1 Department of Cancer Medicine, Division of Medicine, Imperial College School of Medicine, Charing Cross Hospital, Fulham Palace Road, London W6 8RF, 2 Department of Histopathology, Division of Investigative Sciences, Imperial College School of Medicine, Charing Cross Hospital, Fulham Palace Road, London W6 8RF and 3 Directorate of Women's Services, Newham General Hospital, Glen Road, Plaistow, London E13 8SL, UK
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Abstract |
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Key words: biparental complete mole/genomic imprinting/IVF/repetitive mole
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Introduction |
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Materials and methods |
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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 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).
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Results |
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Discussion |
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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 BeckwithWiedemann 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, 19q13.313.4. 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.
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.
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Note added at proof |
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Acknowledgments |
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Notes |
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References |
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Submitted on July 20, 1999; accepted on November 10, 1999.