Heritable skewed X-chromosome inactivation leads to haemophilia A expression in heterozygous females

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 percentageactivated normal X-chromosome was found in HA heterozygous females (R 2 ¼ 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 (480:20 or o20:80) (Po0.05). The distribution of XCIR frequencies within this family was significantly different than predicted by normal population data or models of random XCI (Po0.025), 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.

Familial skewed X-chromosome inactivation linked to a component of the cohesin complex, SA2

“…7,8 Naumova et al 9 used an approach of classic linkage analysis, to show that markers DXS425 (Xq25) and DXS294 (Xq26) are likely linked to the human XCE. Cau et al used a similar approach to identify a candidate region of 4.2Mb cM ranging from DXS8067 (Xq24) to DXS8057 (Xq25), which partially overlaps the region identified by Naumova et al 7 In a previous report, 10 we described a family with three males and three females with clinical symptoms of haemophilia A, a bleeding disorder caused by low factor VIII activity. We showed that the three affected females had XCI ratios skewed toward activation of the mutated X-chromosome and that the degree of skewing correlated with FVIII activity and the severity of disease in all carriers in the family.…”

“…The family studied here (Figure 1) is as we have previously described, 10 with the addition of seven new family members, II.4, II.10, III.4, III.6, III.7, IV.I and IV.2.…”

“…10 In accordance with the IWK Health Sciences Centre guidelines on genetic testing of minors, the F8 genotypes of young females, who were not obligate carriers, were not assessed. Factor VIII activity measurements were taken.…”

“…10 Intraoral buccal mucosa cells were collected with cytology brushes (Orifice Medical AB, Ystad, Sweden). DNA was extracted using a QIAamp DNA Mini Kit (Qiagen, Germantown, MD, USA) according to the manufacturer's instructions.…”

“…10 At this locus, the inactive allele is hypermethylated, 11 therefore the peak with the greatest area is the least active. In some cases, the technique was adapted for analysis by fluorescent column electrophoresis (Supplementary Materials and methods).…”