Combined ankyrin and spectrin deficiency in hereditary spherocytosis

Summary Hereditary spherocytosis is characterized by a reduced spectrin content of the erythrocytes. However, the underlying primary defect remains unclear in the majority of cases. Genetic studies have revealed a linkage to the gene for ankyrin in some families. By means of ELISA we measured the ankyrin, spectrin, and band-3 contents in erythrocytes of 45 patients with typical spherocytosis. They were classified as having mild or moderate spherocytosis, according to clinical severity. Sixteen patients with mild spherocytosis showed slight reductions of ankyrin and spectrin contents. In contrast, 29 patients with moderate spherocytosis exhibited a clear reduction of both ankyrin and spectrin to about 60% of normal. Band 3 and lipid phosphorus, as measures for membrane surface area, were only slightly reduced to 85%. Our results, together with the molecular genetic data indicating the linkage between spherocytosis and the gene for ankyrin, suggest an ankyrin defect or deficiency as the primary lesion in most cases of spherocytosis.Supported by theDeutsche Forschungsgemeinschaft (Schr. 86/18-1)     

Combined spectrin and ankyrin deficiency is common in autosomal dominant hereditary spherocytosis

The common autosomal dominant form of hereditary spherocytosis (HS) has been genetically linked to defects of the erythroid ankyrin gene in a few families; however, the frequency of ankyrin deficiency and its relationship to red blood cell (RBC) spectrin content are unknown. To test these questions, we measured RBC spectrin and ankyrin by radioimmunoassay in 39 patients from 20 families with dominant HS. Normal RBCs contained 242,000 +/- 20,500 spectrin heterodimers and 124,500 +/- 11,000 ankyrins per cell. In dominant HS, RBC spectrin and ankyrin ranged from about 40% to 100% of normal and were continuously distributed. Measurements in the same patient on different occasions were reproducible (+/- 5% to 10%) and RBCs from affected members of a kindred contained similar amounts of spectrin and ankyrin (+/- 3% to 4%). Spectrin and ankyrin levels were almost always less than the assay controls, but were less than the normal range in only 75% and 80% of kindreds, respectively. Remarkably, the degree of RBC spectrin and ankyrin deficiency was very similar in 19 of 20 HS kindreds. One otherwise typical family differed, with marked ankyrin deficiency (45% of control) and a relatively mild spectrin deficit (81%). We conclude that most patients with dominant HS have combined ankyrin and spectrin deficiency and that the two proteins are usually about equally deficient, suggesting that defects in ankyrin expression, ankyrin stability, or ankyrin band 3 (AE1) interactions may be common in dominant HS.     

Hereditary spherocytosis (HS) due to loss of anion exchange transporter.

BACKGROUND. Hereditary spherocytosis encompasses a heterogenous group of inherited disorders due to alteration of r.b.c. surface/volume ratio. Spectrin deficiency is the most common observed defect. We analyzed a case of HS associated with band 3 deficiency without spectrin reduction. METHODS. In the study of a family originating from southern Italy, we show that a 20% deficiency of band 3 with normal spectrin content may be responsible for dominantly inherited hereditary spherocytosis (HS). The proband is a 12 years old girl consulting for jaundice, chronic anaemia and splenomegaly. Her mother had a similar haematologic phenotype. RESULTS. Electrophoretic analysis of erythrocyte membrane proteins showed a deficiency in band 3 protein. Band 3 protein chymotryptic fragments, deglycosylated band 3, and its isolated cytoplasmic domain, all displayed normal electrophoretic migrations. Furthermore, the tryptic peptides profile of the cytoplasmic domain of the protein did not demonstrate any abnormality, nor did the amino acid composition of the peptides. Analysis of the membrane proteins during erythrocyte ageing, evaluated in density-fractionated red cells, showed that band 3 content was normal in the lighter fraction, whereas in the denser fraction band 3 deficiency was more pronounced than in membranes from non fractionated red blood cells. CONCLUSIONS. This case describes HS due to anion exchange transporter deficiency. Our results on fractioned red cells support the hypothesis that the defect was probably due to a band 3 protein loss during cell ageing and not to a primitive quantitative defect.     

Hereditary spherocytosis with high fetal hemoglobin: an interesting case

Raised Hb F is occasionally found in stress erythropoiesis associated with hemolytic anemias. In hereditary spherocytosis (HS), elevation of Hb F by 2-5% may be seen but Hb F in the range of 10-20% has not been reported. We present an interesting case of a child, initially presenting with high Hb F, who showed a spontaneous and progressive decline, and was subsequently diagnosed to have HS with raised fetal hemoglobin (Hb F) using an eosin-5-maleimide flow cytometric test.     

Hereditary spherocytosis in an elderly woman with periodic attacks of jaundice

Hereditary spherocytosis is a disease with chronic hemolytic anemia found mostly in childhood. We encountered a rare case of sporadic hereditary spherocytosis in a 68-year-old woman who developed periodic jaundice caused by hemolytic crises. Since the hemolytic crises were caused by cholelithiasis-related biliary inflammation, administration of ursodeoxycholic acid was useful for the prevention of the hemolytic crises. In the differential diagnosis of periodic increases in indirect bilirubin, the possibility of hemolytic diseases, including hereditary ones, should be considered, even if the patients are elderly.     

Immune deficiency due to high copy numbers of an Ak beta transgene

Because allelic polymorphism of the major histocompatibility complex class II antigens affects the immune response at several levels, we wished to characterize the contribution of a particular alpha or beta chain in vivo using transgenic mice. We have established and characterized 12 lines of H-2s/s mice carrying from 1 to 65 copies of an Ak beta transgene. The transgene was coexpressed with the endogenous allele in a tissue-specific manner, and Ak beta mRNA expression correlated well with transgene copy number. High copy number (extreme overexpression) of the transgene was associated with a variety of defects, including a significant reduction in Ia cell-surface expression, a severe decrease in B-cell number, abnormal extramedullary granulopoiesis, and an increased susceptibility to infection. In this paper we describe in detail the phenotype associated with high copy numbers of the Ak beta transgene. The defects we have observed may be relevant to similar phenomena seen in other transgenic mice. In addition, these mice have fortuitously provided a system in which to assess the effect of various levels of class II cell-surface expression in the thymus on selection of the T-cell repertoire.     

Hereditary spherocytosis associated with mutations in HFE gene

We report on a Spanish family in which three members of different generations were diagnosed with hereditary spherocytosis (HS). Additionally, one of them II-I (44-years-old), presented iron overload with hepatic deposit and needed treatment with periodic phlebotomies. The rest of the family members presented normal analytical values in iron metabolism. To investigate the presence of H63D and C282Y mutations in the HFE gene, patient II-I was found to be compound heterozygous and was the only family member presenting HS and this genetic condition in HFE. We propose a synergistic effect of HS and mutations in HFE as the cause of the iron deposits.     

Elliptocytosis-associated spectrin Rouen (beta 220/218) has a truncated but still phosphorylatable beta chain.

Spectrin Rouen (beta 220/218) is a novel variant, carrying a shortened beta chain with an apparent molecular weight of 218 kDa. It was detected in a French family. All affected members suffered from haemolytic hereditary elliptocytosis. As other shortened beta chain variants described before, the beta Rouen chain is truncated at its carboxyl terminus. Spectrin Rouen is associated with a defect in spectrin dimer self-association and with an abnormally high amount of the alpha I 74 kDa peptide following partial tryptic digestion. Dimer reconstitution experiments from normal and abnormal purified Sp subunits indicated that the increased alpha I 74 kDa fragment is induced by the altered beta chain. However, spectrin Rouen is different from other mutants with a truncated beta chain in several respects: its amount is low (less than 10%) and the spectrin dimer self-associated defect is mild. Critically, the beta Rouen chain has retained the ability of undergoing phosphorylation, even though it is modified in its C-terminal region. These results, compared to those obtained with beta 220/214 spectrin Le Puy and beta 220/216 spectrin Nice, allowed better localization of the beta chain sites that can be phosphorylated by a membrane-bound casein kinase.     

Hemoglobin Cranston, an unstable variant having an elongated beta chain due to nonhomologous crossover between two normal beta chain genes.

An asymptomatic woman was found to have a compensated hemolytic state due to an unstable hemoglobin variant, comprising 35% of the total. Peptide maps of tryptic digests of the abnormal beta chain were identical to those of beta A except that tryptic peptide 15 (Tyr-His-COOH) was absent and a new peptide was detected, containing equivalent amounts of Ser, Ile, Thr, and Lys. Its sequence was determined by manual Edman degradation. An additional hydrophobic peptide isolated by counter-current distribution contained: Asx, Ser, Ala, Tyr, 2 Phe, and 3 Leu. Its sequence was determined on an automatic solid phase sequencer. Digestion with carboxypeptidase A confirmed the C-terminal sequence. Hemoglobin Cranston has an elongated beta chain with the following structure: (see article). This variant is the first "adult" human hemoglobin known to contain isoleucine. It is likely that hemoglobin Cranston arose because of a nonhomologous crossover of two normal beta chain genes, probably during meiosis, with the insertion of two nucleotides (AG) at position 144, resulting in a frame shift. Hemoglobin Cranston provides new information on the structure of the beta chain gene as well as an explanation of both the structure and genetic basis of hemoglobin Tak, the only other elongated beta chain variant that has been described.     

Hereditary spherocytosis is associated with decreased pyruvate kinase activity due to impaired structural integrity of the red blood cell membrane

Hereditary spherocytosis (HS) is characterised by increased osmotic fragility and enhanced membrane loss of red blood cells (RBC) due to defective membrane protein complexes. In our diagnostic laboratory, we observed that pyruvate kinase (PK) activity in HS was merely slightly elevated with respect to the amount of reticulocytosis. In order to evaluate whether impaired PK activity is a feature of HS, we retrospectively analysed laboratory data sets from 172 unrelated patients with HS, hereditary elliptocytosis (HE), glucose-6-phosphate dehydrogenase (G6PD) or PK deficiency, sickle cell or haemoglobin C disease, or β-thalassaemia minor. Results from linear regression analysis provided proof that PK activity decreases with rising reticulocyte counts in HS (R² = 0·15; slope = 9·09) and, less significantly, in HE (R² = 0·021; slope = 8·92) when compared with other haemolytic disorders (R² ≥ 0·65; slopes ≥ 78·6). Reticulocyte-adjusted erythrocyte PK activity levels were significantly lower in HS and even declined with increasing reticulocytes (R² = 0·48; slope = −9·74). In this report, we describe a novel association between HS and decreased PK activity that is apparently caused by loss of membrane-bound PK due to impaired structural integrity of the RBC membrane and may aggravate severity of haemolysis in HS.     

Two distinct truncated variants of ankyrin associated with hereditary spherocytosis

We present two distinct truncated variants of ankyrin associated with mild to moderate hereditary spherocytosis. Ankyrin Saint-Etienne 1 was manifested by an additional band located between bands 2.1 and 2.2. It was associated with a nonsense mutation in exon 39: TGG→TGA; W1721X. Ankyrin Saint-Etienne 2 appeared as two faint bands underlining bands 2.1 and 2.2. It was associated with a nonsense mutation in exon 41: CGA→TGA; R1833X. Overall ankyrin was diminished in splenectomized patients. Messenger RNAs Saint-Etienne 1 and 2 amounted to 20 and 37% of the total ankyrin mRNA, respectively. Ankyrin molecules truncated in their C-terminal region retain some ability to bind to the membrane whereas the bulk of nonsense mutations, located in more upstream regions, result in the mere disappearance of one haploid set of ankyrin. In the present cases, it was not possible to apportion the roles of ankyrin reduction and truncation in the pathogenesis of hereditary spherocytosis. Am. J. Hematol. 58:36–41, 1998. © 1998 Wiley-Liss, Inc.     

Enhanced haemolysis with beta-thalassaemia trait due to the unstable beta chain variant, Hb Gunma, accompanied by hereditary elliptocytosis due to protein 4.1 deficiency in a Japanese family

We identified a Japanese family with a beta-thalassaemia trait and hereditary elliptocytosis (HE). We studied five members of this family. One was normal, one had only the beta-thalassaemia trait, one had heterozygous HE, and two had compound heterozygous beta-thalassaemia trait and HE. The last two had already undergone splenectomy. The molecular profile of beta-thalassaemia was consistent with that of Hb Gunma: codon 127/128CAGGCT(Gln-Ala)--> CCT(Pro). Analysis of erythrocyte membrane proteins revealed a partial deficiency of protein 4.1 in all those with HE, whereas the spectrin content was within the normal range. Each heterozygous family member with either the beta-thalassaemia trait or HE was asymptomatic, whereas the two with both beta-thalassaemia and HE had marked red blood cell deformities and haemolysis. The abnormalities of the red blood cells in patients with the beta-thalassaemia trait might be enhanced by association with HE owing to a protein 4.1 deficiency.     

A case of elliptocytosis associated with a truncated spectrin chain

A case of haemolytic anaemia with elliptocytosis is described, in which a large part of the smaller (beta) subunit of the spectrin is truncated, and has an apparent molecular weight of about 214 000 compared with about 230 000 for the normal chain. It is shown that this is not a product of adventitious proteolysis during lysis or extraction. At the same time about 35% of the total spectrin in the cells is liberated from the membrane as the dimer (which is present in normal cells to the extent of less than 10%). The truncated (beta') chain appears exclusively in this dimer fraction. The beta'-chain is incapable of phosphorylation by the endogenous cAMP-independent membrane kinase, and it may be inferred that the deleted segment of the chain contains both the spectrin self-association site and the residues normally phosphorylated. The alpha beta'-dimer is active with respect to participation in a ternary complex with its partnering proteins in the membrane cytoskeleton, F-actin and 4.1, confirming that the phosphorylation sites are not involved in the primary interaction with the other cytoskeletal proteins at the network junctions. The spectrin alpha-chain generates the terminal tryptic fragment of molecular weight 80 000 characteristic of normal spectrin, rather than the 74 000 molecular weight peptide derived from the alpha-chain in cases of hereditary elliptocytosis and pyropoikilocytosis, associated with anomalous self-association of spectrin dimer. Membrane cytoskeletons, extracted from the patient's red cells, undergo normal gelation on incubation with cAMP-independent kinase and ATP, and thus do not resemble those derived from hereditary spherocytosis cells. The properties of the anomalous spectrin resemble in most respects that described in a French family by Dhermy et al (1982).