Two elliptocytogenic alpha I/74 variants of the spectrin alpha I domain. Spectrin Culoz (GGT----GTT; alpha I 40 Gly----Val) and spectrin Lyon (CTT----TTT; alpha I 43 Leu---Phe). L Morlé, … , B G Forget, J Delaunay J Clin Invest. 1990;86(2):548-554. https://doi.org/10.1172/JCI114743. Research Article Spectrin alpha I/74 elliptocytosis results from abnormalities involving the "head" region of spectrin dimer. Increased susceptibility to trypsin enhances cleavage of the alpha spectrin chain, yielding an increased amount of the alpha I 74-kD fragment at the expense of the alpha I 80-kD parent fragment. Recently we showed that the mutations causing the Sp alpha I/74 abnormality may lie in the alpha- or the beta-chain, and that spectrin Culoz and spectrin Lyon were two (alpha I/74) alpha-variants, respectively. We now show that the spectrin Culoz alpha I domain undergoes prominent tryptic cleavage after Lys 42, whereas cleavage prevails after Arg 39 in spectrin Lyon. Applying the polymerase chain reaction (PCR) technique to exon 2 of the spectrin alpha I domain, we have established that the mutation responsible for spectrin Culoz is alpha I 40 Gly----Val; GGT----GTT. Applying the PCR technique to the cDNA derived from reticulocyte mRNA, we have shown that the mutation responsible for spectrin Lyon is alpha I 43 Leu----Phe; CTT----TTT. Studies of normal controls and of family members using dot blot hybridization with allele-specific oligonucleotide probes confirmed these results. Variants such as spectrin Culoz and spectrin Lyon should provide insight into a region that participates in spectrin dimer self-association and whose susceptibility to proteolysis must reflect subtle conformational changes. Find the latest version: https://jci.me/114743/pdf Two Elliptocytogenic ai174 Variants of the Spectrin al domain Spectrin Culoz (GGT- RGTT; al 40 Gly-oVal) and Spectrin Lyon (CTT--'TTT; al 43 Leu-*Phe) L. Morle,* A.-F. Roux,** N. Alloisio,* B. Pothier,* J. Starck,* L. Denoroy,§ F. Morie,* R.-C. Rudigoz,11 B. G. Forget,' J. Delaunay,* and J. Godett *Centre National de la Recherche Scientifique (CNRS) URA 1171, Faculte de Medecine Grange-Blanche, 69373 Lyon Cedex 08, France; tCNRS UMR 106, Universite Claude-Bernard Lyon-I, 69622 Villeurbanne Cedex, France; OService Central d'Analyse, CNRS, BP 22, 69390 Vernaison, France;, CliniquecGyneologique et Obstetricale, H6pital de la Croix-Rousse, 69317 Lyon Cedex 04, France; and 'Hematology Section, Department ofInternal Medicine, Yale University School ofMedicine, New Haven, Connecticut 06510 Abstract self-association. The al domain appears in the form of an 80-kD fragment, which is further cleaved into a minor 78-kD Spectrin a/74 elliptocytosis results from abnormalities involv- fragment and a prominent 74-kD fragment. The amount of ing the "head" region of spectrin dimer. Increased susceptibil- the 74-kD peptide varies depending on the samples and/or the ity to trypsin enhances cleavage of the a spectrin chain, yield- experimental conditions. In addition, the precise cleavage ing an increased amount of the al 74-kD fragment at the ex- point seems to be heterogeneous, occurring either after arginyl pense of the aI 80-kD parent fragment. Recently we showed residue 39 (3) or iysyl residue 42 (4, 5). The al domain is that the mutations causing the Spa'/74 abnormality may lie in composed of five 106-amino acid repeats (6) designated a- I to the a- or the ,8-chain, and that spectrin Culoz and spectrin a-S. In turn, each repeat can be folded into three helices. Be- Lyon were two (a1/74)a-variants, respectively. We now show tween repeat a-l and the NH2 terminus lies a partial repeat, that the spectrin Culoz al domain undergoes prominent tryptic termed a- 1' and containing part of helix 3 of the following cleavage after Lys 42, whereas cleavage prevails after Arg 39 repeats. At the gene level, the NH2-terminal part of the aI in spectrin Lyon. Applying the polymerase chain reaction domain is encoded by exon I (Met -6 to Thr 2) and exon 2 (PCR) technique to exon 2 of the spectrin aI domain, we have (Val 3 to Gln 82) (7). established that the mutation responsible for spectrin Culoz is Hereditary elliptocytosis (HE)' refers to a set of conditions al 40 Gly-o-Val; GGT- GTT. Applying the PCR technique to in which the red cells have an elliptical shape. In recent years, the cDNA derived from reticulocyte mRNA, we have shown HE has been shown to be associated with a number of abnor- that the mutation responsible for spectrin Lyon is aI 43 malities of spectrin or protein 4.1 (for review, see references 8 Leu-wPhe; CIT -iTTT. Studies of normal controls and of fam- and 9). Many changes involve the al and ftI domains and as a ily members using dot blot hybridization with allele-specific result weaken spectrin dimer self-association. The spectrin oligonucleotide probes confirmed these results. Variants such al/74 (Spa'O4) abnormality is one of these changes. It is charac- as spectrin Culoz and spectrin Lyon should provide insight into terized by an increase of the al 74-kD fragment at the expense a region that participates in spectrin dimer self-association and of the parent al 80-kD fragment (which encompasses nearly whose susceptibility to proteolysis must reflect subtle confor- the entire al domain). The Spa"174 alteration occurs sporadi- mational changes. (J. Clin. Invest. 1990. 86:548-554.) Key cally among both black (10-13) and white populations words: Sp a174 elliptocytosis * spectrin mutants,, spectrin a- (14-16). It generates highly variable clinical and morphologi- gene cal manifestations. Initially it was assumed that the primary mutation would Introduction lie in the a-chain itself. However, several findings put this view in The red cell membrane skeleton is a protein network that in question: the Spa"/74 abnormality was observed associa- underlies the inner surface of the membrane (for review, see tion with 3-chain mutants that carry deletions of variable reference 1). It determines the unique shape and deformability lengths in their COOH-terminal region (17); in other families, of the erythrocyte. Spectrin represents its main component haplotype studies (18) or DNA sequencing (19) ruled out de- and occurs, in vitro, as an a13 dimer. Peptide mapping of spec- fects of the a spectrin gene, or at least of exon 2 of this gene, as trin partial tryptic digests allowed the division of the a- and the cause of the alteration. Yet the possibility remained that into five and four domains, respectively (2). The al the a-gene would be involved in some cases. Using in vitro ,8-chains were able to and ,BI domains, which face each other, participate in dimer spectrin dimer reconstitution, Pothier et al. (20) assign the Spa"174 abnormality to either the a- or ,8-chain, making possible the distinction between (a1/74)a-variants and Portions of this work have been presented at the 31st Annual Meeting (a1/74)13-variants. Accordingly, spectrin Culoz and spectrin of the American Society of Hematology, 2-5 December 1989, and variants found in two unre- in abstract form Blood. 74(Suppl. 1): 60a.). Lyon, which are elliptocytogenic have been published (1989. as )a-variants. Address correspondence to Dr. L. Morle, CNRS URA 1171, lated French Caucasian families, appeared (a'/74 Facult6 de MEdecine Grange-Blanche, 69373 Lyon Cedex 08, France. Some differences in the protein phenotype indicated that these Receivedfor publication 1I December 1989 and in revisedform 9 variants would not be identical. Using amplification of geno- April 1990. mic DNA or of cDNA derived from reticulocyte mRNA, we J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 1. Abbreviations used in this paper: ASO, allele-specific oligonucleo- 0021-9738/90/08/0548/07 $2.00 tides; HE, hereditary elliptocytosis; PCR, polymerase chain reaction; Volume 86, August 1990, 548-554 Spa'1/74, spectrin a1t/74. 548 MorM et al. found that spectrin Culoz is due to an al 40 Gly--Val substi- colate (0.1 mmol/liter) was added to the cathode reservoir, and a high tution (GGT-*GTT) and that spectrin Lyon results from the protein/acrylamide ratio was used. The gels were blotted onto poly- al 43 Leu-*Phe (CTT--l-TTT) substitution. vinylidene fluoride (PVDF) membranes (Immobilon; Millipore/Con- tinental Water Systems, Bedford, MA) as previously described (4). Because the gels had to be overloaded for the purposes of the present Methods experiment (100 ,ug of digest), the separation of the 74a and 74b sub- components was decreased. Whereas both components could be cut out separately in spectrin Culoz with limited cross-contamination, we Case reports had to cut out the a! 74-kD fragment as a single band in spectrin Lyon Case reports concerning spectrin Culoz and spectrin Lyon have been and in the control. All fragments were sequenced using a gas-phase presented before (20). Briefly, spectrin Culoz was found in a Caucasian sequenator (model 470A; Applied Biosystems Inc., Foster City, CA) girl and her father (family CH). It was clinically asymptomatic and coupled on-line with a 120A PTH-amino acid analyzer (Applied Bio- even associated with normal red cell morphology in the father. We systems Inc.). The determination of the NH2-terminal segment of the noted several important features. (a) There was an unexpectedly high mixture of 74a and 74b peptides (in spectrin Lyon and the normal percentage of spectrin dimers in 40C extracts: 67.3% in the proposita, control) will be discussed in Results. presenting with clinically asymptomatic elliptocytosis, and 51.4% in the clinically asymptomatic father with normal morphology (controls: DNA or cDNA amplification, cloning, and DNA sequencing 6.4±1.9% [n = 12]) (20).