产前病毒感染对胎儿基因组免疫功能基因甲基化调控的影响

Viral infection has long been believed as an environmental risk factor and trigger for complex human immune diseases, such as type 1 diabetes. But convincing evidence is still lacking. Epigenetics study of type 1 diabetes demonstrated that altered DNA methylation regulation of immune genes can be a genuine causal risk factor for the disease. It is also known that viruses (like Hepatitis B virus) were able to hijack the epigenetic mechanisms of host cells, which would eventually contribute to the aetiology of cancers. A recent study further demonstrated that significant cross-gestation genome-wide differences in DNA methylation levels in placenta, with the most differentially methylated genes including many immune regulators. It is therefore very important to ask whether prenatal viral infection would disturb such methylation regulation patterns of these immune regulators, which in turn would add to the risk of immune related diseases during an individual's life course. Through further analysis of the same cross-gestation study data, we found that genetic loci known to be associated with complex immune diseases had significantly higher level of methylation and their level of methylation was also more varied across different trimesters, in comparison with average immune genes. These results prompted us to design the current study, to investigate the patterns of genome-wide DNA methylation in placneta and cood blood samples between normal samples and those infected by Hepatitis B virus and cytomegalovirus. Our aim is to understand whether prenatal viral infection can indeed cause abnormal regulation of DNA methylation of immune genes or any groups of important genes at all, hence whether through this epigenetic mechanism prenatal viral infection can be a genuine contributor to the aetiology of complex diseases such as type 1 diabetes.

虽然病毒感染被普遍认为是免疫疾病的触发因子，但分子证据仍然缺乏。复杂疾病表观遗传学研究结果表明，免疫功能基因的甲基化变异确实会引起个体免疫疾病，而在对癌症致病机理的研究中，人们发现包括乙肝病毒在内的病毒感染可以劫持宿主的甲基化机制。最近的一项研究又发现，胎盘组织免疫功能基因是不同妊娠期之间甲基化水平变异最高的基因类群。通过对该数据的进一步分析，我们发现与基因组其它位点相比免疫疾病易感性位点和妊娠早、中、晚期甲基化调控变异有特别显著的相关性。根据这些研究结果，我们推测如果产前病毒等微生物感染能影响此类基因位点甲基化调控，那它们将很可能成为免疫疾病最早的病因之一。为此，我们设计了两种不同病毒（乙肝病毒与巨细胞病毒）在产前感染的情况下胎盘与脐血全基因组甲基化变异的分析研究，希望由此发现产前病毒感染是否确实影响胎儿免疫功能基因，进而影响其出生后相关疾病

表观遗传学的发展增进了人们对疾病发生机制的理解，为了证实产前环境风险因子暴露与个体疾病发生风险这一可能的因果关系，我们针对产前乙肝病毒感染、有机氯农药暴露与胎儿基因组DNA甲基化的问题进行了研究。研究建立在1064对孕妇-胎儿的人群队列，完成了问卷调查和生物样本的采集。在探索阶段：对孕妇外周血和胎儿脐带血进行乙肝检测（乙肝五项和HBV-DNA检测）。并对150对孕妇-胎儿血清进行了有机氯农药DDT含量检测。根据检测结果，选取病例组（发生乙肝宫内感染的胎儿/有机氯农药含量最高）和对照组（母亲和胎儿均未感染乙肝/有机氯农药含量最低）各12例，采用全基因组甲基化芯片技术（Infinium Human Methylation450 Bead Chip Kit）对脐血样本基因组进行甲基化水平检测，从中筛选甲基化变异显著位点，并以此为依据进行一系列的生物信息学分析；在验证阶段：根据全基因组的数据，选择甲基化差异显著且功能明确的1-2个基因，采用焦磷酸测序法检测位点甲基化，同时采用RT-qPCR进行基因mRNA表达水平的检测，分析暴露因子与特定基因甲基化的关系。产前乙肝病毒暴露对胎儿全基因组甲基化影响的研究发现663个甲基化差异位点（FDR调整P值＜0.05且|ΔM|≥1），这些差异位点分布于534个基因上。进一步验证产前乙肝病毒感染与胎儿KLHL35及CPT1B基因甲基化的关系发现，母亲产前乙肝病毒感染并发生宫内感染可能会改变胎儿KLHL35及CPT1B基因甲基化状态，进而可能影响胎儿在生长发育过程中对肝癌等疾病的易感性以及对脂肪酸的代谢能力。产前有机氯农药DDT暴露对胎儿全基因组甲基化影响的研究发现1131个甲基化差异位点（FDR调整P值＜0.05且|ΔM|≥1），这些差异位点分布于598个基因上。进一步验证产前DDT暴露与胎儿BRCA1基因甲基化的关系发现，母亲产前暴露于DDT可能会改变胎儿BRCA1基因甲基化状态，增加其出生后患乳腺癌的风险。该项研究为DNA甲基化可能介导成人疾病在胎儿时期起源的假说增加了新的证据，其研究结果不但可以作为系统性跟踪研究的起点，而且将对国际上以大型出生队列为基础的纵向研究形成有力的支持与补充。