tions were normal in the three patients. The additional band present in the patients' red cell membranes probably represents a proteolytic degradation product. This alter- ation, present both in whole cells and isolated membranes, might affect the intact cells in vivo. We suggest that the patients' erythrocyte membrane instability may be related to the presence of an abnormal protein 4.1 whose modula- tory influence on the spectrin-actin interaction in the skeleton is defective.

tions were normal in the three patients. The additional band present in the patients’ red cell membranes probably represents a proteolytic degradation product. This alteration, present both in whole cells and isolated membranes, might affect the intact cells in vivo. We suggest that the patients’ erythrocyte membrane instability may be related to the presence of an abnormal protein 4.1 whose modulatory influence on the spectrin-actin interaction in the skeleton is defective.

To the Editor:Many mutations in the α- and β-spectrin genes are known to be associated with hereditary elliptocytosis (HE). Spectrin abnormalities are detected as abnormal peptides after limited trypsin digestion of spectrin (Sp).[1][1] We had an opportunity to study a Filipino family living in

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.

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.

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.

Purified PK from human erythrocyte was phosphorylated by cAMP-dependent protein kinase type I from human erythrocyte membrane; this phosphorylation affected only the ‘heavy’ L′ subunit but not the L subunit. On the other hand, the L subunit of liver PK was highly phosphorylated. Thus it appears that the L subunits from erythrocyte and liver PK are not identical protein molecules.

Hereditary deficiency in glutathion- synthetase is a rare disease presenting up to now either with a congenital non-spherocytic anaemia or with a metabolic acidosis, most often neonatal and accompanied by a pyroglutamic amino-aciduria (5-oxoprolinuria). These two syndrome may be present together or exist independently. Pyroglutamic amino-aciduria is the result of extension of the deficiency to non-haematopoietic cells, in particular renal. Two new cases of glutathion-synthetase deficiency are reported: both with haemolytic anaemia and moderate pyroglutamic amino-aciduria, in the absence of clinical signs of metabolic acidosis. The clinical, haematological and biochemical heterogeneity of the deficiency is illustrated by these two cases and datas from the literature.

Two patients with mild chronic haemolytic anaemia, a mother and her son, were found to be heterozygous for erythrocyte pyruvate kinase deficiency. In the red blood cells the enzymatic activity was reduced by about 50% and the residual PK had normal kinetic properties, stability and electrofocusing pattern. The PK antigen concentration was also decreased by half, so that the ratio of the enzymatic activity to the immunological reactivity (i.e. the molecular specific activity) was normal. In the son's liver PK enzymatic activity was slightly reduced and, above all, an abnormal active form, more anodic than normal PK, was detected by electrofocusing. The propositus's liver PK was also slightly thermo-unstable. It is suggested that the patients were heterozygous for an unstable PK variant which is found in liver, nucleated tissue actively synthesizing proteins, but which disappeared from the erythrocytes because of its unstability.

The authors studied red blood cell pyruvate kinase activity of 202 patients with various hemopathies. A PK deficiency of moderate grade was found in 39% of patients with acute myeloblastic leukemias, in 57% of those with primary medullary insufficiency without aplasia, in 61% of those with refractory sideroblastic anemia. The PK deficiency was often associated with deficiencies of other red cell enzymes. The mechanism of such enzyme abnormalities was discussed with the hypothesis of a post-translational molecular alteration.

Erythrocyte pyruvate kinase (PK) from 5 patients with congenital non-spherocytic hemolytic anemia and erythrocyte PK deficiency have been studied by immunological methods and electrofocusing.

A variant of erythrocytic pyruvate kinase, observed in a patient with congenital nonspherocytic hemolytic anemia is characterized by the following properties: a 50-percent decrease in hemolysate enzyme activity; increased affinity for phosphoenolpyruvate; decreased FDP activation; decreased ATP inhibition; very important thermal instability; normal urea denaturation; normal affinity for the second substrate ADP; normal pH of optimal activity. At least all these properties should be studied to identify new variants of red cell pyruvate kinase.