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

Summary

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.

Zusammenfassung

Hämophilie A ist die häufigste erbliche X-chromosomale Blutgerinnungsstörung, die durch einen Mangel des Gerinnungsfaktor VIII (FVIII) verursacht wird. Viele unterschiedliche Mutationen im Faktor-VIII-Gen (F8) wurden als ursächlich für diese Blutgerinnungsstörung beschrieben, u. a. auch einige Promotor-Mutationen. Dennoch kann mit Routinemethoden in ca. 2–5% der Hämophilie-Patienten keine kausale Mutation identifiziert werden. Nach unserem Wissensstand wurden bisher keine epigenetischen Veränderungen in regulatorischen Regionen des Faktor-VIII-Gens in der Krankheitspathogenese berücksichtigt.

Daher haben wir einen Assay basierend auf Bisulfit-Pyrosequenzierung entwickelt, der den Methylierungsstatus von möglichen regulatorischen Bereichen im FVIII-Gen (5’UTR und Intron 1) von Patienten mit un-bekanntem Mutationsstatus bestimmen sollte. Die Methylierungsmuster der Hämophilie-A-Patienten wiesen keinen Unterschied zu den Kontrollen auf. Es konnten aber bei drei Patienten chromosomale Anomalien nach-gewiesen werden, die mit einer defekten FVIII-Synthese in Zusammenhang gebracht werden könnten.

Keywords

Methylation - CpG island - promoter - intron 1

Schlüsselwörter

Methylierung - CpG-Insel - Promotor - Intron 1

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