1st year funding
Wenda L. Greer, PhD
Dalhousie University, Halifax, Nova Scotia
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
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
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:
- 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.
- To 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.
This study will provide answers for this family and insight into the basic biology of X-chromosome inactivation.
2nd year funding
Dr. Paula James
Queen’s University, Kingston, Ontario
CHS Research Program
Von Willebrand disease (VWD) is the
most common known inherited bleeding disorder in humans, affecting as
many as 1% of the population. People with VWD have difficulty with
bleeding from mucous membranes such as the nose, mouth or lining of the
uterus, or can have problems with bleeding after injuries, dental work
or surgical procedures. There are 3 subtypes: Type 1 VWD is the most
common and least severe and is caused by a mild to moderate deficiency
of a blood clotting factor called von Willebrand factor (VWF). Type 3
VWD is the least common and most severe and is caused by a severe
deficiency of VWF. Type 2 VWD is caused by VWF that doesn’t function
Type 1 VWD is inherited from one parent while Type 3 VWD is inherited from both parents. In this study, entitled Genetic Differences Between Obligate Carriers of Type 3 VWD and Individuals with Type 1 VWD,
we are interested in examining the genetic changes in VWD. A person
affected with Type 1 VWD would have inherited it from one parent, while
a person affected with Type 3 VWD must have inherited it from both
parents. A parent of an individual with Type 3 VWD is usually not
affected by any bleeding problem and is referred to as a “carrier”. By
using special techniques that allow us to examine an individual’s
genetic make-up, we hope to improve our understanding of the types of
genetic changes that might lead to Type 1 VWD and those that would lead
to being a carrier for Type 3 VWD.
1st year funding
Dr. Gonzalo Hortelano
McMaster University, Hamilton, Ontario
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
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 investigated.
This transplantation therapy could
reduce and ultimately eliminate the need for regular FVIII injections.
Importantly, the microcapsules can be removed, increasing the safety of