Corey R. Quackenbush

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.

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.

Antipsychotic-induced vacuous chewing movements and extrapyramidal side effects are highly heritable in mice

Pharmacogenomics is yet to fulfill its promise of manifestly altering clinical medicine. As one example, a predictive test for tardive dyskinesia (TD) (an adverse drug reaction consequent to antipsychotic exposure) could greatly improve the clinical treatment of schizophrenia but human studies are equivocal. A complementary approach is the mouse-then-human design in which a valid mouse model is used to identify susceptibility loci, which are subsequently tested in human samples. We used inbred mouse strains from the Mouse Phenome Project to estimate the heritability of haloperidol-induced activity and orofacial phenotypes. In all, 159 mice from 27 inbred strains were chronically treated with haloperidol (3 mg kg−1 per day via subdermal slow-release pellets) and monitored for the development of vacuous chewing movements (VCMs; the mouse analog of TD) and other movement phenotypes derived from open-field activity and the inclined screen test. The test battery was assessed at 0, 30, 60, 90 and 120 days in relation to haloperidol exposure. As expected, haloperidol caused marked changes in VCMs, activity in the open field and extrapyramidal symptoms (EPS). Unexpectedly, factor analysis demonstrated that these measures were imprecise assessments of a latent construct rather than discrete constructs. The heritability of a composite phenotype was ∼0.9 after incorporation of the longitudinal nature of the design. Murine VCMs are a face valid animal model of antipsychotic-induced TD, and heritability estimates from this study support the feasibility of mapping of susceptibility loci for VCMs.

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.

No association of the serotonin transporter polymorphisms 5-HTTLPR and RS25531 with schizophrenia or neurocognition.

A promoter polymorphism in the serotonin transporter gene has been widely studied in neuropsychiatry. We genotyped the 5‐HTTLPR/rs25531 triallelic polymorphism in 728 schizophrenia cases from the CATIE study and 724 control subjects. In a logistic regression with case/control status as dependent variable and 7 ancestry‐informative principal components as covariates, the effect of 5‐HTTLPR/rs25531 composite genotype was not significant (odds ratio = 1.008, 95% CI 0.868–1.172, P = 0.91). In cases only, 5‐HTTLPR/rs25531 was not associated with neurocognition (summary neurocognitive index P = 0.21, working memory P = 0.32) or symptomatology (PANSS positive P = 0.67 and negative symptoms P = 0.46). We were unable to identify association of the triallelic 5‐HTTLPR with schizophrenia, neurocognition, or core psychotic symptoms even at levels of significance unadjusted for multiple comparisons.

Identifying bipolar disorder susceptibility loci in a densely affected pedigree

Bipolar disorder (BIP) is a highly heritable disorder with complex patterns of genetic inheritance, and recent genetic findings highlight the role of numerous common variants each with subtle effects.1 The existence of Mendelian subtypes of BIP (rare variants of very strong effects) has been postulated, particularly as such variants could prove to be more tractable for subsequent biological investigation.2 One way to evaluate this hypothesis is via the study of pedigrees densely affected with BIP in which a genetic variant of strong effect inherited from a common ancestor may be more likely. With the advent of high-throughput technologies, we can now search densely-affected pedigrees for specific variants that may contribute to risk for BIP. We therefore evaluated a Spanish multi-generational pedigree with an exceptional prevalence of BIP using multiple complementary genomic techniques (Table S1). This pedigree contains 18 cases with BIP (including a sibship with six of 11 affected) and seven individuals with recurrent major depressive disorder (Figure 1a). The lifetime prevalence of mood disorders in this large pedigree (6 generations; 120 individuals, 30 with known mood disorders, 42 with DNA) makes it a strong candidate for identifying genetic risk factors of near-Mendelian effects. Our search strategy is depicted in Figure 1b. All protocols were IRB approved and all subjects provided written informed consent. Figure 1 (a) The pedigree was ascertained in Spain, and has a high prevalence of mood disorders, particularly bipolar disorder (type 1) and recurrent major depressive disorder. First, we used genome-wide linkage analysis and an affecteds-only approach to identify genomic regions sharing identity-by-descent. Linkage analysis was performed using microsatellite data from 13 individuals.3 We used data from Illumina HumanOmni Quad genotyping arrays to identify shared segments using Beagle,4 Germline,5 PedIBD,6 and runs of identity-by-state.7 Regions identified by linkage or with two sharing methods were considered candidate regions (Table S2, six regions totaling 93.7Mb). Second, we hypothesized that one of these regions contained a novel, rare functional single nucleotide variant (SNV) of high penetrance. We attempted to identify novel SNVs in these six genomic regions shared by BIP cases using exome sequencing (five BIP cases) and whole genome sequencing (three BIP cases). Whole genome sequence data was screened to identify high-quality homozygous or heterozygous SNVs that were within the candidate regions, novel, of predicted functional consequence, and present in all three BIP cases. This procedure identified 26 SNVs in three olfactory receptors (OR4C3, OR9G9, and OR4C45). Because multiple variants were present in each gene, given the existence of other highly similar members of the large olfactory gene family, and manual review of alignment patterns, we believed all to be due to misalignment. We repeated this process for the exome sequencing data, and identified 21 SNVs (17 were also identified by whole genome sequencing), and all were in olfactory genes (OR4C3, OR9G9, and OR8U8/OR8U1), and likely due to incorrect alignment. We next evaluated CNVs in these regions. Using PennCNV8 calls from Illumina arrays, no novel CNVs were identified in these candidate regions that were >1kb, present in ≥8 of 11 BIP cases, and confirmed via whole genome sequencing. Therefore, we were unable to identify any potential SNVs or CNVs within these candidate regions that were promising for follow-up. Third, we then extended our analysis genome-wide. From the whole genome sequencing of three individuals, we identified novel SNVs of predicted functionality that were present in all three BIP cases (Table S3). We excluded SNVs in olfactory receptors, with questionable alignments, or that were not confirmed in exome sequencing of five BIP cases. This resulted in eight SNVs that we sequenced in 11 affected individuals using Sanger sequencing. Five SNVs verified and had plausible inheritance patterns, and were then genotyped in 42 individuals in the pedigree. Two SNVs were potentially interesting (chr12:52452495 C>T in NR4A1 and chr18:47793974 G>C in MBD1). Neither exhibited unequivocal Mendelian inheritance (Figures S2–S3). The NR4A1 (nerve growth factor IB, NGFIB or Nur77) SNV is novel but common in this pedigree (homozygous or heterozygous in 30/34 individuals descended from the founders, excluding married-in individuals) and did not clearly segregate in BIP cases as the SNV was found in 92% of BIP cases and 86% of subjects without a mood disorder (although the number of unaffecteds is small). The MBD1 (methyl-CpG-binding domain protein 1) SNV tracked with BIP (66%), other mood disorders (88%), and less so in unaffecteds (21%). However, this SNV was recently identified by the 1000 Genomes Project in subjects from the United Kingdom. Therefore, this variant is not completely novel, but has an interesting pattern of segregation. Both variants warrant additional follow-up in a larger case-control samples although neither appears to be a strong novel Mendelian variant. Genome-wide expansion of the CNV search identified no novel CNVs that were present in ≥8 BIP cases, >1kb in size, overlapped an exon, and verified by whole genome sequencing. Fourth, given the absence of compelling results in support of a near-Mendelian variant, we evaluated the contribution of common variation in this pedigree. The presence of many common variant risk alleles in a pedigree is a potential explanation for a dense pedigree.9 This can be due to or exacerbated by assortative mating (in which mental illness in the family of a spouse is more likely to be tolerated if one has it in one’s own family) and result in an accumulation of common risk alleles. We therefore calculated risk profile scores, which are the weighted number of BIP risk alleles in each subject. Based on results from the Psychiatric GWAS Consortium10 (7,481 BIP cases and 9,250 controls), risk profile scores were computed for the GAIN BIP cases (N=1080) and controls (N=1058),11 and the 11 BIP cases in the densely-affected pedigree (Figure 1c). As anticipated, the GAIN BIP cases had significantly higher risk profile scores than the GAIN controls (p < 0.001). Risk profile scores for 11 BIP cases from the Spanish pedigree were also significantly greater than controls (p < 0.001) but not significantly different from GAIN BIP cases (p = 0.19). It is particularly notable that the BIP pedigree cases did not have markedly lower risk profile scores (e.g., low common variant profiles might be consistent with the present of a strong mutation). Rather, the BIP pedigree cases appeared to have common variant risk profile scores similar to European-American BIP cases ascertained clinically without regard to family history. In conclusion, we systematically assessed this large pedigree dense with BIP for genetic variants of strong effect. This comprehensive analysis did not conclusively identify any SNVs or CNVs of near-Mendelian effect. However, we cannot exclude the presence of risk variants with more complex inheritance patterns, variants with more cryptic functional effects, or variants missed due to coverage or individuals sequenced. However, the common variant risk profiles of BIP cases in this pedigree are similar to those of BIP cases ascertained without regard to family history. Therefore, it is possible that the etiology of BIP in this pedigree is more related to multiple common risk variants rather than one or a few variants of extremely strong effect.

Comparative genomic evidence for the involvement of schizophrenia risk genes in antipsychotic effects

Genome-wide association studies (GWAS) for schizophrenia have identified over 100 loci encoding >500 genes. It is unclear whether any of these genes, other than dopamine receptor D2, are immediately relevant to antipsychotic effects or represent novel antipsychotic targets. We applied an in vivo molecular approach to this question by performing RNA sequencing of brain tissue from mice chronically treated with the antipsychotic haloperidol or vehicle. We observed significant enrichments of haloperidol-regulated genes in schizophrenia GWAS loci and in schizophrenia-associated biological pathways. Our findings provide empirical support for overlap between genetic variation underlying the pathophysiology of schizophrenia and the molecular effects of a prototypical antipsychotic.

Erratum: Analyses of allele-specific gene expression in highly divergent mouse crosses identifies pervasive allelic imbalance (Nature Genetics (2015) 47 (353-360))

Nat. Genet. 47, 353–360 (2015); published online 2 March 2015; corrected after print 16 April 2015 In the version of this article initially published, an accession number was not provided for RNA-seq data sets. The RNA-seq data sets that passed quality control are available at the Sequence Read Archive (SRA) under accession SRP056236.