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CHS Research Grants for 2006

The Role of X-inactivation in the Expression of Hemophilia A in Women

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 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:

  1. 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.
  2. 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).
  3. 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.

Genetic Differences Between Obligate Carriers of Type 3 VWD and Individuals with Type 1 VWD

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 properly.

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.

Implantable Microcapsules as Gene Therapy for Hemophilia A

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 hemophilia A.

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 the treatment.