Hereditary pyropoikilocytosis and elliptocytosis in a white French family with the spectrin alpha I/74 variant related to a CGT to CAT codon change (Arg to His) at position 22 of the spectrin alpha I domain

We describe a white French family in which 12 subjects presented with hereditary elliptocytosis (HE) or hereditary pyropoikilocytosis (HPP). Eight of these subjects were shown to be heterozygous for a spectrin (Sp) alpha I/74 variant, as demonstrated by analysis of partial tryptic digestion fragments of spectrin. This abnormal peptide pattern was associated with a decreased ability of Sp dimers to self-associate. In this kindred, in which four generations were available for study, the clinical expression varied from mild HE to HPP with an intermediate status of hemolytic HE. The severity of the disease appeared to be correlated both with the estimated amount of variant Sp (42% to 65%) and the excess of Sp dimers found in the membrane (30% to 51%, with a normal value of 3.7% +/- 1.6%). Reassociation studies using isolated Sp alpha and beta chains from an affected patient and an unaffected control subject showed that the Sp alpha I/74 Kd abnormal tryptic peptide resulted from a defect in the Sp alpha chain. Partial amino acid sequencing showed that the Sp alpha I/74 Kd peptide resulted from cleavage at lysine residue 42 of the Sp alpha I/80 Kd domain. Knowledge of the exon/intron organization of the human alpha Sp gene allowed us to amplify by the polymerase chain reaction the second exon of the alpha Sp gene in total cellular DNA of the HPP proposita. The amplified fragment was subcloned and sequenced. We found a G to A base substitution in the 22nd codon (CAT for CGT), which changes the normal arginine to a histidine. Hybridization of amplified DNAs with allele-specific oligonucleotides corresponding to the normal and mutant sequences confirmed the presence of the mutation in six other HE and HPP members of the family. The identification of this mutation at the DNA level confirmed the transmission of the same molecular defect in Sp through four generations but with different patterns of clinical expression.