K D Chambert

De novo mutations in schizophrenia implicate synaptic networks

Inherited alleles account for most of the genetic risk for schizophrenia. However, new (de novo) mutations, in the form of large chromosomal copy number changes, occur in a small fraction of cases and disproportionally disrupt genes encoding postsynaptic proteins. Here we show that small de novo mutations, affecting one or a few nucleotides, are overrepresented among glutamatergic postsynaptic proteins comprising activity-regulated cytoskeleton-associated protein (ARC) and N-methyl-d-aspartate receptor (NMDAR) complexes. Mutations are additionally enriched in proteins that interact with these complexes to modulate synaptic strength, namely proteins regulating actin filament dynamics and those whose messenger RNAs are targets of fragile X mental retardation protein (FMRP). Genes affected by mutations in schizophrenia overlap those mutated in autism and intellectual disability, as do mutation-enriched synaptic pathways. Aligning our findings with a parallel case–control study, we demonstrate reproducible insights into aetiological mechanisms for schizophrenia and reveal pathophysiology shared with other neurodevelopmental disorders.

A polygenic burden of rare disruptive mutations in schizophrenia

Schizophrenia is a common disease with a complex aetiology, probably involving multiple and heterogeneous genetic factors. Here, by analysing the exome sequences of 2,536 schizophrenia cases and 2,543 controls, we demonstrate a polygenic burden primarily arising from rare (less than 1 in 10,000), disruptive mutations distributed across many genes. Particularly enriched gene sets include the voltage-gated calcium ion channel and the signalling complex formed by the activity-regulated cytoskeleton-associated scaffold protein (ARC) of the postsynaptic density, sets previously implicated by genome-wide association and copy-number variation studies. Similar to reports in autism, targets of the fragile X mental retardation protein (FMRP, product of FMR1) are enriched for case mutations. No individual gene-based test achieves significance after correction for multiple testing and we do not detect any alleles of moderately low frequency (approximately 0.5 to 1 per cent) and moderately large effect. Taken together, these data suggest that population-based exome sequencing can discover risk alleles and complements established gene-mapping paradigms in neuropsychiatric disease.

Characterization of bipolar disorder patient-specific induced pluripotent stem cells from a family reveals neurodevelopmental and mRNA expression abnormalities

Bipolar disorder (BD) is a common neuropsychiatric disorder characterized by chronic recurrent episodes of depression and mania. Despite evidence for high heritability of BD, little is known about its underlying pathophysiology. To develop new tools for investigating the molecular and cellular basis of BD, we applied a family-based paradigm to derive and characterize a set of 12 induced pluripotent stem cell (iPSC) lines from a quartet consisting of two BD-affected brothers and their two unaffected parents. Initially, no significant phenotypic differences were observed between iPSCs derived from the different family members. However, upon directed neural differentiation, we observed that CXCR4 (CXC chemokine receptor-4) expressing central nervous system (CNS) neural progenitor cells (NPCs) from both BD patients compared with their unaffected parents exhibited multiple phenotypic differences at the level of neurogenesis and expression of genes critical for neuroplasticity, including WNT pathway components and ion channel subunits. Treatment of the CXCR4+ NPCs with a pharmacological inhibitor of glycogen synthase kinase 3, a known regulator of WNT signaling, was found to rescue a progenitor proliferation deficit in the BD patient NPCs. Taken together, these studies provide new cellular tools for dissecting the pathophysiology of BD and evidence for dysregulation of key pathways involved in neurodevelopment and neuroplasticity. Future generation of additional iPSCs following a family-based paradigm for modeling complex neuropsychiatric disorders in conjunction with in-depth phenotyping holds promise for providing insights into the pathophysiological substrates of BD and is likely to inform the development of targeted therapeutics for its treatment and ideally prevention.

Copy Number Variation in Bipolar Disorder

Large (>100u2009kb), rare (<1% in the population) copy number variants (CNVs) have been shown to confer risk for schizophrenia (SZ), but the findings for bipolar disorder (BD) are less clear. In a new BD sample from the United Kingdom (n=2591), we have examined the occurrence of CNVs and compared this with previously reported samples of 6882 SZ and 8842 control subjects. When combined with previous data, we find evidence for a contribution to BD for three SZ-associated CNV loci: duplications at 1q21.1 (P=0.022), deletions at 3q29 (P=0.03) and duplications at 16p11.2 (P=2.3 × 10−4). The latter survives multiple-testing correction for the number of recurrent large CNV loci in the genome. Genes in 20 regions (total of 55 genes) were enriched for rare exonic CNVs among BD cases, but none of these survives correction for multiple testing. Finally, our data provide strong support for the hypothesis of a lesser contribution of very large (>500u2009kb) CNVs in BD compared with SZ, most notably for deletions >1u2009Mb (P=9 × 10−4).

Cis-Acting Regulation of Brain-Specific ANK3 Gene Expression by a Genetic Variant Associated with Bipolar Disorder

Several genome-wide association studies for bipolar disorder (BD) have found a strong association of the Ankyrin 3 (ANK3) gene. This association spans numerous linked single-nucleotide polymorphisms (SNPs) in an ∼250-kb genomic region overlapping ANK3. The associated region encompasses predicted regulatory elements as well as two of the six validated alternative first exons, which encode distinct protein domains at the N-terminus of the protein also known as Ankyrin-G. Using RNA ligase-mediated rapid amplification of cDNA ends to identify novel transcripts in conjunction with a highly sensitive, exon-specific multiplexed mRNA expression assay, we detected differential regulation of distinct ANK3 transcription start sites and coupling of specific 5′ ends with 3′ mRNA splicing events in postmortem human brain and human stem cell-derived neural progenitors and neurons. Furthermore, allelic variation at the BD-associated SNP rs1938526 correlated with a significant difference in cerebellar expression of a brain-specific ANK3 transcript. These findings suggest a brain-specific cis-regulatory transcriptional effect of ANK3 that may be relevant to BD pathophysiology.

Evidence of Common Genetic Overlap Between Schizophrenia and Cognition

Cognitive impairment is a core feature of schizophrenia but there is limited understanding of the genetic relationship between cognition in the general population and schizophrenia. We examine how common variants associated with schizophrenia en masse contribute to childhood cognitive ability in a population-based sample, and the extent to which common genetic variants associated with childhood cognition explain variation in schizophrenia. Schizophrenia polygenic risk scores were derived from the Psychiatric Genomics Consortium (n = 69 516) and tested for association with IQ, attention, processing speed, working memory, problem solving, and social cognition in over 5000 children aged 8 from the Avon Longitudinal Study of Parents and Children birth cohort. Polygenic scores for these cognitive domains were tested for association with schizophrenia in a large UK schizophrenia sample (n = 11 853). Bivariate genome-wide complex trait analysis (GCTA) estimated the amount of shared genetic factors between schizophrenia and cognitive domains. Schizophrenia polygenic risk score was associated with lower performance IQ (P = .001) and lower full IQ (P = .013). Polygenic score for performance IQ was associated with increased risk for schizophrenia (P = 3.56E-04). Bivariate GCTA revealed moderate genetic correlation between schizophrenia and both performance IQ (r G = −.379, P = 6.62E-05) and full IQ (r G = −.202, P = 5.00E-03), with approximately 14% of the genetic component of schizophrenia shared with that for performance IQ. Our results support the presence of shared common genetic factors between schizophrenia and childhood cognitive ability. We observe a genetic relationship between schizophrenia and performance IQ but not verbal IQ or other cognitive variables, which may have implications for studies utilizing cognitive endophenotypes for psychosis.

Common alleles contribute to schizophrenia in CNV carriers.

The genetic architecture of schizophrenia is complex, involving risk alleles ranging from common alleles of weak effect to rare alleles of large effect, the best exemplar of the latter being large copy number variants (CNVs). It is currently unknown whether pathophysiology in those with defined rare mutations overlaps with that in other individuals with the disorder who do not share the same rare mutation. Under an extreme heterogeneity model, carriers of specific high-penetrance mutations form distinct subgroups. In contrast, under a polygenic threshold model, high-penetrance rare allele carriers possess many risk factors, of which the rare allele is the only one, albeit an important, factor. Under the latter model, cases with rare mutations can be expected to share some common risk alleles, and therefore pathophysiological mechanisms, with cases without the same mutation. Here we show that, compared with controls, individuals with schizophrenia who have known pathogenic CNVs carry an excess burden of common risk alleles (P=2.25 × 10−17) defined from a genome-wide association study largely based on individuals without known CNVs. Our finding is not consistent with an extreme heterogeneity model for CNV carriers, but does offer support for the polygenic threshold model of schizophrenia. That this is so provides support for the notion that studies aiming to model the effects of rare variation may uncover pathophysiological mechanisms of relevance to those with the disorder more widely.

A family-based study of common polygenic variation and risk of schizophrenia

Recent studies have suggested that a large number of common variants may, in aggregate, underlie a substantial proportion of the heritability of complex traits such as schizophrenia, multiple sclerosis and height, even though the effect of each individual variant is typically very small (1-5). A persistent concern for population-based genome-wide association studies (GWAS), however, is that subtle population stratification could lead to bias. Although the abovementioned studies adhered to “best-practice” for controlling bias in GWAS, some authors have speculated, with respect to the International Schizophrenia Consortium’s schizophrenia study, that “cryptic population stratification could substantially affect [the results]” (6). Here, we repeat the analysis described in (1) utilizing a family-based sample, to ask directly whether this is in fact a viable contention, as family-based designs, by definition, control for “cryptic population stratification” completely. n nWe analyzed GWAS data on 694 parent-offspring schizophrenia trios from Bulgaria. Of these probands, 360 were cases in the original ISC manuscript. All samples were ascertained and genotyped on Affymetrix 6.0 arrays following the protocols described in (1), with the exception that we further required SNPs to have greater than 99% genotyping and no more than 1 Mendel error. The original analysis in (1) created “scores” in case/control target samples, where the score per individual was a weighted sum of “risk alleles”, with the weights and the alleles determined by standard tests of association in an independent “discovery” case/control sample. Here, we designated the entire ISC sample (excluding all Bulgarian individuals) as the “discovery” sample; our target comparison was between the transmitted and untransmitted alleles of the Bulgarian trios. In other words, we asked whether putative risk alleles from a case/control study tend to, on average, be over-transmitted to offspring with schizophrenia. This analysis does not constitute an independent replication of (1) because of the overlapping cases; rather, our current purpose is solely to exclude bias due to cryptic population stratification as a possible source of inflated type I error. n nAlthough within-family association statistics are free from bias due to population stratification, they are in fact susceptible to certain technical biases that do not impact population-based studies, arising from non-random genotyping error (7). Particularly for low frequency variants, heterozygotes are more likely to be misclassified as the common than the rare homozygote. In family-based studies, this leads to a bias in which the common allele is over-transmitted. This is because, if only one parent is heterozygous and transmits the minor allele, a miscall indicating the common homozygote in the parent, will result in a Mendel error whereas the same error in the offspring will result in an apparent transmission of the common allele. We observed this phenomenon in our total trio dataset: using the transmission disequilibrium test (TDT) (8) the mean log of the odds ratio for the minor allele is -.004, which is significantly different from 0 (p 300 out of a total of 525,571 SNPs). We also removed all SNPs with less than complete genotyping (because SNPs with lower call rates tend to have more miscalling errors), or a minor allele frequency less than 2%. The resulting dataset showed no significant bias (P=.34). n nWe designated “score alleles” in the discovery sample (2,794 cases, and 2,976 controls from (1)) for a subset of 45,544 SNPs selected to be in approximate linkage equilibrium, preferentially retaining SNPs with higher association in the discovery sample. In the target sample we calculated the weighted sum of “score alleles” at various discovery p-value thresholds (P<0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5) for the transmitted and untransmitted alleles. n nFigure 1 shows the results of the primary analysis, in which we compared scores between transmitted and untransmitted chromosomes using logistic regression as in (1). At every discovery P-value threshold, transmitted chromosomes had significantly higher rates of the score alleles, (i.e. alleles that were more common in cases compared to controls in the independent discovery analysis). The estimate of variance explained (Nagelkerke’s pseudo-R2) by the observed score reached ~5%, that is similar to values from (1). We confirmed these results using a 1-sided t-test on the difference in score between matched pairs of transmitted and untransmitted chromosomes (data not shown). n n n nFigure 1 n nVariance explained by p-value threshold, corresponding significance at each threshold (p=0.0044, 5.4×10-06, 3.2×10-08, 5.1×10-10, 1.2×10-11, 2.2×10-12, 1.03×10-12) n n n nIn summary, individuals with schizophrenia from distinct European populations show enrichment across a very large number of SNPs for the same sets of common alleles. As previously discussed (1), this observation is consistent with a highly polygenic model of disease risk involving causal common variation. Further to the arguments already presented in (1), we can reject cryptic population stratification as a viable alternative explanation.

Genome-wide association study identifies SESTD1 as a novel risk gene for lithium-responsive bipolar disorder

Lithium is the mainstay prophylactic treatment for bipolar disorder (BD), but treatment response varies considerably across individuals. Patients who respond well to lithium treatment might represent a relatively homogeneous subtype of this genetically and phenotypically diverse disorder. Here, we performed genome-wide association studies (GWAS) to identify (i) specific genetic variations influencing lithium response and (ii) genetic variants associated with risk for lithium-responsive BD. Patients with BD and controls were recruited from Sweden and the United Kingdom. GWAS were performed on 2698 patients with subjectively defined (self-reported) lithium response and 1176 patients with objectively defined (clinically documented) lithium response. We next conducted GWAS comparing lithium responders with healthy controls (1639 subjective responders and 8899 controls; 323 objective responders and 6684 controls). Meta-analyses of Swedish and UK results revealed no significant associations with lithium response within the bipolar subjects. However, when comparing lithium-responsive patients with controls, two imputed markers attained genome-wide significant associations, among which one was validated in confirmatory genotyping (rs116323614, P=2.74 × 10−8). It is an intronic single-nucleotide polymorphism (SNP) on chromosome 2q31.2 in the gene SEC14 and spectrin domains 1 (SESTD1), which encodes a protein involved in regulation of phospholipids. Phospholipids have been strongly implicated as lithium treatment targets. Furthermore, we estimated the proportion of variance for lithium-responsive BD explained by common variants (‘SNP heritability’) as 0.25 and 0.29 using two definitions of lithium response. Our results revealed a genetic variant in SESTD1 associated with risk for lithium-responsive BD, suggesting that the understanding of BD etiology could be furthered by focusing on this subtype of BD.

Analysis of exome sequence in 604 trios for recessive genotypes in schizophrenia.

Genetic associations involving both rare and common alleles have been reported for schizophrenia but there have been no systematic scans for rare recessive genotypes using fully phased trio data. Here, we use exome sequencing in 604 schizophrenia proband–parent trios to investigate the role of recessive (homozygous or compound heterozygous) nonsynonymous genotypes in the disorder. The burden of recessive genotypes was not significantly increased in probands at either a genome-wide level or in any individual gene after adjustment for multiple testing. At a system level, probands had an excess of nonsynonymous compound heterozygous genotypes (minor allele frequency, MAF ⩽1%) in voltage-gated sodium channels (VGSCs; eight in probands and none in parents, P=1.5 × 10−4). Previous findings of multiple de novo loss-of-function mutations in this gene family, particularly SCN2A, in autism and intellectual disability provide biological and genetic plausibility for this finding. Pointing further to the involvement of VGSCs in schizophrenia, we found that these genes were enriched for nonsynonymous mutations (MAF ⩽0.1%) in cases genotyped using an exome array, (5585 schizophrenia cases and 8103 controls), and that in the trios data, synaptic proteins interacting with VGSCs were also enriched for both compound heterozygosity (P=0.018) and de novo mutations (P=0.04). However, we were unable to replicate the specific association with compound heterozygosity at VGSCs in an independent sample of Taiwanese schizophrenia trios (N=614). We conclude that recessive genotypes do not appear to make a substantial contribution to schizophrenia at a genome-wide level. Although multiple lines of evidence, including several from this study, suggest that rare mutations in VGSCs contribute to the disorder, in the absence of replication of the original findings regarding compound heterozygosity, this conclusion requires evaluation in a larger sample of trios.