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 contribut...
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Frontiers Media S.A.
2023-04-01
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| Series: | Frontiers in Psychiatry |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fpsyt.2023.1077415/full |
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| author | Ashish Kumar Ashish Kumar Mark Z. Kos Donna Roybal Donna Roybal Melanie A. Carless Melanie A. Carless Melanie A. Carless |
| author_facet | Ashish Kumar Ashish Kumar Mark Z. Kos Donna Roybal Donna Roybal Melanie A. Carless Melanie A. Carless Melanie A. Carless |
| author_sort | Ashish Kumar |
| collection | DOAJ |
| description | 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. |
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| spelling | doaj.art-dd40fece4cae47c2b57f620b01aea5b52023-04-17T14:13:40ZengFrontiers Media S.A.Frontiers in Psychiatry1664-06402023-04-011410.3389/fpsyt.2023.10774151077415A pilot investigation of differential hydroxymethylation levels in patient-derived neural stem cells implicates altered cortical development in bipolar disorderAshish Kumar0Ashish Kumar1Mark Z. Kos2Donna Roybal3Donna Roybal4Melanie A. Carless5Melanie A. Carless6Melanie A. Carless7Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, NC, United StatesPopulation Health Program, Texas Biomedical Research Institute, San Antonio, TX, United StatesSouth Texas Diabetes and Obesity Institute, Department of Human Genetics, The University of Texas Rio Grande Valley School of Medicine, San Antonio, TX, United StatesTraditions Behavioral Health, Larkspur, CA, United StatesDepartment of Neuroscience, Developmental and Regenerative Biology, The University of Texas at San Antonio, San Antonio, TX, United StatesPopulation Health Program, Texas Biomedical Research Institute, San Antonio, TX, United StatesDepartment of Neuroscience, Developmental and Regenerative Biology, The University of Texas at San Antonio, San Antonio, TX, United StatesBrain Health Consortium, The University of Texas at San Antonio, San Antonio, TX, United StatesIntroductionBipolar 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.https://www.frontiersin.org/articles/10.3389/fpsyt.2023.1077415/fullhydroxymethylationbipolar disorderneuronal differentiationRRHPreduced representation 5-hydroxymethylcytosine profilingneural stem cell (NSC) |
| spellingShingle | Ashish Kumar Ashish Kumar Mark Z. Kos Donna Roybal Donna Roybal Melanie A. Carless Melanie A. Carless Melanie A. Carless A pilot investigation of differential hydroxymethylation levels in patient-derived neural stem cells implicates altered cortical development in bipolar disorder Frontiers in Psychiatry hydroxymethylation bipolar disorder neuronal differentiation RRHP reduced representation 5-hydroxymethylcytosine profiling neural stem cell (NSC) |
| title | A pilot investigation of differential hydroxymethylation levels in patient-derived neural stem cells implicates altered cortical development in bipolar disorder |
| title_full | A pilot investigation of differential hydroxymethylation levels in patient-derived neural stem cells implicates altered cortical development in bipolar disorder |
| title_fullStr | A pilot investigation of differential hydroxymethylation levels in patient-derived neural stem cells implicates altered cortical development in bipolar disorder |
| title_full_unstemmed | A pilot investigation of differential hydroxymethylation levels in patient-derived neural stem cells implicates altered cortical development in bipolar disorder |
| title_short | A pilot investigation of differential hydroxymethylation levels in patient-derived neural stem cells implicates altered cortical development in bipolar disorder |
| title_sort | pilot investigation of differential hydroxymethylation levels in patient derived neural stem cells implicates altered cortical development in bipolar disorder |
| topic | hydroxymethylation bipolar disorder neuronal differentiation RRHP reduced representation 5-hydroxymethylcytosine profiling neural stem cell (NSC) |
| url | https://www.frontiersin.org/articles/10.3389/fpsyt.2023.1077415/full |
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