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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:

  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.

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 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.

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 decision.

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:
To 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 inhibitors.