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)     

Molecular basis of spectrin and ankyrin deficiencies in severe hereditary spherocytosis: evidence implicating a primary defect of ankyrin.

While varying degrees of spectrin deficiency have been found in the majority of patients with hereditary spherocytosis (HS), a combined severe deficiency of both spectrin and the spectrin-binding protein, ankyrin, has been reported only in two patients with severe HS. To elucidate the molecular basis of these protein deficiencies, we have studied the synthesis, assembly, and the mRNA levels of spectrin and ankyrin in peripheral blood reticulocytes in one of the previously reported probands. Pulse-labeling studies showed that in HS reticulocytes, the synthesis of alpha-spectrin was comparable with control reticulocytes while that of beta-spectrin was increased about fourfold, presumably reflecting increased erythropoietic drive. On the HS reticulocyte membrane, the amount of newly assembled spectrin was reduced to about half of the control values, presumably reflecting a decrease in the synthesis of the spectrin binding protein, ankyrin: the ankyrin synthesis was nearly absent in the cytosol and the amounts of membrane-associated ankyrin were reduced to about half of the normal values. The changes in the amounts of spectrin and ankyrin mRNAs quantitated by slot blot and Northern blot analyses were comparable with changes in the synthesis of these proteins: The alpha spectrin mRNA was within a control range and the beta-spectrin mRNA was slightly increased, while the amounts of ankyrin mRNA were reduced to about 50% of control values. We conclude that the primary defect underlying the combined spectrin and ankyrin deficiency is a deficiency of ankyrin mRNA leading to a reduced synthesis of ankyrin which, in turn, underlies the decreased assembly of spectrin on the membrane.     

Ankyrin deficiency in dominant hereditary spherocytosis: report of three cases

We describe three Italian subjects from two unrelated families affected with isolated hereditary spherocytosis (HS) without other clinical abnormalities, associated with partial spectrin and ankyrin deficiency. In both families the propositus has normal biological parents, and thus appears to be the result of a new mutation; in one of them the disease is further transmitted in an autosomal dominant fashion. Cytogenetic analysis of the latter family excluded abnormalities of the short arm of chromosome 8. We speculate that in both kindreds ankyrin deficiency is the primary defect related to ankyrin gene mutation. Several pieces of evidence suggest that ankyrin deficiency is probably the most common molecular defect in HS. It is inherited in a, dominant manner and its clinical and biochemical expression is heterogenous.     

Hematologically important mutations: ankyrin variants in hereditary spherocytosis

The primary defect in the hereditary spherocytosis (HS) syndromes is a qualitative or quantitative alteration in one or more erythrocyte membrane proteins. Mutation of the erythrocyte membrane protein ankyrin are the most common cause of typical, dominant HS. Ankyrin mutations also cause nondominant spherocytosis due to ankyrin gene promoter or de novo mutations. In most cases, HS-related ankyrin mutations are private. A summary of reported HS-associated ankyrin gene mutations is provided in this report.     

Relative iron deficiency in hereditary spherocytosis

Seventy-three patients with hereditary spherocytosis (HS) (58 nonsplenectomized, 15 splenectomized) were studied to evaluate iron status and the adequacy of iron availability for erythropoiesis. Splenectomized patients, who had hemoglobin levels in the normal or upper normal range, had higher levels of serum iron, transferrin saturation, and serum ferritin than normal matched controls and normal zinc protoporphyrin (ZnPP) levels. On the contrary, nonsplenectomized patients presenting with mild to severe anemia had higher red cell ZnPP concentrations than both splenectomized subjects and matched normal controls. ZnPP in nonsplenectomized patients correlated inversely with Hb concentration, mean corpuscular volume (MCV), mean red cell hemoglobin concentration (MCHC), transferrin saturation, and serum iron, and directly with reticulocyte count. At multiple regression analysis only Hb concentration was a significant explanatory variable for high ZnPP. The authors conclude that a number of nonsplenectomized HS patients have relative iron deficiency primarily because of expansion of erythropoiesis caused by anemia.     

Partial spectrin deficiency in hereditary pyropoikilocytosis

Hereditary pyropoikilocytosis (HPP) is a severe hemolytic anemia in which an instability of the red cell membrane skeleton has been correlated with structural and functional defects of spectrin. We now report that 13 unrelated HPP subjects have approximately 30% less spectrin than normal as evidenced by a decreased spectrin/band 3 ratio. We also examine the role of spectrin degradation as an underlying cause of this partial spectrin deficiency. Our studies demonstrate that the reduced spectrin content of HPP red cells remains constant during in vivo aging of the cells in the peripheral blood, as well as during in vitro incubation. Furthermore, immunoblotting experiments using an affinity-purified antispectrin antibody indicate that there is no loss of spectrin during membrane preparation and also that neither whole HPP red cells nor ghosts nor cytosol contains any abnormal spectrin degradation products. These data suggest that spectrin is not degraded and that it is stable on the membrane of the circulating HPP red cell. In contrast, however, incubation of free spectrin with a lysate of nucleated erythroid precursor cells indicates that HPP alpha I/46 spectrin, but not HPP alpha I/74 spectrin, is more susceptible to proteolytic degradation than a control. These data imply that the decreased spectrin content of HPP is not due to a single defect but that a more complex mechanism is involved. In HPP Sp alpha I/46 subjects, an increased proteolytic degradation in bone marrow erythroid precursors of cytosolic spectrin, prior to its assembly on the membrane, could contribute toward the partial spectrin deficiency.     

Homozygosity for dominant form of hereditary spherocytosis

A 6-month-old male infant with hereditary spherocytosis (HS) who was the first child of a cousin marriage is presented. The patient had splenomegaly and severe anaemia. Examination of the peripheral blood smear revealed spherocytes and the osmotic fragility of red blood cells was greatly increased. Physical examination of the parents revealed that both parents had mild anaemia, jaundice and splenomegaly. Their peripheral blood smears showed spherocytes and a few acanthocytes. Osmotic fragility of red blood cells of both parents were increased. Red cell membrane electrophoresis indicated a deficiency of ankyrin in the propositus; mild deficiency was also detected in both parents. Electrophoretic patterns of red cell membrane proteins suggested that the child was homozygous for the dominant form of HS associated with ankyrin deficiency, while both parents had the simple dominant form of the disease. Red blood cell transfusions were given to the patient starting at the age of 1 month until splenectomy was performed at the age of 1 year that resulted in complete haematological response. This observation indicates that homozygosity for dominant type of HS associated with ankyrin deficiency is life compatible and splenectomy may cure the anaemia.     

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.     

Absence of one component of spectrin adenosine triphosphatase in hereditary spherocytosis.

The stimulation by calcium and magnesium of ATPase activity of isolated ghosts, of water-soluble protein (spectrin), and of residual vesicles, derived from normal erythrocytes and from hereditary spherocytes (H.S.), has been measured. The ATPase activity found in normal water-soluble protein (WSP) at low levels of calcium (0.1-2.0 mM) is essentially absent in H.S. water-soluble protein, but the ATPase activity with magnesium and with high levels of calcium (60-100 mM) is the same in H.S. and normal WSP. Compared to normal, H.S. ghosts have increased Mg2+-stimulated activity. This increased activity is retained by the sedimentable vesicles ("residue") after extraction of the ghosts with 0.025 mM EDTA. The Ca2+, Mg2+-ATPase associated with the calcium pump is not significantly different in H.S.     

Molecular basis of spectrin deficiency in hereditary pyropoikilocytosis

Hereditary pyropoikilocytosis (HPP) is a recessively inherited hemolytic anemia characterized by severe poikilocytosis and red blood cell fragmentation. HPP red blood cells are partially deficient in spectrin and contain a mutant alpha or beta-spectrin that is defective in terms of spectrin self-association. Although the nature of the latter defect has been studied in considerable detail and many mutations of alpha-spectrin and beta spectrin have been identified, the molecular basis of spectrin deficiency is unknown. Here we report two mechanisms underlying spectrin deficiency in HPP. The first mechanism involves a thalassemia-like defect characterized by a reduced synthesis of alpha-spectrin as shown by studies involving synthesis of spectrin in two unrelated HPP probands and their parents: One parent carries the elliptocytogenic spectrin mutation, whereas the other parent is fully asymptomatic. Peripheral blood mononuclear cells as a source of erythroid burst-forming unit (BFUe) were cultured in a two-phase liquid culture system that gives rise to terminally differentiated erythroblasts. Pulse-labeling studies of an equal number of erythroblasts or morphologically identical maturity showed that the synthesis of alpha-spectrin as well as the mRNA levels as measured by the competitive polymerase chain reaction (PCR) method are markedly reduced in the presumed asymptomatic carriers and the HPP probands. In contrast, the synthesis and mRNA levels of beta-spectrin were normal. These results constitute a direct demonstration of an alpha-spectrin synthetic defect in a subset of asymptomatic carriers of HPP and HPP probands. The second mechanism underlying spectrin deficiency involves increased degradation of mutant spectrin before its assembly on the membrane. This is evidenced by pulse labeling studies of erythroblasts from a patient with HPP associated with a homozygous state for spectrin alpha I/46 mutation (leu-pro mutation at AA 207 of alpha-spectrin). These studies showed that although spectrin is synthesized in the cytosol in normal amounts, the rate of turnover of alpha-spectrin is faster resulting in about 40% to 50% reduced assembly of alpha-spectrin and beta-spectrin on the membrane. Thus, spectrin deficiency in this case is at least in part caused by increased susceptibility of the mutant spectrin to degradation before its assembly on the membrane. We conclude that at least two separate mechanisms underlie the molecular basis of spectrin deficiency in HPP.     

Diminished extractable spectrin in the erythrocytes of a patient with 'sporadic' hereditary spherocytosis

A 51-year-old white man of Irish extraction was found to have apparent 'sporadic' hereditary spherocytosis with a reticulocyte count of 6%. Twelve of his 13 siblings were examined and found to be haematologically normal. The patient's erythrocytes were found to have a diminished amount of spectrin as compared to his siblings and to unrelated controls. It is suggested that the proband may represent either a new mutant or possibly double heterozygosity for two inherited biochemical variants of the red cell membrane skeleton which individually give no haematological abnormalities.     

Glutathione reductase deficiency in a kindred with hereditary spherocytosis.

A kindred with hereditary spherocytosis was found to have erythrocyte glutathione reductase deficiency. The glutathione reductase was evaluated enzymatically and immunologically. The enzyme activity could not be restored either by flavin adenine dinucleotide (FAD) in vitro, or by oral administration of FAD. Kinetic and electrophoretic studies, heat stability tests, and pH curves revealed no evidence of a varient enzyme. The results of electroimmunodiffusion and neutralization tests using an antiserum indicated that the deficiency is caused by a diminished production of enzyme protein. Hereditary spherocytosis and glutathione reductase deficiency traits segregated independently. The amount of reduced glutathione in the erythrocyte was normal, and the deficiency state without hereditary spherocytosis was asymptomatic. All deficient members in this kindred were heterozygous and the residual enzyme activity in the heterozygote might be sufficient to maintain the reduced glutathione level to prevent oxidative stress.     

Hyperlipoproteinaemia(a) is a common cause of autosomal dominant hypercholesterolaemia

Summary Autosomal dominant hypercholesterolaemias (ADH) are a heterogeneous group of monogenic lipid disorders. The plasma level of lipoprotein(a) (Lp(a)) is a heritable trait associated with increased coronary heart disease (CHD) risk. Objective To evaluate the frequency of elevated Lp(a) as a cause of ADH and the characteristics of subjects with high Lp(a) (hyperLp(a)). Material and methods 200 healthy subjects and 933 unrelated Spanish subjects with a clinical diagnosis of ADH who were screened for low-density lipoprotein receptor (LDLR) and apolipoprotein B (APOB) gene mutations. Standard cardiovascular risk factors and blood lipid levels, including Lp(a), were evaluated. HyperLp(a) was defined as Lp(a) levels ≥95th centile of control values. Results Lp(a) was higher in 263 subjects without LDLR or APOB mutations (nonLDLR/nonAPOB group) than in 670 subjects with mutations (FH group): 40.0 mg/dl (interquartile range (IR) 15.0–89.0) versus 31.0 mg/dl (IR 11.0–73.7) respectively, p = 0.002. HyperLp(a) was present in 23% of ADH subjects (odds ratio (OR) 5.6 (95% CI, 2.9 to 10.7) versus controls) and 29% of nonLDLR/nonAPOB subjects (OR 7.7; 3.9 to 15.4). After adjusting for Lp(a), LDL cholesterol levels were <95th centile in 28 (10.6%) nonLDLR/nonAPOB subjects and in 9 (1.3%) FH subjects. Lp(a) levels were nonsignificantly higher in ADH subjects with early-onset CHD than in those without (43.5 mg/dl, (IR, 12.0–82.0) versus 31.7 mg/dl (11.8–76.5), respectively). Conclusions HyperLp(a) is responsible for ADH in approximately 6% of nonLDLR/nonAPOB subjects. HyperLp(a) would not appear to be a risk factor for early-onset CHD in ADH, independently of whether genetic defects have or have not been demonstrated. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Competing interests: None declared References to electronic databases: Familial hypercholesterolaemia: OMIM #143890. Familial defective apo B: OMIM #144010. PCSK9: OMIM *607786. Neural apoptosis-regulated convertase 1: OMIM *607786. Lipoprotein(a): OMIM +152200.     

Abnormal binding of spectrin to the membrane of erythrocytes in some cases of hereditary spherocytosis

Summary In two cases of hereditary spherocytosis that we have examined, spectrin was bound abnormally tightly to the erythrocyte membrane, and could not be released by low ionic strength dialysis. This type of behaviour occurs in normal red cells only after heating above 50 C. It appears that some cases of spherocytosis may be due to the presence of a protein which is abnormally temperature sensitive.This work was supported by an Australian Government Research Grant     

Marked reduction of spectrinin hereditary spherocytosis in the common house mouse

In contrast to the disease in humans, hereditary spherocytosis in the common house mouse produces an extreme spherocytosis. The cells show a broad distribution in size ranging from microcytic to macrocytic. Of particular interest is the finding of a substantial reduction in the major membrane polypeptide called spectrin, supporting a critical role for this protein in the control of erythrocyte shape and membrane stability.     

??-spectrinBari: a truncated ??-chain responsible for dominant hereditary spherocytosis

We describe a β-spectrin variant, named β-spectrin Bari, characterized by a truncated chain and associated with hereditary spherocytosis. The clinical phenotype consists of a moderately severe hemolytic anemia, splenomegaly, and spherocytes and acanthocytes in the blood smear. The occurrence of the truncated protein, that represents about 8% of the total β-spectrin occurring on the membrane, results in a marked spectrin deficiency. The altered protein is due to a single point mutation at position −2 (A->G) of the acceptor splice site of intron 16 leading to an aberrant β-spectrin message skipping exons 16 and 17 indistinguishable from that reported for β-spectrin Winston-Salem. We provide evidence that the mutated gene is transcribed but its mRNA is less abundant than either its normal counterpart or β-spectrin Winston-Salem mRNA. Our findings are an example of how mutations in different splice sites, although causing the same truncating effect, result in clearly different clinical pictures.