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.
