Blood coagulation factor VIII

When activated by thrombin, factor VIII protein
consists of five subunits (A1, A2, A3, C1, C2) held
together by calcium ions (1). The inactive factor
VIII protein (2) contains three domains (A, B, C).
Domain A occurs in three homologous copies
(A1, A2, A3), domain C in two (C1, C2), and
domain B in one copy. In humans, the gene for
factor VIII (3) maps to the distal long arm of the
X chromosome in region 2, band 8 (Xq28). It
consists of 26 exons and spans 186000 base
pairs (186 kb), corresponding to about 0.1% of
the whole X chromosome. Noteworthy in this
gene are the large exon 14 (3106 base pairs),
which codes for the B domain, and a large intron
of 32000 base pairs between exons 22 and 23.
Most point mutations occur in DNA sequences
involving TCGA, the recognition sequence for
the restriction enzyme TaqI. It contains the
dinucleotide CG, which is easily mutated. Since
the cytosine of this dinucleotide is frequently
methylated and deamination of methyl cytosine
leads to C-to-T transition, mutations in
CG dinucleotide regions are frequent. Mutation
of TCGA to TTGA creates a stop codon (TGA), resulting
in a truncated factor VIII protein. Even a
stop codon at position 2307 leads to severe
hemophilia, although only the last 26 amino
acids are missing (Gitschier et al., 1985).
Polymorphic restriction sites (RFLPs, restriction
fragment length polymorphisms) can be utilized
for molecular genetic diagnosis of
hemophilia A (4). When present, a variant recognition
sequence (B*) for the restriction
enzyme BcII in the region of exons 17 and 18
produces a fragment of 879 base pairs and a
fragment of 286 base pairs; when it is absent, a
single fragment of 1165 base pairs results. This
can be used in RFLP diagnosis (5): The index
patient (II-1) with hemophilia A carries the 879
bp fragment. This fragment indicates the mutation.
His sister (II-2) has an affected son (III-2)
who also carries the 879 bp fragment, inherited
from his mother. A brother (III-1) carries the
1165-bp fragment and thus is not at risk for the
disease, because this is not linked to the mutation.
In addition to point mutations, factor VIII gene
rearrangements involving the long intron 22 are
frequent.