Pedigree, mutation detection, and X chromosome inactivation analysis.

(A) Pedigree of the family and results of the polymorphism analysis with 6 F8 markers. From the top to the bottom: CA repeat ofF8 intron 13, F8 BclI intron 18, CA repeat ofF8 intron 22, XbaI/KpnI F8 intron 22, DXS 52, and DXS 15 mapping 3 recombination units from the F8 gene. (B) X-inactivation analysis at the HUMARA locus. On the top line, results of the PCR amplification of the androgen-receptor polymorphic (CAG) repeat without HpaII digestion. The proband (II.1) is heterozygous at this locus and inherited the allele 186 from her mother and the allele 198 from her father. Her paternally derived allele (198) is completely digested by the methylation-sensitive restriction enzymeHpaII and, therefore, not amplified by PCR. The remaining peak (186) is the maternally derived allele and represents the inactive, methylated X chromosome that resists cleavage byHpaII and thus is successfully amplified. This female patient thus shows complete skewing of X inactivation. (C) Identification of the Q565delC/ter566 mutation in theF8 gene. F8 exon 11 direct nucleotide sequence fragments are shown in an unrelated wild-type control and in the patient with the relevant wild-type and mutant sequences noted to the left. In the mutated allele, a C is deleted (arrow) and results in overriding of the wild-type and mutated sequences 3′ to the deletion point. (D) Exon 11 mutation DGGE screening in the family. Lane 1, migration pattern of the wild-type alleles from a control subject (C). Lane 2 and 4, DNA samples from the proband's mother (I.2) and the proband's father (I.1), respectively, demonstrating a same migration pattern than the control. Lane 3, DGGE profile of the Q565delC/ter566 mutation from the heterozygous female patient (II.1).