產前暴露重金屬對於SNRPN基因甲基化程度與新生兒的影響

Abstract

背景： 重金屬是對人體有害的物質之一，而這些環境中的重金屬很多都具有穿透胎盤的能力，因此會導致胎兒暴露到環境中的重金屬。許多研究已經證實，產前重金屬暴露與新生兒出生情形以及小孩未來發展是高度相關的。另外，DNA甲基化是基因表達的重要調控機制之一，其能夠在不改變基因序列的情況下調節基因表達。過去的研究指出，環境暴露會對基因DNA甲基化程度發生改變，同時也有研究表明，產前重金屬暴露所導致的基因DNA甲基化程度改變，會影響胎兒發育以及新生兒出生情形。小核核糖核蛋白相關蛋白N（SNRPN）是母系印記基因。 SNRPN的印跡控制區（ICR）靠近泛素 - 蛋白質連接酶E3A（UBE3A）的基因啟動子，其與遺傳疾病Angelman綜合徵（AS）和Parder-Willi綜合徵（PWS）相關。因此，SNRPN的基因表達也與AS和PWS的遺傳疾病相關，並且這些遺傳疾病將影響兒童的發育。我們的目的是澄清重金屬暴露是否會導致SNRPN的DNA甲基化改變，並進一步影響出生結果。 目標： 本研究希望能夠了解產前重金屬暴露與SNRPN基因DNA甲基化之間的關係，並分析SNRPN基因DNA甲基化變化是否會影響新生兒出生情形。 方法： 本研究使用台灣出生世代研究(TBPS)於2004年7月至2005年6月於台灣北部四間醫院所收集的486個個案進行分析。我們使用台灣出生世代研究所量測臍帶血中18種重金屬濃度作為重金屬暴露資料並進行分析，其中包含鉛，砷，汞，鈹，鉑，釷，錳，鋅，銅，硒，鈷，鉬和鈾等重金屬。同時，我們使用胎盤作為SNRPN基因DNA甲基化程度分析的檢體，並以焦磷酸測序來進行基因甲基化程度的測量。最後將甲基化測量結果和重金屬暴露濃度，來跟出生時量測的新生兒體重、懷孕週數等出生情形來進行相關性統計分析。 結果： 研究結果顯示，重金屬銅與新生兒體重呈現顯著負相關(β = -192.60; p-value < 0.05)，並且與新生兒神經行為檢查呈現顯著負相關(β = -4.10; p-value < 0.01)。此外，箔與懷孕週數成顯著負相關(β = -0.82; p-value < 0.05)，而產前重金屬鋅暴露則是與懷孕週數成顯著正相關(β = 1.84; p-value < 0.001)。在產前重金屬暴露與SNRPN 基因DNA甲基化的部分，本研究觀察到重金屬汞(β = 1.28; p-value < 0.05)以及釷(β = 1.07; p-value < 0.05)跟SNRPN 基因DNA甲基化程度呈現顯著正相關，而銻(β = -2.04; p-value < 0.05)則與SNRPN 基因DNA甲基化程度呈現顯著負相關。最終，在SNRPN 基因DNA甲基化與新生兒出生情形的部分，本研究並未觀察到任何顯著關係。 結論： 本研究結果發現，產前重金屬暴露與SNRPN基因DNA甲基化程度之間是有高度相關。此外，我們也觀察到產前重金屬暴露與新生兒的出生情形是有顯著相關。儘管在本研究中並未觀察到SNRPN基因DNA甲基化程度與新生兒出生情形間的關聯性，但本研究結果可以明白產前重金屬暴露、新生兒出生情形以及SNRPN基因DNA甲基化程度，三者彼此間的關聯性。

Background: Heavy metals are harmful substances to the human body. Many heavy metals from the environment have the ability to penetrate the placenta and expose the fetus. Some studies have confirmed that prenatal heavy metals exposure is strongly associated with birth outcomes and children future development. DNA methylation is one of the important regulatory mechanisms for gene expression, it can regulate gene expression without changing gene sequence. Some previous studies suggested that the environmental exposure will alter gene DNA methylation. Moreover, the alteration of DNA methylation by prenatal exposure can affect fetus development and infant birth outcomes. Small nuclear ribonucleoprotein-associated protein N (SNRPN) is a maternal imprinted gene. The imprinting control region (ICR) of SNRPN is near by the gene promoter of ubiquitin-protein ligase E3A (UBE3A) which is associated with genetic disease Angelman Syndrome (AS) and Parder-Willi Syndrome (PWS). Therefore, the gene expression of SNRPN was also associated with the genetic disease of AS and PWS, and those genetic disease will affect children developments. We aimed to clarify whether heavy metal exposure causes DNA methylation of SNRPN and further affects birth outcomes. Objective: To investigate the relationship between prenatal exposure heavy metals and SNRPN gene DNA methylation level and clarify whether the alteration of small nuclear ribonucleoprotein-associated protein n (SNRPN) affects birth outcomes. Methods: The Study subjects came from Taiwan Birth Panel Study. A total of 486 mother-infant pairs were recruited from July 2004 to June 2005. We measured related heavy metals data from cord blood. The exposure heavy metals substances were including lead, arsenic, mercury, beryllium, platinum, thorium, manganese, zinc, copper, selenium, cobalt, molybdenum and uranium. For analyzing the DNA methylation level of small nuclear ribonucleoprotein-associated protein N (SNRPN), we used placenta as sample and detected by pyrosequencing. The birth outcome data was collected at birth, included birth weight, birth length, gestational age, etc. Finally, we used different statistical models to analyze the data. Result: Copper showed significant negative association with birth weight (β = -192.60; p-value < 0.05) and neonatal neurobehavioral examination (β = -4.10; p-value < 0.01) in adjusted model. Platinum was significant negative association with gestational age (β = -0.82; p-value < 0.05). Zinc showed significant positive association with gestational age (β = 1.84; p-value < 0.001) in adjusted model. Then, mercury (β = 1.28; p-value < 0.05) and thorium (β = 1.07; p-value < 0.05) were significant positive association with DNA methylation level of SNRPN. Furthermore, antimony (β = -2.04; p-value < 0.05) was significantly negative associated with DNA methylation level of SNRPN. The DNA methylation level of SNRPN was not significantly associated with birth outcomes. Conclusion: Our results suggested that prenatal heavy metals exposure was associated with DNA methylation level of SNRPN gene. In addition, we also observed that prenatal heavy metals significantly associated with birth outcome of infant. Although we didn’t observe significantly association between SNRPN gene DNA methylation and birth outcomes, our study confirmed three correlations including heavy metals exposure and birth outcomes, heavy metals exposure and DNA methylation, DNA methylation and birth outcomes. But, we were unclear about the direct relationship between these individual correlations.