Having completed a clinical fellowship in Hematology/Oncology, I have spent the past two years working with Dr. Jiri Palek's group under the guidance of Drs. Jiri Palek, Ken Sahr and Shih-Chun David Liu. I have spent equal time learning the basics of molecular biology and protein biochemistry of the red cell membrane. This training has lead me, with the guidance of Dr. Jiri Palek to propose the project described in this grant application which relates to the primary molecular defect in hereditary spherocytosis. Hereditary spherocytosis (HS) is caused by abnormalities of the erythrocyte membrane skeleton. In the majority of patients with HS the erythrocyte membrane has a deficiency in spectrin, the major protein of the membrane skeleton. The primary defect responsible for this deficiency appears heterogeneous and has been defined in a small number of patients only. We have identified in our laboratory several distinct subsets of patients with HS, including a subset with deficiency in spectrin but a normal ankyrin content. Because of the growing data linking HS to ankyrin, as well as the important role of ankyrin as the principal attachment site of the spectrin skeleton we have asked whether a subset of HS patients could have a defect of ankyrin leading to a poor binding to spectrin and a subsequent deficiency in spectrin. In preliminary experiments, we have examined the spectrin/ankyrin binding interaction in three unrelated patients in this subset of HS patients. We have found, in two unrelated probands, a decreased binding of normal spectrin to patients' inside-out-vesicles (IOVs) as compared to normal IOVs.
The specific aim of this work is to investigate the possibility that, in this subset of patients with HS, a defect in the spectrin binding domain of ankyrin could be responsible for an abnormal binding and a subsequent deficiency in spectrin. First, we will further characterize this binding abnormality in these 2 patients, focusing on the spectrin binding domain of ankyrin: (i) At the protein level, by examining the binding of normal spectrin to purified ankyrin or its proteolytic 72 kD fragment (which contains the spectrin binding domain) or its subfragments. (ii) At the gene level, we will try to uncover mutations of the portion of the gene coding for the spectrin binding domain using the denaturing gradient gel electrophoresis technique. If a mutation is uncovered, we will examine the co- inheritance of this mutation with the HS phenotype by analyzing DNA from family members, using the DGGE, allele specific oligonucleotide hybridization or restriction enzyme analysis. Second, we will study the frequency of ankyrin defects characterized by a weak binding to spectrin in other HS patients in the same subset. At the protein level, we will examine the spectrin/ankyrin interaction using binding assays as above. At the gene level, we will try to uncover new mutations of the spectrin binding domain of ankyrin using the DGGE. I strongly believe that the group of Dr. Jiri Palek and his colleagues will provide an optimal environment both for my future career development and for a successful execution of this project as it combines strong expertise in protein biochemistry of the red cell membrane, a strong molecular biology training as well as a broad clinical expertise in hemolytic anemias and a wide patient referral population.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08HL002720-04
Application #
2210439
Study Section
Special Emphasis Panel (SRC (FB))
Project Start
1992-04-01
Project End
1997-03-31
Budget Start
1995-04-01
Budget End
1996-03-31
Support Year
4
Fiscal Year
1995
Total Cost
Indirect Cost
Name
St. Elizabeth's Medical Center of Boston
Department
Type
DUNS #
073797292
City
Boston
State
MA
Country
United States
Zip Code
01235
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Hassoun, H; Wang, Y; Vassiliadis, J et al. (1998) Targeted inactivation of murine band 3 (AE1) gene produces a hypercoagulable state causing widespread thrombosis in vivo. Blood 92:1785-92
Hassoun, H; Hanada, T; Lutchman, M et al. (1998) Complete deficiency of glycophorin A in red blood cells from mice with targeted inactivation of the band 3 (AE1) gene. Blood 91:2146-51
Hassoun, H; Vassiliadis, J N; Murray, J et al. (1997) Characterization of the underlying molecular defect in hereditary spherocytosis associated with spectrin deficiency. Blood 90:398-406
Hassoun, H; Palek, J (1996) Hereditary spherocytosis: a review of the clinical and molecular aspects of the disease. Blood Rev 10:129-47
Hassoun, H; Vassiliadis, J N; Murray, J et al. (1996) Hereditary spherocytosis with spectrin deficiency due to an unstable truncated beta spectrin. Blood 87:2538-45
Hassoun, H; Vassiliadis, J N; Murray, J et al. (1995) Molecular basis of spectrin deficiency in beta spectrin Durham. A deletion within beta spectrin adjacent to the ankyrin-binding site precludes spectrin attachment to the membrane in hereditary spherocytosis. J Clin Invest 96:2623-9
Hassoun, H; Coetzer, T L; Vassiliadis, J N et al. (1994) A novel mobile element inserted in the alpha spectrin gene: spectrin dayton. A truncated alpha spectrin associated with hereditary elliptocytosis. J Clin Invest 94:643-8