Biomedical subjects

Martine De Rycke

Publications and source records attributed to Martine De Rycke.

7 recordsLinked to original sources

PubMed2007

Methylation analysis of the intergenic differentially methylated region of DLK1-GTL2 in human.

Imprinting is a non-Mendelian form of inheritance where epigenetic modifications control mono-allelic expression depending on the parental origin. Methylation of CpG-dinucleotides at differentially methylated regions (DMRs) is one of the best-studied mechanisms directing expression to one specific parental allele. We studied the methylation patterns of the intergenic (IG)-DMR of DLK1 and GTL2. The methylation marks of the IG-DMR were analysed in human gametes, preimplantation embryos, amniocytes and blood of babies born after intracytoplasmic sperm injection (ICSI) and blood from adults using a bisulphite sequencing technique. In oocytes, the IG-DMR was mainly unmethylated while in sperm cells a generally methylated pattern was detected. This germ-line specific methylation mark was maintained in the preimplantation embryos until the second cleavage stage. Afterwards in the preimplantation embryos, intermediate methylation patterns (26-74% methylation) occurred, which may point to relaxation of the imprints. Intermediate patterns were also present in amniocytes, blood from ICSI babies and adults. We hypothesise that in the early cleavage stage embryo a strict differential methylation pattern is needed for the correct imprint establishment of surrounding imprinted genes. Once correct imprinting of the involved gene(s) is acquired, a more relaxed state of the IG-region is allowed.

Calcium-Binding Proteins↗

PubMed2006

Methylation analysis of KvDMR1 in human oocytes.

Recently, several reports have been published that showed a higher incidence of assisted reproductive technologies (ART) in patients with Beckwith-Wiedemann syndrome compared with the general population, and in most of these patients, aberrant methylation imprints of KvDMR1 have been found. This has led to the concern that ART might increase the incidence of imprinting syndromes such as Beckwith-Wiedemann syndrome. Not much is known on environmental or genetic factors that may interfere with the processes of imprint maintenance or resetting. A methylation analysis of KvDMR1 was performed in human oocytes at different stages of nuclear maturity and in sperm cells. The results indicate that the maternal methylation imprints were already established at the germinal vesicle stage, whereas all sperm cells were unmethylated, thereby showing that the KvDMR1 carries a germline methylation imprint. For one of the oocytes analysed, an unmethylated pattern was found, which highlights the need for further molecular studies that consider the safety of ART.

Base Sequence↗

PubMed2006

Preimplantation genetic diagnosis for Marfan syndrome.

OBJECTIVE: To develop and apply efficient and reliable protocols for preimplantation genetic diagnosis (PGD) for Marfan syndrome. DESIGN: Two mutation-specific protocols were developed, and the markers D15S1028, D15S992, D15S196, D15S576, D15S123, and D15S143 were used to set up four multiplex polymerase chain reactions (PCRs). SETTING: Research Center Reproduction and Genetics. PATIENT(S): Ten couples carrying mutations in the FBN1 gene. INTERVENTION(S): Six PGD protocols were developed for 10 couples, and 7 of them underwent a total of 16 clinical cycles. MAIN OUTCOME MEASURE(S): Amplification, allele drop-out (ADO), and contamination rates during the preclinical assays. DNA analyses of blastomeres from embryos biopsied during PGD cycles. RESULT(S): Six different protocols were set up, with the main objective being to to use one protocol for several couples. A total of 16 PGD cycles were performed, which resulted in the delivery of an unaffected boy and three ongoing pregnancies. CONCLUSION(S): The development of single-cell multiplex PCRs for linked markers and its use in PGD reduce the workload of the genetic diagnostic laboratory as well as the average waiting time for patients. This approach also allows for the simultaneous and accurate detection of recombination, contamination, and ADO, thereby increasing the reliability of the diagnosis.

Adult↗

PubMed2006

Single-cell chromosomal imbalances detection by array CGH.

Genomic imbalances are a major cause of constitutional and acquired disorders. Therefore, aneuploidy screening has become the cornerstone of preimplantation, prenatal and postnatal genetic diagnosis, as well as a routine aspect of the diagnostic workup of many acquired disorders. Recently, array comparative genomic hybridization (array CGH) has been introduced as a rapid and high-resolution method for the detection of both benign and disease-causing genomic copy-number variations. Until now, array CGH has been performed using a significant quantity of DNA derived from a pool of cells. Here, we present an array CGH method that accurately detects chromosomal imbalances from a single lymphoblast, fibroblast and blastomere within a single day. Trisomy 13, 18, 21 and monosomy X, as well as normal ploidy levels of all other chromosomes, were accurately determined from single fibroblasts. Moreover, we showed that a segmental deletion as small as 34 Mb could be detected. Finally, we demonstrated the possibility to detect aneuploidies in single blastomeres derived from preimplantation embryos. This technique offers new possibilities for genetic analysis of single cells in general and opens the route towards aneuploidy screening and detection of unbalanced translocations in preimplantation embryos in particular.

Aneuploidy↗

PubMed2004

Intergenerational instability of the expanded CTG repeat in the DMPK gene: studies in human gametes and preimplantation embryos.

The CTG repeat at the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene shows marked intergenerational and somatic instability in patients with myotonic dystrophy (DM1), when the repeat is expanded to more than approximately 55 repeats. Intensive research has yielded some insights into the timing and mechanism of these intergenerational changes: (1) increases in expansion sizes occur during gametogenesis but probably not during meiosis, (2) the marked somatic mosaicism becomes apparent from the 2nd trimester of development onward and increases during adult life, and (3) DNA repair mechanisms are involved. We have performed preimplantation genetic diagnosis for DM1 since 1995, which has given us the unique opportunity to study the expanded CTG repeat in affected embryos and in gametes from affected patients. We were able to demonstrate significant increases in the number of repeats in embryos from female patients with DM1 and in their immature and mature oocytes, whereas, in spermatozoa and embryos from male patients with DM1, smaller increases were detected. These data are in concordance with data on other tissues from adults and fetuses and fill a gap in our knowledge of the behavior of CTG triplet expansions in DM1.

Blastocyst↗

PubMed2003

Methylation imprints of the imprint control region of the SNRPN-gene in human gametes and preimplantation embryos.

Imprinting is an epigenetic mechanism leading to mono-allelic expression of imprinted genes. In order to inherit the differential epigenetic imprints from one generation to the next, these imprints have to be erased in the primordial germ cells and re-established in a sex-specific manner during gametogenesis. The exact timing of the imprint resetting is not yet known and the use of immature gametes in assisted reproductive technologies may therefore lead to abnormal imprinting and related diseases. Imprinting is associated with differential allelic methylation in a CpG-context. We studied the methylation patterns of the imprint control (IC) region of the human SNRPN-gene in human spermatozoa, oocytes in different developmental stages [germinal vesicle (GV), metaphase I and metaphase II oocytes] and in preimplantation embryos using the bisulphite sequencing technique. In the spermatozoa, almost all potential methylation sites were unmethylated whereas mainly methylated patterns were found in the oocytes at different developmental stages. In the embryos, an average methylation pattern of 53% was found indicating that the imprints, which have been set during gametogenesis, are stably maintained in the preimplantation embryo. Our results indicate that the maternal imprints for the IC-region of the human SNRPN-gene are already re-established at the GV stage and that they are not re-established in a late oocyte stage or after fertilization as previously reported. Recent advances in assisted reproductive technologies raise questions concerning safety and the epigenetic risks involved. Our study was the first to check the methylation imprints in human pre-implantation embryos and oocytes at different developmental stages.

Autoantigens↗

PubMed2003

Imprinting.

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Alleles↗