(1) Elucidation of the structural defect of HE, HPP spectrin (Sp). Three molecular defects of the yield I domain of Sp, the SpD-SpD self-association site, have been identified by us and others in HPP and a subpopulation of HE. These are characterized by a formation of 74, 65 and 46 kd tryptic peptides generated instead of the normal 80 kd yield I peptide. To elucidate the structure of these Sp and their possible heterogeneity, we will subject the abnormal peptides to further chemical or enzymatic cleavage, followed by HPLC peptide separation. Abnormal peptides will be subjected to amino acid sequence analysis to identify the primary structural defect. We will also employ other enzymatic and chemical cleavages to generate peptides containing the trypsin sensitive sites (the presumptive site of the primary structural defect). Employing a similar strategy we will further characterize recently identified asymptomatic variants of Sp yield III and Beta II domains and recently detected HE(SpD-SpD), with no identifiable abnormalities on limited tryptic peptide maps. (2) Genetics of the Sp I in HE and HPP. Based on functional, structural and clinical expression of the Sp yield defects, we distinguish at least 3 clinical phenotypes: asymptomatic carriers, whose red cells contain about 25% of abnormal Sp, HE containing 40-50% and HPP containing 75-100% of abnormal Sp, respectively.
Our aim i s to define factors that determine the above phenotypes including: (i) instability of HPP Sp, (ii) compound heterozygous state for a structural Sp defect and a variably expressed defect of SP synthesis, and (iii) possible duplication of allelic yield Sp genes. In the latter case, the differences in clinical phenotype may be due to involvement of one, two, three or four of the four SP genes, respectively. To study those hypothetical factors we will (a) provide quantitative analysis of normal and abnormal Sp in the cells, (b) measure relative synthetic rates of yield, Beta Sp in reticulocytes and red cell precursors and Sp mRNA content, and (c) examine the susceptibility of normal and abnormal Sp to proteolytic degradation in bone marrow precursors. (3) Identification of molecular defects in other HE and HS patients. In a subpopulation of HE (11 kindred out of 46) and in all 12 HS kindred studied to date, we find neither a deficiency of one of the major proteins on SDS PAGE, nor a qualitative defect of Spd-Spd. In some of these, we studied patient Sp-control 4.1 and patient Sp-control IOV interaction and found them to be normal. To search for a putative molecular defect in these patients we plan (a) to employ sensitive screening assays to measure Sp-4.1 and Sp-ankyrin, ankyrin-band 3 interactions, (b) to analyze limited tryptic and chymotryptic peptides of 4.1 and ankyrin, (c) to look for a possible deficiency of one of the minor skeletal proteins, and (d) study Sp, 4.1 interactions with PS, PE, the major phospholipids of the inner membrane bilayer.
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