Linda M. Brzustowicz

Genome Scan Meta-Analysis of Schizophrenia and Bipolar Disorder, Part II: Schizophrenia

Schizophrenia is a common disorder with high heritability and a 10-fold increase in risk to siblings of probands. Replication has been inconsistent for reports of significant genetic linkage. To assess evidence for linkage across studies, rank-based genome scan meta-analysis (GSMA) was applied to data from 20 schizophrenia genome scans. Each marker for each scan was assigned to 1 of 120 30-cM bins, with the bins ranked by linkage scores (1 = most significant) and the ranks averaged across studies (R(avg)) and then weighted for sample size (N(sqrt)[affected casess]). A permutation test was used to compute the probability of observing, by chance, each bins average rank (P(AvgRnk)) or of observing it for a bin with the same place (first, second, etc.) in the order of average ranks in each permutation (P(ord)). The GSMA produced significant genomewide evidence for linkage on chromosome 2q (PAvgRnk<.000417). Two aggregate criteria for linkage were also met (clusters of nominally significant P values that did not occur in 1,000 replicates of the entire data set with no linkage present): 12 consecutive bins with both P(AvgRnk) and P(ord)<.05, including regions of chromosomes 5q, 3p, 11q, 6p, 1q, 22q, 8p, 20q, and 14p, and 19 consecutive bins with P(ord)<.05, additionally including regions of chromosomes 16q, 18q, 10p, 15q, 6q, and 17q. There is greater consistency of linkage results across studies than has been previously recognized. The results suggest that some or all of these regions contain loci that increase susceptibility to schizophrenia in diverse populations.

MAOA and the cycle of violence : Childhood abuse and neglect, MAOA genotype, and risk for violent and antisocial behavior

BACKGROUND Two recent studies with white males have shown that genotypes associated with high levels of monamine oxidase A (MAOA) protect against the impact of childhood maltreatment and adversity on the development of antisocial behavior and conduct disorder. METHODS Participants in a prospective cohort design study involving court substantiated cases of child abuse and neglect and a matched comparison group were followed up into adulthood and interviewed (N = 802). Eighty-two percent consented to provide blood and 631 gave permission for DNA extraction and analyses. A composite index of violent and antisocial behavior (VASB) was created based on arrest, self-report, and diagnostic information. RESULTS No main effect was found for the relationship between MAOA genotype and VASB. Genotypes associated with high levels of MAOA activity buffered abused and neglected whites from increased risk of becoming violent and/or antisocial in later life. This protective effect was not found for non-white abused and neglected individuals. CONCLUSIONS Possible explanations for this differential effect for whites and non-whites include differences in contextual factors (e.g., environmental stressors) and a question of the suitability of using the MAOA promoter VNTR polymorphism as a proxy for MAOA levels in non-white populations.

Linkage of Familial Schizophrenia to Chromosome 13q32

Over the past 4 years, a number of investigators have reported findings suggestive of linkage to schizophrenia, with markers on chromosomes 13q32 and 8p21, with one recent study by Blouin et al. reporting significant linkage to these regions. As part of an ongoing genome scan, we evaluated microsatellite markers spanning chromosomes 8 and 13, for linkage to schizophrenia, in 21 extended Canadian families. Families were analyzed under autosomal dominant and recessive models, with broad and narrow definitions of schizophrenia. All models produced positive LOD scores with markers on 13q, with higher scores under the recessive models. The maximum three-point LOD scores were obtained under the recessive-broad model: 3.92 at recombination fraction (theta).1 with D13S793, under homogeneity, and 4.42 with alpha=.65 and straight theta=0 with D13S793, under heterogeneity. Positive LOD scores were also obtained, under all models, for markers on 8p. Although a maximum two-point LOD score of 3.49 was obtained under the dominant-narrow model with D8S136 at straight theta=0.1, multipoint analysis with closely flanking markers reduced the maximum LOD score in this region to 2. 13. These results provide independent significant evidence of linkage of a schizophrenia-susceptibility locus to markers on 13q32 and support the presence of a second susceptibility locus on 8p21.

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

A genome scan meta-a nalysis (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 (PSR) 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.

A Major Susceptibility Locus for Specific Language Impairment Is Located on 13q21

Children who fail to develop language normally-in the absence of explanatory factors such as neurological disorders, hearing impairment, or lack of adequate opportunity-are clinically described as having specific language impairment (SLI). SLI has a prevalence of approximately 7% in children entering school and is associated with later difficulties in learning to read. Research indicates that genetic factors are important in the etiology of SLI. Studies have consistently demonstrated that SLI aggregates in families. Increased monozygotic versus dizygotic twin concordance rates indicate that heredity, not just shared environment, is the cause of the familial clustering. We have collected five pedigrees of Celtic ancestry that segregate SLI, and we have conducted genomewide categorical linkage analysis, using model-based LOD score techniques. Analysis was conducted under both dominant and recessive models by use of three phenotypic classifications: clinical diagnosis, language impairment (spoken language quotient <85) and reading discrepancy (nonverbal IQ minus non-word reading >15). Chromosome 13 yielded a maximum multipoint LOD score of 3.92 under the recessive reading discrepancy model. Simulation to correct for multiple models and multiple phenotypes indicated that the genomewide empirical P value is <.01. As an alternative measure, we also computed the posterior probability of linkage (PPL), obtaining a PPL of 53% in the same region. One other genomic region yielded suggestive results on chromosome 2 (multipoint LOD score 2.86, genomic P value <.06 under the recessive language impairment model). Our findings underscore the utility of traditional LOD-score-based methods in finding genes for complex diseases, specifically, SLI.

Association of the homeobox transcription factor, ENGRAILED 2 , 3 , with autism spectrum disorder

Mouse mutants of the homeobox transcription factor Engrailed2 (En2) and autistic individuals display similar cerebellar morphological abnormalities, which include hypoplasia and a decrease in the number of Purkinje cells.1,2,3,4,5,6,7,8, 9,10,11,12,13,14,15,16, 17,18,19 Human EN2 maps to 7q36, a chromosomal region that has demonstrated suggestive linkage to autism spectrum disorder (ASD).20,21,22 To investigate EN2 for evidence of association with ASD, four single-nucleotide polymorphisms (SNPs) (rs3735653, rs1861972, rs1861973, rs2361689) that span the majority of the 8.0 kb gene were assessed by the transmission/disequilibrium test23,24,25,26. Initially, 138 triads of autistic individuals and their parents were tested. Two intronic SNPs (rs1861972 and rs1861973) demonstrated significant association with autism (rs1861972, P=0.0018; rs1861973, P=0.0003; haplotype, P=0.000005). Flanking exonic SNPs (rs3735653 and rs2361689) did not display association. This analysis was then extended to include 167 small nuclear ASD pedigrees and significant association was again only observed for rs1861972 and rs1861973 under both the narrow and broad diagnostic criteria (narrow: rs1861972 P=0.0290, rs1861973 P=0.0073, haplotype P=0.0009; broad: rs1861972 P=0.0175, rs1861973 P=0.0107, haplotype P=0.0024). These data demonstrate association between a cerebellar patterning gene and ASD, suggesting a role for EN2 as a susceptibility locus and supporting a neurodevelopmental defect hypothesis in the etiology of autism.

Altered microRNA expression profiles in postmortem brain samples from individuals with schizophrenia and bipolar disorder.

BACKGROUND MicroRNAs (miRNAs) are potent regulators of gene expression with proposed roles in brain development and function. We hypothesized that miRNA expression profiles are altered in individuals with severe psychiatric disorders. METHODS With real-time quantitative polymerase chain reaction, we compared the expression of 435 miRNAs and 18 small nucleolar RNAs in postmortem brain tissue samples from individuals with schizophrenia, individuals with bipolar disorder, and psychiatrically healthy control subjects (n = 35 each group). Detailed demographic data, sample selection and storage conditions, and drug and substance exposure histories were available for all subjects. Bayesian model averaging was used to simultaneously assess the impact of these covariates as well as the psychiatric phenotype on miRNA expression profiles. RESULTS Of the variables considered, sample storage time, brain pH, alcohol at time of death, and postmortem interval were found to affect the greatest proportion of miRNAs. Of miRNAs analyzed, 19% exhibited positive evidence of altered expression due to a diagnosis of schizophrenia or bipolar disorder. Both conditions were associated with reduced miRNA expression levels, with a much more pronounced effect observed for bipolar disorder. CONCLUSIONS This study suggests that modest underexpression of several miRNAs might be involved in the complex pathogenesis of major psychosis.

Support for the homeobox transcription factor gene ENGRAILED 2 as an autism spectrum disorder susceptibility locus.

Our previous research involving 167 nuclear families from the Autism Genetic Resource Exchange (AGRE) demonstrated that two intronic SNPs, rs1861972 and rs1861973, in the homeodomain transcription factor gene ENGRAILED 2 (EN2) are significantly associated with autism spectrum disorder (ASD). In this study, significant replication of association for rs1861972 and rs1861973 is reported for two additional data sets: an independent set of 222 AGRE families (rs1861972-rs1861973 haplotype, P=.0016) and a separate sample of 129 National Institutes of Mental Health families (rs1861972-rs1861973 haplotype, P=.0431). Association analysis of the haplotype in the combined sample of both AGRE data sets (389 families) produced a P value of .0000033, whereas combining all three data sets (518 families) produced a P value of .00000035. Population-attributable risk calculations for the associated haplotype, performed using the entire sample of 518 families, determined that the risk allele contributes to as many as 40% of ASD cases in the general population. Linkage disequilibrium (LD) mapping with the use of polymorphisms distributed throughout the gene has shown that only intronic SNPs are in strong LD with rs1861972 and rs1861973. Resequencing and association analysis of all intronic SNPs have identified alleles associated with ASD, which makes them candidates for future functional analysis. Finally, to begin defining the function of EN2 during development, mouse En2 was ectopically expressed in cortical precursors. Fewer En2-transfected cells than controls displayed a differentiated phenotype. Together, these data provide further genetic evidence that EN2 might act as an ASD susceptibility locus, and they suggest that a risk allele that perturbs the spatial/temporal expression of EN2 could significantly alter normal brain development.

Copy Number Variations in Schizophrenia: Critical Review and New Perspectives on Concepts of Genetics and Disease

OBJECTIVE Structural variations of DNA, such as copy number variations (CNVs), are recognized to contribute both to normal genomic variability and to risk for human diseases. For example, schizophrenia has an established connection with 22q11.2 deletions. Recent genome-wide studies have provided initial evidence that CNVs at other loci may also be associated with schizophrenia. In this article, the authors provide a brief overview of CNVs, review recent findings related to schizophrenia, outline implications for clinical practice and diagnostic subtyping, and make recommendations for future reports on CNVs to improve interpretation of results. METHOD The review included genome-wide surveys of CNVs in schizophrenia that included one or more comparison groups, were published before 2009, and used newer methods. Six studies were identified. RESULTS Despite some limitations, these initial genome-wide studies of CNVs provide replicated associations of schizophrenia with rare 1q21.1 and 15q13.3 deletions. Collectively, the results point to a more general mutational mechanism involving rare CNVs that elevate risk for schizophrenia, especially more developmental forms of the disease. Including 22q11.2 deletions, rare risk-associated CNVs appear to account for up to 2% of schizophrenia. CONCLUSIONS The more penetrant CNVs have direct implications for clinical practice and diagnostic subtyping. CNVs with lower penetrance promise to contribute to our genetic understanding of pathogenesis. The findings provide insight into a broader neuropsychiatric spectrum for schizophrenia than previously conceived and indicate new directions for genetic studies.

Linkage disequilibrium mapping of schizophrenia susceptibility to the CAPON region of chromosome 1q22.

Previously, we have reported linkage of markers from chromosome 1q22 to schizophrenia, a finding supported by several independent studies. We have now examined the region of strongest linkage for evidence of linkage disequilibrium (LD) in a sample of 24 Canadian familial-schizophrenia pedigrees. Analysis of 14 microsatellites and 15 single-nucleotide polymorphisms (SNPs) from the 5.4-Mb region between D1S1653 and D1S1677 produced significant evidence (nominal P<.05) of LD between schizophrenia and 2 microsatellites and 6 SNPs. All of the markers exhibiting significant LD to schizophrenia fall within the genomic extent of the gene for carboxyl-terminal PDZ ligand of neuronal nitric oxide synthase (CAPON), making it a prime positional candidate for the schizophrenia-susceptibility locus on 1q22, although initial mutation analysis of this gene has not identified any schizophrenia-associated changes within exons. Consistent with several recently identified candidate genes for schizophrenia, CAPON is involved in signal transduction in the NMDA receptor system, highlighting the potential importance of this pathway in the etiology of schizophrenia.