Molecular Genetics of Hemophilia A 
Jane M. Gitschier, Ph.D. — Assistant Investigator 
Dr. Gitschier is also Assistant Professor of Medicine ( Genetics ) at the University of California, San Fran- 
cisco. She received a B.S. degree in engineering science from Pennsylvania State University, an M.S. in 
applied physics from Harvard University, and a Ph.D. in biology from the Massachusetts Institute of 
Technology. She did postdoctoral research with Richard lawn at Genentech, Inc., before joining the faculty 
at the University of California. 
THE genetic basis of hemophilia has been un- 
derstood since biblical times, when mothers 
of boys who had bled to death at circumcision 
were advised not to have future sons circum- 
cised. The most common form of this disease, he- 
mophilia A, affects 1 male in 5,000 throughout 
the world. It results from mutations in the gene 
coding for a blood coagulation protein called fac- 
tor VIII. The factor VIII gene is located on the X 
chromosome, one of the two sex chromosomes. 
Consequently, hemophilia A is inherited as a 
"sex-linked" trait. Males, having a single X chro- 
mosome, are afflicted with hemophilia if their 
factor VIII gene is mutated, but females are pro- 
tected from manifesting the disease by a second, 
normal X chromosome. Thus females may be si- 
lent carriers of the disease but transmit it geneti- 
cally to their sons. 
Our laboratory is interested in understanding 
what types of mutations lead to hemophilia A and 
how these mutations are generated. The disease is 
well suited to studies on mutagenesis because it 
is clinically heterogeneous, implicating a wide 
variety of mutations in the factor VIII gene. 
Correlating the types of mutations with particular 
clinical findings may be very helpful in under- 
standing the role of factor VIII in coagulation. In 
addition, many cases of hemophilia occur sporad- 
ically in families without any history of the dis- 
ease. This observation suggests that many muta- 
tions arise anew in affected individuals or their 
mothers. Knowledge of mutations allows accu- 
rate prenatal diagnosis and detection of female 
carriers by analysis of the mutation itself. 
Because the factor VIII gene is unusually large, 
one of the major obstacles in this project lies in 
finding the mutations. We have experimented 
with several different mutation screens and have 
had considerable success with a technique called 
denaturing gradient gel electrophoresis. This 
technique is based on the principal that DNA 
fragments differing by a single base pair (e.g., 
normal vs. mutated) also differ in their thermal 
stability. In practice, the thermal stability of DNA 
fragments from normal and hemophilic individ- 
uals is assayed on gels containing a chemical de- 
naturant, which has the same destabilizing effect 
as temperature. 
We have focused on finding mutations in DNA 
samples from patients with mild and moderately 
severe hemophilia. Our experience suggested 
that these patients would be likely to have amino 
acid coding changes. We developed a strategy of 
screening for mutations in factor VIII coding re- 
gions by denaturing gradient gel electrophoresis. 
Mutations were found in 21 of the 35 patients 
screened to date. These mutations are scattered 
throughout the factor VIII gene and appear to be 
quite diverse. 
Having identified the mutations responsible 
for hemophilia in some patients whose disease is 
sporadic, we have had the opportunity to investi- 
gate the origin of the mutations. In the past, mu- 
tations were assumed to occur as isolated events 
in either eggs or sperm. However, we have evi- 
dence that some mutations occur much earlier, at 
some point during parental development, ren- 
dering the parent "mosaic" for mutant and nor- 
mal cells in different parts of the body. Conse- 
quently, many cells in the parent's germline may 
carry the mutation, but his or her blood cells, in 
which the presence of the mutation is tested, may 
not. Mosaicism has also been found associated 
with other X-linked and dominantly inherited 
disorders. These findings demonstrate that the 
unaffected parent of a child with sporadic disease 
may be at substantial risk of having a second child 
with the disease. 
In a second line of research, our laboratory is 
exploring DNA sequences near the factor VIII 
gene. These are of great interest because many 
genetic diseases map near hemophilia A. For ex- 
ample, two of the most common X-linked dis- 
eases, red and green color blindness and glucose- 
6-phosphate dehydrogenase (G6PD) deficiency, 
are tightly linked to the hemophilia locus. By 
physical mapping methods, we found that the 
genes for factor VIII, visual pigment, and G6PD 
are located within a million base pairs of each 
other. In collaboration with Barbara Trask at 
Lawrence Livermore National Laboratory, we 
were able to establish the relative "orientation" 
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