Cancer Genetics Laboratory, Department of Biochemistry, University of Otago, Dunedin, New Zealand.
Fertil Steril. 2012 Jan;97(1):147-53.e7. doi: 10.1016/j.fertnstert.2011.10.027. Epub 2011 Nov 23.
To examine an IVF cohort for imprinted and genome-wide DNA methylation abnormalities.
Retrospective study.
Research laboratory.
PATIENT(S): DNA samples from a previously described IVF cohort that comprised 66 IVF-conceived prepubertal children (IVF, n = 34; intracytoplasmic sperm injection, n = 32) and 69 matched naturally conceived controls.
INTERVENTION(S): DNA methylation was examined at four imprinted gene loci (H19, SNRPN, KCNQ1OT1, and IGF2) and satellite 2 using methylation-sensitive quantitative polymerase chain reaction (MSQ-PCR) followed by bisulfite sequencing at H19, SNRPN, and KCNQ1OT1. Methylated DNA immunoprecipitation (MeDIP) microarray with validation using the Sequenom MassARRAY EpiTYPER(®) platform was also used.
MAIN OUTCOME MEASURE(S): Percentage of DNA methylation by MSQ-PCR, differential methylation based on microarray signal intensity, and percentage DNA methylation as determined by Sequenom MassARRAY EpiTYPER were compared.
RESULT(S): No differences in percentage of methylation between the IVF and control group were observed at H19, KCNQ1OT1, SNRPN, or IGF2. Absence of aberrant imprinting was confirmed using bisulfite sequencing. Methylation of satellite 2 repeats (a surrogate for global methylation) showed no difference between the IVF and control groups. MeDIP was used to screen for differences in promoter methylation. Subsequent quantification of methylation of eight candidate genes using the Sequenom MassARRAY EpiTYPER system did not reveal any differential methylation.
CONCLUSION(S): Low-level imprinting errors are not common in the IVF population.
检查体外受精（IVF）队列中是否存在印迹和全基因组 DNA 甲基化异常。
回顾性研究。
研究实验室。
来自先前描述的 IVF 队列的 DNA 样本，该队列包括 66 名 IVF 受孕的青春期前儿童（IVF，n=34；胞浆内精子注射，n=32）和 69 名匹配的自然受孕对照。
使用甲基化敏感定量聚合酶链反应（MSQ-PCR）检测四个印迹基因座（H19、SNRPN、KCNQ1OT1 和 IGF2）和卫星 2 的 DNA 甲基化，然后对 H19、SNRPN 和 KCNQ1OT1 进行亚硫酸氢盐测序。还使用甲基化 DNA 免疫沉淀（MeDIP）微阵列并用 Sequenom MassARRAY EpiTYPER（®）平台进行验证。
MSQ-PCR 检测的 DNA 甲基化百分比、微阵列信号强度差异的差异甲基化和 Sequenom MassARRAY EpiTYPER 确定的 DNA 甲基化百分比。
在 H19、KCNQ1OT1、SNRPN 或 IGF2 基因座，IVF 组与对照组之间的甲基化百分比没有差异。亚硫酸氢盐测序证实不存在异常印迹。卫星 2 重复（整体甲基化的替代物）的甲基化在 IVF 组和对照组之间没有差异。使用 MeDIP 筛选启动子甲基化差异。随后使用 Sequenom MassARRAY EpiTYPER 系统对八个候选基因的甲基化进行定量，未发现任何差异甲基化。
在 IVF 人群中，低水平的印迹错误并不常见。
