Coinheritance of two alpha-spectrin gene defects in a recessive spherocytosis family.

We studied a recessive hereditary spherocytosis (HS) family from Norway in which all four children had haemolytic spherocytosis while spectrin (Sp) deficiency was detected in the proband. Molecular analysis demonstrated that all affected children had inherited the low expression alpha-Sp allele LEPRA (Low Expressed PRAgue) from the father. Haplotyping with a polymorphic dinucleotide repeat for the alpha-Sp gene (alphaVNTR) located in the 3' untranslated region of mRNA showed that all recessive children had inherited the same maternal alpha-spectrin allele. The paternal Sp-alphaLEPRA allele was found in cis of the polymorphic alpha-Sp Bughill allele (alphaBH) characterized by the A970D point mutation in the Sp alpha-chain. This mutation was identified on two-dimensional electrophoresis of Sp tryptic digests as an acidic shift of the alphaII tryptic domains (spots alphaIIa). Analyses of the relative expression of the paternal alpha-Sp Bughill polymorphism in the proband showed that the product of the maternal alpha-Sp gene is almost completely absent from the mature erythrocyte membrane. Comparative analysis between alphaVNTR PCR-amplified from genomic DNA and from cDNA showed that the maternal low expression alpha-Sp allele is associated with a decreased amount of mRNA. Results from molecular and biochemical studies showed that all the affected children of this family are compound heterozygous for two different low expression alpha-Sp alleles: an uncharacterized defective alpha-Sp allele on the maternal side and an alphaLEPRA allele tagged by the alphaIIa polymorphism on the paternal side.

Spectrin Tunis (Sp alpha I/78), an elliptocytogenic variant, is due to the CGG----TGG codon change (Arg----Trp) at position 35 of the alpha I domain.

Spectrin Tunis (Sp alpha I/78) is an alpha l domain variant that causes asymptomatic elliptocytosis in the heterozygote state. It is manifested by a reduction of spectrin dimer self-association and by the development of a major 78-Kd fragment at the expense of the alpha l 80-Kd fragment upon spectrin-limited digestion. Amino acid sequence analysis, following peptide transfer onto Immobilon membranes, showed that the 78-Kd fragment results from a sensitized cleavage after lysyl residue 10. Using a 13.5-kb genomic alpha-spectrin probe and the Xbal, Pvull, and Mspl polymorphic sites detected with this probe, we concluded that spectrin Tunis is associated with the + - + haplotype (in the above order). Twenty mer oligonucleotides, complementary to genomic segments from introns 2 and 3, respectively, were synthesized. We then performed DNA amplification and sequencing. In the two investigated carriers of spectrin Tunis, we found the C----T base substitution of the codon corresponding to position 35 of the alpha l domain (CGG----TGG; Arg----Trp). The mutation lies in the last part of an alpha helix that extends from residues 9 to 44 of partial repeat alpha 1' and is comparable with helix 3 of full repeats 1 to 5. The modified proteolytic site, located 25 amino acid residues upstream, occurs at the beginning of the helix.