[Show abstract][Hide abstract] ABSTRACT: Hemophilia A (HA) is caused by partial or total deficiency of F8 protein activity. In a small group, about 1.8% of patients with HA, no mutation is found in the F8 gene. Among this group, we report here on one patient with severe HA in whom no mRNA of the F8 gene was detected. Using 2 common polymorphisms in F8 exon 14, we were able to show that the same allele shared by the patient, his mother, and his sister was not detected by reverse transcription-polymerase chain reaction (RT-PCR) from total blood mRNA. Skewed X-chromosome inactivation in both the mother and the sister was excluded by studying the methylation profile of the androgen receptor gene (HUMARA locus). These findings strongly suggest that the cause of HA in this patient is either absence or rapid degradation of the F8 mRNA, which points to a novel mechanism leading to HA.
[Show abstract][Hide abstract] ABSTRACT: haemophilia A (HA) is characterized by partial or total deficiency of factor VIII (FVIII) protein activity. It is caused by a broad spectrum of mutations in the FVIII gene. Despite tremendous improvements in mutation screening methods, in about 2% of HA patients no DNA change could be found, even after sequencing the whole coding part of the FVIII gene including the flanking splice sites, as well as the promotor and the 3' UTR regions. OBJECTIVES, PATIENTS AND METHODS: In the present study we performed a detailed RNA analysis of three groups of patients. The first included control patients with known splicing defects, the second included two patients with already identified nucleotide changes close to splicing sites, that could potentially alter the normal splicing process, and a third group of 11 unrelated patients whose genomic DNA have already been screened for mutations by DHPLC and direct sequencing with no mutation being identified.
Both candidate splice site mutations were shown to result in either skipping or alternative splicing of at least one exon, therefore these DNA changes must be considered as causal for the patients' HA phenotype. In contrast, no abnormalities on the RNA level were observed in any of 11 unrelated patients without mutations in the FVIII gene.
These findings exclude mutations that could be located deep in the introns and affecting either normal splicing or lead to mechanisms causing some unknown rearrangements of the FVIII gene. In fact, our results point to the presence of still unknown factor(s) causing HA, which might be either allelic or in the close proximity of the FVIII gene or non-allelic associated with other genetic loci that are involved in the processing of the FVIII protein.
Journal of Thrombosis and Haemostasis 03/2005; 3(2):332-9. DOI:10.1111/j.1538-7836.2005.01140.x · 5.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Human Molecular Genetics 07/2003; 12(12):1405-13. · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Nucleic Acids Research 04/2002; 30(6):e25. · 9.11 Impact Factor