1. ¹Department of Pathology, Dalhousie University, Halifax, Canada
2. ²Department of Medical Genetics, IWK Health Sciences Centre and Dalhousie University, Halifax, Canada
3. ³Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Canada
4. ⁴Department of Medical Genetics, Hôpital Sainte-Justine and Université de Montréal, Montréal, Canada
5. ⁵Department of Cancer-Genetics and Developmental Biology, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada
6. ⁶Department of Pathology and Laboratory Medicine, QEII Health Sciences Centre and Dalhousie University, Halifax, Canada

Correspondence: Dr WL Greer, Division of Hematology, QEII Health Sciences Centre, 5788 University Avenue, McKenzie Building, Rm 223B, Halifax, Nova Scotia, Canada B3H 1V8. Tel: +1 902 473 6691; Fax: +1 902 473 4113; E-mail: w.greer@dal.ca

Received 6 June 2006; Revised 23 January 2007; Accepted 31 January 2007; Published online 7 March 2007.

Abstract

Factor VIII gene, F8, mutations cause haemophilia A (HA), an X-linked recessive disorder. Expression in heterozygous females has been ascribed to skewed X-chromosome inactivation (XCI). To investigate the cause of HA in three heterozygous females within an Atlantic Canadian kindred, the proband (severely affected girl, FVIII activity: 2%) and 17 relatives across three generations were studied. F8 genotype, FVIII activity, XCI ratio (XCIR) (paternal active X: maternal active X), karyotype, submegabase resolution tiling set array competitive genome hybridization (competitive genomic hybridization (SMRT)), and microsatellite analyses were utilized. A positive linear relationship between FVIII activity and percentage-activated normal X-chromosome was found in HA heterozygous females (R²=0.87). All affected, but no unaffected females, had an XCIR skewed toward activation of the mutant X-chromosome (proband 92:8, SD 2). Unexpectedly, high numbers of females have dramatically skewed XCIRs (>80:20 or <20:80) (P<0.05). The distribution of XCIR frequencies within this family was significantly different than predicted by normal population data or models of random XCI (P<0.025), with more females having higher degrees of skewing. Known causes of skewing, such as chromosomal abnormalities, selection against deleterious alleles, and X-inactive-specific transcript mutations, are not consistent with our results. This study shows that FVIII activity in HA heterozygous females can be directly related to XCI skewing, and that low FVIII activity in females in this family is due to unfavourable XCI skewing. Further, the findings suggest that these XCI ratios are genetically influenced, consistent with a novel heritable human X controlling element (XCE) functioning similarly to the mouse Xce.