Abstract
Haemophilia A is classically due to a defect in the factor 8 gene. Mutations are heterogeneous, and located throughout the gene. Other than the IVS 22 inversion mutation, causative of approximately 46% of severe haemophilias, there are few mutation hot spots. It has been established that approximately 2% of patients with confirmed Haemophilia A have no detectable genetic abnormality in the factor 8 gene. Genetic analysis at the RNA level has generally failed to identify mutations not seen at the genomic level. Splicing abnormalities appear to be restricted to the splice junctions as no cryptic splice sites have yet been identified deep within introns.
One possible approach in the investigation of these non-F8 associated Haemophilia patients, is to look at up/down regulation of other genes via genome wide gene expression analysis. To this end we have subjected a small number of confirmed Haemophilia A patients with no identifiable mutation to microarray analysis, utilising an Affymetrix GeneChip® array system comprising 54676 genes. Their gene expression pattern was then compared to that of patients with similar phenotype in whom mutations have been defined. Initially, data analysis has focussed on the X chromosome, as the sex distribution in this group of patients does not appear to differ from classical F8-associated Haemophilia.
Preliminary results suggest a significant down regulation of ARHGAP6, a Rho GTPase activating protein. This gene is located at Xp22, comprises 14 exons, and encodes a 974 amino acid protein, with multiple splice variants. Interestingly, ARHGAP shares with factor 8 the possession of an embedded gene within a large intron. The F8A gene located within intron 22 of F8 is involved in the recombination event which results in the IVS22 inversion mutation. Intron 1of the ARHGAP gene spans more than 280kb and contains the amelogenin gene (AMELX). Like F8A, AMELX is transcribed from the opposite strand to the ’host gene’. This unusual phenomenon has only been reported in a very small number of human genes.
ARHGAP6 is known to have two independent functions, one as a GTPase-activating protein (GAP) involved in signal transduction, and another as a GTPase activator, regulating the interaction of signalling molecules with the actin cytoskeleton. It is possible that such interactions affect the secretion of the factor VIII protein from hepatocytes.
The genetic basis of Haemophilia A becomes more complex as the patient base investigated increases. It is likely that the number of genes that have an affect on plasma FVIII levels will continue to grow. ARHGAP6, and other GTPase activating proteins, may be worthy of closer examination in the search for genetic modifiers of FVIII levels.
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