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Abstract：
Human serotonin receptor 4 (HTR4) encodes a 5-HT4 receptor involved in learning, memory, depression, anxiety, and feeding behavior. The aim of this study was to investigate the association between the deoxyribonucleic acid (DNA) methylation of HTR4 promoter and autism spectrum disorder (ASD), a disease characterized by communication disorder and repetitive or restrictive behavior.Peripheral blood DNA was obtained from 61 ASD children and 66 healthy children, and the DNA methylation of HTR4 promoter was assessed by quantitative methylation-specific polymerase chain reaction. We used percentage of methylated reference (PMR) to represent DNA methylation level.Due to significant age differences between ASD cases and controls (3 [2, 5] years and 6 [5, 6] years, P = 3.34E-10), we used binary logistic regression analysis for adjustment. Our results showed that the DNA methylation levels of HTR4 promoter were significantly lower in children with ASD than in healthy children (median PMR: 66.23% vs 94.31%,P = .028, age-adjusted P = .034). In addition, the DNA methylation of HTR4 promoter was inversely associated with age in male ASD cases (total cases: r = -0.283, P = .027; male cases: r = -0.431, P = .002; female cases: r = -0.108, P = .752). Dual-luciferase reporter gene assay showed that the reporter gene expression in the strain with recombinant pGL3-promoter-HTR4 plasmid was significantly higher than that in the strain with pGL3-promoter plasmid (fold change = 2.01, P = .0065), indicating that the HTR4 promoter fragment may contain transcription factors to upregulate promoter activity.Our study suggested that hypomethylation of the HTR4 promoter is a potential biomarker for predicting the risk of male ASD.
摘要：
人类5-羟色胺受体4（HTR4）编码参与学习的5-HT4受体，记忆,抑郁症,焦虑,和喂养行为。这项研究的目的是研究HTR4启动子的脱氧核糖核酸（DNA）甲基化与自闭症谱系障碍（ASD）之间的关系。一种以沟通障碍和重复或限制性行为为特征的疾病。从61名ASD儿童和66名健康儿童获得外周血DNA，并通过定量甲基化特异性聚合酶链反应评估HTR4启动子的DNA甲基化。我们使用甲基化参考(PMR)的百分比来表示DNA甲基化水平。由于ASD病例和对照组之间的显着年龄差异（3[2，5]岁和6[5，6]岁，P=3.34E-10),我们使用二元逻辑回归分析进行调整。我们的结果表明，ASD儿童的HTR4启动子的DNA甲基化水平显着低于健康儿童（PMR中位数：66.23％vs94.31％，P=.028，年龄调整后的P=.034）。此外,在男性ASD病例中,HTR4启动子的DNA甲基化与年龄呈负相关(总病例:r=-0.283,P=.027;男性病例:r=-0.431,P=.002;女性病例:r=-0.108,P=.752)。双荧光素酶报告基因检测显示,重组pGL3-启动子-HTR4质粒菌株的报告基因表达显著高于pGL3-启动子质粒菌株(倍数变化=2.01,P=.0065),这表明HTR4启动子片段可能含有上调启动子活性的转录因子。我们的研究表明，HTR4启动子的低甲基化是预测男性ASD风险的潜在生物标志物。
