A pilot investigation of differential hydroxymethylation levels in patient-derived neural stem cells implicates altered cortical development in bipolar disorder

Introduction: Bipolar disorder (BD) is a chronic mental illness characterized by recurrent episodes of mania and depression and associated with social and cognitive disturbances. Environmental factors, such as maternal smoking and childhood trauma, are believed to modulate risk genotypes and contribute to the pathogenesis of BD, suggesting a key role in epigenetic regulation during neurodevelopment. 5-hydroxymethylcytosine (5hmC) is an epigenetic variant of particular interest, as it is highly expressed in the brain and is implicated in neurodevelopment, and psychiatric and neurological disorders. Methods: Induced pluripotent stem cells (iPSCs) were generated from the white blood cells of two adolescent patients with bipolar disorder and their same-sex age-matched unaffected siblings (n = 4). Further, iPSCs were differentiated into neuronal stem cells (NSCs) and characterized for purity using immuno-fluorescence. We used reduced representation hydroxymethylation profiling (RRHP) to perform genome-wide 5hmC profiling of iPSCs and NSCs, to model 5hmC changes during neuronal differentiation and assess their impact on BD risk. Functional annotation and enrichment testing of genes harboring differentiated 5hmC loci were performed with the online tool DAVID. Results: Approximately 2 million sites were mapped and quantified, with the majority (68.8%) located in genic regions, with elevated 5hmC levels per site observed for 3\u27 UTRs, exons, and 2-kb shorelines of CpG islands. Paired t-tests of normalized 5hmC counts between iPSC and NSC cell lines revealed global hypo-hydroxymethylation in NSCs and enrichment of differentially hydroxymethylated sites within genes associated with plasma membrane (FDR = 9.1 × 10-12) and axon guidance (FDR = 2.1 × 10-6), among other neuronal processes. The most significant difference was observed for a transcription factor binding site for the KCNK9 gene (p = 8.8 × 10-6), encoding a potassium channel protein involved in neuronal activity and migration. Protein-protein-interaction (PPI) networking showed significant connectivity (p = 3.2 × 10-10) between proteins encoded by genes harboring highly differentiated 5hmC sites, with genes involved in axon guidance and ion transmembrane transport forming distinct sub-clusters. Comparison of NSCs of BD cases and unaffected siblings revealed additional patterns of differentiation in hydroxymethylation levels, including sites in genes with functions related to synapse formation and regulation, such as CUX2 (p = 2.4 × 10-5) and DOK-7 (p = 3.6 × 10-3), as well as an enrichment of genes involved in the extracellular matrix (FDR = 1.0 × 10-8). Discussion: Together, these preliminary results lend evidence toward a potential role for 5hmC in both early neuronal differentiation and BD risk, with validation and more comprehensive characterization to be achieved through follow-up study

Dopamine perturbation of gene co-expression networks reveals differential response in schizophrenia for translational machinery.

The dopaminergic hypothesis of schizophrenia (SZ) postulates that positive symptoms of SZ, in particular psychosis, are due to disturbed neurotransmission via the dopamine (DA) receptor D2 (DRD2). However, DA is a reactive molecule that yields various oxidative species, and thus has important non-receptor-mediated effects, with empirical evidence of cellular toxicity and neurodegeneration. Here we examine non-receptor-mediated effects of DA on gene co-expression networks and its potential role in SZ pathology. Transcriptomic profiles were measured by RNA-seq in B-cell transformed lymphoblastoid cell lines from 514 SZ cases and 690 controls, both before and after exposure to DA ex vivo (100 μM). Gene co-expression modules were identified using Weighted Gene Co-expression Network Analysis for both baseline and DA-stimulated conditions, with each module characterized for biological function and tested for association with SZ status and SNPs from a genome-wide panel. We identified seven co-expression modules under baseline, of which six were preserved in DA-stimulated data. One module shows significantly increased association with SZ after DA perturbation (baseline: P = 0.023; DA-stimulated: P = 7.8 × 10-5; ΔAIC = -10.5) and is highly enriched for genes related to ribosomal proteins and translation (FDR = 4 × 10-141), mitochondrial oxidative phosphorylation, and neurodegeneration. SNP association testing revealed tentative QTLs underlying module co-expression, notably at FASTKD2 (top P = 2.8 × 10-6), a gene involved in mitochondrial translation. These results substantiate the role of translational machinery in SZ pathogenesis, providing insights into a possible dopaminergic mechanism disrupting mitochondrial function, and demonstrates the utility of disease-relevant functional perturbation in the study of complex genetic etiologies

Whole genome sequence data implicate RBFOX1 in epilepsy risk in baboons

Background: Baboons exhibit a genetic generalized epilepsy (GGE) that resembles juvenile myoclonic epilepsy and may represent a suitable genetic model for human epilepsy. The genetic underpinnings of epilepsy were investigated in a baboon colony at the Southwest National Primate Research Center (San Antonio, TX) through the analysis of whole-genome sequence (WGS) data. Methods: Baboon WGS data were obtained for 38 cases and 19 healthy controls from the NCBI Sequence Read Archive and, after standard QC filtering, two subsets of variants were examined: (1) 20,881 SNPs from baboon homologs of 19 candidate GGE genes; and (2) 36,169 protein-altering SNPs. Association tests were conducted in SOLAR, and gene set enrichment analyses (GSEA) and protein-protein interaction (PPI) network construction were performed on genome-wide significant association results (Pn= 441 genes). Results: Heritability for epileptic seizure in the pedigreed baboon sample was estimated at 0.76 (SE=0.77; P=0.07). A significant association was detected for an intronic SNP in RBFOX1 (P=5.92 × 10-6; adjusted P=0.016). For protein-altering variants, GSEA revealed significant positive enrichment for genes involved in the extracellular matrix structure (ECM; FDR=0.0072) and collagen formation (FDR=0.017). Conclusions: SNP association results implicate RBFOX1 in baboon epilepsy, a gene that plays a key role in neuronal excitation and transcriptomic regulation, and has been previously linked to human epilepsy, both focal and generalized. Moreover, protein-damaging variants from across the baboon genome exhibit a wider pattern of association that links collagen-containing ECM to epilepsy risk. These findings suggest a shared genetic etiology between baboon and human forms of GGE

Dopamine perturbation of gene co-expression networks reveals differential response in schizophrenia for translational machinery

The dopaminergic hypothesis of schizophrenia (SZ) postulates that positive symptoms of SZ, in particular psychosis, are due to disturbed neurotransmission via the dopamine (DA) receptor D2 (DRD2). However, DA is a reactive molecule that yields various oxidative species, and thus has important non-receptor-mediated effects, with empirical evidence of cellular toxicity and neurodegeneration. Here we examine non-receptor-mediated effects of DA on gene co-expression networks and its potential role in SZ pathology. Transcriptomic profiles were measured by RNA-seq in B-cell transformed lymphoblastoid cell lines from 514 SZ cases and 690 controls, both before and after exposure to DA ex vivo (100 μM). Gene co-expression modules were identified using Weighted Gene Co-expression Network Analysis for both baseline and DA-stimulated conditions, with each module characterized for biological function and tested for association with SZ status and SNPs from a genome-wide panel. We identified seven co-expression modules under baseline, of which six were preserved in DA-stimulated data. One module shows significantly increased association with SZ after DA perturbation (baseline: P = 0.023; DA-stimulated: P = 7.8 × 10-5; ΔAIC = −10.5) and is highly enriched for genes related to ribosomal proteins and translation (FDR = 4 × 10−141), mitochondrial oxidative phosphorylation, and neurodegeneration. SNP association testing revealed tentative QTLs underlying module co-expression, notably at FASTKD2 (top P = 2.8 × 10−6), a gene involved in mitochondrial translation. These results substantiate the role of translational machinery in SZ pathogenesis, providing insights into a possible dopaminergic mechanism disrupting mitochondrial function, and demonstrates the utility of disease-relevant functional perturbation in the study of complex genetic etiologies

Whole Genome Sequence Data From Captive Baboons Implicate RBFOX1 in Epileptic Seizure Risk

In this study, we investigate the genetic determinants that underlie epilepsy in a captive baboon pedigree and evaluate the potential suitability of this non-human primate model for understanding the genetic etiology of human epilepsy. Archived whole-genome sequence data were analyzed using both a candidate gene approach that targeted variants in baboon homologs of 19 genes (n = 20,881 SNPs) previously implicated in genetic generalized epilepsy (GGE) and a more agnostic approach that examined protein-altering mutations genome-wide as assessed by snpEff (n = 36,169). Measured genotype association tests for baboon cases of epileptic seizure were performed using SOLAR, as well as gene set enrichment analyses (GSEA) and protein–protein interaction (PPI) network construction of top association hits genome-wide (p \u3c 0.01; n = 441 genes). The maximum likelihood estimate of heritability for epileptic seizure in the pedigreed baboon sample is 0.76 (SE = 0.77; p = 0.07). Among candidate genes for GGE, a significant association was detected for an intronic SNP in RBFOX1 (p = 5.92 × 10–6; adjusted p = 0.016). For protein-altering variants, no genome-wide significant results were observed for epilepsy status. However, GSEA revealed significant positive enrichment for genes involved in the extracellular matrix structure (ECM; FDR = 0.0072) and collagen formation (FDR = 0.017), which was reflected in a major PPI network cluster. This preliminary study highlights the potential role of RBFOX1 in the epileptic baboon, a protein involved in transcriptomic regulation of multiple epilepsy candidate genes in humans and itself previously implicated in human epilepsy, both focal and generalized. Moreover, protein-damaging variants from across the genome exhibit a pattern of association that links collagen-containing ECM to epilepsy risk. These findings suggest a shared genetic etiology between baboon and human forms of GGE and lay the foundation for follow-up research

A pilot investigation of differential hydroxymethylation levels in patient-derived neural stem cells implicates altered cortical development in bipolar disorder

IntroductionBipolar disorder (BD) is a chronic mental illness characterized by recurrent episodes of mania and depression and associated with social and cognitive disturbances. Environmental factors, such as maternal smoking and childhood trauma, are believed to modulate risk genotypes and contribute to the pathogenesis of BD, suggesting a key role in epigenetic regulation during neurodevelopment. 5-hydroxymethylcytosine (5hmC) is an epigenetic variant of particular interest, as it is highly expressed in the brain and is implicated in neurodevelopment, and psychiatric and neurological disorders.MethodsInduced pluripotent stem cells (iPSCs) were generated from the white blood cells of two adolescent patients with bipolar disorder and their same-sex age-matched unaffected siblings (n = 4). Further, iPSCs were differentiated into neuronal stem cells (NSCs) and characterized for purity using immuno-fluorescence. We used reduced representation hydroxymethylation profiling (RRHP) to perform genome-wide 5hmC profiling of iPSCs and NSCs, to model 5hmC changes during neuronal differentiation and assess their impact on BD risk. Functional annotation and enrichment testing of genes harboring differentiated 5hmC loci were performed with the online tool DAVID.ResultsApproximately 2 million sites were mapped and quantified, with the majority (68.8%) located in genic regions, with elevated 5hmC levels per site observed for 3’ UTRs, exons, and 2-kb shorelines of CpG islands. Paired t-tests of normalized 5hmC counts between iPSC and NSC cell lines revealed global hypo-hydroxymethylation in NSCs and enrichment of differentially hydroxymethylated sites within genes associated with plasma membrane (FDR = 9.1 × 10−12) and axon guidance (FDR = 2.1 × 10−6), among other neuronal processes. The most significant difference was observed for a transcription factor binding site for the KCNK9 gene (p = 8.8 × 10−6), encoding a potassium channel protein involved in neuronal activity and migration. Protein–protein-interaction (PPI) networking showed significant connectivity (p = 3.2 × 10−10) between proteins encoded by genes harboring highly differentiated 5hmC sites, with genes involved in axon guidance and ion transmembrane transport forming distinct sub-clusters. Comparison of NSCs of BD cases and unaffected siblings revealed additional patterns of differentiation in hydroxymethylation levels, including sites in genes with functions related to synapse formation and regulation, such as CUX2 (p = 2.4 × 10−5) and DOK-7 (p = 3.6 × 10−3), as well as an enrichment of genes involved in the extracellular matrix (FDR = 1.0 × 10−8).DiscussionTogether, these preliminary results lend evidence toward a potential role for 5hmC in both early neuronal differentiation and BD risk, with validation and more comprehensive characterization to be achieved through follow-up study

Using genetic information from candidate gene and genome-wide association studies in risk prediction for alcohol dependence

Family-based and genome-wide association studies (GWAS) of alcohol dependence (AD) have reported numerous associated variants. The clinical validity of these variants for predicting AD compared with family history information has not been reported. Using the Collaborative Study on the Genetics of Alcoholism (COGA) and the Study of Addiction: Genes and Environment (SAGE) GWAS samples, we examined the aggregate impact of multiple single nucleotide polymorphisms (SNPs) on risk prediction. We created genetic sum scores by adding risk alleles associated in discovery samples, and then tested the scores for their ability to discriminate between cases and controls in validation samples. Genetic sum scores were assessed separately for SNPs associated with AD in candidate gene studies and SNPs from GWAS analyses that met varying P-value thresholds. Candidate gene sum scores did not exhibit significant predictive accuracy. Family history was a better classifier of case-control status, with a significant area under the receiver operating characteristic curve (AUC) of 0.686 in COGA and 0.614 in SAGE. SNPs that met less stringent P-value thresholds of 0.01-0.50 in GWAS analyses yielded significant AUC estimates, ranging from mean estimates of 0.549 for SNPs with P < 0.01 to 0.565 for SNPs with P < 0.50. This study suggests that SNPs currently have limited clinical utility, but there is potential for enhanced predictive ability with better understanding of the large number of variants that might contribute to risk

Transforming Growth Factor β Receptor Type 1 Is Essential for Female Reproductive Tract Integrity and Function

The transforming growth factor β (TGFβ) superfamily proteins are principle regulators of numerous biological functions. Although recent studies have gained tremendous insights into this growth factor family in female reproduction, the functions of the receptors in vivo remain poorly defined. TGFβ type 1 receptor (TGFBR1), also known as activin receptor-like kinase 5, is the major type 1 receptor for TGFβ ligands. Tgfbr1 null mice die embryonically, precluding functional characterization of TGFBR1 postnatally. To study TGFBR1–mediated signaling in female reproduction, we generated a mouse model with conditional knockout (cKO) of Tgfbr1 in the female reproductive tract using anti-Müllerian hormone receptor type 2 promoter-driven Cre recombinase. We found that Tgfbr1 cKO females are sterile. However, unlike its role in growth differentiation factor 9 (GDF9) signaling in vitro, TGFBR1 seems to be dispensable for GDF9 signaling in vivo. Strikingly, we discovered that the Tgfbr1 cKO females develop oviductal diverticula, which impair embryo development and transit of embryos to the uterus. Molecular analysis further demonstrated the dysregulation of several cell differentiation and migration genes (e.g., Krt12, Ace2, and MyoR) that are potentially associated with female reproductive tract development. Moreover, defective smooth muscle development was also revealed in the uteri of the Tgfbr1 cKO mice. Thus, TGFBR1 is required for female reproductive tract integrity and function, and disruption of TGFBR1–mediated signaling leads to catastrophic structural and functional consequences in the oviduct and uterus