title: Assignment of Sp alpha I/74 hereditary elliptocytosis to the alpha- or beta-chain of spectrin through in vitro dimer reconstitution.
abstract: Partial digestion of spectrin dimers in vitro has allowed the definition of domains. For example, the portions of the dimers that are involved in spectrin self-association are represented by the alpha I and the beta I domains. The alpha I domain (80 Kd) is further cleaved into a minor 78 Kd fragment and, more substantially, into a 74 Kd fragment. The intensity of the latter, which we expressed as the 74:(80 + 78 + 74) ratio, or the 74:alpha I ratio, is variable depending on the experimental conditions, eg, in fine, on the conformation of the alpha I domain. A number of cases of hereditary elliptocytosis (HE) are associated with an increase of the 74:alpha I ratio, also referred to as the Sp alpha I/74 abnormality. Several lines of evidence have suggested that the causal mutations may lie in the alpha- or the beta-chain, a point of importance before one undertakes studies at the gene level. In order to address this question, we reconstituted spectrin dimers in vitro, combining alpha- and beta-chains of various origins, and then carried out partial digestion and assayed the Sp alpha I/74 abnormality. The patterns obtained with reconstituted dimers were nearly identical to those of native dimers. We applied the assay to three spectrin variants that cause Sp alpha I/74 HE: (1) a variant that we previously designated spectrin Nice and whose beta-chain lacks a 4 Kd fragment in its C-terminal region; and two distinct variants that we found in two unrelated white families and that we provisionally designated spectrin Lyon and spectrin Culoz. The Sp alpha I/74 abnormality appeared in all kinds of dimers that harbored the beta-chain of spectrin Nice, or the alpha-chain of spectrin Lyon or spectrin Culoz, respectively. Therefore, we confirmed that spectrin Nice is a (alpha I/74) beta-variant, and established that both spectrin Lyon and spectrin Culoz are (alpha I/74) alpha-variants. The present assay may be extended to any spectrin variant displaying the Sp alpha I/74 abnormality.
authors.affiliation: Faculté de Médecine Grange-Blanche, Centre National de la Recherche Scientifique URA, Lyon, France.
authors.forename: B
authors.initials: B
authors.lastname: Pothier
authors.forename: N
authors.initials: N
authors.lastname: Alloisio
authors.forename: J
authors.initials: J
authors.lastname: Maréchal
authors.forename: L
authors.initials: L
authors.lastname: Morlé
authors.forename: M T
authors.initials: MT
authors.lastname: Ducluzeau
authors.forename: C
authors.initials: C
authors.lastname: Caldani
authors.forename: N
authors.initials: N
authors.lastname: Philippe
authors.forename: J
authors.initials: J
authors.lastname: Delaunay
mesh_terms: D000293:Adolescent; D015153:Blotting, Western; D002648:Child; D002869:Chromosome Aberrations / Q000235:genetics; D002872:Chromosome Deletion; D025063:Chromosome Disorders; D004591:Electrophoresis, Polyacrylamide Gel; D004612:Elliptocytosis, Hereditary / Q000209:etiology / Q000235:genetics*; D005260:Female; D014644:Genetic Variation; D006801:Humans; D009154:Mutation; D013049:Spectrin / Q000032:analysis / Q000235:genetics*
chemical_list: D013049:Spectrin