Biparental hydatidiform moles: a maternal effect mutation affecting imprinting in the offspring

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Human Reproduction Update, Vol.12, No.3 pp. 233–242, 2006 doi:10.1093/humupd/dmk005

Advance Access publication March 15, 2006

Biparental hydatidiform moles: a maternal effect mutation affecting imprinting in the offspring

I.B.Van den Veyver1,2,4 and T.K.Al-Hussaini3

1Department of Obstetrics and Gynecology, 2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA and 3Department of Obstetrics and Gynecology, Assiut University, Assiut, Egypt

4To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Baylor College of Medicine, 1709 Dryden, Suite 1100, Houston, TX 77030, USA. E-mail: [email protected]

Highly recurrent hydatidiform moles (HMs) studied to date are not androgenetic but have biparental genomic contri-bution (BiHM). Affected women have an autosomal recessive mutation that causes their pregnancies to develop intoHM. Although there is genetic heterogeneity, a major locus maps to chromosome 19q13.42, but a mutated gene hasnot yet been identified. Molecular studies have shown that maternal imprinting marks are deregulated in the BiHMtrophoblast. The mutations that cause this condition are, therefore, hypothesized to occur in genes that encode trans-acting factors required for the establishment of imprinting marks in the maternal germline or for their maintenancein the embryo. Although only DNA methylation marks at imprinted loci have been studied in the BiHM, the mutationmay affect genes that are essential for other forms of chromatin remodelling at imprinted loci and necessary for cor-rect maternal allele-specific DNA methylation and imprinted gene expression. Normal pregnancies interspersed withBiHM have been reported in some of the pedigrees, but affected women repeatedly attempting pregnancy should becounselled about the risk for invasive trophoblastic disease with each subsequent BiHM.

Key words: embryology/genetic disorders/germ cells/imprinting/pregnancy

Introduction

A hydatidiform mole (HM) is an abnormal pregnancy with exces-sive proliferation of placental villi but severely stunted or absentembryonic development. HM can be classified into complete HM(CHM) or partial HM (PHM). Most CHM are androgenetic(AnCHM) with a diploid genome that is entirely paternallyderived. PHM have a triploid genome, with three copies of eachchromosome, two of which are paternally inherited (Szulman andSurti, 1978). When two of the three copies of each chromosomeare of maternal origin, triploid pregnancies have an underdevel-oped placenta. Hence, an excess of paternally inherited genesresults in molar pregnancy, which suggests that deregulatedexpression of imprinted genes plays an important role in thisabnormal development of the human trophoblast. HM is consid-ered a sporadic disorder with 1% recurrence risk, but severalfamilial and sporadic cases of women with highly recurrent CHM(and sometimes PHM) have been described (Wu, 1973; Ambaniet al., 1980; Federschneider et al., 1980; Nalini et al., 1989;Kircheisen and Schroeder-Kurth, 1991; Narayan et al., 1992;Sunde et al., 1993; Seoud et al., 1995; Helwani et al., 1999;Moglabey et al., 1999; Sensi et al., 2000; Al-Hussaini and Abdel-Alim, 2001; Ozalp et al., 2001; Fisher et al., 2002; Judson et al.,2002; Al-Hussaini et al., 2003; Fallahian, 2003; Hodges et al.,2003). Where studied, the molar pregnancies of these women are

of normal diploid biparental inheritance (BiHM) and do not con-tain excess of paternally inherited genetic material (Helwani et al.,1999) but show abnormal expression (Fisher et al., 2002; Hodgeset al., 2003) or epigenetic marking of imprinted genes (Judsonet al., 2002; El-Maarri et al., 2003). The pedigrees are consistentwith the affected women having an autosomal recessive mutation(Moglabey et al., 1999; Sensi et al., 2000; Judson et al., 2002;Hodges et al., 2003; Panichkul et al., 2005; Slim et al., 2005) thatappears to disturb normal imprinting in their pregnancies. An activesearch for the mutated gene, hypothesized to encode an essentialtransacting factor for the regulation of imprinting in oocytes and/or the preimplantation embryo, is ongoing in several laboratories.Here, we review current research progress on BiHM and speculateon possible functions of the candidate gene, based on recentlyaccumulated knowledge about epigenetic processes in germ cells,fertilized zygotes and preimplantation embryos.

Aetiology and genetics of sporadic CHMs and PHMs

The incidence of HM is variable, complicating from 1/1000 to 1/2000pregnancies in the United States (Benirschke and Kaufmann,1990; Szulman, 1999) and to 1/125 in some high-incidence areasin Southeast Asia. In the Middle East, where many familial casesof highly recurrent BiHM originate, the incidence is about 1/500

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(Al-Hussaini and Abdel-Alim, 2001). CHM is characterized byembryo degeneration probably between 15 and 31 days post con-ception (dpc) (Szulman, 1999) and varying degrees of abnormalproliferation of both the cytotrophoblastic and the syncytiotro-phoblastic layers. The initially oedematous and hypercellular mes-enchymal villar stroma develops central cisternae that coalesceinto larger cystic cavities compressing the stroma and centralblood vessels. The result is a mass of clear vesicles that vary insize from microscopic to a few centimetres and no discerniblefetus or amnion. One should differentiate between true CHM andhydropic degeneration, which is not considered gestational tro-phoblastic disease (Berkowitz et al., 1991; Szulman, 1999). Theterm PHM is used when the molar changes are focal and lessadvanced, and fetal development is preserved. PHM have slowlyprogressive avascular chorionic villi, whereas vascular villi with afunctioning fetal–placental circulation are spared (Shapter andMcLellan, 2001).

Haplotype analysis with polymorphic markers has determinedthat most CHM are AnCHM (Kajii and Ohama, 1977; Wake et al.,1978). The majority have a 46,XX karyotype, resulting from mon-ospermic fertilization of an oocyte with ‘inactivated genome’ (alsoreferred to as ‘empty oocyte’) with duplication of the male haploidgenome contributed by the fertilizing sperm (Figure 1a). Althougha 46,YY conceptus could also be formed from these events, thiskaryotype has never been observed in CHM, suggesting thatabsence of the X chromosome is not compatible with early devel-opment. About 20% of AnCHM result from dispermic fertilizationof an oocyte with an inactivated genome and can have a 46,XY or46,XX karyotype (Szulman and Surti, 1978; Kovacs et al., 1991)(Figure 1b). Most PHM are diandric triploid (69,XXY, 69,XXX or69,XYY) and are formed by dispermic fertilization of a haploid

oocyte, resulting in a conceptus with two paternal and one mater-nal haploid set of chromosomes (Szulman and Surti, 1978; Danielet al., 2001) (Figure 1c). Although PHM are associated withlonger development of a viable fetus, triploidy is a highly lethalchromosomal abnormality and most affected embryos die within afew weeks of conception. Of those surviving longer, more than70% have severe symmetrical intrauterine growth restriction, and93% have structural anomalies including three to four syndactylyof hands, hydrocephalus, heart anomalies and micrognathia(Jauniaux, 1998). Digynic triploidy with two maternal haploid setsof chromosomes results in a small underdeveloped placenta(McFadden and Kalousek, 1991). Interestingly, ovarian teratomas,which are tumours that originate from parthenogenetically acti-vated female germ cells, contain a wide variety of tissues butnever trophoblast (Varmuza and Mann, 1994). These data clearlysupport an imprinting effect wherein the parental genomes con-tribute unequally to the development of the placenta: paternallyexpressed genes promote trophoblast proliferation and differentia-tion, and maternally expressed genes are essential to counterbal-ance this effect (Falls et al., 1999).

BiHMs

Kindreds wherein several women have recurrent HM have nowbeen reported (Ambani et al., 1980; Kircheisen and Schroeder-Kurth, 1991; Sunde et al., 1993; Seoud et al., 1995; Helwani et al.,1999; Moglabey et al., 1999; Sensi et al., 2000; Fisher et al., 2002;Judson et al., 2002; Al-Hussaini et al., 2003; Fallahian, 2003;Hodges et al., 2003; Panichkul et al., 2005). Most molar gesta-tions of these affected women are CHM, but some PHM havebeen described. In three of the reported pedigrees (Ambani et al.,1980; Sunde et al., 1993; Seoud et al., 1995; Fallahian, 2003;Fisher et al., 2004), the women also had liveborn offspring, but inthe other families and some sporadic cases of recurrent HM, theyonly have large numbers of consecutive HM pregnancies (Wu,1973; Ozalp et al., 2001; Judson et al., 2002; Al-Hussaini et al.,2003; Fisher et al., 2004; Panichkul et al., 2005). Using shorttandem-repeat markers spread throughout the genome, Helwaniet al. (1999) studied the parental genomic contribution of molarpregnancy tissues from affected women in a large consanguineouspedigree described by Seoud et al. (1995). They established thatthe pregnancies were diploid with one paternal and one maternalallele at each of the informative loci (Figure 1d). Using differentmethods of analysis, Sunde et al. (1993) had already found sup-port for maternal and paternal genomic contribution in the recur-rent HM of another member of this pedigree (Helwani et al.,1999). Hence, although phenotypically these pregnancies werelike AnCHM (Helwani et al., 1999) or diandric PHM (744; Sundeet al., 1993), they were biparentally inherited (BiHM). This obser-vation led the authors to suggest that the HM phenotype may becaused by the deregulation of one or more imprinted genes inthose pregnancies (Sunde et al., 1993; Helwani et al., 1999).

Evidence for an autosomal recessive mutation in women with BiHM

The familial cases of BiHM are extremely rare, but the inheritancepattern in several reported pedigrees support that BiHM is an auto-somal recessive Mendelian disorder (Ambani et al., 1980; Kircheisen

Figure 1. Genetics of hydatidiform moles (HMs). (a) Most sporadic completeHMs (CHMs) are androgenetic (AnCHM), resulting from monospermic fertili-zation of an anuclear oocyte with duplication of the paternal haploid genome.(b) About 20% result from dispermic fertilization of an anuclear oocyte.(c) Most partial HMs are androgenetic triploid with two paternal and one mater-nal haploid set of chromosomes. (d) Recurrent HMs are biparentally inherited(BiHM) with one maternal and one paternal haploid set of chromosomes.



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and Schroeder-Kurth, 1991; Seoud et al., 1995; Sensi et al., 2000;Al-Hussaini and Abdel-Alim, 2001; Ozalp et al., 2001; Fisheret al., 2002; Judson et al., 2002; Al-Hussaini et al., 2003; Fallahian,2003; Hodges et al., 2003; Slim et al., 2005). The family structureof these kindreds often shows consanguinity in the parents of theaffected women as well as between these women and their part-ners. If the BiHM would have resulted from an autosomal reces-sive mutation in the conceptus, transmitted by heterozygote carrierparents, only 25% of pregnancies would be expected to be affec-ted. In the majority of described pedigrees (8/11), the pregnanciesof affected women are only confirmed HMs (Al-Hussaini et al.,2003), spontaneous miscarriages that are not all pathologicallyverified or occasionally a stillbirth. In others (3/11), there aresome normal pregnancies interspersed with the BiHM (Ambaniet al., 1980; Sunde et al., 1993; Seoud et al., 1995; Fallahian,2003; reviewed in Fisher et al., 2004). Furthermore, in some fam-ilies, a few of the affected women have recurrent molar pregnan-cies with different partners, who themselves were fathers ofhealthy offspring from previous or subsequent relationships(Fisher et al., 2000; Al-Hussaini et al., 2003). These combinedobservations indicate that the women themselves are affected withthe autosomal recessive mutation that causes the recurrent BiHMand that the paternal genotype does not contribute to the pathogen-esis. However, it is currently not well understood why in somepedigrees, not all pregnancies of these women are BiHM (Slimet al., 2005).

The first linkage study to map the gene mutated in women withBiHM was performed in a large Middle-Eastern pedigree withextensive consanguinity (Seoud et al., 1995) as well as a secondnorthern European pedigree with affected sisters (Kircheisen andSchroeder-Kurth, 1991). These genetic linkage studies revealedthat the affected women were homozygous by descent for a 15.2 cMregion on chromosome 19q13.3-19q13.4, flanked by markersD19S924 and D19S890 (Moglabey et al., 1999). This interval wasfirst narrowed by 2.8 cM at the telomeric end in an unrelated fam-ily (Sensi et al., 2000) and confirmed in a fourth familial case(Fisher et al., 2002). A more recent study refined the candidateregion to a 1.1 Mb interval in 19q13.42 between centromericmarker D19S418 and a new marker at the telomeric end(AAAT11138) (Hodges et al., 2003). Our own data on four newlydescribed Middle-Eastern pedigrees are consistent with this regionbeing a major candidate locus (Panichkul et al., 2005). In otherpedigrees, linkage to chromosome 19q13.42 could not be estab-lished, supporting that there is genetic heterogeneity for this con-dition. Additional candidate loci for these other families have notbeen reported (Judson et al., 2002; Slim et al., 2005).

The original 15.2 cM critical interval encompassed theimprinted PEG3/ZIM2 locus (paternally expressed gene 3/zinc-finger gene 2 from imprinted domain) (Kim et al., 2000; Van denVeyver et al., 2001). PEG3 is the human homologue of theimprinted mouse Peg3 gene, is highly expressed in placenta and issilenced from the maternal allele in both human and mouse(Kuroiwa et al., 1996; Hiby et al., 2001). Along with X-linkedloci, Peg3 has been implicated in the placental hypertrophy foundin the inter-species hybrid crosses between the monogamousPeromyscus polionotus (PO) and the polyandrous Peromyscusmaniculatus (BW) mice (Vrana et al., 1998, 2000). The subse-quent fine mapping has now entirely excluded this imprinted locusfrom the candidate region (Hodges et al., 2003; Panichkul et al.,

2005). However, altered imprinting of PEG3 may still contributeto the placental phenotype of the BiHM via a mutation in a trans-acting factor that deregulates maternal epigenetic marking (Judsonet al., 2002; El-Maarri et al., 2003) of this and other imprintedgenes (see below).

BiHM have a global defect in reprogramming or maintenance of maternal imprinting marks

Most imprinted genes lie in clusters of interspersed maternally andpaternally expressed genes, which often contain cytosine–guanine(CpG) islands, groups of CpG dinucleotides. Cytosine residueswithin CpG dinucleotides can become methylated at position 5 ofthe pyrimidine ring. CpG islands located in imprinted gene clus-ters are marked by differentially methylated regions (DMRs) oneither the maternally or the paternally inherited chromosome, andthe disruption of DNA methylation at such DMRs has been usedto diagnose patients with defects in genomic imprinting (Kubotaet al., 1996). CpG-island methylation is typically associated withsilencing in cis of the nearby gene, but some DMRs act as long-range imprinting control centres and may show more complexrelationships between methylation and gene expression. In regionswith more than one regulatory DMR, the primary imprints whichare established in the developing gametes may influence the CpGmethylation status of secondary imprints. Secondary imprints canoccur from the post-fertilization to the blastocyst stage (Weinsteinet al., 2004).

Because genomic imprinting was implicated in the pathogenesisof BiHM, two groups have evaluated CpG methylation of DMRsat several imprinted loci in tissues from a total of five BiHM preg-nancies (Judson et al., 2002; El-Maarri et al., 2003). Judson et al.(2002) studied tissues derived from a patient with familial recur-rent BiHM that did not map to the 19q13.42 region. In the secondstudy, two tissues were obtained from the original pedigree thatdemonstrated linkage to 19q13.42, whereas two were derivedfrom sporadic BiHM (El-Maarri et al., 2003).

Abnormal CpG methylation at imprinted genes after passagethrough the maternal germline was found by both, but subtle dif-ferences were seen at specific loci. Judson et al. (2002) found lossof cytosine methylation in BiHM of the DMRs at KCNQ1OT1,PEG1, PEG3/ZIM2 and 5¢ SNRPN that are normally methylatedon the maternal allele and unmethylated on the paternal allele.Similarly, El-Maarri et al. (2001) demonstrated a loss of methyla-tion at the PEG3 and 5¢ SNRPN DMRs (Figure 2a). In the mouse,all of these sites are primary imprints that normally become meth-ylated in the oocyte. In humans, it is not clear whether 5¢ SNRPNis a primary DNA methylation imprint or is established as a sec-ondary imprint in the embryo (El-Maarri et al., 2001). Single-nucleotide polymorphism analysis of the 5¢ SNRPN DMR on DNAfrom parents and from the BiHM revealed that CpG methylationwas indeed lost from the maternal allele (El-Maarri et al., 2003).

CpG methylation at a subset of the DMRs at the GNAS1imprinted locus was also examined (Figure 2b). GNAS1 encodesthe heterotrimeric G protein α-subunit, GSα, the overlappinglonger XLαs protein, and the unrelated chromogranin-like proteinNESP55. GNAS1 has a complex structure and imprinting pattern(for review, see Beaudet, 2004; Weinstein et al., 2004). Recentdata suggest that there are two imprinting control regions atGNAS1 (Bastepe et al., 2005; Liu et al., 2005a), and we will



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discuss the reported findings at the analysed GNAS1 DMRs inBiHM in this context. GSα is biparentally expressed in most tis-sues but paternally silenced in some (Weinstein et al., 2004). TheDNA at the GSα promoter at exon 1 is unmethylated on both alle-les, but in mice there is differential histone H3 lysine 4 methyla-tion (H3K4me) at this exon (Sakamoto et al., 2004). The primaryimprinting mark for tissue-specific paternal imprinting of GSα isthe 2.5 kb upstream exon 1A. CpG methylation of its maternalallele is established in oogenesis. The unmethylated paternal alleleexpresses a non-coding RNA (1A or A/B transcript) that may parti-cipate in the regulation of imprinting of GSα but not of the otherGNAS1 transcripts (Williamson et al., 2004; Liu et al., 2005a).The latter depends on the other primary maternal methylationimprinting mark, 47 kb upstream, at the NESPAS/XLas promoterwhich controls the expression of the XLas mRNA and NESPAS,an antisense RNA to NESP55 (Coombes et al., 2003). The firstexon of XLas (35 kb upstream of GSα exon 1) is also methylatedon the maternal allele, but this is established later in developmentthan at the NESPAS/XLas promoter, and some randomness inmethylation is seen in mouse blastocysts (Liu et al., 2000). TheXLas mRNA and the NESPAS antisense RNA are transcribed from

the unmethylated paternal allele (Weinstein et al., 2004). There isevidence from the analysis of mouse models as well as DNA frompatients with primary pseudohypoparathyroidism type IB thatthere can be dissociation between the imprinting at the first exonof XLas and the NESPAS/XLas promoter (Liu et al., 2005b). TheNESP55 DMR is methylated on the paternal chromosome andunmethylated and transcribed from the maternal chromosome.This is a secondary imprint, established in post-implantationdevelopment that appears co-regulated with and dependent on theprimary imprinting mark at NESPAS/XLas (Weinstein et al.,2004).

Judson et al. found that exon 1A is unmethylated in BiHM,which is as expected if this is a primary maternal imprint that failsto be established. A few of the control chorionic villus samples (2/10)and 1/3 AnCHM showed some aberrant methylation patterns. Thisis currently difficult to explain but does not detract from the sig-nificance of the finding that there is a failure to establish a primarymaternal methylation mark at this exon in BiHM. The NESPAS/XLas promoter also behaved as expected, with loss of the primarymaternal methylation mark, whereas the data at the first exon ofXLas showed an unexpected variable mixed pattern of methyla-tion. This is not completely understood but could be related to thefact that this is a later developing and not primary methylationmark (see above). The NESP55 DMR was analysed in both studiesand showed complete methylation on both alleles, i.e. a paternalepigenotype. Polymorphism analysis confirmed that the gain ofmethylation was on the maternally inherited allele (El-Maarriet al., 2003). Judson et al. concluded that this gain occurred,because maternal lack of methylation at the NESP55 DMR (aswell as presence of methylation at the NESPAS/XLas DMR) is asecondary mark dependent on the primary maternal methylationmark at exon 1A, while El-Maarri et al., who also found somematernal methylation gain at the H19 DMR (discussed below),concluded that there is a primary gain of methylation. Eventhough more recent data now show that DMR methylation at NES-PAS/XLas, at least in mice, appears to be a primary mark withwhich NESP55 is co-regulated and independent of exon 1A meth-ylation (see above), both interpretations are still plausible.

Both studies also examined the H19 DMR, which carries aprimary CpG methylation imprint on the paternal allele (Figure 2c).In the familial BiHM that was not linked to 19q13.42, CpG meth-ylation at H19 was completely normal (Judson et al., 2002), asexpected if the underlying defect would only cause failure toestablish primary maternal methylation imprints. However, incontrast to the Judson study, El Maarri et al. found that the H19DMR demonstrated variable CpG methylation (El-Maarri et al.,2003). Two of four BiHM samples aberrantly acquired methyla-tion on the maternal allele: one showed a mixed variable patternand a sporadic BiHM had normal methylation. These results sug-gested to the authors that there is not only a loss of methylation onprimary maternally methylated alleles but also a gain of primarypaternal-specific methylation on maternal alleles in the samplesthey analysed.

The DNA methylation defect in BiHM appears to be specific toimprinted genes and not the result of a global disruption of DNAmethylation, because there is normal methylation at non-imprintedCpG sites (Judson et al., 2002) and at an X-linked gene that is sub-ject to X chromosome inactivation (El-Maarri et al., 2003).Expression analysis by immunohistochemistry on BiHM molar

Figure 2. Abnormal DNA methylation patterns at imprinted genes in bipa-rentally inherited hydatidiform mole (BiHM). The left of each panel repre-sents the normal DNA methylation patterns at the analysed loci; the rightrepresents the methylation patterns found in BiHM. The maternal allele is onthe top and in pink; the paternal allele is on the bottom and in blue. Open andfilled circles represent unmethylated and methylated cytosine–guanine (CpG)sites, respectively. Different shades of hatching indicate complete or partialgain of a paternal epigenotype. (a) Differentially methylated regions (DMRs)at PEG3, 5¢ SNRPN, PEG1 and KCNQ1OT1 lose methylation marks and gaina paternal epigenotype (indicated by dark blue hatching) on the maternal allelein BiHM. (b) DMRs at the GNAS1 locus. The exon 1A (1A) and the NESPAS/XLas (AS) promoter DMRs are primary imprints (1st) and normally methyl-ated on the maternal allele. The NESP55 and XLaS first exon (XLaS) DMRsare secondary imprints (2nd) and are normally unmethylated and methylated,respectively, on the maternal allele. Black arrows indicate the co-regulation ofsecondary with primary imprint. Hatched arrow indicates that XLas methyla-tion occurs later than AS methylation and can sometimes be dissociated fromthe methylation status of AS. Hence, if the AS DMR is unmethylated as inBiHM, the maternal NESP55 DMR becomes methylated. XLaS is expected tobe unmethylated but had variable methylation on both alleles in BiHM (*).(c) The H19 DMR, normally only methylated on the paternal allele, showsdifferent results between studies: one study found that it remains unmethyl-ated (Judson et al., 2002), whereas the second study found variable patternsof methylation: ranging from normal absence of maternal methylation tovariable increased methylation (partial lighter hatching) to hypermethylation(El-Maarri et al., 2003).



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trophoblast tissues for p57KIP2, which is normally expressedfrom the maternally inherited copy and silenced from the pater-nally inherited copy, showed markedly decreased expression(Fisher et al., 2002; Hodges et al., 2003). This is also expected,because imprinted expression of this gene depends on the maternalmethylation of the KCNQ1OT1 DMR. Finally, the DNA methyla-tion status of investigated DMRs at imprinted genes is normal inthe mothers of the affected pregnancies (El-Maarri et al., 2005),further supporting the observation that the imprinting defects arisede novo during female gametogenesis.

What might cause the different results at the H19 DMR betweenthese studies? One explanation might be genetic heterogeneity: theinvestigated tissues were from BiHM that were not linked to19q13.42 (Judson et al., 2002), linked to this region (El-Maarriet al., 2003), or from sporadic BiHM cases of unknown geneticlinkage (El-Maarri et al., 2003). Thus, it is possible that differentbasic components of the machinery that establishes or maintainsimprinting marks may be disrupted in different cases: one result-ing in loss of DNA methylation at DMRs of primary maternalmethylation imprints, whereas another also causes gain of DNAmethylation on the maternal allele of DMRs that have a primarymethylation imprint on the paternal allele. There may also beinfluence from genetic modifiers or environmental factors on thephenotypic expression of the underlying defect (Slim et al., 2005).Other potential factors explaining the different results could be theuse of short-term culture of the studied trophoblast (Judson et al.,2002) or unreported pathological confirmation of the diagnosis insporadic cases. This information was not provided by El-Maarriet al. (2003), and one of the BiHMs in their study appeared tohave normal methylation marks at several of the analysed loci.Hence, although those differences in the results of methylationanalysis at imprinted loci between the two studies should ideallybe confirmed on additional independent samples, they may haveimplications for hypotheses on the nature and timing of the molec-ular defects resulting in BiHM.

Chromatin dynamics at imprinted genes

It has become clear in recent years that allele-specific cytosinemethylation at CpGs of DMRs is only one aspect of the multiplechromatin modifications associated with imprinted gene expres-sion. The methylated DMRs of imprinted loci have other hall-marks of repressive chromatin, such as post-translationalmodifications of core histone proteins that make up the nucleo-somes around which the DNA double helix is wound. MethylatedDMRs also show hypoacetylation of histones H3 and H4, dimeth-ylation of H3 at lysine 9 (H3K9me2) and, in at least one case, tri-methylation of H3 at lysine 27 (H3K27me3). The converse hasbeen shown for the unmethylated alleles of DMRs, which containhyperacetylated H3 and H4 and H3K4me, all of which are hall-marks of transcriptionally permissive DNA (Fournier et al., 2002;Perk et al., 2002; Soejima and Wagstaff, 2005). Experiments inplants (Arabidopsis) and yeast (Neurospora crassa) indicate thatin these organisms, the repressive histone modification H3K9me2may induce cytosine methylation. The absence of histone modifi-cations in mutant mouse models with inactivated DNA methyl-transferase enzymes and the reduced DNA methylation in micewith inactivation of the major H3K9 methyltransferases, Suv39h1and Suv39h2, suggests that this relationship is conserved in

mammals (Soejima and Wagstaff, 2005). Furthermore, H3K9methylation has been shown to be necessary for CpG methylationand allele-specific expression at Snrpn (Xin et al., 2003). Theobservation that for some genes, imprinted expression in placentaoccurs without DNA methylation, but in the presence of H3K9methylation, has led to the suggestion that H3K9 methylation maybe the initial chromatin modification, sufficient to maintainimprinting in the short lifespan of the placenta, but requiring stabi-lization by DNA methylation in the longer-lived embryo proper(Lewis et al., 2004; Morgan et al., 2005). This is interesting andimplies that in the BiHM pregnancies, deregulated DNA methyla-tion at imprinted loci could be secondary to a defect of anothertype of chromatin modification essential for imprinted geneexpression. Unfortunately, histone modifications have not yetbeen studied in BiHM. Such experiments may be difficult toaccomplish considering the scarceness and archival nature of mostsamples.

Molecular mechanisms and timing of reprogramming of imprinted genes

Although imprinting at specific loci is not universally conservedbetween humans and mice [e.g. Igf2r and Mash2/Ascl2 areimprinted in mouse, but not in humans (Riesewijk et al., 1996;Miyamoto et al., 2002)], the basic molecular mechanisms and rel-ative order of events are most likely conserved between the twospecies. Most of our current knowledge about the critical stepsrequired to correctly establish and maintain imprinted geneexpression derives from analysis of various mouse models but ispertinent to this review.

There are four critical steps in this process that overlap withdramatic genome-wide DNA methylation and epigenetic modifi-cations of chromatin-associated proteins (Figure 3): (i) erasure ofprevious imprints in the developing germ cells, (ii) re-establishmentof parent-specific imprints either in the germ cells or in the earlyembryo, (iii) postzygotic maintenance of imprints in the preim-plantation embryo and (iv) maintenance of imprints after lineagecommitment of the embryonic and extra-embryonic tissues.

In the mouse, primordial germ cells (PGCs) are set aside from theepiblast at 7.2 dpc (Hajkova et al., 2002), proliferate and begin tomigrate into the genital ridge by 8.5 dpc, where they start arriving atabout 10.5–11.5 dpc. This timing corresponds to the 5- to 11-weekhuman embryo (Onyango et al., 2002). During PGC migrationfrom the genital ridge, complete demethylation of all CpG dinu-cleotides, including those at imprinted DMRs, occurs in both maleand female germ cells (Labosky et al., 1994; Tada et al., 1997,1998; Kato et al., 1999; Hajkova et al., 2002; Lee et al., 2002;Onyango et al., 2002; Sato et al., 2003; Yamazaki et al., 2003).This process appears to be encoded within the gamete itself and iscompleted by 13.5 dpc; the speed by which it occurs suggests anas yet unidentified active demethylase enzyme (Hajkova et al.,2002; Morgan et al., 2005). The number of germ cells increases toabout 25 000 at 13.5 dpc, the time when gender-specific differ-ences become apparent in the primitive gonads and female PGCsare arrested in meiotic prophase I, whereas male PGCs undergomitotic arrest in G0 (Labosky et al., 1994; Hajkova et al., 2002).Subsequent re-establishment of the epigenetic marks associatedwith parent-of-origin–specific imprinting initiates once the germcells start differentiating (Ferguson-Smith and Surani, 2001) and



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occurs earlier in the male gamete than in the female gamete withvariable timing for specific imprinted genes in gametes of bothsexes (El-Maarri et al., 2001; Obata and Kono, 2002; Li et al.,2004; Lucifero et al., 2004). In male gametes, it occurs at the pros-permatogonia stage before the start of meiosis when the cells arein G0 arrest (Davis et al., 1999, 2000; Sato et al., 2003) and is usu-ally complete by 18.5 dpc but can continue until the mature spermstage. In contrast, female gametes initiate remethylation later (afterbirth in the mouse) in the growing oocyte, and this may continueafter fertilization in the female pronucleus of the zygote (Obataet al., 1998; Davis et al., 2000; El-Maarri et al., 2001; Ferguson-Smith and Surani, 2001; Lucifero et al., 2004).

A few essential proteins are known to be necessary for the cor-rect reprogramming of DNA methylation imprints. Conditionalinactivation in germ cells of the de novo DNA methyltransferases,Dnmt3a and Dnmt3b, has revealed that Dnmt3a is important forboth maternal and paternal imprinting (Kaneda et al., 2004) whileDnmt3b may primarily regulate DNA methylation of retrotrans-posons and pericentromeric heterochromatic regions. Mouse DNAmethyltransferase 3-like protein (Dnmt3l), a homologue ofDnmt3a and Dnmt3b that lacks the catalytic domain, is requiredfor the establishment of imprinting in female, but not male gam-etes. In the male, Dnmt3l is necessary for the methylation of retro-transposons and pericentromeric heterochromatic regions inprospermatogonia, and its absence leads to meiotic arrest and steril-ity (Bourc’his et al., 2001; Hata et al., 2002; Bourc’his and Bestor,

2004; Webster et al., 2005). Dnmt3l directly interacts via itsC-terminal domain with the catalytic regions of various isoformsof Dnmt3a and Dnmt3b. In cell-culture experiments, interactionsenhance the efficiency of Dnmt3a and Dnmt3b to methylate DNAin vitro and in vivo and may participate in targeting these proteinsto the DMRs of imprinted genes slated for de novo methylation(Chedin et al., 2002; Suetake et al., 2004; Chen et al., 2005). Onlythe Dnmt3a2 and the Dnmt3b1 isoforms appear co-expressed withDnmt3l during the critical stages of germ cell development, butthe relevance of this co-expression for asymmetric DNA methyla-tion of imprinted DMRs is unknown.

In the oocyte, not all imprinted genes acquire their methylationmarks at the same time. For example, the Peg3 DMR acquiresDNA methylation earlier than the Peg1 DMR (Obata and Kono,2002; Lucifero et al., 2004). Within the developing oocyte, thereis also asynchrony between the parental alleles: gametes with thematernally inherited Snrpn allele acquire DNA methylation fasterthan those with the paternally inherited Snrpn allele (Luciferoet al., 2004). This observation supports the existence of other, notyet characterized, epigenetic modifications at imprinted loci thatendow them with an epigenetic parent-of-origin–specific memorydespite the total erasure of DNA methylation in PGC.

During the first cleavage divisions, the genomes are once moresubject to massive changes in chromatin organization and DNAmethylation. Immediately after fertilization, the paternal pro-nucleus undergoes reprogramming whereby the DNA-bound

Figure 3. The establishment and maintenance of imprinting marks. The four critical steps are represented by black double arrows above the graph: 1, erasure; 2, re-establishment of parent-specific imprints (hatched line indicates that some secondary imprints can be set in the embryo after fertilization); 3, postzygotic mainten-ance of imprints; 4, maintenance of imprints after lineage commitment. The graph shows the levels of cytosine–guanine (CpG) (DNA) methylation over time. Equalevents in paternal and maternal genomes are represented as black lines. Unequal events are represented as coloured lines or coloured text (male or paternal genomein blue; female or maternal genome in pink). Solid coloured lines represent whole-genome events, and dotted coloured lines represent events at imprinted differen-tially methylated regions. Genome-wide histone modifications are listed by their abbreviations at their approximate timing (see text for details). The yellow arrow-head indicates the variability of the timing at this stage, as progress to the next phase is dependent on fertilization. Red brackets and arrows indicate the most likelytiming of the putative BiHM mutation. The hatched arrow represents a low possibility of a trophoblast-specific mutation.



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protamines are exchanged for highly acetylated histones, whichimmediately acquire H3K4, H3K9 and H3K27 monomethylation.During the transition from paternal protamines to maternal his-tones, the paternal genome rapidly undergoes active demethyla-tion of CpG dinucleotides within the first 6 h after fertilization,except at methylated DMRs of imprinted genes, which appear pro-tected and remain methylated. The maternal genome also becomesdemethylated, but much slower and over several cell divisions, ina manner dependent on DNA replication. This suggests a passiveprocess, caused by the lack of maintenance methylation. Recentdata also indicate that different histone modifications in paternaland maternal genomes (Kourmouli et al., 2004; Santos et al.,2005) might explain their different timing of global demethylationin the preimplantation embryo. Maternally methylated DMRs atimprinted genes are also protected during global demethylation ofthe maternal genome (Morgan et al., 2005). Mice with a mutationthat selectively removes Dnmt1o, encoding an oocyte-specificisoform of the primary maintenance methyltransferase Dnmt1 thatis only in the nucleus at the 8-cell stage of embryonic development(Mertineit et al., 1998), show loss of methylation of maternallymethylated DMRs with consequent bi-allelic expression of mater-nally imprinted genes. This supports that Dnmt1o is essential forprotecting the maternally methylated DMRs during globaldemethylation of the genome in the preimplantation embryo(Howell et al., 2001). However, because the mutation in severalBiHM families does not map to the genomic region containingDNMT1, it is likely not sufficient. Hence, all the mechanisms ormolecular modifications that maintain the differential methylationof imprinted DMRs are unknown.

Hypotheses for the molecular defect underlying the formation of BiHM

Current data show abnormal methylation of maternally methylatedDMRs in BiHM and support that the defect is not in erasure butrather in the establishment or maintenance of maternal imprints(Fisher et al., 2002; Judson et al., 2002; El-Maarri et al., 2003). Itcannot be unequivocally excluded that maternal imprints aremaintained in the early embryo but are subsequently lost in thetrophoblast as it proliferates along its molar phenotype. However,this has been considered a less likely hypothesis, because there isrelative homogeneity of the pathological and molecular findings inaffected CHM samples (El-Maarri et al., 2003, 2005). AlthoughJudson et al. (2002) concluded that the primary defect may lie in afailure to acquire or maintain primary DNA methylation imprint-ing marks in the maternal germ line, El-Maarri et al. (2003) intheir study of imprinting in different BiHM samples also foundevidence for a gain of methylation on the maternally inheritedalleles of a primary paternal methylation mark. Samples that canbe analysed are scarce, and, until the gene has been found and ani-mal models can be created, this will remain an open question.

What are the most likely attributes of the candidate gene(s)mutated in women with recurrent BiHM? They should beexpressed either in the female gamete during the critical windowwhen imprinting marks are established or in the preimplantationembryo when imprinting marks have to be protected fromgenome-wide reprogramming and CpG demethylation. Theknown pedigrees strongly suggest a true maternal-effect mutation.Hence, even though the candidate gene may encode a protein that

carries out its function in the zygote or the embryo during the first-cleavage divisions, the mRNA should be produced from thematernal genome. The absence of a male reproductive phenotypein the pedigrees does not preclude the gene’s expression in malegametes but argues that its function is not essential for their nor-mal development and function.

For those cases where the mutation does not map to the majorlocus on chromosome 19q13.42, one can only evaluate candidategenes based on their known role in imprinting mechanisms. Anal-ysis of DNMT1o, DNMT3L, DNMT3a and DNMT3B in a BiHMfamily not linked to 19q13.4 did not reveal any mutations (Haywardet al., 2003). This is not surprising considering newer informationon the function of these proteins. DNMT3L has no methyltrans-ferase activity on its own but co-operates with DNMT3A to estab-lish imprinting marks. Although theoretically an excellentcandidate, recent data suggest that DNMT3L also participates inother de novo methylation events, as well as in the establishmentof paternal DNA methylation imprints. DNMT3A is also a de novoDNA methyltransferase for many other regions of the genome(Chen et al., 2004), and its DNMT3A2 isoform is expressed ingerm cells and specifically affects imprinting, but not in a mater-nal allele-specific manner (Kaneda et al., 2004). Because theestablishment and maintenance of DNA methylation imprints maybe preceded by the methylation of specific lysine residues on corehistones, it is possible that proteins that perform these histonemodifications or other chromatin remodelling factors are mutatedin BiHM. Yet, the major histone methyltransferases, G9A,SUV39H1 and SUV39H2, are probably not involved, becausethey have a more widespread role in the genome (Tachibana et al.,2002; Rice et al., 2003). A novel histone-modifying enzyme with aunique specificity towards imprinted loci on the maternal genomeor a factor that specifically targets more general histone-modifyingenzymes to these regions would be a good candidate.

The currently established minimal candidate region on chromo-some 19q13.4 contains more than 60 transcripts. By in silico ana-lysis, a subset is uniquely or primarily expressed incomplementary DNA libraries from germ cells, embryonic stemcells or ovaries. The presence of a transcript in the candidateregion that encodes a protein with a SET domain, which is foundin many known or putative histone methyltransferases, is veryinteresting. However, no mutations in the coding region of thisgene have been identified in women affected with BiHM (Shaoand Van den Veyver, unpublished data). Furthermore, the murinehomologue of this gene has been shown to be a H4K20 methyl-transferase that primarily acts on pericentromeric heterochromatin,and a role of this protein for histone methylation at imprinted regionshas not been described (Schotta et al., 2004; van der Heijden et al.,2005).

Clinical implications for patients with recurrent HM

BiHM have a similar clinical course and management as AnCHM.Patients with CHM classically present with vaginal bleeding,abnormally high levels of maternal serum HCG and uterine sizegreater than expected for the gestational age. The wide use ofultrasonography and serum HCG levels now allows for early diag-nosis in asymptomatic women. The sonographic features of a uterinecavity filled with multiple sonolucent areas of varying size andshape (‘snowstorm’ appearance) and absence of embryonic or



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fetal structures combined with an elevated serum HCG level arehighly indicative of the presence of CHM, even before the finalhistopathological diagnosis is confirmed (Jauniaux, 1998). Medi-cal complications of CHM include pregnancy-induced hyperten-sion, anaemia, hyperemesis, thyrotoxicosis, embolization and thedevelopment of ovarian theca lutein cysts. Treatment optionsrange from the simple suction evacuation with follow up to chem-otherapy, hysterotomy or hysterectomy depending on the patient’sage, risk factors and completion of their family. Patients withCHM have an increased risk of persistent gestational trophoblasticdisease, invasive mole and choriocarcinoma development, requir-ing treatment with methotrexate or combination chemotherapy.Although the total number of studied cases is limited, our ownobservations of women from two consanguineous pedigrees andother published reports are consistent with an increased risk forinvasive trophoblastic disease after a BiHM that is at least as highas in sporadic AnCHM (Al-Hussaini et al., 2003), indicating thattheir clinical courses are largely comparable. These observationsalso support that it is not the apparent homozygosity for recessivemutations in AnCHM that predisposes to malignant transforma-tion but rather the unbalanced expression of imprinted genes inboth AnCHM and BiHM.

For patients with recurrent HM, reproductive options are cur-rently very limited. In some pedigrees, pregnancies with normaloutcomes have been reported in affected women with BiHM(Moglabey et al., 1999), but in the majority of families, includingthose we study, there are no normal pregnancy outcomes. Attemptsto avert recurrent molar pregnancies with IVF combined withdonor sperm and preimplantation genetic diagnosis have had poorsuccess (Edwards et al., 1990, 1992; Reubinoff et al., 1997). In thereported cases, the affected pregnancies were not genotyped forbiparental inheritance, as most were done before the reports ofbiparental inheritance of recurrent HMs. In one case, thereappeared to be a higher incidence of triploid embryos (Pal et al.,1996) based on morphological analysis of the fertilized oocytes,which indicated that they contained three pronuclei. This suggeststhat some cases of recurrent HM may have a different aetiologythan BiHM, but it remains to be proven whether they result from adefect in the paternal gametes for which it would be valid toattempt insemination with donor sperm as a therapy. Others havesuggested that because some affected women have normal preg-nancies interspersed with the recurrent moles, the disease may beunder similar influences from environment or genetic modifiers assporadic AnCHM and propose that once identified, these factorscould potentially be modulated and open avenues for treatment inthe future (Slim et al., 2005). Until then, patients with highly recur-rent BiHM, who may repeatedly attempt spontaneous pregnancies(Al-Hussaini et al., 2003), should be counselled about the risk forpersistent trophoblastic disease or invasive choriocarcinoma witheach molar pregnancy. Once the underlying genetic defect is identi-fied and is unequivocally proven to be oocyte-specific, a potentialalternative strategy for those women with recurrent BiHM could beIVF with donor oocytes, especially in those families where affectedwomen have only HMs and never a normal pregnancy outcome.

Conclusion

BiHM is an autosomal recessive disorder with sex-limited expres-sion that only affects females. More than one genetic locus may be

involved, and the putative mutated genes most likely encode trans-acting factors required for the correct establishment of imprintingmarks in the maternal germline at multiple imprinted loci or for theirmaintenance after fertilization. Identification of these genes will bedifficult because of the small number of affected families andwomen that can be studied and the lack of a known animal model forBiHM. Yet, we predict that their discovery will have ramificationsfar beyond the identification of the cause for this rare condition. Ithas consequences for the understanding of imprinting dynamics ingametes and preimplantation embryos. It will also benefit our under-standing of a possible link between assisted reproductive technolo-gies and imprinting disorders, as well as the epigenetic disturbancesobserved in cloned embryos and embryonic stem cells.

Acknowledgements

The authors thank Amy Lossie, Scott Dindot and Ying Chuck Kou forcritical reading and helpful suggestions during preparation of thismanuscript. This work is supported by a grant from the National Insti-tute for Child Health and Human Development to I.B.V. (HD045970).

Note added in proof

Murdoch et al. (2006) very recently reported that autosomal recessivemutations in the NALP7 gene on chromosome 19q13.4 have been iden-tified in the women with recurrent hydatidiform moles whose mutationmaps to this region. Interestingly, NALP7 is widely expressed andbelongs to a larger family of genes that encode proteins with a role inthe intracellular inflammatory response to bacterial infection and apop-tosis. The known function of NALP7 does not suggest a direct role inthe molecular events that regulate establishment or maintenance ofimprinting in the germline or early embryo. Murdoch et al. (2006)therefore propose that the disruption of epigenetic markings atimprinted genes seen in recurrent hydatidiform moles may be secondaryto a general defect in the oocyte growth and maturation around thetime imprinting marks are reset. The possibility of an abnormal inflam-matory response further suggests that this gene as well as genes encod-ing other members of this protein family could play a wider role inreproductive failure. This would be supported by the fact that somewomen with mutations in NALP7 not only have biparental hydatidiformmoles, but also have other reproductive loss and intrauterine growthretardation. This exciting discovery opens up an entire area of researchinto the causes of various forms of reproductive loss and must make usreconsider potential molecular mechanisms for gene–environment inter-actions that can result in disrupted genetic imprinting. Because there isgenetic heterogeneity in recurrent hydatidiform moles, it remains to bedetermined whether other causative genes have similar functions.

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Submitted on August 18, 2005; resubmitted on December 3, 2005; accepted onJanuary 3, 2006



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Biparental hydatidiform moles: a maternal effect mutation affecting imprinting in the offspring