Yunjung Kim

Genome-wide association analysis identifies 13 new risk loci for schizophrenia

Schizophrenia is an idiopathic mental disorder with a heritable component and a substantial public health impact. We conducted a multi-stage genome-wide association study (GWAS) for schizophrenia beginning with a Swedish national sample (5,001 cases and 6,243 controls) followed by meta-analysis with previous schizophrenia GWAS (8,832 cases and 12,067 controls) and finally by replication of SNPs in 168 genomic regions in independent samples (7,413 cases, 19,762 controls and 581 parent-offspring trios). We identified 22 loci associated at genome-wide significance; 13 of these are new, and 1 was previously implicated in bipolar disorder. Examination of candidate genes at these loci suggests the involvement of neuronal calcium signaling. We estimate that 8,300 independent, mostly common SNPs (95% credible interval of 6,300–10,200 SNPs) contribute to risk for schizophrenia and that these collectively account for at least 32% of the variance in liability. Common genetic variation has an important role in the etiology of schizophrenia, and larger studies will allow more detailed understanding of this disorder.

Schizophrenia Genetics: Where Next?

The purpose of this invited review is to summarize the state of genetic research into the etiology of schizophrenia (SCZ) and to consider options for progress. The fundamental uncertainty in SCZ genetics has always been the nature of the beast, the underlying genetic architecture. If this were known, studies using the appropriate technologies and sample sizes could be designed with an excellent chance of producing high-confidence results. Until recently, few pertinent data were available, and the field necessarily relied on speculation. However, for the first time in the complex and frustrating history of inquiry into the genetics of SCZ, we now have empirical data about the genetic basis of SCZ that implicate specific loci and that can be used to plan the next steps forward.

Analyses of allele-specific gene expression in highly divergent mouse crosses identifies pervasive allelic imbalance

Complex human traits are influenced by variation in regulatory DNA through mechanisms that are not fully understood. Because regulatory elements are conserved between humans and mice, a thorough annotation of cis regulatory variants in mice could aid in further characterizing these mechanisms. Here we provide a detailed portrait of mouse gene expression across multiple tissues in a three-way diallel. Greater than 80% of mouse genes have cis regulatory variation. Effects from these variants influence complex traits and usually extend to the human ortholog. Further, we estimate that at least one in every thousand SNPs creates a cis regulatory effect. We also observe two types of parent-of-origin effects, including classical imprinting and a new global allelic imbalance in expression favoring the paternal allele. We conclude that, as with humans, pervasive regulatory variation influences complex genetic traits in mice and provide a new resource toward understanding the genetic control of transcription in mammals.

Hypothesis-driven candidate genes for schizophrenia compared to genome-wide association results

BACKGROUND Candidate gene studies have been a key approach to the genetics of schizophrenia (SCZ). However, the results of these studies are confusing and no genes have been unequivocally implicated. The hypothesis-driven candidate gene literature can be appraised by comparison with the results of genome-wide association studies (GWAS). METHOD We describe the characteristics of hypothesis-driven candidate gene studies from the SZGene database, and use pathway analysis to compare hypothesis-driven candidate genes with GWAS results from the International Schizophrenia Consortium (ISC). RESULTS SZGene contained 732 autosomal genes evaluated in 1374 studies. These genes had poor statistical power to detect genetic effects typical for human diseases, assessed only 3.7% of genes in the genome, and had low marker densities per gene. Most genes were assessed once or twice (76.9%), providing minimal ability to evaluate consensus across studies. The ISC studies had 89% power to detect a genetic effect typical for common human diseases and assessed 79% of known autosomal common genetic variation. Pathway analyses did not reveal enrichment of smaller ISC p values in hypothesis-driven candidate genes, nor did a comprehensive evaluation of meta-hypotheses driving candidate gene selection (SCZ as a disease of the synapse or neurodevelopment). The most studied hypothesis-driven candidate genes (COMT, DRD3, DRD2, HTR2A, NRG1, BDNF, DTNBP1 and SLC6A4) had no notable ISC results. CONCLUSIONS We did not find support for the idea that the hypothesis-driven candidate genes studied in the literature are enriched for the common genetic variation involved in the etiology of SCZ. Larger samples are required to evaluate this conclusion definitively.

Genome-Wide Association Mapping Of Loci for Antipsychotic-Induced Extrapyramidal Symptoms in Mice

Tardive dyskinesia (TD) is a debilitating, unpredictable, and often irreversible side effect resulting from chronic treatment with typical antipsychotic agents such as haloperidol. TD is characterized by repetitive, involuntary, purposeless movements primarily of the orofacial region. In order to investigate genetic susceptibility to TD, we used a validated mouse model for a systems genetics analysis geared toward detecting genetic predictors of TD in human patients. Phenotypic data from 27 inbred strains chronically treated with haloperidol and phenotyped for vacuous chewing movements were subject to a comprehensive genomic analysis involving 426,493 SNPs, 4,047 CNVs, brain gene expression, along with gene network and bioinformatic analysis. Our results identified ~50 genes that we expect to have high prior probabilities for association with haloperidol-induced TD, most of which have never been tested for association with human TD. Among our top candidates were genes regulating the development of brain motor control regions (Zic4 and Nkx6-1), glutamate receptors (Grin1 and Grin2a), and an indirect target of haloperidol (Drd1a) that has not been studied as well as the direct target, Drd2.

Deep resequencing and association analysis of schizophrenia candidate genes.

In 2005, we selected 10 genes for which there was reasonable evidence for involvement in the etiology of schizophrenia (COMT, DAOA, DISC1, DRD2, DRD3, DTNBP1, HTR2A, NRG1, SLC6A3, SLC6A4, Table S1)1. Although these genes have not received support from far larger and comprehensive subsequent studies, and may not contain etiological common variation2, it is possible that they contain uncommon variation of etiological importance. To test this hypothesis, we conducted a multistage resequencing study. In Stage 1, we used Sanger methods to sequence the exons, 5′ and 3′ UTRs, splice sites, promoters and conserved intronic regions of these 10 genes in 727 cases with schizophrenia from CATIE3 and 733 controls of European (EUR) and African (AFR) ancestry. In Stage 2, we validated single nucleotide variants (SNVs) using Roche 454 sequencing in the same samples. In Stage 3, we genotyped prioritized SNVs in independent samples (Supplemental Material and Figure S1). In Stage 1, Sanger sequencing identified 782 variants, including 587 novel SNVs not found in dbSNP132 (Tables S2–S4). As expected, the number of novel variants discovered per individual was higher in those of AFR (1.46) than EUR ancestry (0.95) but cases and controls did not differ (EUR cases/controls: 0.920/0.980; AFR cases/controls: 1.492/1.430). The numbers of SNVs per gene were also similar although DISC1 showed a non-significant excess in cases (EUR cases/controls: 0.138/0.119; AFR cases/controls: 0.243/0.167) mostly due to novel variants in AFR subjects (cases/controls: 0.173/0.099). Three unrelated cases, but zero controls, were found to each have a single novel nonsense mutation: two for DISC1 (truncating only the “Es” splice variants) and one for SLC6A4 (Tables S2–S4). The ratio of nonsynonymous to synonomous variants was similar in cases and controls (1.25 vs 1.16). In Stage 2, we prioritized 254 of the 782 variants for technical replication since they met at least one of the following criteria: 1) novel nonsense, missense or splice site variant, 2) novel intronic variant in ≥1 EUR case, 3) novel variant with an odds ratio >2 in the EUR cohort, 4) dbSNP nonsense, missense or splice site variant in ≥1 EUR case. Validation sequencing by Roche 454 revealed 225 true variants and 29 false positives (accuracy rate of 89%, Figure S2). In Stage 3, we selected 92 of the 225 SNVs (Table S5) from Stage 2 for genotyping in an independent sample of 2,191 cases and 2,659 controls (EUR and AFR). We included: 1) all novel nonsense, missense or splice site variants seen in ≥1 case, 2) all variants seen in >1 case, 3) three nonsense variants observed in one case each. After genotyping 92 SNVs in the replication samples, 29 were monomorphic (22 of these were seen in only one case in Stage 1), six had low quality genotypes, and 57 SNVs were tested for association with schizophrenia (logistic regression, separately for EUR and AFR subjects). Table 1 lists the SNVs with the smallest p-value in each gene (complete results in Table S6). No gene contained a SNV reaching criteria for genome-wide significance (p < 5 × 10−8). We then tested the aggregate effects of uncommon variants within a gene for 35 non-intronic SNVs with MAF <0.01 (Table 1). No gene was significant following correction for multiple testing. For example, of the 20 uncommon variants in DISC1 (Figure S3), there were 32 minor alleles in EUR cases and 40 in EUR controls. Table 1 Summary of Stage 3 replication genotyping results. Thus, multistage resequencing of ten schizophrenia candidate genes did not yield support for uncommon exonic variation. This result is consistent with common variation results that do not, to date, provide support for these genes despite a sample size of 21,856 individuals. The replication sample had 80% power to detect a genotypic relative risk of 3.2 in the EUR cohort and 5.1 in the AFR cohort, with MAF of 0.01 and significance level of P = 5×10−8. For a relaxed threshold P = 0.001, there would have been >99% power to detect genotypic relative risk of 2.9 in the EUR cohort and 4.6 in the AFR cohort. The Stage 1 and 2 results hinted that DISC1 might contain an excess of uncommon variants, and DISC1 was thus the main focus in Stage 3 replication. However, there was no evidence in our results to support the hypothesis that DISC1 contains uncommon variants of relevance to schizophrenia as no single-SNV or aggregate test approach was even of nominal significance. Although DISC1 nonsense variants were present in two Stage 1 cases and 0 controls, no additional cases or controls in the larger Stage 3 sample had those particular nonsense mutations. DISC1 has been the focus of dozens of genetic studies4. However, a recent comprehensive meta-analysis of common variation did not support its role in schizophrenia susceptibility5. To our knowledge, four groups have sequenced DISC1 in cases with schizophrenia6–9, and all had discovery samples far smaller than reported here (34, 90, 198 and 288 cases). Of the association results in these studies, none met criteria for genome-wide significance. Only one study had a replication component, and the initial finding did not replicate9. Therefore, despite employing a replication sample three times the size of the discovery sample, DISC1 was not found to contain common or uncommon variants individually or in aggregate associated with schizophrenia. The results of this study suggest that classical schizophrenia candidate genes do not harbor uncommon coding region variation of etiological importance.

Candidate risk factors and mechanisms for tolvaptan-induced liver injury are identified using a collaborative cross approach

Clinical trials of tolvaptan showed it to be a promising candidate for the treatment of Autosomal Dominant Polycystic Kidney Disease (ADPKD) but also revealed potential for idiosyncratic drug-induced liver injury (DILI) in this patient population. To identify risk factors and mechanisms underlying tolvaptan DILI, 8 mice in each of 45 strains of the genetically diverse Collaborative Cross (CC) mouse population were treated with a single oral dose of either tolvaptan or vehicle. Significant elevations in plasma alanine aminotransferase (ALT) were observed in tolvaptan-treated animals in 3 of the 45 strains. Genetic mapping coupled with transcriptomic analysis in the liver was used to identify several candidate susceptibility genes including epoxide hydrolase 2, interferon regulatory factor 3, and mitochondrial fission factor. Gene pathway analysis revealed that oxidative stress and immune response pathways were activated in response to tolvaptan treatment across all strains, but genes involved in regulation of bile acid homeostasis were most associated with tolvaptan-induced elevations in ALT. Secretory leukocyte peptidase inhibitor (Slpi) mRNA was also induced in the susceptible strains and was associated with increased plasma levels of Slpi protein, suggesting a potential serum marker for DILI susceptibility. In summary, tolvaptan induced signs of oxidative stress, mitochondrial dysfunction, and innate immune response in all strains, but variation in bile acid homeostasis was most associated with susceptibility to the liver response. This CC study has indicated potential mechanisms underlying tolvaptan DILI and biomarkers of susceptibility that may be useful in managing the risk of DILI in ADPKD patients.

Genetics of Adverse Reactions to Haloperidol in a Mouse Diallel: A Drug–Placebo Experiment and Bayesian Causal Analysis

Haloperidol is an efficacious antipsychotic drug that has serious, unpredictable motor side effects that limit its utility and cause noncompliance in many patients. Using a drug–placebo diallel of the eight founder strains of the Collaborative Cross and their F1 hybrids, we characterized aggregate effects of genetics, sex, parent of origin, and their combinations on haloperidol response. Treating matched pairs of both sexes with drug or placebo, we measured changes in the following: open field activity, inclined screen rigidity, orofacial movements, prepulse inhibition of the acoustic startle response, plasma and brain drug level measurements, and body weight. To understand the genetic architecture of haloperidol response we introduce new statistical methodology linking heritable variation with causal effect of drug treatment. Our new estimators, “difference of models” and “multiple-impute matched pairs”, are motivated by the Neyman–Rubin potential outcomes framework and extend our existing Bayesian hierarchical model for the diallel (Lenarcic et al. 2012). Drug-induced rigidity after chronic treatment was affected by mainly additive genetics and parent-of-origin effects (accounting for 28% and 14.8% of the variance), with NZO/HILtJ and 129S1/SvlmJ contributions tending to increase this side effect. Locomotor activity after acute treatment, by contrast, was more affected by strain-specific inbreeding (12.8%). In addition to drug response phenotypes, we examined diallel effects on behavior before treatment and found not only effects of additive genetics (10.2–53.2%) but also strong effects of epistasis (10.64–25.2%). In particular: prepulse inhibition showed additivity and epistasis in about equal proportions (26.1% and 23.7%); there was evidence of nonreciprocal epistasis in pretreatment activity and rigidity; and we estimated a range of effects on body weight that replicate those found in our previous work. Our results provide the first quantitative description of the genetic architecture of haloperidol response in mice and indicate that additive, dominance-like inbreeding and parent-of-origin effects contribute strongly to treatment effect heterogeneity for this drug.

Temporal variability of glucocorticoid receptor activity is functionally important for the therapeutic action of fluoxetine in the hippocampus

Previous studies have shown inconsistent results regarding the actions of antidepressants on glucocorticoid receptor (GR) signalling. To resolve these inconsistencies, we used a lentiviral-based reporter system to directly monitor rat hippocampal GR activity during stress adaptation. Temporal GR activation was induced significantly by acute stress, as demonstrated by an increase in the intra-individual variability of the acute stress group compared with the variability of the non-stress group. However, the increased intra-individual variability was dampened by exposure to chronic stress, which was partly restored by fluoxetine treatment without affecting glucocorticoid secretion. Immobility in the forced-swim test was negatively correlated with the intra-individual variability, but was not correlated with the quantitative GR activity during fluoxetine therapy; this highlights the temporal variability in the neurobiological links between GR signalling and the therapeutic action of fluoxetine. Furthermore, we demonstrated sequential phosphorylation between GR (S224) and (S232) following fluoxetine treatment, showing a molecular basis for hormone-independent nuclear translocation and transcriptional enhancement. Collectively, these results suggest a neurobiological mechanism by which fluoxetine treatment confers resilience to the chronic stress-mediated attenuation of hypothalamic–pituitary–adrenal axis activity.

Assessment of gene expression in peripheral blood using RNAseq before and after weight restoration in anorexia nervosa

We examined gene expression in the blood of six females with anorexia nervosa (AN) before and after weight restoration using RNAseq. AN cases (aged 19-39) completed clinical assessments and had blood drawn for RNA at hospital admission (T1,<~75% ideal body weight, IBW) and again at discharge (T2,≥ ~ 85% IBW). To examine the relationship between weight restoration and differential gene expression, normalized gene expression levels were analyzed using a paired design. We found 564 genes whose expression was nominally significantly different following weight restoration (p<0.01, 231 increased and 333 decreased). With a more stringent significance threshold (false discovery rate q<0.05), 67 genes met criteria for differential expression. Of the top 20 genes, CYP11A1, C16orf11, LINC00235, and CPA3 were down-regulated more than two-fold after weight restoration while multiple olfactory receptor genes (OR52J3, OR51L1, OR51A4, and OR51A2) were up-regulated more than two-fold after weight restoration. Pathway analysis revealed up-regulation of two broad pathways with largely overlapping genes, one related to protein secretion and signaling and the other associated with defense response to bacterial regulation. Although results are preliminary secondary to a small sample size, these data provide initial evidence of transcriptional alterations during weight restoration in AN.