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Supplemental MATERIALS AND METHODS
Samples
In The Danish Mole Project, fresh tissue is collected from molar conceptuses. All of the
16 departments of gynaecology and obstetrics in the western part of Denmark participate
in the project. Each department posses a “mole sampling kit”, including a 100 ml widenecked flask with sterile cell culture medium (RMP1 1640, Boehringer). The
gynaecologists are instructed to watch out for conceptuses presenting with vesicular villi
on ultrasound or in the evacuated tissue. From such conceptuses, one representative
sample is fixed in formalin and forwarded for morphologic diagnostics at the local
department of pathology, whereas another representative sample, up to 50 ml, is
transferred sterilely to the cell culture medium and forwarded for the genetic laboratory.
At the laboratory the fresh tissue is transferred to a Petri dish and inspected with the
naked eye and using a dissection microscope (x25). Only samples presenting with more
than 10 vesicular villi with diameters of at least 1 mm, are included. The samples are
freed from decidua and blood, and representative parts are used for preparation of
chromosomes without previous culture (approximately 20 µl) and after culture
(approximately 100 µl), and stored at -80 ○C for DNA preparation (2 x 1 ml) and for flow
cytometry (2x 15 µl). For small samples the amount stored for DNA preparation is
reduced. In the period April 1986-June 2003, samples from 309 conceptuses were
received.
Histopathology
Previously, the histopathology was reviewed blindly with respect to the results of the
genetic analyses.17 In brief: The diagnosis HM was made when the evacuated tissue
displayed vesicular villi with diameters of at least 2 mm and trophoblastic hyperplasia.
The diagnosis “complete hydatidiform mole” (CHM) was made when the villi were
diffusely hydropic, the trophoblastic hyperplasia was frequent, and no foetal tissue
(except from stromal nuclear debris in the villi) was noted. The diagnosis “partial
hydatidiform mole” (PHM) was made when both normal and hydropic villi were
identified, the trophoblastic hyperplasia was focal, and invaginations of the villous
surface and/or trophoblastic pseudoinclusions and/or foetal tissue were noted. In order to
preserve the possibility of comparing phenotype and genotype, we deliberately based the
diagnoses on the macroscopic and microscopic morphology. We did not include
immunostaining of p57KIP2 as the presence/absence of this antigen is not a morphologic
parameter in a strict sense; it merely indicates the presence/absence of a maternally
imprinted chromosome 11. Material was successfully retrieved for histopathological
revision from 294 of the 309 conceptuses and 270 samples were classified as originating
in hydatidiform moles.17
Ploidy
The ploidies have been determined by karyotyping of uncultured and/or cultured cells
and/or by measurement of the nuclear DNA contents by flow cytometry of unfixed
nuclei, using trout and chicken erythrocytes as controls (DNA-ploidy).36
Of the 270 HMs, 162 were diploid, 105 were triploid and three were tetraploid. Eight of
the diploid HMs were part of multiple pregnancies (seven cases of twinning: HM and
normal conceptus, and one case of HM and two normal conceptuses).17
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Parental origin of the genome
The parental origins of the genome have been determined by comparing DNA markers in
the moles, with those in the parents. DNA was prepared from parental leucocytes and
from vesicular villi using standard techniques. In the early project period RFLP markers
were analysed, later mini- and microsatellite markers were used. A HM was classified as
having an androgenetic genome when at least two unlinked markers exclusively showed
alleles not present in the mother. 9 diploid HMs were classified as having genomes from
both parents as the markers in all loci analysed were compatible with one originating
from the father and one from the mother, and in at least in two unlinked loci one allele in
the molar DNA was identical with a maternal allele that was not present in the father and
one allele in the molar DNA was identical with a paternal allele that was not present in
the mother. Two diploid HMs (HM269 and HM525) were classified as having genomes
from both parents although paternal DNA was not available, since one allele was
identical with one of the maternal alleles in all of 10 loci and one allele was different
from both maternal alleles in 10 and 7 loci, respectively.
One cell population or mosaicism; analysis of tissue
The 11 moles showing signs of having genome from both parents were subjected to
microsatellite analysis, using a panel of at least 10 markers located on chromosome 13,
18, 21, X and Y with heterozygosity frequencies between 67% and 93% (Supplemental
Table 1, online). The PCR products were visualized using an ABI prism 310 Genetic
Analyzer and analyzed with ABI prism GeneScan software (Applied Biosystems).
In an electropherogram displaying the PCR product of a heterozygous microsatellite
locus, the peak representing to the shorter allele normally is higher than the peak
representing to the longer allele, due to preferential amplification of shorter fragments. In
some cases, the peak representing the shorter allele was smallest or there were three
peaks. We designated this “imbalanced peaks”. For the interpretation of imbalanced
peaks, we constructed an “expected peak pattern for a biparental cell population” (P1M)
by first identifying the peak representing the maternal allele and then assigning a
“corresponding paternal peak”. The corresponding paternal peak was identified either as
one of the paternal peaks with the appropriate height or as an appropriate part of (one of)
the paternal peak(s), taking into account that the “remaining peak pattern” should be
compatible with either homozygosity for one of the paternal alleles or heterozygosity for
both paternal alleles. Three remaining peak patterns were observed:
1) In all loci analyzed: One remaining peak, only, representing an allele identical with the
paternal allele in the biparental cell population, as if the paternal alleles in both cell
populations originated in one spermatozoon (P1M+P1P1) (Fig. 1A).
2) In all loci analyzed: One remaining peak, only, but (in at least one locus) this peak
represented a paternal allele that was different from the paternal allele in the biparental
cell population, as if the paternal genome in the androgenetic cell population originated
from an independent spermatozoon (P1M+P2P2) (Fig. 1B).
3) In some loci: Two remaining peaks with heights corresponding to what one would
expect from a paternally derived, heterozygous cell population (P1M+P1P2) (Fig. 1C).
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Although the PCR reactions were not optimized for quantification, we made rough
estimates of the frequencies of the androgenetic cells by visual evaluation of the relative
heights of the paternal peak(s), including data from all loci where the HM showed two or
three peaks.18
One cell population or mosaicism; analysis of single cells
For single cell isolation, approximately 15 l of unfixed frozen vesicular villi were
thawed, washed with PBS, spun down and incubated with collagenase D (Roche) at 10
mg/ml at 37C for one hour. After centrifugation and washing in PBS, the cells were resuspended in RPMI-1640 (buffered with 5 mM HEPES, pH 7.2). The single cells were
isolated while inspected through a Leica MZ 12,5 microscope (x 160) and rinsed five
times in cell culture medium (Medicult). From each cell, DNA was prepared by
incubation at 45 C for 40 min with proteinase K. DNA was amplified with primer pairs
for the markers D6S105 and D6S2443, in a multiplexed analysis. The PCR products were
analyzed using an ABI prism 3100 Genetic Analyzer and ABI prism Gene Scan software
(Applied Biosystems). We estimated the allele drop-out frequencies by analyzing 34
lymphocytes. For marker D6S105 the frequency was 6%, whereas for marker D6S2443 it
was 0%.
In the interpretation of the analysis of single cells, we classified a cell as contaminated
with alien DNA, when the PCR product contained an allele that was not seen in the DNA
prepared from the villous tissue. PCR products that for one locus showed the allele(s)
expected for one of the predicted cell populations and for the other locus showed no
alleles or only one of two expected alleles, were classified as showing “no product in one
locus” or “allele drop-out”, respectively.