Download Repetitive complete hydatidiform mole can be biparental in origin

Human Reproduction vol.15 no.3 pp.594–598, 2000
Repetitive complete hydatidiform mole can be
biparental in origin and either male or female
R.A.Fisher1,4, R.Khatoon1, F.J.Paradinas2,
A.P.Roberts3 and E.S.Newlands1
1Department
of Cancer Medicine, Division of Medicine, Imperial
College School of Medicine, Charing Cross Hospital, Fulham
Palace Road, London W6 8RF, 2Department of Histopathology,
Division of Investigative Sciences, Imperial College School of
Medicine, Charing Cross Hospital, Fulham Palace Road,
London W6 8RF and 3Directorate of Women’s Services, Newham
General Hospital, Glen Road, Plaistow, London E13 8SL, UK
4To
whom correspondence should be addressed
Complete hydatidiform mole (CHM) is an abnormality in
pregnancy due to a diploid conception which is generally
androgenetic in origin, i.e. all 46 chromosomes are
paternally derived. We have examined the genetic origin
of repetitive hydatidiform moles in a patient having three
CHM by two different partners, and no normal pregnancies. Using fluorescent microsatellite genotyping, we have
shown all three CHM to be biparental, rather than
androgenetic, in origin. Examination of informative
markers for each homologous pair of chromosomes, in
two of the CHM, failed to reveal any evidence of unipaternal disomy, suggesting that the molar phenotype might
result from disruption of normal imprinting patterns due
to a defect in the maternal genome. It has been suggested
that intracytoplasmic sperm injection (ICSI), followed by
selection of male embryos, can prevent repetitive CHM;
but examination of sex chromosome-specific sequences in
the three CHM described here, showed that, while two
were female, the first CHM was male. Selection of male
embryos is therefore unlikely to prevent repetitive CHM
in this patient. Our results suggest that the genetic origin
of repetitive CHM should be determined prior to in-vitro
fertilization (IVF) and that current strategies for the
prevention of repetitive CHM may not be appropriate
where the CHM are of biparental origin.
Key words: biparental complete mole/genomic imprinting/IVF/
repetitive mole
Introduction
The premalignant condition, hydatidiform mole (HM), can be
classified on the basis of histological examination and genetic
origin as complete (CHM) or partial (PHM) (Szulman and
Surti, 1978a,b). Genetically CHM are diploid, but abnormal
in that they are usually androgenetic, i.e. all 46 chromosomes
are paternal in origin (Kajii and Ohama, 1977; Wake et al.,
1978). PHM also have two paternal contributions to the nuclear
genome but, in contrast to CHM, also have a maternal
594
contribution and are consequently triploid. A patient who has
had a molar pregnancy is at an increased risk of a subsequent
pregnancy resulting in HM (Bagshawe et al., 1986). Evidence
of molar tissue in the uterus of a patient a few months after
termination of one molar pregnancy may, therefore, be a second
HM. Alternatively, this may represent persistent trophoblastic
disease from the first HM. In this situation genetic analysis
can distinguish between a second, repetitive HM, arising from
a new conceptus, or persistent trophoblastic disease from the
previous HM (Roberts and Mutter, 1994; Fisher, 1997). In
performing genetic analysis of molar tissue in a patient with
a second CHM, 5 months after evacuation of a previous CHM,
we found that, rather than being androgenetic in origin, both
CHM arose from apparently normal, biparental concepti (Fisher
and Newlands, 1998). This patient has subsequently had a
third molar pregnancy. This report describes genetic studies
of the three HM, which confirm that all three are biparental
in origin. The implications for patients undergoing in-vitro
fertilization (IVF) following repetitive HM are discussed.
Materials and methods
Patient
The patient was a 30 year old Asian woman who, during a 4 year
period, had three molar pregnancies terminated by evacuation of the
uterus at 16, 6 and 9 weeks gestation respectively. The patient has
had no other pregnancies. There was no consanguinity between the
patient and her partner and karyotyping did not reveal any chromosomal abnormalities in either the patient or her partner. Molecular
genetic studies were carried out to determine the genetic origin of
each of the three HM.
DNA preparation
DNA was prepared from blood samples of the patient and her
husband using standard techniques. DNA from each HM was prepared
from pathological blocks of formalin-fixed, paraffin-embedded tissue.
In each case molar tissue was identified and microdissected from a
5 µm unstained section of tissue with reference to a consecutive section
stained with haematoxylin and eosin. DNA was then prepared from this
tissue using a modification of a previously described method (Wright
and Manos, 1990). Briefly, the tissue was extracted with octane,
washed with 100% ethanol and dried under vacuum. When dry, the
tissue was incubated in 25 µl of digestion buffer (50 mmol/l Tris
pH 8.5, 1 mmol/l EDTA, 0.5 % Tween 20) containing 200 µg/ml of
proteinase K. After 3 h at 55°C the tube was incubated for 8 min at
95°C to inactivate the proteinase K. The supernatant (1 µl) was then
used as template for polymerase chain reaction (PCR) amplification.
PCR amplification
In order to determine the sex chromosome complement of each of
the three HM, 1 µl DNA from each HM was amplified using both
© European Society of Human Reproduction and Embryology
Biparental complete mole
Table I. Microsatellite polymorphisms identified in DNA from the patient, her partner and the three consecutive CHM. Allele sizes for each polymorphism
are given in bp
Microsatellite
polymorphism
Reference
Chromosome
location
Patient
CHM1
CHM2
CHM3
Partner
Mfd50
D10S89
TH
VWA
D22S264
Weber et al. (1990)
Weber and May (1990)
Polymeropoulos et al. (1991)
Kimpton et al. (1992)
Reed et al. (1994)
7
10
11
12
22
190
146–156
199
138–142
188–198
190–192
150–156
195–199
138–142
188–198
188*–190
144*–146
187*–199
142–146
188–204*
190–192
150–156
195–199
138
188–198
176–192
148–150
195
138–146
188
*Alleles in CHM 2 not present in either the patient or her partner.
Table II. Informative microsatellite polymorphisms in the patient, her partner, CHM1 and CHM3. Allele
sizes for each polymorphism are given in bp
Chromosome
Microsatellite
Patient
CHM1
CHM3
Partner
1
2
3
4
5
6
7
8
9
10
11
12
13
13
14
15
16
17
18
19
20
21
22
X
D1S228
D2S165
D3S1262
D4S415
D5S210
D6S290
Mfd50
D8S552
D9S176
D10S192
D11S925
D12S97
D13S158
D13S170
D14S74
D15S207
D16S516
D17S807
D18S61
D19S220
D20S120
D21S270
IL2RB
DXS996
121–123
141–151
108–116
191–193
219
260
191
169–173
254–260
239–259
269–280
263–273
110–116
137–151
296–298
163–168
282–284
121–123
220–222
277
212
211–217
133
153–159
123–125
151–155
116–118
169–191
217–219
256–260
191–193
173–176
256–260
239–255
269–284
267–273
110–118
ND
298–306
163–170
280–284
119–123
218–222
264–277
212–230
215–217
133–141
159
123–125
151–155
108–118
169–193
219–225
256–260
191–193
173–176
252–254
239–255
269–284
263–267
NI
137–147
294–298
163–164
280–282
119–121
222–224
264–277
212–239
215–217
133–135
128–159
125
155–159
106–118
169
217–225
252–256
177–193
176
252–256
249 –255
284–286
259–267
116–118
147
294–306
164–170
280–288
119
218–224
264–270
230–239
211–215
135–141
128
NI ⫽ not informative; ND ⫽ not done.
X and Y chromosome-specific primers (Witt and Erickson, 1989).
50 ng of DNA from the patient and her partner were amplified with
the same primers as controls.
To determine the genetic origin of each HM, 50 ng DNA from the
patient and her partner and 1 µl DNA from each of the HM was
amplified using five pairs of primers which flank polymorphic
microsatellite repeat (two tetranucleotide and three dinucleotide)
sequences on different chromosomes (Table I). One of each pair of
primers was labelled with the fluorescent dye FAM (blue) or TAMRA
(yellow) Applied Biosystems, Warrington, UK. Following amplification, 5 µl of each PCR reaction product was analysed by
electrophoresis in a 1% agarose gel to assess the yield of product.
PCR products were diluted as appropriate and subsequently resolved
by capillary electrophoresis using an ABI PRISM 310 Genetic
Analyser (Applied Biosystems Ltd). Analysis and sizing of the
microsatellite polymorphisms was performed using ABI PRISM
GeneScan software (Applied Biosystems Ltd).
Further analysis of CHM1 and CHM3 was carried out by amplification of DNA from the maternal, paternal and molar DNA for at least
one polymorphic marker on each of the 22 autosomes and the X
chromosome (Reed et al., 1994) (Table II). Where a marker was
uninformative with respect to the origin of the molar DNA, a further
marker for that chromosome was examined. Primers were labelled
with FAM (blue), TAMRA (yellow), HEX (green) or TET (green)
and analysis of fluorescent products performed using ABI PRISM
GeneScan software.
Results
All three hydatidiform moles showed cistern formation, excess
circumferential trophoblast, karyorrhexis and polyploid villi
characteristic of CHM in early gestation (Paradinas, 1994;
Paradinas et al., 1996) (Figure 1).
The allele sizes of five microsatellite polymorphisms on
different chromosomes are shown in Table I for the patient,
her partner and the three HM. Analysis of DNA from CHM1
and CHM3 showed that in each case one allele for the
microsatellite polymorphisms Mfd50, D10S89 and TH was
maternal in origin while the other allele was inherited from
her partner demonstrating that these concepti were biparental
in origin (Figure 2). For the markers VWA and D22S264,
595
R.A.Fisher et al.
Figure 1. Microscopic appearance of complete hydatifiorm mole
(CHM3), evacuated at 9 weeks gestation, showing the branching
villi with excess trophoblast characteristic of complete hydatidiform
mole (CHM) in early gestation. Haematoxylin and eosin staining.
Bar ⫽ 500 µm; V ⫽ branching villi; T ⫽ excess trophoblast
(arrows).
alleles found in the CHM, while not fully informative, were
compatible with a contribution from both parents. Examination
of the alleles found in CHM2, identified several alleles which
were not present in either parent (Figure 2). However, for each
marker, the results were compatible with one of the two alleles
being maternal in origin showing that CHM2 was also of
biparental origin but arose from a conception with a different
partner to the first and third CHM. In all three HM the relative
heights of the peaks representing each of the alleles in
the molar tissue were consistent with disomy, i.e. a single
maternally derived and a single paternally derived allele. No
evidence of trisomy was observed for any of the polymorphisms
examined.
Analysis of products following amplification with X and Y
chromosome-specific primers showed that tissue from CHM1
was male (Y-positive), while CHM2 and CHM3 were female
(Y-negative) (Figure 3).
Further informative microsatellite markers examined in the
patient, her partner, CHM1 and CHM3 are shown in Table II.
For each marker examined, CHM1 and CHM3 were shown to
be biparental in origin. In CHM1 a single polymorphism of
maternal origin was identified in the molar tissue for markers
on the X chromosome, compatible with a male genotype.
CHM3 had two X-specific alleles, one maternal and one
paternal in origin, confirming a female genotype. No evidence
of unipaternal disomy was found for any of the autosomes or
the sex chromosomes.
Discussion
CHM are generally diploid (Szulman and Surti, 1978a)
and androgenetic in origin (Kajii and Ohama, 1977; Wake
et al., 1978), all 46 chromosomes being derived from the
father. They may be monospermic, arising by fertilization of an
enucleate egg by a single spermatozoon which then doubles to
596
Figure 2. Fluorescently labelled polymerase chain reaction (PCR)
products identified following amplification of the microsatellite TH
in the patient, her partner, and the three hydatidiform moles (HM)
(CHM1, CHM2, CHM3). The patient was homozygous for the
199 bp allele while her partner was homozygous for the 195 bp
allele. The maternal 199 bp allele (shaded) was present in all three
HM. While CHM1 and CHM3 each had the 195 bp allele identified
in the partner, in CHM2 the second, 187 bp, allele was not present
in either the patient or her partner.
Figure 3. Polymerase chain reaction (PCR) products produced
following amplification of DNA with X and Y-chromosome specific
primers in the patient, her partner, and the three hydatidiform mole,
(HM) (CHM1, CHM2, CHM3). A 130 bp, X-specific product was
present in all samples. A 170 bp, Y-specific product was present
only in the partner and CHM1.
provide a diploid chromosome complement, or dispermic,
arising from fertilization of an enucleate egg by two
spermatozoa (Ohama et al., 1981). PHM are usually triploid
(Szulman and Surti, 1978a) and generally arise by dispermy
(Jacobs et al., 1982; Lawler et al., 1982). In both CHM and
Biparental complete mole
PHM, the trophoblastic hyperplasia, characteristic of a molar
pregnancy, is associated with the presence of two paternal
genomes and thus involves imprinted genes, that is genes
which are normally only expressed from the maternally or
paternally derived allele. Further evidence that the trophoblastic
hyperplasia typical of molar pregnancies results from increased
expression of paternally derived genes is provided by studies
of the development of reconstituted mouse eggs. While those
embryos which received a male and a female pronuclei
developed to term following implantation (Surani et al., 1984),
androgenetic embryos with two male pronuclei showed much
greater trophoblastic development than those with two female
pronuclei (Barton et al., 1984) in which fetal development
was favoured. HM is therefore an imprinted condition in that
the pathology is dependent on the parental origin of the genome.
Although the majority of CHM are androgenetic in origin,
occasionally CHM have been shown to be biparental in origin
(Vejerslev et al., 1987; Ko et al., 1991; Kovaks et al., 1991;
Sunde et al., 1993; Fisher et al., 1997). These unusual CHM
have only one chromosome complement from the father, the
second set of chromosomes being inherited from the mother
as in a normal pregnancy. The rarity of these cases makes it
difficult to estimate their true frequency. However, a recent
study of two families in which several sisters had one or more
CHM, found that all CHM examined were biparental in origin
(Moglabey et al., 1999) suggesting that familial repetitive HM
is of biparental origin. In one family in particular there was a
high degree of consanguinity. The case described here represents an unrelated couple who had three CHM and no normal
pregnancies. This case is of particular interest in that the
second of the three CHM was conceived with a different
partner to the first and third CHM.
In androgenetic CHM, where the whole paternal chromosome complement is over-represented, it is difficult to
specifically identify those genes that contribute to the
abnormal development. Since biparental CHM are pathologically indistinguishable from the more common androgenetic
CHM the underlying mechanism giving rise to these CHM is
also likely to be an over-expression of paternally transcribed
genes. These rare biparental CHM are, therefore, potentially
valuable for identifying the imprinted genes involved in molar
development, since much smaller regions of the genome are
likely to be abnormal in these cases. There are a number of
human genetic disorders involving imprinted genes such as
Angelman syndrome and Beckwith–Wiedemann syndrome
(BWS) which result from a number of different mechanisms
including duplication of a single paternal chromosome with
loss of the corresponding maternal chromosome (Hall, 1997).
In order to investigate whether biparental CHM might result
from unipaternal disomy of a specific chromosome, at least
one informative microsatellite polymorphism was examined
for each pair of autosomes in CHM1 and CHM3. No evidence
of unipaternal disomy was found in either CHM. This suggests
that the molar pathology in these cases results from uniparental
disomy of only a small region of the paternal genome.
Alternatively two active copies of the genes involved in molar
development might result from expression of maternal genes
which are normally imprinted and therefore not transcribed.
While some cases of BWS are due to the presence of two
copies of paternal genes, others have a normal genotype and
result instead from loss or relaxation of imprinting of the
maternally inherited gene (Hall, 1997).
That the defect may be in the maternal, rather than paternal,
genome in diploid, biparental molar pregnancies is suggested
by the fact that two different partners were involved in the
three molar pregnancies in this study. This is supported by a
recent report (Moglabey et al., 1999) of two families in which
several sisters have repetitive HM. The repetitive HM in both
the families described by Moglabey et al. were also shown to
be biparental in origin. Linkage and homozygosity analysis
suggested that, in their families, there is a defective gene
located on chromosome, 19q13.3–13.4. This region of the
genome has recently been shown to be the location of at least
two imprinted genes, PEG3, a paternally expressed gene (Kim
et al., 1997) involved in maternal behaviour and offspring
growth in mice (Li et al., 1999) and Zim1, a Kruppel-type
zinc-finger gene which is maternally expressed (Kim et al.,
1999). These observations suggest the presence of an imprinted
domain in human chromosome 19q13.4 in which other
imprinted genes involved in molar development might be
located. Cases of familial HM, for further linkage analysis,
are extremely rare. However, a number of cases of recurrent
HM are available. Although some cases of repetitive HM are
androgenetic (Roberts and Mutter, 1994; Fisher, 1997), others
are clearly biparental in origin. A pathological and genetic
review of patients with repetitive CHM may identify
additional patients with biparental HM for further investigation and identification of the genes involved in trophoblastic
development.
Identification of the genetic origin of repetitive HM is also
important for patients considering IVF to avoid repetitive
CHM. It has been suggested that in patients with repetitive
HM, intracytoplasmic sperm injection (ICSI) followed by
preimplantation genetic diagnosis (PGD) can be used to
prevent recurrent HM (Reubinoff et al., 1997). ICSI of oocytes
ensures that only a single spermatozoon enters the egg, thus
preventing CHM, or PHM, which arise by dispermy. Following
fertilization, the sex of the embryo can be determined from
one or two blastomeres. Although CHM which arise by
dispermy may be male or female (Fisher and Lawler, 1984;
Wake et al., 1984) the 75% of CHM (Fisher et al., 1989)
which arise following fertilization of an anucleate egg by a
haploid spermatozoon are always female with a 46,XX karyotype. A 46,YY karyotype is presumed to be non-viable.
Subsequent rejection of 46,XX embryos in favour of 46,XY
embryos will therefore eliminate CHM which arise by doubling
of a haploid spermatozoon (Reubinoff et al., 1997). However,
selection of male embryos would not prevent a further CHM
in cases of repetitive CHM of biparental origin which we have
shown may be either male or female. Thus ICSI followed
by PGD may prevent the recurrence of triploid PHM and
androgenetic CHM but not repetitive biparental CHM. Patients
with recurrent HM have been reported in which some HM are
CHM and others PHM (Rice et al., 1989); we are unaware of
any cases of repetitive CHM in which patients have both
androgenetic and biparental CHM.
597
R.A.Fisher et al.
Studies of fertilization, syngamy and cleavage in eggs from
patients with a history of repetitive CHM have revealed a
variety of abnormal pronuclei (Edwards et al., 1992; Edwards,
1994). In one case a small number of eggs were observed to
have only a single pronucleus. These cells which later cleaved
into normal 2-cell embryos and continued to divide, were
interpreted as androgenetic diploid embryos. Observation of
pronuclear development would also identify any embryo with
three polar bodies that might represent a triploid PHM.
It would obviously be of interest to examine pronuclear
development in cases of biparental repetitive CHM which
might be expected to have two pronuclei.
Further genetic studies of repetitive HM are therefore
important in the clinical management of patients with repetitive
HM and for a better understanding of the role of imprinted
genes in the development of trophoblast.
Acknowledgements
Facilities for 310 analysis were provided through funds from the
Wellcome Trust and the Trustees of Charing Cross Hospital. This
work was supported by grants from the Cancer Treatment and
Research Trust.
Note added at proof
Since submitting this manuscript we have examined the genetic origin
of the HM in two further cases of repetitive HM in which the patient
had 3 or more CHM. In both cases all CHM were shown to be
biparental in origin.
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Received on July 20, 1999; accepted on November 10, 1999