Zilkha Neurogenetic Institute, Keck School of Medicine of USC, 1501 San Pablo Street, Los Angeles, CA 90089, USA.
J Neurodev Disord. 2013 Jun 11;5(1):15. doi: 10.1186/1866-1955-5-15.
Several proteins involved in epigenetic regulation cause syndromic neurodevelopmental disorders when human genes are mutated. More general involvement of epigenetic mechanisms in neurodevelopmental phenotypes is unclear.
In an attempt to determine whether DNA methylation differentiates clinical subgroups, profiling was performed on bisulfite converted DNA from lymphoblastoid cell lines (LCLs) in discovery (n = 20) and replication (n = 40) cohorts of females with Rett syndrome (RTT; n = 18), autism (AUT; n = 17), seizure disorder (SEZ; n = 6), and controls (CTL; n = 19) using Illumina HumanMethylation27 arrays. TAC1 CpGs were validated using a Sequenom EpiTYPER assay and expression was measured in LCLs and postmortem brain. Chromatin immunoprecipitation was performed in HEK cells. Cells were treated with valproic acid and MeCP2 binding was assessed.
Two female-only cohorts were analyzed. DNA methylation profiling in a discovery cohort identified 40 CpGs that exhibited statistically significant differential methylation (≥15%) between clinical groups (P <0.01). Hierarchical clustering and principal components analysis suggested neurodevelopmental groups were distinct from CTL, but not from each other. In a larger and more heterogeneous replication cohort, these 40 CpG sites suggested no clear difference between clinical groups. Pooled analysis of DNA methylation across all 60 samples suggested only four differentially methylated CpG sites (P <0.0005), including TAC1. TAC1 promoter CpG hypermethylation was validated in AUT and SEZ (P <0.005). Analyzed for the first time in postmortem brain, TAC1 expression was reduced in cingulate cortex in RTT and AUT+SEZ (P = 0.003). However, no significant difference in TAC1 promoter CpG methylation was detected in RTT and AUT+SEZ brains. Additional molecular analyses revealed that MeCP2 binds directly to the TAC1 promoter and is sensitive to antiepileptic drug treatment.
These data suggest that DNA methylation is not widely altered in RTT, consistent with subtle changes in gene expression previously observed. However, TAC1 may be an important target for further functional analyses in RTT. Studies of larger sample cohorts using primary cells that also consider shared clinical features and drug treatments may be required to address apparent subtle disruptions of DNA methylation in neurodevelopmental disorders.
当人类基因发生突变时，几种参与表观遗传调控的蛋白质会导致综合征性神经发育障碍。然而，表观遗传机制在神经发育表型中的更普遍作用尚不清楚。
为了确定 DNA 甲基化是否能区分临床亚组，我们对来自雷特综合征（RTT；n=18）、自闭症（AUT；n=17）、癫痫发作障碍（SEZ；n=6）和对照组（CTL；n=19）女性的淋巴母细胞系（LCL）的 bisulfite 转化 DNA 进行了 Illumina HumanMethylation27 芯片的分析。在发现队列（n=20）和复制队列（n=40）中，使用 Sequenom EpiTYPER 测定法对 TAC1 CpG 进行了验证，并在 LCL 和死后脑中测量了表达。在 HEK 细胞中进行了染色质免疫沉淀实验。用丙戊酸处理细胞，并评估 MeCP2 结合情况。
分析了两个仅女性的队列。在发现队列的 DNA 甲基化分析中，有 40 个 CpG 显示出统计学上显著的差异甲基化（≥15%），存在于临床组之间（P<0.01）。层次聚类和主成分分析表明，神经发育组与 CTL 不同，但彼此之间没有差异。在更大、更具异质性的复制队列中，这些 40 个 CpG 位点提示临床组之间没有明显差异。对所有 60 个样本的 DNA 甲基化进行汇总分析表明，只有 4 个差异甲基化 CpG 位点（P<0.0005），包括 TAC1。TAC1 启动子 CpG 高甲基化在 AUT 和 SEZ 中得到验证（P<0.005）。TAC1 的表达首次在 RTT 和 AUT+SEZ 的扣带回皮层中进行了分析（P=0.003），但在 RTT 和 AUT+SEZ 大脑中未检测到 TAC1 启动子 CpG 甲基化的显著差异。进一步的分子分析表明，MeCP2 直接结合 TAC1 启动子，并且对抗癫痫药物治疗敏感。
这些数据表明，RTT 中 DNA 甲基化没有广泛改变，与先前观察到的基因表达的细微变化一致。然而，TAC1 可能是 RTT 中进一步功能分析的重要靶点。使用原发性细胞进行更大样本队列的研究，同时考虑共同的临床特征和药物治疗，可能需要解决神经发育障碍中 DNA 甲基化的明显细微破坏。
