Mosaics and Moles Lone Sunde, Isa Niemann, Estrid Stæhr Hansen, Johnny Hindkjaer, Birte Degn, Uffe Birk Jensen, Lars Bolund To cite this version: Lone Sunde, Isa Niemann, Estrid Stæhr Hansen, Johnny Hindkjaer, Birte Degn, et al.. Mosaics and Moles. European Journal of Human Genetics, Nature Publishing Group, 2011, 10.1038/ejhg.2011.93. hal-00649448 HAL Id: hal-00649448 https://hal.archives-ouvertes.fr/hal-00649448 Submitted on 8 Dec 2011 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. 1 TITLE: Mosaics and moles AUTHORS: Lone Sunde1,2 Department of Clinical Genetics, Aarhus University Hospital, Aalborg Sygehus, DK- 9000 Aalborg, Denmark3 Department of Human Genetics, Aarhus University, DK-8000 Aarhus, Denmark Phone: 0045 9932 8952 Mobil phone: 0045 4014 4364 and 0045 4020 3199 FAX: 0045 9932 8940 E-mail: [email protected] and [email protected] Isa Niemann2 Department of Gynaecology and Obstetrics, Aarhus University Hospital, Skejby Sygehus, DK-8200 Aarhus, Denmark Estrid Staehr Hansen University Insitute of Pathology, Aarhus University Hospital, Aarhus Hospital, DK-8000 Aarhus, Denmark Johnny Hindkjaer The Fertility Clinic, Department of Gynaecology and Obstetrics, Aarhus University Hospital, Skejby Sygehus, DK-8200 Aarhus, Denmark Birte Degn The Fertility Clinic, Department of Gynaecology and Obstetrics, Aarhus University Hospital, Skejby Sygehus, DK-8200 Aarhus, Denmark Uffe Birk Jensen Department of Human Genetics, Aarhus University, DK-8000 Aarhus, Denmark3, and Department of Clinical Genetics, Aarhus University Hospital, Skejby Sygehus, Brendstrupgaardsvej 21C, DK-8200 Aarhus, Denmark Lars Bolund Department of Human Genetics, Aarhus University, DK-8000 Aarhus, Denmark3 and, Beijing Genomics Institute/HuaDa-Shenzhen, Shenzhen 518000, China 1Corresponding author 2These two authors contributed equally to this work. 3Current address RUNNING TITLE: Mosaics and moles 1 2 1 ABSTRACT 2 Hydatidiform mole is an abnormal human pregnancy, where the placenta presents with 3 vesicular swelling of the chorionic villi. A foetus is either not present, or malformed and 4 not viable. Most moles are diploid androgenetic as if one spermatozoon fertilized an 5 empty oocyte, or triploid with one maternal and two paternal chromosome sets as if two 6 spermatozoa fertilized a normal oocyte. However, diploid moles with both paternal and 7 maternal markers of the nuclear genome have been reported. Among 162 consecutively 8 collected diploid moles, we have earlier found indications of both maternal and paternal 9 genomes in 11. In the present study, we have performed detailed analysis of DNA- 10 markers in tissue and single cells from these 11 HMs. In 3/11 we identified one 11 biparental cell population, only, whereas in 8/11 we demonstrated mosaicism: one 12 biparental cell population and one androgenetic cell population. One mosaic mole was 13 followed by Persistent Trophoblastic Disease. In seven of the mosaics, one 14 spermatozoon appeared to have contributed to the genomes of both cell types. Our 15 observations make it likely that mosaic conceptuses, encompassing an androgenetic cell 16 population, result from various postzygotic abnormalities including paternal pronuclear 17 duplication, asymmetric cytokinesis, and postzygotic diploidization. This corroborates 18 the suggestion that fertilization of an empty egg is not mandatory for the creation of an 19 androgenetic cell population. Future studies of mosaic conceptuses may disclose details 20 about fertilization, early cell divisions and differentiation. Apparently only a minority of 21 diploid moles with both paternal and maternal markers are “genuine” diploid biparental 22 moles. 23 3 1 KEYWORDS 2 Hydatidiform mole [C13.703.720.949.416.875] 3 Mosaicism [G05.365.590.175.595] 4 Biparental diploidy 5 Triploidy 6 Genomic imprinting [G05.355.315.203.500] 7 Persistent Trophoblastic Disease 8 4 1 INTRODUCTION 2 Hydatidiform mole (HM) is an abnormal human pregnancy, characterized by vesicular 3 swelling of the chorionic villi and hyperplasia of the trophoblastic layer. By far most 4 HMs are diploid or triploid. In most diploid HMs, analyses of markers of the nuclear 5 genome identify markers that are identical with markers in the paternal genome, only 6 (diploid androgenetic hydatidiform moles, DiAndHMs). Most DiAndHMs show 7 homozygosity in all loci analyzed, as if an empty oocyte was fertilized by one 8 spermatozoon, followed by duplication of the haploid paternal genome. Fewer 9 DiAndHMs show heterozygosity in some loci as if an empty oocyte was fertilized by 10 two independent spermatozoa from the same father.1 However, over the years diploid 11 hydatidiform moles with biparental contributions to the genome have been reported.2-7 12 13 A molar phenotype in a diploid conceptus with biparental alleles may be caused by 14 paternal methylation patterns on maternally inherited chromosomes.8-11 Another possible 15 explanation is the co-existence of two diploid cell populations, one androgenetic and the 16 other biparental.12-16 17 18 In The Danish Mole Project we have consecutively been collecting samples from HMs 19 since 1986. In the present study we have subjected 11 diploid HMs showing signs of 20 having biparental genomes to detailed genetic analyses and found indications that only a 21 minority of these have “true” biparental genomes, whereas most are mosaics. 22 23 MATERIALS AND METHODS 24 5 1 In the Danish Mole Project, fresh tissue is collected from conceptuses presenting with 2 vesicular villi. In the period April 1986-June 2003, samples from 309 conceptuses were 3 received. Previously, material was successfully retrieved for histopathological revision 4 from 294 of these conceptuses and 270 samples were classified as originating in 5 hydatidiform moles. Ploidy was determined by karyotyping of uncultured and/or cultured 6 cells and/or by measurement of the nuclear DNA contents by flow cytometry of unfixed 7 nuclei. The parental origin of the genome was determined by comparing DNA markers in 8 the moles with those in the parents. Of the 270 HMs 162 were diploid, and of these 11 9 were classified as having genomes from both parents.17 10 11 In the present work, DNA from tissue samples from the 11 moles showing signs of 12 having genome from both parent and DNA from the parents were subjected to detailed 13 analysis, using a panel of at least 10 microsatellite markers. The patterns of peaks in the 14 molar electropherograms were interpreted by comparing to the electropherograms that 15 would be expected for various hypothetical offspring of the parents: a diploid 16 androgenetic conceptus, a diploid biparental conceptus and a mixture of these two. Rough 17 estimates of the frequencies of the androgenetic cells were made by visual evaluation of 18 the heights of the peaks.18 19 20 From four moles, DNA from single cells was prepared by incubating unfixed vesicular 21 villi with collagenase D. After rinsing five times in cell culture medium, DNA was 22 prepared by incubation with proteinase K and amplified with primers for the markers 23 D6S105 and D6S2443, in a multiplexed analysis. 24 6 1 For details on materials, methods, and interpretation see, Supplemental Materials and 2 Methods, and Supplemental Table 1, online). 3 4 Clinical data were obtained from a questionnaire filled in by the parents and from the 5 medical records. 6 7 The regional Committee on Biomedical Research Ethics approved the study. All 8 participants gave informed consent. 9 7 1 RESULTS 2 11 diploid HMs displaying both maternal and paternal alleles, identified in a consecutive 3 cohort of 270 HMs, were subjected to genetic analyses. The results are summarized in 4 Table 1, along with clinical and morphological data. The mother of HM131 has had a 5 total of six pregnancies, all showing molar morphology. She is member of a 6 consanguineous family and she is the first cousin of two sisters with repetitive diploid 7 moles with alleles from both parents. Only HM131 was part of the cohort of 270 HMs. 8 Observations in this family have been published before.6, 19, 20 The parents of the 10 other 9 moles were Danish, and had no personal or familial history of moles. 10 11 In four HMs, both uncultured and cultured cells were karyotyped. In two of these, we 12 observed a discrepancy between the karyotypes of uncultured cells and cultured cells: In 13 HM269 the karyotype in uncultured cells was 46,XY, whereas the karyotype after culture 14 was 46,XX. In HM192 the uncultured cells had a surplus derivative chromosome 7, 15 which was not found in the cultured cells. In both HMs, comparison of chromosomal 16 heteromorphisms made it unlikely that these discrepancies were caused by maternal 17 overgrowth in the cell culture. 18 19 Analyzing microsatellite markers we found imbalances in the signals in eight of the 11 20 HMs. None of the moles had more than one maternally derived allele at any locus, 21 excluding maternal contamination. Rather we either observed a disproportion between 22 the heights of the peaks representing two alleles (Fig. 1A-C) and/or three peaks at the 23 same locus (Fig. 1B-C). See Supplemental Table 1, online, for details. In all cases the 24 third peak, or the surplus height of one or two peak(s), represented one, or both, paternal 8 1 allele(s). The most probable explanation of these findings is the presence of two cell 2 populations, one androgenetic cell population and one cell population with balanced 3 biparental contributions to the genome (DiAnd/BiparHM).