Methylation analysis of the promoter region and intron 1 of the factor VIII gene in haemophilia A patients.

Methylation, CpG island, promoter, intron 1 Haemophilia A is the most common X-linked inherited coagulation disorder caused by a deficiency of the factor VIII protein (FVIII). A plethora of different mutations in the factor VIII gene (F8) have been identified as causative for this bleeding disease including a few promoter mutations. However, in approximately 2-5% of all haemophilic patients, the causal mutation still remains unknown. To our knowledge, epigenetic abnormalities in regulatory regions of the F8 gene have not yet been implicated in the disease pathogenesis. We therefore developed bisulfite pyrosequencing assays to screen patients with unknown mutation status for their methylation patterns in presumed regulative regions of the F8 gene (5'UTR and intron 1). The methylation patterns of haemophilia A patients did not differ from that of controls. In three patients, chromosomal aberrations were identified which could be associated with a defective FVIII synthesis.

Epigenetic profile of developmentally important genes in bovine oocytes.

Assisted reproductive technologies are associated with an increased incidence of epigenetic aberrations, specifically in imprinted genes. Here, we used the bovine oocyte as a model to determine putative epigenetic mutations at three imprinted gene loci caused by the type of maturation, either in vitro maturation (IVM) in Tissue Culture Medium 199 (TCM) or modified synthetic oviduct fluid (mSOF) medium, or in vivo maturation. We applied a limiting dilution approach and direct bisulfite sequencing to analyze the methylation profiles of individual alleles (DNA molecules) for H19/IGF2, PEG3, and SNRPN, which are each associated with imprinting defects in humans and/or the mouse model, and are known to be differentially methylated in bovine embryos. Altogether, we obtained the methylation patterns of 203 alleles containing 4,512 CpG sites from immature oocytes, 213 alleles with 4,779 CpG sites from TCM-matured oocytes, 215 alleles/4,725 CpGs in mSOF-matured oocytes, and 78 alleles/1,672 CpGs from in vivo-matured oocytes. The total rate of individual CpGs and entire allele methylation errors did not differ significantly between the two IVM and the in vivo group, indicating that current IVM protocols have no or only marginal effects on these critical epigenetic marks. Furthermore, the mRNA expression profiles of the three imprinted genes and a panel of eight other genes indicative of oocyte competence were determined by quantitative real-time PCR. We found different mRNA expression profiles between in vivo-matured oocytes versus their in vitro-matured counterparts, suggesting an influence on regulatory mechanisms other than DNA methylation.

Characterization of differentially methylated regions in 3 bovine imprinted genes: a model for studying human germ-cell and embryo development.

Correct imprinting is crucial for normal fetal and placental development in mammals. Experimental evidence in animal models and epidemiological studies in humans suggest that assisted reproductive technologies (ARTs) can interfere with imprinted gene regulation in gametogenesis and early embryogenesis. Bos taurus is an agriculturally important species in which ARTs are commonly employed. Because this species exhibits a similar preimplantation development and gestation length as humans, it is increasingly being used as a model for human germ-cell and embryo development. However, in contrast to humans and mice, there is relatively little information on bovine imprinted genes. Here, we characterized the bovine intergenic IGF2-H19 imprinting control region (ICR) spanning approximately 3 kb. We identified a 300-bp differentially methylated region (DMR) approximately 6 kb upstream of the H19 promoter, containing a CpG island with CTCF-binding site and high sequence similarity with the human intergenic ICR. Additional differentially methylated CpG islands lie -6 kb to -3 kb upstream of the promoter, however these are less conserved. Both classical bisulfite sequencing and bisulfite pyrosequencing demonstrated complete methylation of the IGF2-H19 ICR in sperm, complete demethylation in parthenogenetic embryos having only the female genome, and differential methylation in placental and somatic tissues. In addition, we established pyrosequencing assays for the previously reported bovine SNRPN and PEG3 DMRs. The observed methylation patterns were consistent with genomic imprinting in all analyzed tissues/cell types. The identified IGF2-H19 ICR and the developed quantitative methylation assays may prove useful for further studies on the relationship between ARTs and imprinting defects in the bovine model.

Cross-species chromosome painting corroborates microchromosome fusion during karyotype evolution of birds.

The stone curlew, also known as thick-knee (Burhinus oedicnemus, BOE), represents a phylogenetically young species of the shorebirds (Charadriiformes) that exhibits one of the most atypical genome organizations known within the class of Aves, due to an extremely low diploid number (2n = 42) and only 6 pairs of microchromosomes in its complement. This distinct deviation from the 'typical' avian karyotype is attributed to repeated fusions of ancestral microchromosomes. In order to compare different species with this atypical avian karyotype and to investigate the chromosome rearrangement patterns, chromosome-specific painting probes representing the whole genome of the stone curlew were used to delineate chromosome homology between BOE and 5 species belonging to 5 different avian orders: herring gull (Charadriiformes), cockatiel (Psittaciformes), rock pigeon (Columbiformes), great gray owl (Strigiformes) and Eurasian coot (Gruiformes). Paints derived from the 20 BOE autosomes delimited 28 to 33 evolutionarily conserved segments in the karyotypes of the 5 species, similar to the number recognized by BOE paints in such a basal lineage as the chicken (28 conserved segments). This suggests a high degree of conservation in genome organization in birds. BOE paints also revealed some species-specific rearrangements. In particular, chromosomes BOE1-4 and 14, as well as to a large extent BOE5 and 6, showed conserved synteny with macrochromosomes, whereas homologous regions for BOE7-13 are found to be largely distributed on microchromosomes in the species investigated. Interestingly, the 6 pairs of BOE microchromosomes 15-20 appear to have undergone very few rearrangements in the 5 lineages investigated. Although the arrangements of BOE homologous segments on some chromosomes can be explained by complex fusions and inversions, the occurrence of homologous regions at multiple sites may point to fission of ancestral chromosomes in the karyotypes of the species investigated. However, the present results demonstrate that the ancestral microchromosomes most likely experienced fusion in the stone curlew lineage forming the medium-sized BOE chromosomes, while they have been conserved as microchromosomes in the other neoavian lineages.