Michel Garbarz

We report an unusual case of congenital dyserythropoietic anaemia (CDA). The propositus is a 25-year-old gipsy female presenting with a recessively inherited haemolytic anaemia. The diagnosis of CDA was based on erythrokinetic data and the morphological appearance of the erythroid precursors. The direct assay of HEMPAS antigen was negative. In peripheral blood there were 15% dacryocytes. The red cell membrane protein pattern was dramatically altered, with four major aberrant bands. Band a (mol wt 86 000) was at the lower edge of band 3, band b (mol wt 82 000) was below band 3, band c (68 000) and band d (67000) were below band 4.2. In addition, there was an array of aberrant minor bands below band d. Gel densitometric determinations and immunological characterization showed that these bands did not derive from any of the major components of the membrane. In fact, membrane proteins appeared normal in many respects, although periodic acid-Schiff staining revealed an apparent decrease of sialoglycoproteins. The major aberrant bands a, b and c occur in very low amounts in controls. These bands, as well as band d, also exist in normal cytosol and were strongly increased in the propositus.

An electrophoretically fast-moving variant of the spectrin beta-chain was discovered in the erythrocyte membranes of a woman and her father who both exhibited elliptocytosis and mild hemolytic anemia. This abnormal beta'-subunit (Mr = 214,000) co-existed with a decreased normal beta-chain and represented about half of the total beta-chains in the membrane. In contrast to the spectrin beta-chain, the beta'-chain was phosphorylated neither in the membrane by endogenous protein kinases nor in solution by pure membrane casein kinase whether or not the spectrin was dephosphorylated by erythrocyte cytosolic spectrin phosphatase. The presence of the beta'-chain was associated with a defective self-association of spectrin dimer to form tetramer as manifested by: (a) an excess of spectrin dimer in the 4 degrees C spectrin crude extract, (b) a defective self-association of the spectrin dimer in the 37 degrees C crude spectrin extracts. Gel electrophoretic analysis of the tetramer and dimer species isolated from the proband's 4 degrees C extract showed that the tetramer contained trace amounts of the beta'-chain, whereas in contrast, a large proportion of beta'-chain was present in the dimer. These results demonstrated the responsibility of the beta'-chain for the defective reassociation of spectrin dimer into tetramer. The study of this abnormal spectrin confirms the participation of spectrin beta-chain in dimer-dimer association and strongly suggests that the phosphorylation sites of the normal beta-chain are located at the end of the molecule involved in the dimer-dimer interactions.

Summary. Allele αLELY is a low-expression allele of the erythroid spectrin α-gene. It carries mutations in exon 40 (αV/41 polymorphism) and intron 45, respectively, and is associated with partial skipping of exon 46. The latter phenomenon is thought to impair the recruitment of α-chains by β-chains, and would eventually account for the low-expression character. When it occurs in trans to an α-allele responsible for hereditary elliptocytosis (αHE allele; αHE/αLELY diplotype), allele αLELY enhances the severity of elliptocytosis. Because allele αLELY is widespread, we anticipated that it would occasionally carry HE determinants. These variants of allele αLELY will be designated αHE-LELY alleles. We report two families with the same αHE-LELY allele. The HE component was the known α28 Arg His mutation. This αHE-LELY allele was investigated within the αHE-LELY/αLELY diplotype, a diplotye not described before. Except for the neonatal period, the presentation was mild. In a consistent manner, the αLELY component in cis of the αHE mutation counteracted the like component in trans.

Limited tryptic digestion of native spectrin (Sp) has revealed several variants in hereditary pyropoikilocytosis (HPP) and in a subset of patients with hereditary elliptocytosis (HE). In most cases, tryptic peptide corresponding to the αI (N-terminal) 80 kD domain is wholly or partially replaced by smaller fragments. These variants are provisionally designated according to the molecular weight of the most prominent new peptide. Partial amino acid sequences of the abnormal peptides and DNA analysis of the α-spectrin gene have shown that most variants result from substitution or insertion of an amino acid in the αI-domain. However, similar investigations did not detect any such abnormality in the spectrin αI-domain of an HE black kindred with one of the spectrin variants called Sp αI/74. In this kindred, restriction fragment length polymorphism studies and transmission of the genetic polymorphism relative to the αII-domain excluded the involvement of the α-chain in the pathological process. To ascertain whether the abnormal αI 74 kD peptide might be caused by a β-chain mutation, we reconstituted hybrid dimers combining normal and HE Sp-chains. The tryptic peptide patterns of spectrin hybrid dimers containing HE α-chain and control β-chain showed a normal 80 kD tryptic product. In contrast, the hybrid dimer containing normal α-chain and HE β-chain gave rise to increased 74 kD peptide at the expense of the 80 kD, demonstrating that the mutation in this family resides in the β-chain. The same method was used to show that in two other unrelated white kindreds, the elevated 74 kD peptide arised from a Sp α-chain defect. Thus an alteration in tryptic susceptibility within the N-terminal domain of the spectrin α-chain can be directed by a mutation in the β-chain. The hybridization technique affords a definitive means of distinguishing between α- and β-chain mutants.

EREDITARY elliptocytosis (HE) is a heterogeneous group of disorders characterized by the presence of elliptical red blood cells.'.* The clinical presentation of HE can range from an asymptomatic condition to severe hemoly- sis referred to as hereditary pyropoikilocytosis (HPP).3 In all cases, the underlying alterations involve the red blood cell membrane skeleton.'.' Spectrin (Sp), the main component of the membrane skeleton, is composed of two subunits, a and p, which are encoded by separate The a- and p-Sp chains intertwine in an antiparallel manner to form rod-like heterodimers, which, in turn, self-associate head to head to form tetramers and higher order oligomers. Using partial trypsin digestion, Sp chains have been dissected into do- mains (a1 to aV, and PI to pIV domains)? Subsequent work has shown that Sp a- and @-chains consist of 22 and 17 repeating segments, respectively, with each repeat folded into a triple-helical structure containing three helices (3, 1, and 2, in the N to the C dire~tion).~.~.' Helices 1 and 2 of one repeat also combine with helix 3 of the following repeating segment to give the molecule a rod-like structure.* In the formation of a0 dimers, the N-terminal a1 domain (in partic- ular, the isolated helix 3 at the N-terminal end of the mole- cule) is thought to associate with the C-terminal @I domain, which, unlike the other repeat units, contains only a diad of helices 1 and 2.9 Many of the mutations identified in patients with HE are located in regions coding for the Sp heterodimer contact site formed by the head-to-head contact of a- and &chains, and affect the ability of the molecule to self-associate into tetra- mers and higher order oligomer^.'.'^ Among them, the Spam4 mutations" represent a group of elliptocytogenic mu- tations of Sp resulting in the formation of a 74-kD peptide, due to the cleavage after arginine at position 45 or lysine at position 48 of the a-chain."." The Spam4 point mutations, which have been characterized so far, are located at the 5' coding region of the a-Sp gene (exon 2) or at the 3' coding end (exon 30) of the 8-Sp gene."*I3 The effect of these mutations provided the basis for a model of Sp self-associa- tion in which helices 1 and 2 of repeating segment p17 lie in direct apposition to helix 3 of repeating segment a 1 .I4,l5 In the present study, we applied the polymerase chain reaction H Blood, Vol 84, No 1 (July l), 1994: pp 303-308 containing exon 2 was observed, and led to the detection of a new mutation (lle24Ser) in helix 3 of repeating segment al. In the two other families, an abnormal pattern of migra- tion of PCR-amplified fragments containing exon 30 was ob- served in affected individuals, and sequencing led to the identification of two new mutations (Ala2023Val and Trp2024Arg) in helix 1 of repeating segment p17. The ellipto- genic potential of these mutations emphasizes the impor- tance of the conformational integrity of each of the three helices involved in the formation of the Sp heterodimer con- tact site, and will help identify critical amino acids involved in this interaction.

A severe neonatal haemolytic anaemia was observed in a propositus from the West Indies. Frequent blood transfusions were required until complete splenectomy was carried out. Blood smears showed predominant poikilocytosis with spherocytes and microcytes as observed in hereditary pyropoikilocytosis. Erythrocytes were completely fragmented after incubation at 45°C. The two asymptomatic parents had normal haematological profiles. The erythrocyte membrane electrophoretic patterns of the splenectomized propositus and her parents were normal. The propositus had a moderate defect in the spectrin (Sp) dimer self-association. Limited tryptic digestion of the propositus’ Sp under standard conditions (0°C, 20 h, enzyme-substrate ratio of 1/100) revealed an increased sensitivity to tryptic digestion. The major features detected by one- and two-dimensional electrophoresis gels of Sp tryptic digests were the absence of high molecular weight peptides from the SpαII (48 kDa and 35 kDa peptides) and SpαIII (52 kDa peptide) domains with increased amounts of the lower molecular weight peptides from the SpαII and SpαIII (29 kDa peptide and 47 kDa peptide) domains respectively. Kinetic studies of Sp tryptic digestion (10 min to 36 h) confirmed the increased tryptic susceptibility of Sp. Immunodetection with specific anti-α-chain domain antibodies showed that the highest molecular weight peptides from the αII and αIII domains are released early in digestion, but disappear quickly, with an increase in their corresponding smaller peptides. The molecular weights of the peptides corresponding to the 48 kDa and 35 kDa peptides from the αII domain are slightly modified. Peptides from the αI and αIV domains showed no significant abnormalities. The Sps of both parents were normal. These results indicate that the patient has a novel Spα chain defect, which is probably located in the region of the αII domain which adjoins the αIII domain.

We present the study of a black family in which the proband suffered from a severe neonatal hemolytic anemia with poikilocytosis. Both the parents, sister's, and brother's proband were clinically normal. The presence of poikilocytes in proband led to a search for a red cell membrane skeleton defect. Owing to recent improvements in the erythrocyte membrane knowledge, it is now possible to approach the diagnosis by means of biochemical evaluation of both parents, even if they are asymptomatic. So, the first time discovery of a spectrin self-association defect in both parents allowed us to suspect double inheritance of this abnormality in the proband. A complete morphological and biochemical evaluation of the family allowed us to propound the diagnosis of heterozygous type I hereditary elliptocytosis (HE) for both parents and the sister and the diagnosis of homozygous type I HE for the proband owing to the following reasons: slight ovalocytosis was present in both parents and the sister; cell deformability ektacytometric studies gave the same profiles of curve as those observed in patients with HE. Defective spectrin dimer self-association found in both parents was also observed in the sister and proband, associated with the same abnormal spectrin digest pattern, namely a decrease in the amount of a 80,000-dalton peptide and a corresponding increase in a 74,000-dalton peptide. However, clinical presentation of the proband was consistent either with hereditary pyropoikilocytosis or homozygous hereditary elliptocytosis; erythrocyte thermal sensitivity studies in the proband could not be conclusive because of the presence of transfused cells. Both these diagnoses are discussed in detail.(ABSTRACT TRUNCATED AT 250 WORDS)

Hereditary elliptocytosis (HE) is a genetically determined disorder of the red cell membrane. The main protein which composes the proteinaceous skeleton of the membrane is an elongated molecule named spectrin which is a heterodimer composed of two chains, α and β. In the membrane spectrin dimers are associated head-to-head to form tetrameric structures. We and other authors have reported that spectrin studied from many HE patients exhibited a dimer self-association defect (type I HE). A mutation in the head of the spectrin α chain was mostly found in type I HE. We have previously described one of the three known spectrin pathological variants shown on mild tryptic digest pattern. This variant was characterized by the appearance of an abnormal 65,000-dalton peptide (Sp αI/65). Using nondenaturating gel electrophoresis, we describe in this paper a triplicated pattern of the spectrin tetramer bands which is found in heterozygous HE cases displaying the 65,000-dalton variant. Study of a homozygous case allowed us to characterize the electrophoretic mobility of the abnormal symmetrical spectrin tetramer (α2I/65-β2) and to study the correlation between the fraction of this abnormal symmetrical tetramer found in heterozygous patients and the amount of the 65,000-dalton peptide observed in spectrin tryptic digests.

According to recent works, hereditary elliptocytosis (HE) appears to be related in some instances, to a defective self-association of spectrin (type I HE). We report a new case of type I HE observed in a white patient. Study of limited tryptic digestion of a spectrin dimer showed modification of a peptide involved in the dimer self-association process.

In the process of generating transgenic mice, inserted foreign DNA can cause insertional inactivation of the flanking genetic locus and simultaneously provide a molecular tag for localizing and cloning the inactivated gene. We describe the case of an insertional mutation leading, in animals homozygous for the insertion, to severe anaemia that was lethal within a few days after birth. The haemolytic anaemia and microspherocytosis of the red cells strongly suggested membrane abnormalities of the erythrocytes. Byin situ localization of the integration site, protein analysis of the red cell membranes, northern and Southern blot analyses, we were able to demonstrate that the integrated transgene had affected the α-spectrin gene locus.

We describe a new spectrin variant with a truncated $-chain. It was discovered in a 1 7-year-old white boy presenting with intermittent jaundice and spleen enlarge- ment. He also displayed numerous smooth elliptocytes. On sodium dodecyl sulfate-polyacrylamide gel. the truncated /9-chain (/9'-chain) appeared as an additional band of approximately 21 6 kilodaltons, migrating between spectrin $-chain and ankyrin. It represented

Summary. AlleleαLELY low-expression allele of erythroid spectrin a-chain. It carries mutations both in exon 40 and intron 45 and is associated with partial skipping of exon 46. Allele αLELY remains asymptomatic by itself. In contrast, it enhances the expression level of deleterious α-alleles occurring in trans, and as such has clinical importance. The aim of this study was to evaluate the incidence of allele QLBLY mvarjous ethnic groups, i.e. Caucasians, African Blacks, Japanese and Chinese. Allele QLELY occurred in all groups investigated with a fairly uniform frequency: 31%, 21%, 20% and 22%, respectively. Mutations in exon 40 and intron 45 appeared linked to one another without exception. Partial skipping of exon 46 or the low-expression feature, whenever they could be assessed, were invariably observed. Allele αLELY appears to be an ancient and stable allele.

Summary. The impaired ability of spectrin dimers to self-associate into tetramers is one of the most frequent defects associated with hereditary elliptocytosis (HE) and its more serious form, hereditary pyropoikylocytosis (HPP). We previously described four proteic variants of the spectrin (Sp) a1 tryptic domain associated with the Sp dimer self-association defect (Sp α1/78, Sp α1/74, Sp α1/65, Sp α1/46 variants). Following the characterization of proteic variants, genomic molecular defects were identified and most of the mutations appeared to lie either in or near the self-association site, i.e. in the αI tryptic domain or in the βI tryptic domain.The clinical severity of these different mutations varies considerably and ranges from asymptomatic to severe hae-molytic disease such as in heterozygous HPP patients and in some homozygous HE patients.Studies of 113 patients from 61 HE families showed a correlation among parameters and showed which factors modulate the clinical expression of the molecular defect. Our analysis indicated that the clinical expression was directly correlated with the severity of the spectrin dimer self-association defect as evaluated by the increase in the Sp dimmer percentage found in the 4°C extract. A critical threshold of 40–50% of unassembled Sp dimer was determined; above that, patients exhibited severe haemolysis requiring splenec-tomy. The percentage of Sp dimer depends, in turn, on two factors: (i) the nature of the variant in relation to the position of the mutation versus the tetramerization site: (ii) the relative amount of mutant spectrin present in the membrane (ranging from 15% to 80% in heterozygous patients). As for the severity of haemolysis, the ghost mechanical stability to shear stress, as measured by ektacyometer, was also found to depend on the Sp dimer self-association defect. In contrast, the decrease in erythrocyte deformability was not related to the amount of unassembled Sp dimer but appeared to be correlated with the amount of mutant spectrin whatever the variant.Concerning erythrocyte morphology and the number of elliptocytes, the Sp α1/65 variant appears to be the most ‘elliptocytogenic’ variant, indicating that erythrocyte shape abnormality is not linked to the Sp dimer self-association defect.

Five patients with hereditary elliptocytosis (HE) from two unrelated black families were studied. The patients had prominent elliptocytosis and a decreased erythrocyte resistance to heat treatment. In one infant blood smears showed elliptocytosis and poikilocytosis; his erythrocytes fagmented at a lower temperature than those of his mother and sister, both having typical mild HE. Defective dimer-dimer association was present in all patients. Limited tryptic digestion of spectrin and subsequent analysis by one-and two-dimensional electrophoresis revealed a similar and reproducible decrease in the 80,000-dalton peptide (αI domain) and the comcomitant appearance of a 46,000-dalton peptide. All the patients had the polymorphism of the spectrin αII domain commonly observed in black populations. In addition, modifications relative to the αIII domain were detected; similar variants were found in one black control subject out of 136 and are likely related to a genetic polymorphism of the αIII domain. No differences were observed between the peptide patterns in the infant with poikilocytosis and those of his HE sister and mother.

Hemolytic anemia with red cell fragmentation. poikilocyto- sis. and elliptocytosis was discovered in a 6-week-old black infant. Both parents and a brother of the propositus had compensated mild Hereditary Elliptocytosis (HE). Elliptocy- tosis was prominent in the proband's father with the presence of numerous rod-shaped cells whereas. in the proband's mother, elliptocytosis was less marked and cells were less elongated

Hereditary elliptocytosis (HE) is, in the heterozygous state, a common mild congenital hemolytic disease. In contrast, homozygous elliptocytosis is a severe transfusion-dependent hemolytic anemia. The major determinant of red cell membrane shape and stability is a two-dimensional proteinaceous meshwork named membrane skeleton. Spectrin, the most important protein of the membrane skeleton, is basically a heterodimer composed of alpha and beta chains. Within the membrane, spectrin dimers self-associate to form tetramers. In type I HE spectrin dimer self-association is defective and an excess of spectrin dimer is present in the patient's red cell membranes. The defective self-association is often correlated with an abnormality of the spectrin alpha chain which is depicted by limited tryptic digest of spectrin. In a family previously studied by us (Dhermy et al., 1984), the search for a spectrin defect in the red cells of the fetus of the pregnant mother was indicated for the following reasons: the diagnosis of heterozygous type I HE with the same spectrin variant had been made in the mother as well as in the father. Moreover, homozygous HE had been recognized in one of the children born two years previously with a persistent and severe transfusion dependent hemolytic anemia. Preliminary studies of normal fetal erythrocytes at twenty weeks gestation have shown that fetal and adult spectrin molecules are identical. The results obtained in the fetus at risk allowed us to diagnose type I HE (though elliptocytes were not present in the blood) for the following reasons: (i) erythrocyte deformability was decreased (ii) spectrin self-association was defective with an excess of dimer species in the membrane (iii) limited tryptic digest of spectrin showed the same abnormal pattern as seen in the heterozygous mother, with a decrease in the 80,000-dalton peptide and a concomitant increase in the 74,000-dalton peptide. The heterozygous state, strongly suspected on the tryptic digest pattern of fetal spectrin, was confirmed when the mother gave birth to a baby who did not have hemolytic anemia during the first 18 months of life.

We studied a French kindred with typical hereditary spherocytosis (HS). Studies of erythrocytes and erythrocyte membranes from HS individuals revealed abnormal erythrocyte membrane mechanical stability as well as 15-20% deficiency of band 3, the anion transporter. Anion transport studies of red cells from two affected individuals revealed decreased sulfate flux. Nucleotide sequence of cDNA encoding the distal third of the cytoplasmic domain and the entire transmembrane domain of band 3 obtained by RT-PCR of reticulocyte RNA of an affected family member was normal. Sequence analysis of genomic DNA from an HS individual identified a nonsense mutation of the band 3 gene, Q330X, near the end of the band 3 cytoplasmic domain. This mutation was present in genomic DNA of all HS family members and absent in DNA of unaffected family members. Using an RT-PCR-based assay, a marked quantitative decrease in accumulation of the mutant band 3 RNA was detected. Thus the codon 330 nonsense mutation is responsible for the decreased accumulation of mutant band 3 RNA and the deficiency of band 3 protein in this kindred. These results have important implications for the role of band 3 defects in the membrane pathobiology of HS as well as for the techniques used in detection of HS mutations.

The α207 Leu→Pro mutation in spectrin has recently been identified as a cause of αI/50-46a hereditary elliptocytosis (HE) or pyropoikilocytosis among Black people. We have found this mutation in a Moroccan family in both the heterozygous and homozygous states. The mutated α-spectrin allele carried, in cis, the αV/41 polymorphism, a common polymorphism altering the peptide maps and associated with a low-expression level. This is the first report of the cis combination of an HE mutation and the αV/41 polymorphism. Presumably, such a combination accounts for the very low expression of the abnormal allele in the heterozygous state.

Several polymorphic mutations are located on the spectrin α-chain; among these the variant termed αIIa is characterized by an acid shift in the isoelectric point of the tryptic digest peptides 46 kDa and 35 kDa. In this variant a single amino acid substitution (alanine to aspartic acid) occured at position 972 of the spectrin α-chain due to a point mutation (GCT to GAT) in the DNA. This variant, which seemed very rare in normal people, could be related to the recessive form of hereditary spherocytosis (HS) and could be absent in the dominant form of the disease. We have studied the αIIa variant by denaturing electrophoresis of the spectrin tryptic digest peptides from 179 subjects: 46 controls, 78 patients with dominant (d) or non dominant (nd) HS and 55 relatives of the patients. The confirmation of the results was obtained at the DNA level in 41 subjects. The frequency of the chromosome bearing the αIIa mutation was 7.6% in controls and higher (about 12–14%) in members of families with dHS as well ndHS. However, the family trees clearly showed that the mutation and the HS disease gene(s) were located on different chromosomes and inherited independently from each other. Furthermore, our study allows the conclusion that in most (if not all) cases of dHS, the αIIa the variant is not the cause, is not a marker, and does not influence the phenotypic expression of the disease.

Hereditary pyropoikilocytosis (HPP) is a severe hemolytic anemia characterized by a material instability of the red cell membrane leading to cell fragmentation. This fragility may be correlated with functional and structural defects of spectrin. Most HPP patients have been black. We now report three HPP patients from a Caucasian family, the proposita and her two maternal uncles. The proposita's mother and daughter presented mild type I hereditary elliptocytosis (HE), while the proposita's father was clinically and hematologically normal. Our studies revealed a defective ability of spectrin to self-associate, resulting in an excess of spectrin dimer in 4°C extracts in the three HPP patients and to a similar extent in HE relatives. Limited tryptic digestion of spectrin showed a molecular variant in the αI domain as expressed by a decreased amount of 80 000-dalton peptide with a concomitant increase in the 74 000-dalton peptide. Investigations in the proposita's father revealed no abnormalities of the erythrocyte membrane. The co-transmission of HPP and HE phenotypes in the same lineage might suggest variability in the clinical expression of the same molecular defect and lead us to discuss the hypothesis of a double heterozygosity in HPP patients.