CHS Research Grants for 2007
The role of X-inactivation in the expression of hemophilia A in women
Dr. Wenda L. Greer, FCCMG
Professor, Department of Pathology
Dalhousie University - Halifax, Nova Scotia
Second year funding
Hemophilia A is an X-linked recessive bleeding disorder resulting from
mutations in the F8 gene. It is usually expressed in males who inherit
only one X chromosome from their mother. Females inherit one X from
each parent. Those who inherit only one mutated f8 gene usually do not
express the disease. Rare examples of hemophilia A manifesting in
heterozygous females occur due to an unusual pattern of X chromosome
inactivation. This is a mechanism that causes one X in every female
cell to be inactivated early in development. It is a mechanism which
compensates for the fact that females have a double dose of X chromatin
compared to males. In most females, approximately half of the cells
inactivate their maternal X and half their paternal X. In rare cases, X
chromosome inactivation is skewed. If it is skewed toward the
expression of a mutated X chromosome, a heterozygous female can be
affected with an X-linked recessive disease.
A family has presented with several males and several
females affected with hemophilia A. Analysis of one female showed that
most of her cells were expressing the mutated paternal X chromosome. We
therefore hypothesized that affected females in this family are
expressing hemophilia A due to nonrandom X inactivation patterns. It is
unlikely that random chance could account for the putative dramatic
skewing of X chromosome inactivation leading to 3 affected females.
This led us to consider that these females have inherited a
predisposition to skewed X chromosome inactivation patterns.
XCI is controlled in cis by an untranslated RNA coded by
the XIST gene. Xist is regulated by the Tsix RNA that is antisense to
Xist. It is believed from studies in mice that there is an X chromosome
controlling element (XCE) that down regulates Tsix expression and
alters the probability of an X chromosome being inactivated.
Our objective is to understand why females in this family
are expressing hemophilia A. Our hypothesis is that their X chromosomes
containing the normal F8 gene have been selectively inactivated,
leaving only the mutated f8 available for expression. More
specifically, we propose to test the hypothesis that there is a region
on the X chromosome that contains an XCE that influences selection and
accounts for disease in this family.
Our specific aims are:
This study will provide answers for this family and insight into the basic biology of X-chromosome inactivation.
- To use polymorphic micosatellite markers
at 5cm intervals to compare the X chromosomes of affected and
unaffected female siblings with skewed and random X-inactivation
patterns, respectively. Hypothetically, regions where they differ
should define the critical region of the putative XCE.
further compare these X chromosomes using microarray CGH to look for
regions of duplication, deletion and differential methylation
(collaboration with Dr Wan Lam, Toronto).
- To develop a cell
culture model system to study the process of X-chromosome inactivation
in females. With this testable system, we will determine if
X-inactivation is under genetic control. It will also provide a tool to
localize the XCE gene.
Gene therapy of hemophilia A
Dr. Gonzalo Hortelano
Assistant Professor, Department of Pathology
McMaster University - Hamilton, Ontario
Second year funding
We will evaluate the feasibility of cell transplantation therapy to
reverse severe hemophilia A in mice. Although current factor VIII
(FVIII) products are safe, patients must endure life-long regular FVIII
infusions. Thus, a safe and more economic treatment is desirable.
Gene therapy is an alternative. Gene therapy strategies use
virus as vehicles to introduce the FVIII gene, but they are associated
with undesirable immune responses. Alternatively, transplanted cells
producing FVIII are only temporarily functional. We propose the
transplantation of non-autologous cells (not from the patient)
genetically engineered to continuously produce FVIII. To avoid
rejection of the transplanted cells, they are enclosed in tiny
microcapsules (less than 1mm in diameter) before being transplanted.
The microcapsules allow the free flow of FVIII, but are impermeable to
immune cells, therefore protecting the enclosed cells.
We found that mice transplanted with microcapsules
containing muscle cells engineered to secrete factor IX contained high
amounts of factor IX in the blood for at least 120 days and did not
mount an immune response to human FIX. More importantly, this treatment
was able to reverse the disease in severe hemophilia B mice. If this
were achieved in humans, it would eliminate severe and moderate
hemophiliacs. Therefore, we will apply the same strategy to hemophilia
Initially, we will engineer muscle cells to produce FVIII,
and determine the amount of FVIII they produced. Second, we will
enclose FVIII-producing cells in microcapsules that will then be
transplanted into mice to determine how much FVIII is found in blood,
and for how long. Any immune responses to FVIII will be studied.
Finally, the correction of the disease in hemophilia A mice will be
This transplantation therapy could reduce and ultimately
eliminate the need for regular FVIII injections. Importantly, the
microcapsules can be removed, increasing the safety of the treatment.
Platelet type von Willebrand disease: An underdiagnosed cause of excessive mucocutaneous bleeding?
Dr. Maha Othman
Adjunct Assistant Professor, Department of Pathology and Molecular Medicine
Queen’s University - Kingston, Ontario
First year funding
The overall aim of this project is to investigate the occurrence of
platelet type (PT)-VWD among patients who are provisionally diagnosed
as type 2B VWD based on clinical and laboratory data. This study will
address the question of whether platelet type VWD is being under- or
misdiagnosed in this cohort of patients. The correct discrimination
between the two disorders is critical as it directs the treatment
We hypothesize that the apparent rarity of PT-VWD is at
least partly due to the fact that we are missing the diagnosis of this
condition because of the high degree of similarity with type 2B VWD. We
also hypothesize that the molecular genetic approach is essential to
make a conclusive discrimination. Finally, we emphasize that this issue
carries a significant implication in terms of treatment decisions.
Specific Project Aims:
discriminate type 2B VWD from the closely similar platelet type VWD
using a molecular approach: evaluation of sequence variations in each
of the exon 28 of the VWF gene (platelet binding region) and the
platelet GPIBA gene.
To identify the percentage of misdiagnosed PT-VWD among clinically diagnosed type 2B VWD cases.
To functionally characterize novel mutations, if any, in PT-VWD cases.
Factor VII(a) clearance behaviour
Dr. William P. Sheffield
Associate Professor, Department of Pathology and Molecular Medicine
McMaster University - Hamilton, Ontario
First year funding
The hemophilia community knows all too well that the replacement
therapy that can restore hemophiliacs to an active life is not always
problem-free. For individuals with hemophilia A, replacement therapy
with purified recombinant factor VIII (fVIII) can sometimes be
complicated by inhibitor formation. Inhibitors are antibodies directed
against the injected fVIII. Fortunately, administration of recombinant
factor VIIa (fVIIa) can bypass this potentially serious problem.
However, fVII is one of the coagulation factors that leaves the
circulation the most rapidly. This clearance behaviour is not
completely understood. For this reason, Drs. Bill Sheffield and Bryan
Clarke, of McMaster University and Canadian Blood Services have
received a CHS research grant to study the clearance of fVII(a) in mice
genetically altered to have hemophilia A. The investigators will use
DNA and cell culture technology to make both human fVII identical to
current clinical products, and new forms of fVII in their laboratories.
Once purified, how long the fVII-related proteins last in the mouse
circulation, and how well they control bleeding will be determined. The
most unusual products to be tested are “fusions” - in which fVII and
human serum albumin, ordinarily separate proteins, are combined into a
single chain. The researchers propose that fVII will continue to work
in this new format, and will remain in the circulation for much longer
than the current product. The longer it remains, the more effective it
should be in combating bleeding. Sheffield and Clarke hope that their
research will lead to better understanding of the mechanism of action
of rfVIIa, and to the design of improved rfVII(a) products to provide
caregivers with novel tools to help hemophiliac patients with