful, the diagnostic systems currently employed are not well equipped to capture the substantial clinical heterogeneity observed for most psychiatric disorders, as exemplified by the complex psychotic disorder(s) that Bleuler aptly labeled the "Group of Schizophrenias". The clinical heterogeneity associated with schizophrenia has likely frustrated decades of attempts to illuminate the underlying genetic architecture, although recent genome-wide association studies have begun to provide valuable insight into the role of common genetic risk variants. Here we demonstrate the importance of using diagnostic information to identify a core form of the disorder and to eliminate potential comorbidities in genetic studies. We also demonstrate why applying a diagnostic screening procedure to the control dataset to remove individuals with potentially related disorders is critical. Additionally, subjects may participate in multiple studies at different institutions or may have genotype data released by more than one research group. It is thus good practice to verify that no identical subjects exist within or between samples prior to conducting any type of genetic analysis to avoid potential confounding of results. While the availability of genomic data for large collections of subjects has facilitated many investigations that would otherwise not have been possible, we clearly show why one must use caution when acquiring data from publicly available sources. Although the broad vs. narrow debate in terms of phenotype definition in genetic analyses will remain, it is likely that both approaches will yield different results and that both will have utility in resolving the genetic architecture of schizophrenia.

Whole brain expression of bipolar disorder associated genes: structural and genetic analyses.

Studies of bipolar disorder (BD) suggest a genetic basis of the illness that alters brain function and morphology. In recent years, a number of genetic variants associated with BD have been identified. However, little is known about the associated genes, or brain circuits that rely upon their function. Using an anatomically comprehensive survey of the human transcriptome (The Allen Brain Atlas), we mapped the expression of 58 genes with suspected involvement in BD based upon their relationship to SNPs identified in genome wide association studies (GWAS). We then conducted a meta-analysis of structural MRI studies to identify brain regions that are abnormal in BD. Of 58 BD associated genes, 22 had anatomically distinct expression patterns that could be categorized into one of three clusters (C1-C3). Brain regions with the highest and lowest expression of these genes did not overlap strongly with anatomical sites identified as abnormal by structural MRI except in the parahippocampal gyrus, the inferior/superior temporal gyrus and the cerebellar vermis, regions where overlap was significant. Using the 22 genes in C1-C3 as reference points, additional genes with correlated expression patterns were identified and organized into sets based on similarity. Further analysis revealed that five of these gene sets were significantly associated with BD, suggesting that anatomical expression profile is correlated with genetic susceptibility to BD, particularly for genes in C2. Our data suggest that expression profiles of BD-associated genes do not explain the majority of structural abnormalities observed in BD, but may be useful in identifying new candidate genes. Our results highlight the complex neuroanatomical basis of BD, and reinforce illness models that emphasize impaired brain connectivity.

Evidence for association of bipolar disorder to haplotypes in the 22q12.3 region near the genes stargazin, IFT27 and parvalbumin.

We have previously reported genome-wide significant linkage of bipolar disorder to a region on 22q12.3 near the marker D22S278. Towards identifying the susceptibility gene, we have conducted a fine-mapping association study of the region in two independent family samples, an independent case-control sample and a genome-wide association dataset. Two hundred SNPs were first examined in a 5 Mb region surrounding the D22S278 marker in a sample of 169 families and analyzed using PLINK. The peak of association was a haplotype near the genes stargazin (CACNG2), intraflagellar transport protein homolog 27 (IFT27) and parvalbumin (PVALB; P = 4.69 × 10(-4)). This peak overlapped a significant haplotype in a family based association study of a second independent sample of 294 families (P = 1.42 × 10(-5)). Analysis of the combined family sample yielded statistically significant evidence of association to a rare three SNP haplotype in the gene IFT27 (P = 8.89 × 10(-6)). Twelve SNPs comprising these haplotypes were genotyped in an independent sample of 574 bipolar I cases and 550 controls. Statistically significant association was found for a haplotype window that overlapped the region from the first two family samples (P = 3.43 × 10(-4)). However, analyses of the two family samples using the program LAMP, found no evidence for association in this region, but did yield significant evidence for association to a haplotype 3' of CACNG2 (P = 1.76 × 10(-6)). Furthermore, no evidence for association was found in a large genome-wide association dataset. The replication of association to overlapping haplotypes in three independent datasets suggests the presence of a bipolar disorder susceptibility gene in this region.

Local anesthetics induce chondrocyte death in bovine articular cartilage disks in a dose- and duration-dependent manner.

PURPOSE: The purpose of this study was to evaluate the effect of various local anesthetics on chondrocyte viability in articular cartilage by use of a bovine disk model. METHODS: Full-thickness bovine cartilage disks were isolated from the condylar surfaces of the radial-carpal joint by use of a 4-mm biopsy punch and were incubated in various concentrations of local anesthetics (e.g., bupivacaine) for varying amounts of time and stained for membrane integrity by use of ethidium bromide and SYTO 13 stain (Molecular Probes, Carlsbad, CA). Cell and nuclear morphology was assessed by transmission electron microscopy. RESULTS: The addition of local anesthetics (i.e., 0.25% bupivacaine, 1% lidocaine, and 0.5% ropivacaine) to bovine articular cartilage disks had a negative effect on chondrocyte viability. Culturing bovine articular cartilage disks for increasing periods of time decreased chondrocyte viability for each of the local anesthetics, with significant negative correlations being shown between time of exposure to the drug and chondrocyte viability. These effects were also affected by the presence or absence of epinephrine in local anesthetic preparations. CONCLUSIONS: Our results suggest that local anesthetics (i.e., bupivacaine, lidocaine, or ropivacaine) can have a detrimental effect on chondrocyte viability in bovine articular cartilage disks in a dose- and duration-dependent manner. CLINICAL RELEVANCE: After arthroscopic surgery, it has been common practice to inject various local anesthetics into the joint for pain relief. Because adult chondrocytes have little or no capacity to regenerate, these results suggest that high-dose, long-term intra-articular administration of local anesthetics should be performed with caution.

The possible influence of osmotic poration on cell membrane water permeability.

It has been hypothesized that pores in the plasma membrane form under conditions of rapid water efflux, allowing extracellular ice to grow into the cytoplasm under conditions of rapid freezing. When cells with intracellular ice are thawed slowly, the transmembrane ice crystal expands through recrystallization causing the cell to lyse. One of the implications of this hypothesis is that osmotic pores will provide an alternative route for water movement under conditions of osmotically induced flow. We show that the plasma membrane water permeability of a fibroblast cell changes as a function of the osmotic pressure gradient that is used to drive water movement. It is further shown that cell volume is more important than the magnitude of water flux in causing this departure from a uniform water permeability. We suggest that these data provide evidence of a transient route for water movement across cell membranes.

Further evidence for association of GRK3 to bipolar disorder suggests a second disease mutation.

OBJECTIVES: Two genome-wide linkage surveys suggest chromosome 22q12 may contain a susceptibility locus for bipolar disorder (BPD) in the immediate region of the gene G protein receptor kinase-3 (GRK3). We previously published evidence that a single nucleotide polymorphism (SNP) in the promoter region of GRK3, designated P5, was associated with BPD. This SNP, however, was too rare (allele frequency 0.007) to explain the evidence for linkage. METHODS: To identify other SNPs or haplotypes associated with illness, we have now sequenced an additional 28-kb genomic segment of GRK3 and tested an additional 35 SNPs for association with BPD in 181 Caucasian nuclear families. RESULTS: Transmission disequilibrium test analyses identified two closely related disease-associated haplotypes defined by four SNPs located upstream of the promoter region: transmission to nontransmission ratios=54:22 and 20:9, odds ratios=2.50 and 2.36, and P values=0.0009 and 0.05. The best P value remained significant after correction for multiple testing. These two haplotypes were found on an entirely different set of chromosomes from the previously identified SNP P5. They had a combined frequency of approximately 0.10 and, therefore, a much greater population attributable risk for disease than the previously identified P5 haplotype. CONCLUSIONS: These data provide evidence that at least two distinct haplotypes, and possibly two or more different underlying mutations, in GRK3 might be associated with BPD. These new findings add support for the hypothesis that a dysregulation in GRK3 expression alters signaling desensitization and thereby predisposes to the development of BPD.

A linkage disequilibrium study of bipolar disorder and microsatellite markers on 22q13.

Bipolar disorder is a major psychiatric disorder characterized by extreme mood states that alternate between mania and depression. Family, twin, and adoption studies indicate a genetic component to the disease, but the etiology is suspected to be complex, with multiple genes contributing to an increased susceptibility to the disorder. We have previously reported a genome scan in which a genome-wide maximum LOD score indicated evidence of linkage at the marker D22S278 at 22q13. This area is of particular interest since it is also implicated in schizophrenia, and thus may harbor a susceptibility gene common to both disorders. In our further efforts to fine map this region, we examined 10 microsatellite markers spanning an interval of 2.3 MB in a set of 142 parent-proband triads. Linkage disequilibrium to illness was tested using the Transmission Disequilibrium Test. Haplotypes were determined and marker-to-marker linkage disequilibrium across the region was examined. D22S281 and D22S685 yielded suggestive evidence of linkage disequilibrium to bipolar disorder (empirical values of 0.023 and 0.036, respectively), but a marker-to-marker analysis indicates that a higher density screen is needed to adequately analyze this region.