胎兒暴露體與孩童發展：以印痕基因甲基化程度作為測量指標

Abstract

背景 母親於懷孕時所受到的各種暴露，一直都被認為與不好的新生兒發展、孩童神經系統及行為等有關係。然而，其中的主要機制仍須進一步的闡明。印痕基因甲基化是一種重要的表基因修飾，可提供量化的篩檢指標，進而了解產前暴露對於新生兒發展、神經行為發展的影響，更甚而往後發展成某些疾病。 研究目的 本研究之目的為透過測量表基因甲基化程度，了解其與環境暴露及嬰兒出生結果、兩歲和七歲孩童神經行為發展的關連性。 方法 本研究採用2004至2005年間所蒐集的Taiwan Birth Panel Study (TBPS)世代研究族群，經篩選後共有465對母親嬰兒納入研究。由母親臍帶血及第三孕期時尿液樣本中量測共32種胎兒暴露體，包含古丁尼、18種金屬、2種有機磷殺蟲劑、4種全氟碳化物、3種酚類及4種塑化劑代謝產物，除此之外，MEST及PEG3印痕基因的甲基化是由母親臍帶血中的白血球所萃取測量。本研究使用母親產後結構性問卷、胎兒暴露體及印痕基因DNA甲基化程度等資料，運用PLS回歸分析及廣義線性混合模型，進而探討母親孕期暴露、基因甲基化以及孩童出生及神經行為發展之間的關連性。 結果 本研究於32種胎兒暴露體中，找出11種相對較重要的因子。其中，較高濃度的銅(pos1:-0.34, P=0.0357; pos3:-0.35, P=0.0277), 鉬(pos1:β= -0.38, P=0.026; pos2:β= -0.35, P=0.0358), 不同濃度的鋇(pos5: L-β=0.48, P=0.0359; M-β=0.66, P=0.0071; H-β=0.54, P=0.0322)以及全氟辛烷磺酸(L-β=-0.39, P=0.0148: H-β= -0.41, P=0.0128)均可能會改變MEST (啟動子區) 的甲基化程度，然而不同濃度的銅(L-β=0.59, P=0.0042;M-β=0.57, P=0.0075;H-β=0.64, P=0.0022),低濃度的鋅(β=0.45, P=0.0471)、鋇(β=0.51, P=0.0151)、鈷(pos3:β=-0.46, P=0.0294;pos5: β=-0.49, P= 0.032)以及低濃度古丁尼 (β=0.58, P=0.0267)也與不同程度的PEG3基因甲基化顯著有關。除此之外，本研究也發現高或低的MEST (CTCF結合區)基因甲基化都有可能增加較小嬰兒身長的風險(<Q1: OR=2.41, 95% CI=1.12-5.16; >Q3:OR=2.73, 95% CI=1.20-6.25)。另外，在調整過孩童年齡、母親教育程度、母親BMI以及胎兒小於孕期年齡等干擾因子後，MEST (啟動子區)上2個CpGs的高度甲基化也顯著較參考組的孩童行為問題的風險高(pos3: OR=4.12, 95% CI=1.34-12.66; pos4: OR=3.79, 95% CI=1.21-11.89)。同樣現象也在MEST (CTCF結合區)上低甲基化組觀察到(pos1: OR=5.02, 95% CI=1.49-16.98; pos4: OR=4.71, 95% CI=1.36-16.27)。然而，在PEG3上的某些CpGs中，高或低甲基化可能對不好的孩童神經行為發展等有保護的效果。 結論 本研究指出，在32種胎兒暴露體中，有11種是主要解釋嬰兒出生體重的因子。而這11種胎兒暴露體中，三種金屬(銅、鉬、鋇)和全氟碳化物(全氟辛烷磺酸)與MEST印痕基因甲基化程度是有關係性存在的，而四種金屬(銅、鋅、鈷、鋇)及古丁尼(尼古丁代謝產物)則與PEG3甲基化程度相關。另外，由於不同的胎兒暴露體濃度而增加或減少的印痕基因甲基化程度，都有可能對於孩童的出生結果及2歲時神經行為發展有不好的影響。

Background In utero exposures have been suggested to be linked to adverse birth outcomes, neurodevelopment or child behavior, but the underlying mechanism remains elusive. DNA methylation, an essential epigenetic modification, of candidate imprinted genes might provide a quantitative screening marker for the effects of prenatal exposures on the development and neurobehavioral development of the infants, even the risk of developing certain disease in later life. Objective The objective of this study is to investigate the relationship between multiple fetal exposomes during pregnancy, epigenetic modifications and child birth and neurobehavioral outcomes at follow-up 2 and 7 years old, by quantifying DNA methylation levels of imprinted genes. Methods A total of 465 mother-infant pairs were included in this study from Taiwan Birth Panel Study (TBPS), collecting from 2004 to 2005. Fetal exposomes, including cotinine, 18 metals, 2 organophosphorous Pesticides, 4 perfluorinated compounds (PFCs) and 3 phenols, 4 phthalate metabolites were detected in umbilical cord blood and spot mother’s urine samples. Besides, DNA methylation levels of MEST and PEG3 imprinted gene were measured in leukocytes from umbilical cord blood. This study made use of data from structured questionnaires、fetal exposomes and DNA methylation levels to estimate the association between prenatal exposures, DNA mehtlyation levels of imprinted genes as well as child outcomes by partial least squares (PLS) regression and generalized linear mixed model. Results This study identified 11 relatively important factors among 32 fetal exposomes. Among these 11 exposomes, higher level group of Cu (pos1:-0.34, P=0.0357; pos3:-0.35, P=0.0277), Mo (pos1:β= -0.38, P=0.026; pos2:β= -0.35, P=0.0358), all level group of Ba (pos5: L-β=0.48, P=0.0359; M-β=0.66, P=0.0071; H-β=0.54, P=0.0322), and PFOS (L-β=-0.39, P=0.0148: H-β= -0.41, P=0.0128) were likely to alter methylation levels of MEST gene, whereas all level group of Cu (L-β=0.59, P=0.0042; M-β=0.57, P=0.0075; H-β=0.64, P=0.0022), low level group of Zn (β=0.45, P=0.0471), Ba (β=0.51, P=0.0151), Co (pos3:β=-0.46, P=0.0294; pos5: β=-0.49, P= 0.032) and low level group of cotinine (β=0.58, P=0.0267) might have differential methylation effects on PEG3 gene. Beside, hypo- or hypermethylation of MEST (CTCF binding region) might have increased risk of low birth size (<Q1: OR=2.41, 95% CI=1.12-5.16; >Q3:OR=2.73, 95% CI=1.20-6.25).Further, hypermethylation of 2 CpGs on MEST ( promoter region) gene had increased risk of having behavior problem (pos3: OR=4.12, 95% CI=1.34-12.66; pos4: OR=3.79, 95% CI=1.21-11.89), compared with reference group, after adjusting for child sex, maternal education, maternal BMI and SGA. The same phenomenon was observed in hypomethylation of position 1 and 4 of MEST (CTCF binding region) (pos1: OR=5.02, 95% CI=1.49-16.98; pos4: OR=4.71, 95% CI=1.36-16.27). At some CpGs of PEG3, hyper- or hypermethylation might have protective effects on adverse child outcomes. Conclusion Our study indicates that there were 11 out of 32 fetal exposomes accounting for infant birth weight. Among them, three metals (Cu、Mo、Ba) and perfluorinated compounds(PFOS) might be associated with methylation levels of MEST imprinted gene, while four metals (Cu、Zn、Co、Ba) and cotinine (metabolite of nicotine) were correlated to methylation levels of PEG3. Moreover, either the increased or decreased methylation of imprinted genes as a result of different levels of fetal exposomes was likely to have increased risk of child birth outcomes and child neurodevelopment at 2 years old.