■双相情感障碍(BD)是一种慢性精神疾病,其特征是躁狂症和抑郁症的反复发作,并与社交和认知障碍有关。环境因素,比如母亲吸烟和童年创伤,被认为可以调节风险基因型,并有助于BD的发病机理,提示在神经发育过程中的表观遗传调控中的关键作用。5-羟甲基胞嘧啶(5hmC)是一种特别感兴趣的表观遗传变异体,因为它在大脑中高度表达,并与神经发育有关,以及精神和神经系统疾病。
■从两名患有双相情感障碍的青少年患者及其同性别年龄匹配的未受影响的兄弟姐妹(n=4)的白细胞中产生了诱导多能干细胞(iPSC)。Further,将iPSC分化成神经元干细胞(NSC)并使用免疫荧光表征纯度。我们使用减少代表性羟甲基化分析(RRHP)来进行iPSC和NSC的全基因组5hmC分析,模拟神经元分化过程中5hmC的变化,并评估其对BD风险的影响。用在线工具DAVID进行具有分化的5hmC基因座的基因的功能注释和富集测试。
■大约200万个站点被绘制和量化,大多数(68.8%)位于基因区域,每个站点观察到3个UTR的5hmC水平升高,外显子,和CpG群岛的2kb海岸线。iPSC和NSC细胞系之间的归一化5hmC计数的配对t检验显示,在NSC中整体低羟甲基化,以及与质膜相关的基因中差异羟甲基化位点的富集(FDR=9.1×10-12)和轴突导向(FDR=2.1×10-6)。在其他神经元过程中。对于KCNK9基因的转录因子结合位点观察到最显著的差异(p=8.8×10-6),编码参与神经元活动和迁移的钾通道蛋白。蛋白质-蛋白质相互作用(PPI)网络显示,由具有高度分化的5hmC位点的基因编码的蛋白质之间存在显着的连通性(p=3.2×10-10)。与轴突导向和离子跨膜转运相关的基因形成不同的亚簇。BD病例和未受影响的兄弟姐妹的NSC比较揭示了羟甲基化水平的其他分化模式,包括与突触形成和调节相关的基因中的位点,如CUX2(p=2.4×10-5)和DOK-7(p=3.6×10-3),以及与细胞外基质有关的基因的富集(FDR=1.0×10-8)。
■一起,这些初步结果为5hmC在早期神经元分化和BD风险中的潜在作用提供了证据,通过后续研究可以实现验证和更全面的表征。
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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\' 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).
UNASSIGNED: 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.