Limited tryptic digestion of spectrin (Sp) from seven related individuals manifesting hereditary elliptocytosis (HE) or hereditary pyropoikilocytosis (HPP) phenotypes revealed the presence of a novel peptide with a molecular weight of 78 Kd and a concomitant decrease in the alpha I domain (80-Kd peptide), which is the domain involved in the dimer self-association process. Sp from the normal members of this white family exhibited a normal peptide pattern, as compared with controls. The abnormal peptide pattern was associated with a decreased ability of Sp dimer to self-associate. In this kindred in which three generations were available for study, the clinical manifestations were quite variable and ranged from the asymptomatic HE carrier state to hemolytic HE or to severe anemia requiring splenectomy. The severity of the disease appeared to be correlated both with the amount of mutant spectrin (31% to 69%) and with the excess of the Sp dimer found in the membrane (26% to 60%, compared with a normal value of 5.6% +/- 2.2%). Partial amino acid sequencing showed that the alpha I/78-Kd peptide resulted from cleavage at lysine residue 10 of the alpha I/80-Kd domain. Knowledge of the exon/intron structure of cloned genomic DNA encoding the alpha I domain allowed us to amplify in vitro a DNA fragment containing the third exon of the alpha-spectrin gene. The amplified fragment was subcloned and sequenced. A G to T transversion was found in the 39th codon (AGT for AGG), which changed the normal arginine to a serine. Hybridization of amplified DNAs with allele- specific oligonucleotides corresponding to the normal and mutant sequences confirmed the presence of the mutation in three other HE members of the family (the propositus mother, brother, and sister).

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