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Summary. Nonsense/stop mutations in the ankyrin-1 gene (ANK1 ) are a major cause of dominant HS (dHS) (frequency of 23% in German dHS patients). To date, no common mutation has been found and therefore a simple mutation screening is not feasible. The reduced expression of one cDNA allele in the (AC)n microsatellite polymorphism of the ankyrin-1 gene, as seen in about 20% of Czech patients with dHS, may identify candidates with a possible frameshift/nonsense mutation. In order to verify the efficiency of this screening we screened the ankyrin-1 gene of 22 Czech dHS patients for both the reduced cDNA allele expression in the frequent (AC)n and the common exonic 26/39 polymorphisms, as well as for polymerase chain reaction (PCR) single-stranded conformation polymorphisms in any one of the 42 exons of ANK1 . Anomalous PCR products were sequenced. We found seven new ANK1 frameshift/nonsense mutations in nine patients with, but in none of six patients without, a reduced cDNA allele expression (efficiency of 78%). We conclude that screening of dHS patients for such a reduced allele expression in common ANK1 polymorphisms is an efficient procedure for the identification of candidates for frameshift/nonsense mutations in the ankyrin-1 gene. [ABSTRACT FROM AUTHOR]

Provides information on red blood cell membrane disorders. Composition of red cell membrane; Impact of defective vertical interactions on hereditary spherocytosis (HS); Use of ankyrin in the reduction of expression in HS patients.

The binary interaction between human erythrocyte protein 4.1 and rabbit skeletal muscle F-actin was examined by rapid pelleting of the binary complexes. The binding curves show that the reaction was saturable at approximately one protein 4.1 molecule/actin monomer. The reaction was highly co-operative, displaying a Hill coefficient close to 2. Using a fixed concentration of radiolabelled protein 4.1, and varying the concentration of F-actin, the apparent molar association constant, Ka, was observed to range from 5×104 M-1 to > 106 M-1. The binary interaction between erythrocyte spectrin and actin was also observed to be co-operative under the same conditions. The rate of reaction between protein 4.1 and actin was temperature sensitive in a manner consistent with a high energy of activation. The pelleting assay also showed that the concentration of actin was reduced in the supernatant in the presence of protein 4.1 compared with actin alone, indicating that the critical concentration of actin was lowered in the presence of protein 4.1. Polyvalent anions disrupted the binary interaction between F-actin and protein 4.1, the disruption being consistent with the number of negative charges on these anions at pH 7.5. We postulate that the co-operativity of the binding of protein 4.1 to actin results from a protein 4.1 molecule binding to a single monomer within the filament structure which then promotes conformational changes allowing further protein 4.1 binding. The demonstration of a specific binary association between protein 4.1 and actin suggests that this interaction contributes significantly to the stabilization of the spectrin—actin—protein-4.1 ternary complex. [ABSTRACT FROM AUTHOR]

Erythrocyte protein 4.1 plays a major role in stabilizing the spectrin-actin junction of the erythrocyte membrane skeleton. The particular sites on spectrin responsible for the binding of actin and protein 4.1 have not been specifically defined, although the general region of the ‘tail’ end, opposite the self-association site, has been deduced by electron microscopy. Using a photoactivatable, radiolabel-transfer cross-linker, 1-[N-(2-hydroxy-5-azidobenzoyl)-2-aminoethyl]-4-(N-hydroxysuccinimidyl)-succinate, we have determined that the binding site for protein 4.1 on spectrin resides in the N-terminal region of Β spectrin within a sequence homologous to the actin-binding region of α actinin. Moreover, this technique provided clear evidence for a direct binding interaction between actin filaments and protein 4.1 that was confirmed by rapid-sedimentation assays. In summary, use of radiolabel-transfer cross-linking has enabled assignment of the protein-4.1-binding site on erythrocyte spectrin and has identified a previously ill-defined binary interaction between protein 4.1 and F-actin. [ABSTRACT FROM AUTHOR]

Nonsense/stop mutations in the ankyrin-1 gene (ANK1) are a major cause of dominant HS (dHS) (frequency of 23% in German dHS patients). To date, no common mutation has been found and therefore a simple mutation screening is not feasible. The reduced expression of one cDNA allele in the (AC)n microsatellite polymorphism of the ankyrin-1 gene, as seen in about 20% of Czech patients with dHS, may identify candidates with a possible frameshift/nonsense mutation. In order to verify the efficiency of this screening we screened the ankyrin-1 gene of 22 Czech dHS patients for both the reduced cDNA allele expression in the frequent (AC)n and the common exonic 26/39 polymorphisms, as well as for polymerase chain reaction (PCR) single-stranded conformation polymorphisms in any one of the 42 exons of ANK1. Anomalous PCR products were sequenced. We found seven new ANK1 frameshift/nonsense mutations in nine patients with, but in none of six patients without, a reduced cDNA allele expression (efficiency of 78%). We conclude that screening of dHS patients for such a reduced allele expression in common ANK1 polymorphisms is an efficient procedure for the identification of candidates for frameshift/nonsense mutations in the ankyrin-1 gene.