Hydatidiform mole and triploidy: the role of genomic imprinting in placental development

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1 Human Reproduction Update, Vol.11, No.2 pp , 2005 Advance Access publication January 27, 2005 doi: /humupd/dmh060 Hydatidiform mole and triploidy: the role of genomic imprinting in placental development Koen Devriendt Center for Human Genetics, Herestraat 49, B-3000 Leuven, Belgium. Genomic imprinting, the differential expression of paternal and maternal alleles, is involved in the regulation of embryonic and fetal growth and development. In this review, we focus on the genetics of a disorder caused by a global defect in genomic imprinting, the hydatidiform mole. The ratio between the maternal and paternal genomes is critical in determining the development of both the embryonic and extraembryonic tissues, with an excess of paternally derived chromosomes leading to a complete (no maternal genome) or partial (lower amount of maternal chromosomes) mole. The recent identification and molecular studies in biparental complete moles may yield more insight into the regulation of imprinting during gametogenesis. Key words: genomic imprinting/hydatidiform mole/methylation/pregnancy/triploidy Introduction Different theories have existed to explain embryonic development. According to the preformation theory, which was initially proposed by Aristotle, development was nothing more than the growth of pre-existing structures that were already present in the germ cells. This theory, which gained general acceptance, was corroborated when the first microscopes apparently showed sperm cells as miniature, preformed creatures. However, a second, competing theory was put forward by Harvey in His epigenetic theory stated that all living beings derive from the ovum by the gradual building up and aggregation of its parts. This theory became known by the saying ex ovo omnia. However attractive, both theories are not supported by experimental evidence. In the mouse, one can remove the paternal pronucleus from a fertilized oocyte, and replace this by a second maternal pronucleus, thus creating an embryo where all chromosomes have a maternal origin (reviewed in Solter, 1988). These so called gynogenotes (which are equivalent to parthenogenesis) fail to complete normal development. No extraembryonic components are formed and the embryoblast develops into a teratoma (Surani et al., 1984, 1986). In contrast to this, the opposite is observed in androgenotic embryos, containing paternal chromosomes only. They fail to develop an embryoblast whereas the trophoblast proliferates excessively, resulting in a hydatidiform mole (McGrath and Solter, 1984). These early experiments indicated that both maternal and paternal genes are essential for normal embryonic development. Certain maternal genes are required for development of the embryo proper, whereas extraembryonic components depend on the presence of active paternal genes. This is regulated by a process called genomic imprinting, whereby certain genes are differently expressed, solely depending on whether they are on the maternal or paternal chromosome (reviewed by Reik and Walter, 2001). Several genetic diseases have been delineated in humans caused by abnormal genomic imprinting, such as Angelman syndrome, Silver Russel and Prader Willi syndrome. These disorders are caused by a dysfunction of one or more imprinted genes. In this review, we will focus on the genetics of a disorder caused by a more global defect in genomic imprinting, the hydatidiform mole. Two different forms of hydatidiform mole with a different appearance and aetiology exist, the partial and complete hydatidiform mole. In the complete mole, all villi are cystic and embryonic development is usually absent. In the partial mole, embryonic development is observed, and a range of normal to abnormal, cystic villi are present. The complete mole is almost always caused by the absence of maternal chromosomes, and has thus an exclusively paternal origin of the genetic material. The partial mole has another genetic defect, a triploidy. Complete hydatidiform mole A complete hydatidiform mole is diploid, i.e. the presence of 46 chromosomes, but all chromosomes are of paternal origin. As always in the case of the uniparental origin of one or more chromosomes, this requires at least two sequential errors. The most frequent mechanism of origin is the fertilization of an oocyte without nucleus (or with inactivated nucleus) by a single sperm, followed by duplication of the haploid genome (Lawler et al., 1982) (Figure 1). In the remainder (,20 25%), an enucleated oocyte is fertilized by two sperm cells (Kovacs et al., Human Reproduction Update vol. 11 no. 2 q European Society of Human Reproduction and Embryology 2005; all rights reserved 137

2 K.Devriendt Molecular investigations on DNA extracted from the villi of a hydatidiform mole can confirm the uniparental paternal origin of the chromosomes. In both instances, only paternal alleles are seen, but the two different causes described above can be distinguished. In the monospermic mole, only one single allele is detected for each locus analysed, and this is called a homozygous mole. In contrast, for loci for which the father is heterozygous, the dispermic or heterozygous mole has two different alleles (Figure 1). It has been suggested that heterozygous moles have a higher risk of malignancy (Wake et al., 1984), but follow-up studies could not confirm this (Lawler and Fisher, 1987; Lawler et al., 1991). Partial hydatidiform mole Figure 1. Origin of hydatidiform mole. The hydatidiform mole most frequently results from the fertilization of an oocyte without active nucleus, followed by duplication of the paternal chromosomes (homozygous mole). In 20 25% of cases, two sperm cells fertilize an oocyte without active nucleus (heterozygous mole). 1991). A third possible cause, the fertilization of an empty oocyte by a diploid sperm cell, is extremely rare (Zaragoza et al., 2000). How an enucleated oocyte is generated is not clear, but one possible error is a non-disjunction of all chromosomes during meiosis, with all chromosomes ending up in one of the polar bodies. Since 46,YY has never been observed (and thus probably non-viable), cytogenetic investigation usually reveals a 46,XX karyotype and in a minority a 46,XY karyotype is found. As expected also in the hydatidiform mole, all mitochondria have an exclusively maternal origin (Azuma et al., 1991). The partial mole is caused by a triploidy, the presence of three copies of each chromosome (Figure 2). Triploidy is one of the most common chromosomal anomalies, with an incidence of 10% in spontaneous abortions (Hassold et al., 1980). The extra haploid set of chromosomes can have either a maternal origin (and then called digynic triploidy) or paternal (diandric triploidy). The majority of triploidies are sporadic, but a few cases have been reported with recurrent triploidy in three pregnancies (Pergament et al., 2000; Brancati et al., 2003; Huang et al., 2004). In all three cases, the origin of the triploidy could be investigated at least in one pregnancy and all were of maternal origin (digynic). This observation could be explained by a genetic defect in the maternal meiosis. So far, no proven cases of familial recurrent triploidy have been reported. The majority of triploid fetuses ascertained through a spontaneous abortion are diandric. Typically, the fetus has a relatively normal growth, with either a normal head size or microcephaly (McFadden and Kalousek, 1991; Zaragoza et al., 2000). The placenta usually (but not always) has the appearance Figure 2. Partial mole is caused by a diandric triploidy. The parental origin of the extra chromosomes in a triploidy determines the phenotype and outcome of the fetus and placenta. A partial mole is only observed in the diandric triploidy. IUGR ¼ intrauterine growth retardation; rel ¼ relative. 138

3 Role of imprinting in hydatidiform mole of a partial mole (Zaragoza et al., 2000). The most common mechanism causing diandric triploidy is dispermy, i.e. the fertilization of a single normal oocyte by two sperm cells (Zaragoza et al., 2000). In digynic triploidy, no partial mole is observed, and these fetuses usually are growth-retarded, macrocephalic and have a small placenta (McFadden and Kalousek, 1991). The mechanism of origin is mostly a meiosis II error, and thus the fertilization of an diploid oocyte (Zaragoza et al., 2000). The difference in phenotype between diandric and digynic triploidies is highly reminiscent of experimental observations in the mouse. To gain better insight in the development of androgenetic or gynogenetic embryos, chimeras have been constructed, where only part of the cells are androgenetic or gynogenetic. This is achieved through the fusion of cells from an androgenetic or gynogenetic embryo with a normal embryo. These embryos survive, and thus allow the analysis of the phenotype. Androgenetic and gynogenetic cells appear to contribute to different parts of the developing embryo and fetus (Surani et al., 1988). Gynogenetic cells are found in ectodermal derivatives, i.e. the central nervous system, but are absent from the placenta and yolk sac. As in digynic triploidies, these embryos are growthretarded, but often display an enhanced growth of the brain. In contrast, androgenetic cells are preferentially found in trophoblast, primitive endoderm lineages (the yolk sac), mesodermal derivatives (muscles and heart) and in specific parts of the brain (such as the hypothalamus). These fetuses display an overgrowth, often with a reduced brain development. Interestingly, a few cases have been reported with recurrent triploidy in three pregnancies (Pergament et al., 2000; Brancati et al., 2003; Huang et al., 2004). In all three cases, the origin of the triploidy could be investigated at least in one pregnancy and all were of maternal origin (digynic). This observation could be explained by a genetic defect in the maternal meiosis. So far, no proven cases of familial recurrent triploidy have been reported. Mosaic triploidy Whereas triploidies are almost uniformly lethal, some children carry a triploidy in only part of their cells, with the remaining cells having the normal 46 chromosomes. This is called a mosaic triploidy. Such a mosaicism can only be explained by a postzygotic error, occurring after the first cell division. Genetic studies tracing the origin of the extra set of chromosomes have indicated a paternal origin in some, leading to the suggestion that the mosaicism may originate from the incorporation of a second sperm pronucleus into one embryonic blastomere (Daniel et al., 2003). In others, a maternal origin was detected, most likely through the fusion of one of the (haploid) second polar bodies with an early blastomere (Muller et al., 1993; Brems et al., 2003). In one instance, the most likely explanation was chimaerism with fusion of two separate zygotes developing into a single individual (Daniel et al., 2003). Mosaic triploidy can be viable, depending on the percentage of abnormal cells and their tissue distribution. Clinically, these infants have a recognizable pattern of malformations, including pre- and postnatal growth retardation, peculiar facial features, cutaneous syndactyly of fingers and toes, typically fingers 3 and 4 and toes 2 and 3 and mental retardation. Typically, body asymmetry and linear skin hyper- or hypopigmentation lesions following the Blaschko lines are seen, indicative of mosaicism (Devriendt et al., 2004). Interestingly, some children have precocious puberty, reminiscent of the maternal UPD14 phenotype, also characterized by pre- and postnatal growth retardation and precocious puberty (Kotzot, 2004). Diagnosis can be challenging, since in peripheral white blood cells, the starting point for classical karyotype analysis, the triploid cells have usually been selected out. Therefore, a skin biopsy with chromosome analysis on cultured fibroblasts is necessary to confirm the clinical diagnosis. Pathogenesis of complete and partial hydatidiform mole In the complete mole, the hyperplasia of the placenta stands in sharp contrast to the absence of embryonic development. This suggests that for embryoblast development, the contribution of maternally inherited genes is necessary. As stated in the Introduction, the opposite is observed in the teratoma, with an exclusively maternal origin of the genomes and development of tissue characteristic of the three germ layers of the embryo, but not of the extraembryonic components. Likewise, the digynic triploidy does not develop features of a partial mole, but has a small placenta, and the fetus is growth-retarded (McFadden and Kalousek, 1991). Thus, the ratio between the maternal and paternal genomes is critical in determining the development of both the embryonic and extra-embryonic tissues, with an excess of paternally derived chromosomes leading to a complete (no maternal genome) or partial (lower amount of maternal chromosomes) mole. An increasing number of imprinted genes is being identified in mammals, and of these, several influence placental growth in the mouse (reviewed by Reik et al., 2003). In humans, the prototype of an imprinting disorder featuring overgrowth is the Beckwith Wiedemann syndrome (BWS). This syndrome is characterized by an intrauterine overgrowth, with visceromegaly (kidneys, liver, spleen, adrenal glands), a high birthweight, Figure 3. An imprinted region on chromosome 11p15.5. The chromosomal region 11p15.5 harbours several genes with a different imprinting pattern. The p57kip2, IPL and H19 genes are paternally imprinted or silenced, whereas the IGF2 and KCNQIOT1 genes are maternally imprinted. In a hydatidiform mole, the presence of paternally derived chromosomes only leads to an abnormal expression pattern of these imprinted genes. off indicates absent expression; on indicates active gene. 139

4 K.Devriendt macroglossia and omphalocoele (Elliott and Maher, 1994). The placenta often is enlarged (McCowan and Becroft, 1994). In addition, these children have an increased risk of developing paediatric tumours, typically Wilms tumour, but also a variety of others such as hepatoblastoma, adrenal adenoma and carcinoma, fibromas of the heart, brain stem glioma, ganglioneuroma, rhabdomyosarcoma, lymphoma, pancreatoblastoma and hepatic haemangioepitheliomas (Drut et al., 1992). The disorder is caused by abnormalities in the chromosome 11p15 region, where a cluster of imprinted genes is located. Some of these genes are maternally imprinted (i.e. silenced on the maternal chromosome), such as the IGF2 and KCNQIOT1 (or LIT1) genes, whereas others including the p57 kip2, H19 and IPL genes are paternally imprinted (Figure 3) (reviewed by Weksberg et al., 2003). Among the different causes of the BWS, a frequent mechanism includes biallelic expression of the IGF2 or KCNQIOT1 genes. In,5% of cases, a mutation is found in the maternally inherited p57 kip2 gene, which is expressed from the maternal chromosome only (O Keefe et al., 1997). In normal placental villi, p57 kip2 is expressed from the maternal allele in both cytotrophoblast and chorion cells. As expected, in the hydatidiform mole, most villi do not express this gene, whereas in the partial mole, p57 kip2 expression is detected, since these cells contain a maternal genome. P57 kip2 is a cyclin-dependent kinase inhibitor and functions as a negative regulator of cell proliferation. This gene therefore appeared to be a good candidate gene involved in the pathogenesis of hydatidiform moles (Matsuoka et al., 1995). However, recently, a single case of complete mole Figure 4. (A) Imprinting switch during gametogenesis. During gametogenesis the imprinting pattern of genes is altered according to the sex of the parent. This is accompanied by changes in the methylation pattern. (B) Genetic differences between paternal and biparental hydatidiform moles. In the paternal hydatidiform mole, all imprinted genes carry an exclusively paternal imprinting or methylation pattern. In the biparental mole, methylation but not demethylation is defective in oogenesis, leading to an abnormal methylation of maternally imprinted genes such as KCNQIOT1. 140

5 Role of imprinting in hydatidiform mole with p57 kip2 immunoreactivity has been reported, explained by the selective retention in the cells of human chromosome 11. This argues against an exclusive role of this gene, or other imprinted genes such as IGF2, H19 or IPL, on chromosome 11p in the pathogenesis of complete hydatidiform mole, as well as other genes elsewhere in the genome (Fisher et al., 2004b). Biparental complete hydatidiform mole In exceptional cases (, 10 families have been reported) of histologically typical complete hydatidiform mole, a biparental origin of the chromosomes has been found (reviewed by Fisher et al., 2004b). The interpretation is that the maternal chromosomes behave as if they were of paternal origin. Genomic imprinting is a reversible process, whereby the imprint is reset during gametogenesis according to the sex of the parent. Thus, during oogenesis, genes silenced on the paternal chromosome must be reactivated, whereas genes active on the paternal chromosome must be silenced, and vice versa for spermatogenesis (Figure 4A). Clearly, this process can go wrong. In some instances, this results from an accidental failure of reprogramming, as observed in certain cases of Beckwith Wiedemann syndrome, with loss of imprinting of the KCNQIOT1 gene (Weksberg et al., 2003). Likewise, failure of establishing an imprinting switch in the 15q11-13 region can cause the Prader Willi or Angelman syndrome (Buiting et al., 1998). Recent observations have suggested that in vitro fertilization may confer an increased risk to such imprinting defects (Halliday et al., 2004). Molecular studies of biparental moles have revealed a paternal imprinting pattern in these mole placentas (see below) (El-Maarri et al., 2003). As in classical moles, p57 kip2 expression was found to be absent (Fisher et al., 2002). In several instances, familial occurrence of recurrent biparental moles was observed, and inheritance was compatible with an autosomal recessive disorder (e.g. Helwani et al., 1999). At the DNA level, the signal mediating genomic imprinting is methylation of CpG residues in specific chromosomal regions. This modification of DNA is reversible, i.e. the methyl groups can be removed, and de novo methylation can occur. For many genes, methylation results in silencing, whereas demethylation leads to reactivation of the gene expression. In the normal situation, a differentially methylated region (DMR) in the KCNQIOT1 gene becomes methylated during oogenesis and demethylated during spermatogenesis (Weksberg et al., 2003) (Figure 4A). In biparental molar placentas, as in the common paternal molar pregnancies, both alleles are demethylated (Judson et al., 2002; El-Maari et al., 2003). This indicates that this DMR cannot become methylated and thus acquires a paternal imprinting pattern (Figure 4B). These data also show that the methylated maternal allele is first demethylated, and must be remethylated. In contrast to this, a different pattern has been observed for the H19 gene, which is normally methylated on the paternal chromosome and unmethylated on the maternal chromosome. As expected, in the classical paternal moles the H19 gene is methylated on both alleles. In contrast, in biparental moles, the maternal allele remains unmethylated, the paternal allele is methylated (Figure 4B) (Judson et al., 2002). Also for other imprinted genes, located on different chromosomes, the same was observed. On the one hand, the DMR of the SNRPN gene (chromosome 15q11-13), the PEG1 (chromosome 7q32) and PEG3 (chromosome 19q13.4) genes are methylated on the maternal chromosome in the normal situation. In biparental moles, they were unmethylated on both alleles (Judson et al., 2002; El-Maari et al., 2003). On the other hand, as for the H19 gene, the DMR of NESP55 (located on chromosome 20q13.2) is methylated on the paternal chromosome, and in biparental moles the maternal allele remains unmethylated (Judson et al., 2002; El-Maari et al., 2003). Thus, there appears to be a global methylation defect leading to a switch from maternal to paternal methylation pattern, causing a biparental mole. The demethylation process takes place in a normal way but there is a failure of establishing the normal maternal methylation pattern on several genes located on different chromosomes. However, not all instances of biparental complete hydatidiform moles studied revealed the same pattern of methylation defect (El-Maari et al., 2003). For instance, in two biparental moles, hypomethylation at the SNRPN DMR was not observed and the H19 gene was not hypermethylated. This suggests that the genetic defect has a reduced penetrance, and that the methylation defect can sometimes be partial or even overcome. This would explain why certain females with the disorder nevertheless had a normal pregnancy, despite a theoretical recurrence risk of 100% (Fisher et al., 2004a). Methylation depends on specific enzymes, DNA methyltransferases (Dnmt) and these are therefore excellent candidate genes for the disorder. However, so far, no mutations have been reported in these genes (Hayward et al., 2003). Meanwhile, as an alternative means to identify the molecular basis of this autosomal recessive disorder, linkage analysis has been performed in families with the disorder. This has led to the identification of a relatively small region on chromosome 19q (Moglabey et al., 1999; Hodges et al., 2003). 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