Beckwith-Wiedemann syndrome (BWS) is a model imprinting disorder resulting from mutations or epigenetic events involving imprinted genes at chromosome 11p15.5. Thus, germline mutations in CDKN1C, uniparental disomy (UPD), and loss of imprinting of IGF2 and other imprinted genes have been implicated. Many familial BWS cases have germline CDKN1C mutations. However, most BWS cases are sporadic and UPD or putative imprinting errors predominate in this group. We have identified previously a subgroup of sporadic cases with loss of imprinting (LOI) of IGF2 and epigenetic silencing of H19 proposed to be caused by a defect in a distal 11p15.5 imprinting control element (designated BWSIC1). However, many sporadic BWS patients show biallelic IGF2 expression in the presence of normal H19 methylation and expression patterns. This and other evidence suggested the existence of a further imprinting control element (BWSIC2) at 11p15.5. Recently, we showed that a subgroup of BWS patients have loss of methylation (LOM) at a diVerentially methylated region (KvDMR1) within the KCNQ1 gene centromeric to the IGF2 and H19 genes. We have now analysed a large series of sporadic cases to define the frequency and phenotypic correlates of epigenetic abnormalities in BWS. LOM at KvDMR1 was detected by Southern analysis or a novel PCR based method in 35 of 69 (51%) sporadic BWS without UPD. LOM at KvDMR1 was often, but not invariably associated with LOI of IGF2. KvDMR1 LOM was not detected in BWS patients with putative BWSIC1 defects and cases with KvDMR1 LOM (that is, putative BWSIC2 defects) invariably had a normal H19 methylation pattern. The incidence of exomphalos in putative BWSIC2 defect patients was not significantly diVerent from that in patients with germline CDKN1C mutations (20/29 and 13/15 respectively), but was significantly greater than that in patients with putative BWSIC1 defects (0/5, p=0.007) and UPD (0/22, p<0.0001). These findings are consistent with the hypothesis that LOM of KvDMR1 (BWSIC2 defect) results in epigenetic silencing of CDKN1C and variable LOI of IGF2. BWS patients with embryonal tumours have UPD or a BWSIC1 defect but not LOM of KvDMR1. This study has further shown how (1) variations in phenotypic expression of BWS may be linked to specific molecular subgroups and (2) molecular analysis of BWS can provide insights into mechanisms of imprinting regulation. (J Med Genet 2000;37:921-926)

Maternally imprinted PEG10 and SGCE, separated by approximately 2.15 Mb from Syncytin (HERV-W) gene at 7q21.3, are implicated in choriocarcinoma and Silver-Russell syndrome. Here we have analyzed the temporal regulation of mRNA expression of these genes in placenta and demonstrate that Syncytin gene activation is highest in term placenta, PEG10, downregulated at early hypoxic phase, and highly activated at 11-12 wk of gestation. In contrast, transcription from SGCE remained unchanged throughout pregnancy, suggesting two neighboring imprinted genes are differentially regulated at very early pregnancy. Additionally, accumulation of two major species of mRNA (8 kb and 3.1 kb) encoded by HERV-W in placenta is regulated: 3.1 kb mRNA level remained unchanged throughout pregnancy, whereas the production of 8 kb species was highest in term placenta. Western blot and immunohistochemical staining of placental tissues with monoclonal antibodies revealed a marked reduction of syncytin glycoprotein synthesis in late pregnancy. Therefore, the relative levels of 3.1 kb and 8 kb mRNAs in trophoblasts could regulate syncytin protein synthesis, possibly by competition of the two mRNA species for translational apparatus.

The inability of the mother to switch from T helper cell type 1 (Th1) to Th2 cytokine profiles at the fetal-maternal interface has been proposed as one of the primary causes of miscarriage, intrauterine growth restriction (IUGR), and preeclampsia (PE). The Th1 [interferon-gamma (IFN-gamma), TNF-alpha, and IL-12] and Th2 (IL-4 and IL-10) cytokines have opposite effects on human pregnancy. Leukemia inhibitory factor (LIF) promotes embryo implantation and sustains pregnancy, whereas IFN-gamma and TNF-alpha are detrimental to pregnancy. Both IFN-gamma and LIF are produced by maternal cells and tissues at the fetal-maternal interface, whereas the IFN-gamma receptors (IFN-gamma R1 and IFN-gamma R2) and LIF receptor are abundantly expressed on the surface of placental trophoblasts. The effect of IFN-gamma on T lymphocyte activation is influenced by the relative membrane density of its two receptors, particularly IFN-gamma R2. In this study we report that in PE (25-40 wk gestation) and PE complicated by IUGR, IFN-gamma R2 protein expression is severely down-regulated and is similar to that observed in early placenta (7-10 wk gestation) developing under low O(2) tension. IFN-gamma production was found to be inversely related to the IFN-gamma R2 protein expression, and LIF receptor protein expression in PE mimicked that in early placental development. These results show that in PE, placental trophoblasts fail to establish an early to late switch with respect to IFN-gamma and IFN-gamma R2 expression. This supports the hypothesis that trophoblasts control the polarization of maternal immune effectors and cytokine profiles at the fetal-maternal interface that could be subject to oxidative stress in PE.

Human chorionic gonadotrophin (hCG) is released from placental trophoblasts and is involved in establishing pregnancy by maintaining progesterone secretion from the corpus luteum. Serum hCG is detected in the maternal circulation within the first 2-3 wks of gestation and peaks at the end of the first trimester before declining. In Down's syndrome (DS) pregnancies, serum hCG remains significantly high compared to gestation age-matched uncompromised pregnancies. It has been proposed that increased serum hCG levels could be due to transcriptional hyper-activation of the CGB (hCG beta) gene, or an increased half life of glycosylated hCG hormone, or both. Another possibility is that serum hCG levels remain high due to reduced availability of the hormone's cognate receptor, LHCGR, leading to lack of hormone utilization. We have tested this hypothesis by quantifying the expression of the hCG beta (CGB) RNA, LHCGR RNA and LHCGR proteins in chorionic villous samples. We demonstrate that chorionic expression of hCG beta (CGB) mRNA directly correlates with high serum hCG levels. The steady-state synthesis of LHCGR mRNA (exons 1-5) in DS pregnancies was significantly higher than that of controls, but the expression of fulllength LHCGR mRNA (exons 1-11) in DS was comparable to that of uncompromised pregnancies. However, the synthesis of high molecular weight mature LHCGR proteins was significantly reduced in DS compared to uncompromised pregnancies, suggesting a lack of utilization of circulating hCG in DS pregnancies.

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Incubation of gradient purified human spermatozoa, which are routinely maintained in media prior to IVF and intracytoplasmic sperm injection (ICSI), induced DNA strand breaks (up to 89 nicks x 10(-3) bp) and chromatin release. Unlike highly dispersed Alu repeat sequences, the centromeric heterochromatin was much less susceptible to endonuclease attack. In addition to chromatin release, the permeability of the sperm membrane was altered as evidenced by reduced accessibility of sperm nuclei to decondensation factors in mouse embryo extracts. Hybridization of cDNA microarrays with DNA released from spermatozoa revealed a consistent hypersensitivity of certain genes to endogenous cleavage including TP53, VHL (tumour suppressors), BRCA1 (breast cancer), NOS1 (neurotransmitter), PECAM1, FLT1 (angiogenesis) and CDKN1C (cell cycle/imprinted). N-tert-butyl hydroxylamine (NTBH), a derivative of the anti-teratogenic alpha-phenyl-N-t-butyl nitrone (PBN) and synthetic superoxide dismutase (SOD)/catalase mimetics inhibited chromatin release and sustained or dissipated relative mitochondrial membrane potential. Together, these results show a link between the hyperactivation of sperm mitochondria and chromosomal damage of specific genes in vitro, and that the potential risk of disruption of paternally contributed genes can be circumvented by antioxidants which are known to target mitochondria.

The allele-specific epigenetic markings of endogenously imprinted genes in placental mammals occur during gametogenesis. The identification of the molecular nature of gametic imprints is the first step towards understanding the mechanistic basis of epigenesis in embryonic and adult somatic tissues. The specific question addressed in this work is whether the closely positioned but oppositely imprinted insulin-like growth factor 2 (IGF 2) and H19 genes, which have similar temporal regulation during development, differ in chromatin structure in mammalian spermatozoa. During terminal differentiation of mammalian spermatozoa, about 3-15% of the haploid genome retains a quasisomatic-type chromatin structure, whereas the remaining genomes interact with protamines that are further cross-linked by-S-S- bridges. Micrococcal nuclease (MNase) and DNase I digestions of human (HSN) and porcine sperm nuclei (PSN) showed that the IGF 2 gene in both types of nuclei retained somatic-type nucleosomes that were close-packed with a periodicity of 150 bp. However, the H19 gene in both species was predominantly organised by unique structural repeats, which were 650-674 bp in PSN and 438-522 bp in HSN, condensing at least 20 kb of chromatin. These results, together with previous studies, suggest that epigenetic chromatin modification leading to preferential condensation of the paternal H19 allele in embryonic tissues is already present in the germ cells.