Causing Severe Elliptocytosis in the Homozygous State. C 1988 by Grune & Stratton. Inc. HE was associated with modified tryptic digestion of the cr1

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1 Spectrin Oran (a 121), a New Spectrin Variant Concerning the all Domain and Causing Severe Elliptocytosis in the Homozygous State By N. Alloisio, L. Morl#{233},B. Pothier, A.-F. Roux, J. Marichal, M.-T. Ducluzeau, Z. Benhadji-Zouaoui, and J. Delaunay We report on spectrin Oran (a 21). a new spectrin variant found in an Algerian family. It was characterized by the absence of the spots that classically correspond to the all domain using two-dimensional analysis of spectrin limit digests. On the contrary, the abnormal domain was reprosented by a new set of spots in the 21 -Kd and 1 8-Kd regions. as demonstrated by Western blots using anti-all domain polyclonal antibodies. Spectrin Oran (a21) was found in the homozygous state in two children belonging to two separate branches of the family. ft yields a severe elliptocytosis. Spectrin self-association was altered. The variant was much more difficult to prove in the heterozygous state. in which it results in no clinical and virtually no morphological symptom. In all four parents involved. however. electrophoretic analysis and Western blots showed the existence of the all 21 -Kd and 16-Kd peptides. In one parent, who combines spectrin Oran (am/zl) and the all type-2 polymorphism, the two-dimensional spots (52, 39, 34, and 29 Kd) were quantified and appeared reduced by 30%: there was an intermediary decrease of spectrin self-association in this person. In the three other parents. spectrin Oran combined with the all type-i polymorphism. The all typo-i spots ( and 25 Kd) appeared in normal range, and spectrin self-association was normal. Along with previous observations, the present data emphasize the large fluctuations of the a-variant percentage. Provided spectrin Oran was present in a sufficient proportion. we found an associated alteration of the ll domain (that faces the all domain in the spectrin dimer): the ll 65-Kd fragment was reduced and the fill 52-Kd fragment was reciprocally increased. C 1988 by Grune & Stratton. Inc. S TUDIES on morphologically normal and abnormal RBCs have lead us to establish that the membrane skeleton plays an important role in determining the shape and the stability of the erythrocyte. 2 It is composed of four major proteins: spectrin, actin, protein 4.1, and protein 4.9, linked together to form a submembranous network by two types of interconnections. Heterodimers of a- and /3-spectrin chains are joined head-to-head to form tetramers and probably higher oligomers. Tails of heterodimers are linked together by oligomers of actin. Protein 4.1 promotes the interactions between spectrin and actin. Spectrin structure has been analyzed by limited tryptic digestion. It can be depicted as a linear arrangement of proteolysis-resistant domains that have specific structure and function. There are five domains on the a-chain, called al to av, and four on the 3 chain, termed 3I to f3iv. The COOH-terminal region of the fi subunit is highly susceptible to tryptic digestion and is considered part of the /31 domain.3 The al domain and probably the COOH-terminal region of the fi chain, localized at the head of the heterodimer, are involved in the spectrin dimer-dimer association.4 This protein network is attached to the lipid bilayer by a protein, named ankyrin, that binds spectrin to the anion channel transmembrane protein. The ankyrin-binding function is localized to the (31 to fill junction region.5 Recent studies indicate that hereditary elliptocytosis (HE), which is a heterogeneous disorder both in terms of clinical expression and red cell morphology, is also heterogeneous from a biochemical point of view.6 Several molecular defects of skeletal proteins have been described in different ethnic groups. HE can be associated with a complete or partial absence of protein 4.1 in whites and in Algerian people.7 In a white HE family a variant of protein 4.1 characterized by an abnormal migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been described. 2 In another white family, HE was associated with a defective binding of spectrin to ankyrin. 3 In most of the cases reported, however, HE was accompanied by defective spectrin self-association. 4 6 In three independent cases of whites the functional defect was related to variants of the fi chain with a shortened COOH-terminal end)719 Otherwise HE was associated with modified tryptic digestion of the cr1 domain and was referred to as ( 1 ) the Sp& 74 abnormality in black or white HE famili&#{176}22 (and unpublished data), (2) the Spa abnormality in black HE families, and (3) the Sp& 65 abnormality in black or North African HE families.2528 In addition, spectrin self-association defect was related to a unique variant of the a! domain discovered in a South African black kindred and manifested by 43- and 42-Kd fragments29 and, more recently, in a Tunisian family carrying spectrin Tunis (a 78; manuscript submitted). Intriguingly, the functional defect can be associated with structural alterations concerning more remote domains. Coetzer et a13#{176} reported on abnormalities involving the cr111 domain. Finally, an a-spectrin variant migrating between the normal a and fi spectrin bands in SDS gel was reported in a white kindred, the causal alteration having been tentatively located in the aiv domain.3 We herein report on two children belonging to two branches of the same West Algerian family. They present with a severe hemolytic anemia that displayed a nadir in their early life. Defective spectrin association was present. From CNRS UA 1 171, G#{233}n#{234}tique Mol#{232}culaire de la Membrane Erythrocytaire, Facult#{233}de M#{234}decine Grange-Blanche, Lyon, France; CNRS UA 92, Recherches en Biologie Cellulaire, Universit#{233} Lyon I, Villeurbanne, France; and Service d H#{234}matologie. Oran, Algeria. Submitted January 20, 1987; accepted November 27, /987. Supported by the Universit#{232}Claude Bernard Lyon I. the Centre National de Ia Recherche Scientifique (UA I 171), and the Institut National de Ia Sante et de la Recherche M#{234}dicale(CRE ). Address reprint requests to N. Alloisio, CNRS UA I 1 7/, G#{233}n#{234}tique Mol#{233}culaire de la Membrane Erythrocytaire, Facult#{232} de M#{233}decine Grange-Blanche, Lyon Cedex 08, France. The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C solely to indicate this fact by Grune & Stratton, lnc /88/ / 7$3.00/0 Blood, Vol 71, No4 (April), 1988: pp

2 1040 ALLOISIO ET AL ri,-- I I ) r i- - rl. r-i - L,J -r Lr! L...j LJ I - - I L_ - -!.L, - L I I r i,., I I i i I 1.,,..!.,- 1_,J I,,j.._ L,.J CS I I I L_1...J r L..J L_J BRANCH I.E I B Cs II.Lb.2 #{149}LI Ii Homozygosity for a new spectrin abnormality involving the all domain was found. This variant is clinically and morphologically silent or nearly so in the heterozygous state. Even biochemically, its presence was virtually undetectable in the heterozygous state unless anti-all domain-specific antibodies were used. CASE REPORT The two branches, B and B, of family HA have been investigated. They live in Remchi and Tlemcen, respectively, in the Western part of Algeria. They display a complex pattern ofconsanguinity (Fig 1). Some members of branch B were mentioned in a previous series, concerning 4.1(-) HE, beingjust defined as free of4.1(-) HE. The children, 11.2 (the proposita), Ill, and 11.3, present with a severe hemolytic syndrome was born in Cutaneous pallor and recurrent bronchitis developed in the first months of life. By the age of nearly 2 years, growth appeared delayed (- 1DS), and scleral icterus and dyspnea were present. The spleen was felt 1 1 cm below the costal margin. Initial hematologic data revealed a severe hemolytic anemia (Hemoglobin [Hb], 4.1 g/dl; RBC, 1.33 T/L; packed cell volume [PCV], 10.4%; mean corpuscular volume [MCV], 84 fl; reticulocyte count, 109 GL ). She received then three transfusions; however, the latter became no longer necessary later on. Splenectomy could be postponed. Hematologic parameters spontaneously improved (Table 1). Blood smears showed a prominent elliptocytosis (nearly 100%) with anisocytosis, some rod-shaped cells, and poikilocytes (Fig 2). Children 11.1 and 11.3 belong to another kindred and are second cousins of was born in She now presents with cutaneous pallor and a moderate splenomegaly. She required two blood transfusions at age 2 years. Blood smears revealed 100% elliptocytes. 11.3, who was born in 1986, rapidly developed a very severe hemolytic picture. After the age of 5 months, transfusion became necessary every three weeks, leaving visible, however, a 10% elliptocytosis. Partial splenectomy was performed at 8 months. Since we only obtained samples modified by transfusions in 11.3, hematologic and Hb parameters are not shown in Table 1,and no conclusive biochemical work could be carried out thus far. Hb appeared normal in 11.2 and Ill (Table I; in 11.3 recent transfusion again masked the native phenotype). G6PDH and pyruvate kinase were assayed in 11.2 and found normal. The parents (I.! and 1.2 in branch B; 1.1 and 1.2 in branch B ) were clinically normal, as were children 11.1 and In general Fig 1. Genetic tree of the branch B and branch B of family HA. All persons of branch B have BRANCH B primed numbers. CS. consanguineous marriage; ne, not examined. Years of birth, when known, are indicated. : a21 gene; 0. gene encoding the all type-2 polymorphism; /, proposita;. pendent diagnosis due to frequent transfusions. their hematologic and hemoglobin data were normal. Nevertheless 1.2 presented some degree of anisocytosis with rare elliptocytes. Isolated microcytic erythrocytosis in I.! with normal serum iron suggests heterozygous a-thalassemia-2, a trait affecting 10% of Algerian people.32 The hemoglobin pattern in the affected children and their parents allowed us to rule out homozygous and heterozygous $-thalassemia, respectively, -thalassemia being also frequently encountered in Algeria. MATERIALS AND METHODS To control the minor variations arising from one experiment to another, samples from normal persons were always treated in parallel to patient samples. Controls were from whites or people of North African origin. Preparation of erythrocyte membrane and spectrin extracts. Ghosts were prepared according to Dodge et al,33 except that 0.3 mmol/l phenylmethylsulfonylfluoride (PMSF) was added to the lysis buffer. In one experiment two other protease inhibitors, pepstatin (2 sg/ml) and bestatin (1 ig/ml), were added to the lysis and washing buffers. Crude spectrin extract (4#{176}C) was obtained at low ionic strength34 by washing the ghosts twice in a 0.3 mmol/l phosphate buffer, ph 8.00 containing 0.1 mmol/l PMSF, 0.1 mmol/l EDTA, and 0.1 mmol/l 9-mercaptoethanol, and by incubating them overnight at 4#{176}C. Crude spectrin extract (37#{176}C) was obtained similarly, but incubation was carried out at 37#{176}C (for 30 minutes). Pepstatin, Boehringer Mannheim (2 Mg/mL) and bestatin, Boehringer Mannheim (1 g/ml) were included in the extracting Date OfBIIh. Hematologic RBC T/L Hb g/dl MCV ft. Serum won pmol/l Hb A % ll Ii Hb F % Data were collected in April The reticulocyte count (GIL) was det&mined previously in some members of the family (branch B): 48, 28, 27, and 1 27 GIL in I. 1, l.2, II. 1, and ll.2, respectively.

3 SPECTRIN ORAN: A VARIANT OF THE all DOMAIN 1041 Fig 2. Red cell morphology in persons of branch B. Ii. father; 1.2. mother; 11.1, sister of the proposita; proposita. (37#{176}C)medium in one experiment. Spectrin extracts were separated from the membrane residue by centrifugation at 37,000 g at 4#{176}C for one hour. Membrane protein electrophoresis. Membrane proteins were analyzed by SDS-PAGE according to Laemmli,35 using a 7% homogeneous acrylamide gel, and to Fairbanks et al,36 using a 3.5% to I 7% exponential gradient gel. The Coomassie blue-stained gels were scanned at 570 nm in a Sebia Cellosystem densitometer. Studies on spectrin dimer self-association. Spectrin crude extract (4#{176}C) was used to determine the percentage of spectrin dimer. It was prepared as previously described. 9 Spectrin species (dimer, tetramer, and higher oligomers) were separated by nondenaturing composite gel electrophoresis according to Peacock et al37 and Liu et al.38 Spectrin crude extract (37#{176}C) was used to calculate the association constant of the tetramerization reaction.39 Technical details have been presented elsewhere. 9 Structural studies on spectrin. Spectrin limit digests were obtained according to Speicher et al,3 with some modifications. 9 Kinetic analyses were performed in a number of cases, the digestion times varying from 0.5 to 44.0 hours. One-dimensional gels were carried out with a 7% to 15% polyacrylamide gradient gel using the discontinuous system described by Laemmli.35 The peptides were stained and scanned as described above. Two-dimensional analysis was carried out according to O Farrell,#{176}as modified by Speicher et al.3 To quantify spectrin peptide spots on two-dimensional gels, the fixation, staining, and destaining times were standardized. Each spot was then excised and eluted overnight in 650 L of 50 mmol/l Tris/HCI buffer, 150 mmol/l NaC1, 0.1 mmol/l EDTA, 0.1% SDS, ph 8.00, with stirring.4 Optical density at 590 nm of the eluate was measured in a 8 10 Uvikon spectrophotometer. Immunologic analysis. Rabbit polyclonal antibodies against the all 46-Kd peptide ofspectrin were prepared by immunizing a white New Zealand rabbit with the all 46-Kd spot (nearly 100 g) purified by two-dimensional gel electrophoresis. Three booster injections were administered at two-week intervals. Spectrin peptides separated by one- or two-dimensional electrophoresis, as described above, were electrophoretically blotted onto a nitrocellulose sheet (0.45 mm pore size; Bio-Rad Laboratories, Richmond, CA) using a Trans Blot Cell (Bio Rad Laboratories) as described by Towbin et al.42 Blotting was performed for 2.5 hours at 250 ma. The nitrocellulose filters were soaked overnight at room temperature in PBS containing 5% bovine serum albumin (BSA) and then for 30 minutes at 40#{176}C in the same solution. The filters were washed three times for ten minutes in phosphate-buffered saline (PBS) and then incubated three hours with polyclonal rabbit anti-all 46-Kd peptide antibody (1/30 vol/vol) in PBS containing 0.045% vol/vol Tween 20. After three washes in PBS containing 0.3% vol/vol Tween 20, the filters were flooded for two hours with a 1/ 1,000 solution of peroxidaseconjugated swine immunoglobulins to rabbit immunoglobulins (DAKO) in PBS-Tween. After three additional ten-minute washes the filters were exposed for a few minutes to the substrate solution (0.06% 4-chloro-1-naphthol, 0.033% H2O2, 20% methanol). The reaction was stopped by rinsing the filters in distilled water. DNA mapping. Procedures of DNA isolation, blot hybridization, and probe 32P labeling by random oligoprimer were carried out as presented.43 Spectrin a-gene was mapped using the genomic probe described by Linnenbach et al RESULTS Basic structural and functional parameters. The patterns of membrane proteins separated by SDS-PAGE according to Laemmli35 and Fairbanks et al36 were quantitalively and qualitatively normal (not shown), except that the 4.la/4.lb ratio was reduced in persons (underlined) with hemolytic manifestations: 2.45 (1.1), 1.56 (1.2),2.31 (Ill), 1.16 (11.2), 1.77 (1.1 ), 1.83 (1.2 ), 1.07 (11.1 ), and 2.01 (11.3, transfused); controls = 2.23 ± 0.12 (n = 6). 1.2 was borderline, which fits with her biochemical symptoms (see below). Spectrin self-association was studied with crude spectrin extracts electrophoresed on nondenaturing gels (Fig 3). The percentage of SpD and the association constant appeared the most disturbed in 11.2 and III (Table 2), who turned out to carry spectrin Oran in the homozygous state, as we shall see. It was moderately altered in 1.2, who is a compound heterozygote for spectrin Oran and the all type-2 polymorphism, as will be described. In the other parents (1.1, Fig 3. Nondenaturing gels of spectrin-crude extracts in persons of branch B father; 1.2. mother; ll.1. sister of the proposita; proposita. C. control. a. 4 C extract; b. 37 C extract after tetramerization; 0, oligomer; T. tetramer; D. dimer; 4. increase of dimer band; 44, decrease of tetramer band;, abnormal band.

4 1042 ALLOISIO ET AL ll.1 Table 2. Spectrin Functional Studies Percentage of Spectrin Dimer in 4#{176}C Extracts 6.3(N-3) 5.2 (N - 2) 5.1(N-2) 12.1 (N - 2) 8.2 (N - 2) 7.6±0.6(N-3) 14.2 ± 1.0 (N - 3) Association Constant (Ka) in Solution (105M ) 3.5(N-2) 2.7 (N - 2) 4.7(N-2) 1.3 (N -2) 4.6 ± 0.2 (N -4) 5.4±0.3(N-4) 1.5 ± 0.1 (N - 3) Controls 6.4 ± 1.9 (n - 12) 4.7 ± 0.6 (n - 33) Abbreviations: N. number of determinations on the same sample; n, number of individuals. The protein composition of the spectrin crude extract was controlled using SDS-PAGE. It was found normal and free of any detectable protein contaminant. Significantly different from controls at P <.01. I. 1, and 1.2 ), who proved to be compound heterozygotes for spectrin Oran and the all type-i polymorphism (see below), spectrin self-association parameters appeared normal. They were also normal in 11.1 (an all type-i/all type-2 compound heterozygote). On nondenaturing gels we observed an unidentified faint band below the tetramer band in the 37#{176}C extract ofii.2 (Fig 3) and of 11.1 (not shown). Bidimensional analysis. In 11.2 any spot known to represent the all domain was dramatically absent (Fig 4). These spots were similarly missing in 11.1 (not shown). On the contrary, two new spots in the 2i-Kd region appeared as possible representatives of the all domain, as will be demonstrated below. In 1.2 only these spots corresponding to the all type-2 polymorphism (52, 39, 34, and 29 Kd; increased p1) were observed,45 although in a seemingly decreased amount (Fig 4). Quantitation of these spots confirmed a 30% reduction (Table 3), suggesting that the all type-2 spots witness but 1.2. I C.44 ** 1 lvl4-... #{149}L - 1*.zJ * Ic I 6 5 Fig 4. Two-dimensional analysis of limited tryptic digests (20 hours) of spectrin in persons of branch B. C. control; l.1. father; 1.2. mother; sister of the proposita; proposita. all type-i spots are surrounded by circles and are linked together. all type-2 spots are surrounded by squares and are linked together. There were no all type-i or type-2 spots in ll displayed only all type-2 spots. and 11.1 presented all type-i and all type-2 spots. Full-line squares in the bottom of the gel (l.2 and ll.i ) surround two spots (21 Kd) that are associated with all type-2 polymorphism. Dotted squares ( and 11.2) surround another set of two spots (21 Kd; slightly lower p1). %. reduced intensity of the fill 65-Kd spot in 1.2 and 11.2 (and also of the cxlll 52-Kd spot in 11.2); ti. increased intensity of the $11 52-Kd spot in 1.2 and 11.2, as confirmed by quantification and kinetic analysis (see Table 3).

5 SPECTRIN ORAN: A VARIANT OF THE all DOMAIN Table 3. Quantification of all and ll Spots From Two-Dimensional Peptide Maps of Spectrin at Different Digestion Times all/lll % fill 66/ll6i62 % 1.5h 4h 20h 20h 1.5h 4h 20h 20h i Controls i67 13i ioi ± 9 (n-5) i iii ± 14 (n-4) LU 72 6i 31 ± 4 (n-5) 36 ii z 36 ± 7 all refers to the sum of the spots with the following mol wt (Kd): (type-i polymorphism) andlor (type-2 polymorphism). The $111 spot, the intensity of which appears stable, was taken as a reference. fill spots included the spots of 65 and 52 Kd. the latter being derived from the former. In all cases did the sum of both spots remain constant (not shown). Underlined figures indicate abnormal (reduced) values. Distinct experiment. one haploid stock of spectrin. Kinetic studies indicated that cr11 type-2 spots (1.2) appeared synchronously with their type-i counterparts (control, not shown). Still in 1.2, the missing haploid set of spectrin seemed, again, to be represented by two spots in the 21-Kd region. More precisely, these spots flanked rightward (slightly lower p1) a distinct 2i-Kd doublet that we have constantly seen in association with the all type-2 polymorphism. In the three other parents (1.1, 1.1, and 1.2 ) we only saw the all spots corresponding the type-i polymorphism. Paradoxically, they appeared to occur in normal amount. In addition, they appeared in normal amount at various digestion times (as studied in I.i, not shown). Again we noted the (n-3) presence of spots, although very faint, in the 21-Kd region; since all type-l polymorphism is not known to be accompanied by spots in this region, we raised the possibility that the 2i-Kd new spots were the same as those seen in 11.2 and II.i, as well as the most acidic 2i-Kd spots in 1.2. II. 1, who is free of clinical and hematologic symptoms, turned out to be a compound heterozygote for all type 1 and all type-2 polymorphisms; accordingly, she displayed the less acidic doublet in the 2l-Kd region. In no person did we note any abnormality of the cr1 domain; in particular, the a! 80-Kd and the cr1 74-Kd fragments had normal percentages. The use of additional antiproteases (see Methods ) to prepare the ghosts and the crude spectrin extract (37#{176}C)did not alter the one-dimensional pattern, as studied in Altogether it looked as if children 11.2 and 11.1 were homozygous for a unique abnormality defined at the beginning as the absence of any all spots of the usual type. In 1.2 two-dimensional analysis and quantitative determination allowed us to show heterozygosity for this abnormality and the all type-2 polymorphism. On the other hand, the new abnormality was barely visible in the three other parents. This is the reason why further studies using immunochemical methods and DNA mapping were undertaken. Western blots. Spectrin limit digests at 20 hours were analyzed. Following two-dimensional analysis of a control sample, the anti-all domain polyclonal antibodies that we obtained appeared clearly specific of the all domain (spots at 46, 35, 30, and 25 Kd; Fig 5). In the proposita (11.2) the absence of all spots of any known type (from 52 to 25 Kd) was confirmed. It also appeared that the unique spectrin abnormality is manifested by two all spots in the 2i Kd (p1 5.00), as could be anticipated from the Coomassie bluestained gels (see above) and, additionally, by two spots at 16 Kd (p1 5.45) not seen before with Coomassie blue unless short digestion times were used. Following the conventional nomenclature the present spectrin variant, not previously Fig 5. Western blots following two-dimensional analysis of spectrin limit digests at 20 hours in persons of branch B. C. control; proposita. In C the patterns showed the specificity for all of the antibodies obtained. In ll.2 the absence of all spots of any known type was confirmed. In addition the presence of all spots at 2i Kd and i 6 Kd was demonstrated.

6 1044 ALLOISIO ET AL reported so far as we are aware, will thereafter be designated spectrin Oran (a 121). Because the 21-Kd and the i6-kd peptides were too faint in the heterozygous state after a 20-hour digestion but appeared more clearly visible for shorter digestion periods (kinetic analysis not shown), 90-minute digestion times were used. All members of the family were investigated using one-dimensional electrophoresis and then Western blots (Fig 6). Homozygosity for spectrin Oran (a 21) was manifested in 11.1, as in 11.2, by the absence ofany all bands in the 52 to 25-Kd region and by clearcut bands at 21 and 16 Kd. Critically, heterozygosity for spectrin Oran was manifested in all parents (1.1 and 1.2; 1.1 and 1.2 ) by the presence of these two bands, although with a lower intensity. The presence of the all type-2 polymorphism was also confirmed in 1.2 and II.i (bands at 52 Kd and 39 Kd). After a 90-minute digestion the all type-2 polymorphism (occurring free of spectrin Oran in II.i) did not produce the 2i-Kd fragment mentioned in previous sections; however, the latter became visible at and after 20-hour digestion experiments (not shown); therefore the 2i-Kd spots pertaining to spectrin Oran or those relevant to all type-2 polymorphism have a different kinetic behavior. DNA mapping. Restriction fragment length polymorphism (RFLP) autoradiograms in branch B of the family revealed the same polymorphism as those described by Hoffman et a147 concerning Mspl, Pvull, and Xbal restriction sites. The proposita turned out to be a homozygote; Ai/Ai, A2/A2, A 1/A 1 (writing the restriction sites in the above order). Her sister was homozygote for the reciprocal combination of alleles. The parents (I. 1 and 1.2) were heterozygotes. These patterns, that demonstrate homozygosity of the proposita for the portion of chromosome 1 corresponding to cr1 domain, indirectly support homozygosity for the all domain. Abnormalities ofthe fill and the all! domain. In addition to the a11 2 alteration, another alteration was recorded. It consists of a reduction of the / Kd spot with a reciprocal increase of the / Kd spot (Table 3, Fig 4). Visualization of the reduction of the fill 65-Kd peptide was best seen using one-dimensional gels (Fig 7). This additional alteration appeared essentially in the two homozygotes for spectrin Oran (11.2 and 11.1 ) and in the compound heterozygote for spectrin Oran and the all type-2 polymorphism (1.2). Kinetic analysis was not carried out in the homozygotes but was done in 1.2: at all times did the / Kd fragment appear reduced with respect to the controls (not shown). The fill abnormality appeared absent in the persons combining spectrin Oran and the cr1 type-i polymorphism. It will be discussed whether the fill change is an independant event or is a transeffect of the a11 2 abnormality. Finally, we also noted a modification of the all! domain: the cr Kd peptide was absent in 11.2 and II.i at 20-hour digestion time and was present in all parents. No kinetic analysis could be done. DISCUSSION We have presented spectrin Oran (a 21), an apparently novel spectrin variant involving the all domain. It is primarily manifested as an accelerated and abnormal proteolytic degradation of this domain. That the reduced dimer self-association and, downstream, the clinical and morphological signs are related to this structural change is supported by the following correlations: (1) the defects are the most severely expressed in the two homozygotes; (2) they appear to some extent in 1.2, who presents a noticeable amount of spectrin Oran; (3) they lack in the other heterozygous parents, in whom spectrin Oran was hardly detectable unless immunologic means were used. It may sound unusual that a Fig 6. Western blots following onedimensional analysis of spectrin limit digests at i.5 hours in persons of branches B and B. C. control. In branch B. l.i. father; 1.2. mother; ll.i. proposita s sister; proposita. In branch B father; l.2. mother; ll.i. one child. Western blots showed (or confirmed) that , 1.1, and 1.2 are heterozygous for spectrin Oran. that ll.2 and Il.i are homozygous for spectrin Oran. and that ll.i is a compound heterozygote for all type-i and all type-2 polymorphisms.

7 SPECTRIN ORAN: A VARIANT OF THE all DOMAIN 1045 Fig 7. The fill abnormality seen as the reduction of the ll 65-Kd peptide following one-dimensional analysis. The reciprocal increase of the $11 52-Kd fragment was masked by additional bands in the 52-Kd region. C. control; 1.1. father; 1.2. mother; proposita. disturbed tetramerization is associated with no visible change of the cr1 domain but with alterations of more remote domains; all and also, as we have seen, cr111 and fill (concerning the latter domains, one should note that proteolysis appeared accelerated but not diverted). Lane et a13 have reported on a variety of elliptocytosis in which a reduced dimer self-association was associated with a possible modification of the aiv domain (total a-chain appeared shortened). Wherever the initial event occurs in the present case, it seems to affect functionally and/or structurally a wide region of spectrin: cr1 to all (fill) and cr111. It is possible that distinct functional abnormalities, such as an altered binding of ankyrin, also exist and play an important role. Another interesting point concerns the large fluctuation of spectrin Oran percentages in the heterozygotes. When the allele facing the aoran gene encodes the all type-2 polymorphism (1.2), the latter appears to sensitize the effects of the former, yielding an anisocytosis, a small spectrin selfassociation defect and a reduction (- 30%) of the existing all spots. On the contrary, these manifestations are held out when the a0 gene is faced by an allele encoding the all type-l polymorphism. Spectrin Oran is not associated with a reduction of whole spectrin. We would therefore consider that the proportion of spectrin Oran varies within a pool of total spectrin that remains constant. The present results fit with previous data concerning the variability of spectrin a-mutant percentages. In North African Spa 65 HE, we have reported28 a correlation between ( 1 ) the amount of the cr1 65-Kd peptide and (2) the intensity of the morphological changes and of the spectrin self-association defect. More recently Lane et al3 observed that the percentage of their shortened a-chain variant (from 9.9% to 45.2%) correlated with the reduction of membrane stability and with that of spectrin self-association. Both in avian and mammalian erythroid precursors, spectrin a chain is synthesized in excess of /3 chain.485#{176}this gives way for compensation by one type of a chain in case of a quantitative deficiency (depressed transcription) and/or a qualitative defect (instability) of the other a chain. Under such conditions a number of potentially elliptocytogenic a genes may remain undetected as long as homozygous or compound heterozygous combinations have not been realized. In a sense the concept of HE as being a morphologically dominant condition deserves revision. The third point to be discussed is the status of the fill domain abnormality. We cannot formally rule out the possibility of a distinct genic change. Since the fill alteration was visible in those with a high or noticeable level of spectrin Oran (11.2, Iii, 1.2) but not in those with a low level (1.1, 1.1, 1.2 ), we rather hypothesized that it may represent a transeffect of the a-chain abnormality. A similar situation was encountered (from fi to a chain) in spectrin Nice (fl220!216)19 in which the deletion removing the fl-chain C-terminal region was assumed to generate a concomitant increase of the al 74-Kd fragment. One extreme hypothesis would be that the fill change is primary in spectrin Oran. However, the cotransmission of spectrin Oran and of several polymorphic sites clustered on chromosome I (even though these sites correspond to neighbour al domain) rather favors the view that the initial lesion involves spectrin a gene. Anyhow, further studies using limited digestion of isolated a chain will be necessary to ascertain this hypothesis. Spectrin Oran (a 21) is a new variant that could be detected at the clinical and routine laboratory level only in the homozygous state. Its identification relied on Western blots, using anti-all domain antibodies. Emphasis was placed on the variable percentages of spectrin Oran in the heterozygous state. The possibility of a transeffect on the fill domain was discussed. ACKNOWLEDGMENT We thank family HA for kind cooperation; Dr A. Fischer and Pr. R. Girot for providing us with some samples and clinical data; Pr. B.G. Forget for his generous gift of an a-spectrin probe ; Pr. J. Godet, Dr F. Morl#{233}, and A. Soun for helpful discussions; MA. Dorier for immunizing the rabbit; M. Bouledieb for technical help; and M. Anzilutti for preparing the manuscript. REFERENCES 1. Cohen CM: The molecular organization of the red cell membrane skeleton. 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10 : Spectrin Oran (alpha II/21), a new spectrin variant concerning the alpha II domain and causing severe elliptocytosis in the homozygous state N Alloisio, L Morle, B Pothier, AF Roux, J Marechal, MT Ducluzeau, Z Benhadji- Zouaoui and J Delaunay Updated information and services can be found at: Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: Information about ordering reprints may be found online at: Information about subscriptions and ASH membership may be found online at: Blood (print ISSN , online ISSN ), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC Copyright 2011 by The American Society of Hematology; all rights reserved.