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Biomedical subjects

Osman El-Maarri

Publications and source records attributed to Osman El-Maarri.

8 recordsLinked to original sources
PubMed2005

Patients with familial biparental hydatidiform moles have normal methylation at imprinted genes.

In molar tissues from patients with recurrent biparental hydatidiform moles, we could previously demonstrate that differentially methylated regions (DMRs) of four imprinted genes are abnormally methylated on the maternal alleles. It remained unclear if this abnormal methylation originated de novo in the molar tissues or if it is even recognizable in the patient somatic tissues. To address this question, we investigated the DNA methylation of four imprinted genes in total blood from the two sister-patients. Here, we show that both patients retain normal methylation levels at the DMRs of the four genes in blood tissues. For two maternally expressed genes, we could use informative SNPs to follow the inheritance of the abnormally methylated maternal alleles in the molar tissues. We find that the transmitted abnormally methylated maternal alleles to the moles originated from the maternal grandmother and that the same alleles are not methylated in the patients. Our data suggest that the abnormal methylation in familial biparental molar tissues was acquired de novo in the patients'germline as a result of a false reprogramming or during the postzygotic development of the conceptuses that led to moles.

Alleles↗
PubMed2004

SIRPH analysis: SNuPE with IP-RP-HPLC for quantitative measurements of DNA methylation at specific CpG sites.

This chapter describes a detailed protocol using single-nucleotide primer extension (SNuPE) for quantitative analysis of DNA methylation on specific CpG sites. The first step DNA sample to be studied is treated with sodium bisulfite, which converts selectively unmethylated cytosines to uracil, while methylated cytosines remain unconverted. Subsequently, a SNuPE reaction is performed, with an oligo just flanking a CpG site, using a purified polymerase chain reaction product derived from bisulfite-treated DNA as a template. The oligo is extended by either ddCTP or ddTTP depending on whether the site is methylated or unmethylated, respectively. The reaction is quantitative and linear, and two to three sites can be studied simultaneously in a multiple reaction. The SNuPE product, without further purification, is separated by ion-pair reverse-phase (IP RP) high-performance liquid chromatography (using an alkylated nonporous polysterene-divinylbenzene cartridge) that allows an easy, semiautomated method for separation of the extended and unextended products and an accurate quantification of the extended products. The ratio of the ddCTP to the ddTTP gives the fraction of the methylated cytosines at that specific CpG site.

Chromatography, High Pressure Liquid↗
PubMed2003

Maternal alleles acquiring paternal methylation patterns in biparental complete hydatidiform moles.

We previously mapped a maternal locus responsible for biparental complete hydatidiform moles (BiCHMs) to 19q13.4. The two index patients had a total of 14 molar pregnancies, eight abortions at various developmental stages, and one 16-year-old healthy offspring. We suggested that the defective gene deregulates the expression of imprinted genes. Here, we report the methylation status of four imprinted genes in two BiCHMs from the two sisters, the 16-year-old normal offspring, and two sporadic BiCHMs from unrelated patients. Using two bisulfite-based methods, we demonstrate a general trend of abnormal hypomethylation at the paternally expressed genes, PEG3 and SNRPN, and hypermethylation at the maternally expressed genes, NESP55 and H19, in two to four BiCHMs. Using single nucleotide polymorphisms, we provide the first evidence that SNRPN, NESP55 and H19 are abnormally methylated on the maternal alleles in BiCHMs. We show, in the BiCHMs from the two sisters, that the abnormally methylated H19 allele is inherited from either the maternal grandmother or the maternal grandfather. These data suggest that the abnormal methylation in BiCHMs is not due to an error in erasing the parental imprinting marks but rather in the re-establishment of the new maternal marks during oogenesis or their postzygotic maintenance. The defective 19q13.4 locus may have led to the development of variable degrees of 'faulty' paternal marks on the maternal chromosomes.

Adolescent↗
PubMed2002

A rapid, quantitative, non-radioactive bisulfite-SNuPE- IP RP HPLC assay for methylation analysis at specific CpG sites.

The precise mapping and quantification of DNA methylation as an epigenetic parameter during development and in diseased tissues is of great importance for functional genomics. Here we describe a rapid, quantitative method to assess methylation levels at specific CpG sites using PCR products of bisulfite-treated genomic DNA. Using single nucleotide primer extension (SNuPE) assays in combination with ion pair reverse phase high performance liquid chromatography (IP RP HPLC) separation techniques, methylated and unmethylated CpGs can be discriminated and quantified based on the different masses and hydrophobicities of the extended primer products. The assay is linear, highly reproducible and several sites can be measured simultaneously in one reaction. It can be semi-automated and eliminates the need for cloning and sequencing of individual bisulfite PCR products.

Chromatography, High Pressure Liquid↗
PubMed2002

DNA methylation at promoter regions regulates the timing of gene activation in Xenopus laevis embryos.

The levels of genomic DNA methylation in vertebrate species display a wide range of developmental dynamics. Here, we show that in contrast to mice, the paternal genome of the amphibian, Xenopus laevis, is not subjected to active demethylation of 5-methyl cytosine immediately after fertilization. High levels of methylation in the DNA of both oocyte and sperm are maintained in the early embryo but progressively decline during the cleavage stages. As a result, the Xenopus genome has its lowest methylation content at the midblastula transition (MBT) and during subsequent gastrulation. Between blastula and gastrula stages, we detect a loss of methylation at individual Xenopus gene promoters (TFIIIA, Xbra, and c-Myc II) that are activated at MBT. No changes are observed in the methylation patterns of repeated sequences, genes that are inactive at MBT, or in the coding regions of individual genes. In embryos that are depleted of the maintenance methyltransferase enzyme (xDnmt1), these developmentally programmed changes in promoter methylation are disrupted, which may account for the altered patterns of gene expression that occur in these embryos. Our results suggest that DNA methylation has a role in regulating the timing of gene activation at MBT in Xenopus laevis embryos.

Animals↗
PubMed2002

Epigenetic reprogramming in mouse primordial germ cells.

Genome-wide epigenetic reprogramming in mammalian germ cells, zygote and early embryos, plays a crucial role in regulating genome functions at critical stages of development. We show here that mouse primordial germ cells (PGCs) exhibit dynamic changes in epigenetic modifications between days 10.5 and 12.5 post coitum (dpc). First, contrary to previous suggestions, we show that PGCs do indeed acquire genome-wide de novo methylation during early development and migration into the genital ridge. However, following their entry into the genital ridge, there is rapid erasure of DNA methylation of regions within imprinted and non-imprinted loci. For most genes, the erasure commences simultaneously in PGCs in both male and female embryos, which is completed within 1 day of development. Based on the kinetics of this process, we suggest that this is an active demethylation process initiated upon the entry of PGCs into the gonadal anlagen. The timing of reprogramming in PGCs is crucial since it ensures that germ cells of both sexes acquire an equivalent epigenetic state prior to the differentiation of the definitive male and female germ cells in which new parental imprints are established subsequently. Some repetitive elements, however, show incomplete erasure, which may be essential for chromosome stability and for preventing activation of transposons to reduce the risk of germline mutations. Aberrant epigenetic reprogramming in the germ line would cause the inheritance of epimutations that may have consequences for human diseases as suggested by studies on mouse models.

Animals↗