Analysis of gamete-specific methylation mechanism in human by taking advantage of benign ovarian teratomas

在人类中，女性生殖细胞的研究受到伦理学的限制。目前的研究探讨了良性卵巢畸胎瘤作为卵子替代品在印迹基因分析中的有用性。应用45个微卫星DNA标记对25例卵巢良性畸胎瘤进行分型，并根据其基因型特征进行分类。然后选择两个相反的印记基因H19和SNRPN，用于分析它们在这些肿瘤中的甲基化状态。这些分析表明，良性卵巢畸胎瘤由遗传和表观遗传异质性细胞群的混合物组成。与以前的报道相反，我们只能记录一例由减数分裂-II不分离生殖细胞产生的病例。H19和SNRPN在个体畸胎瘤中甲基化程度不同，从正常体细胞到预期的卵子水平。H19残留甲基化的等位基因与患者血液DNA中甲基化的等位基因一致，因此为父系起源。H19低甲基化和SNRPN高甲基化的程度随着肿瘤细胞起源在卵子发生中的进展而增加，并且在个体畸胎瘤中密切相关。本研究通过RT-PCR克隆测序的方法，检测了人内源性逆转录病毒K（HERV-K）家族6个成员基因在胎盘、睾丸、子宫内膜和子宫内膜中的转录本，并对这些基因的转录本进行了分析。外周血白细胞和胎肺。其中三个，这是至少在两个组织中表达的分析，从BAC库中分离。

In humans, studies of female germ cells are very limited by ethics. The current study investigated the usefulness of benign ovarian teratomas as a substitute for ova in analyses of imprinted genes. Twenty-five human benign ovarian teratomas were typed with 45 microsatellite DNA markers and classified according to their genotypic features. Two oppositely imprinted genes, H19 and SNRPN, were then chosen for analysis of their methylation states in these tumors. These analyses revealed that benign ovarian teratomas consist of a mixture of genetically and epigenetically heterogeneous cell populations. In contrast to previous reports, we could document only one case rising from germ cells by meiosis-II nondisjunction. H19 and SNRPN were methylated in individual teratomas to various degrees, ranging from normal somatic cell to expected ovum levels. The allele with residual methylation of H19 was consistent with that methylated in the patient's blood DNA, thus being of paternal origin. Degrees of H19 hypomethylation and SNRPN hypermethylation increased as the cellular origin of the tumors advanced in oogenesis and were closely correlated in individual teratomas. These results could be best explained by the assumption that the primary imprinting is a progressively organized process and suggest that the establishment of primary imprints on different genes might be mechanistically linked, even when those genes are oppositely imprinted.Transcripts of six genes of human endogenous retrovirus K (HERV-K)-family members were detected by sequencing of RT-PCR clones in the placenta, testis, peripheral blood leukocytes and fetal lung. Three of them, which were expressed at least in the two tissues analysed, were isolated from a BAC library.