Clinical and molecular genetic analysis of a Chinese family with hereditary elliptocytosis caused by a novel mutation in the EPB41 gene.

BACKGROUND: Hereditary elliptocytosis (HE) is a heterogeneous red blood cell membrane disorder characterized by the presence of elliptocytes on a peripheral blood smear. Clinical manifestations of HE vary widely from asymptomatic carriers to patients with severe transfusion-dependent anemia. Most patients are asymptomatic or have mild anemia, which hinders diagnosis. The proband in this case had mild anemia and jaundice over a period of 4 years, the etiology of which was unclear. Hence, he was admitted to our hospital for further diagnosis. METHODS: Peripheral blood smears and routine blood tests were performed and biochemical parameters of the proband, and his family members were determined. To confirm the diagnosis, gene mutations were screened in the proband using next-generation sequencing (NGS) and verified by Sanger sequencing in other family members. RESULTS: A novel mutation (c.1294delA, p.Ser432 fs) in exon 15 of the EPB41 gene was detected in the proband and his family members. This mutation results in a frameshift and a premature stop codon at position 455, encoding a truncated protein. The variant was likely pathogenic according to the criteria of the American College of Medical Genetics and Genomics. SWISS-MODEL protein structure prediction indicated partial loss of the spectrin and actin binding and C-terminal domains. CONCLUSION: A heterozygous mutation 1294delA in exon 15 of the EPB41 gene was identified using NGS and Sanger sequencing in members of a Chinese family. This identification expands the spectrum of EPB41 mutations and contributes to the genetic diagnosis of families with HE.

Glucose-6-phosphate dehydrogenase deficiency and Southeast Asian ovalocytosis in asymptomatic Plasmodium carriers in Sumba island, Indonesia.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency and Southeast Asian ovalocytosis (SAO) caused by a 27-bp deletion in the band 3 gene (Band3Delta 27) are well-documented genetic traits resistant to malarial diseases; however, relationships between these traits and asymptomatic malaria infection hitherto had not been investigated. Filter-blotted blood samples were collected from a total of 210 healthy individuals, 100 males and 110 females, aged 6-17 years, in Sumba island, Indonesia, to survey for the presence of Plasmodium parasites, G6PD activity and the Band3Delta 27 mutation. Presence of P. falciparum and/or P. vivax was identified in 25 subjects (11.9%). In all, 24 subjects (11.4%) showed Band3Delta 27 heterozygously. In males and females, eight and nine subjects, respectively, showed G6PD deficiency. There was no significant difference in the prevalence of asymptomatic malaria infection between individuals with or without these traits (P>0.05). No alterations in the prevalence of asymptomatic malaria infection suggest that parasite invasion into erythrocytes is unlikely to be a target phase in which the two polymorphisms demonstrate possible protective effects against malaria.

Protein 4.1 deficiency and deletion of chromosome 20q are associated with acquired elliptocytosis in myelodysplastic syndrome.

We report a case of myelodysplastic syndrome (MDS), associated with prominent elliptocytosis. A 66-year-old male presented with peripheral pancytopenia, and was diagnosed with MDS [refractory anaemia (RA)]. Apart from marked elliptocytosis, dyshaematopoietic features were not evident in his peripheral blood or hypercellular bone marrow. After 18 months, he had progressed to RA with excess blasts in transformation. Analysis of red blood cell membrane proteins by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a reduced quantity of protein 4.1 (30% of control). Deletion of chromosome 20q was identified by conventional cytogenetic analysis and fluorescence in situ hybridization. Marked elliptocytosis, persistent for more than 17 months, decreased strikingly after chemotherapy with idarubicin and Ara-C. These findings suggest that acquired elliptocytosis occurred as an unusual morphological feature of MDS, associated with abnormalities of protein 4.1 and chromosome 20q.

Assignment of Sp alpha I/74 hereditary elliptocytosis to the alpha- or beta-chain of spectrin through in vitro dimer reconstitution.

Partial digestion of spectrin dimers in vitro has allowed the definition of domains. For example, the portions of the dimers that are involved in spectrin self-association are represented by the alpha I and the beta I domains. The alpha I domain (80 Kd) is further cleaved into a minor 78 Kd fragment and, more substantially, into a 74 Kd fragment. The intensity of the latter, which we expressed as the 74:(80 + 78 + 74) ratio, or the 74:alpha I ratio, is variable depending on the experimental conditions, eg, in fine, on the conformation of the alpha I domain. A number of cases of hereditary elliptocytosis (HE) are associated with an increase of the 74:alpha I ratio, also referred to as the Sp alpha I/74 abnormality. Several lines of evidence have suggested that the causal mutations may lie in the alpha- or the beta-chain, a point of importance before one undertakes studies at the gene level. In order to address this question, we reconstituted spectrin dimers in vitro, combining alpha- and beta-chains of various origins, and then carried out partial digestion and assayed the Sp alpha I/74 abnormality. The patterns obtained with reconstituted dimers were nearly identical to those of native dimers. We applied the assay to three spectrin variants that cause Sp alpha I/74 HE: (1) a variant that we previously designated spectrin Nice and whose beta-chain lacks a 4 Kd fragment in its C-terminal region; and two distinct variants that we found in two unrelated white families and that we provisionally designated spectrin Lyon and spectrin Culoz. The Sp alpha I/74 abnormality appeared in all kinds of dimers that harbored the beta-chain of spectrin Nice, or the alpha-chain of spectrin Lyon or spectrin Culoz, respectively. Therefore, we confirmed that spectrin Nice is a (alpha I/74) beta-variant, and established that both spectrin Lyon and spectrin Culoz are (alpha I/74) alpha-variants. The present assay may be extended to any spectrin variant displaying the Sp alpha I/74 abnormality.

Spectrin Oran (alpha II/21), a new spectrin variant concerning the alpha II domain and causing severe elliptocytosis in the homozygous state.

We report on spectrin Oran (alpha II/21), a new spectrin variant found in an Algerian family. It was characterized by the absence of the spots that classically correspond to the alpha II domain using two-dimensional analysis of spectrin limit digests. On the contrary, the abnormal domain was represented by a new set of spots in the 21-Kd and 16-Kd regions, as demonstrated by Western blots using anti-alpha II domain polyclonal antibodies. Spectrin Oran (alpha II/21) was found in the homozygous state in two children belonging to two separate branches of the family. It 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 alpha II 21-Kd and 16-Kd peptides. In one parent, who combines spectrin Oran (alpha II/21) and the alpha II 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 alpha II type-1 polymorphism. The alpha II type-1 spots (46, 35, 30, 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 alpha-variant percentage. Provided spectrin Oran was present in a sufficient proportion, we found an associated alteration of the beta II domain (that faces the alpha II domain in the spectrin dimer): the beta II 65-Kd fragment was reduced and the beta II 52-Kd fragment was reciprocally increased.

Hereditary elliptocytosis, spherocytosis and related disorders: consequences of a deficiency or a mutation of membrane skeletal proteins.

The membrane skeleton, a protein lattice that laminates the internal side of the red cell membrane, contains four major proteins: spectrin, actin, protein 4.1 and ankyrin. By mass, the most abundant of these proteins is spectrin, a fibre-like protein composed of two chains, alpha and beta, which are twisted along each other into a heterodimer. At their head region, spectrin heterodimers are assembled into tetramers. At their distal end, these tetramers are interconnected into a two dimensional network by their linkage to oligomers of actin. This interaction is greatly strengthened by protein 4.1. The skeleton is attached to the membrane by ankyrin, a protein that connects the spectrin beta chain to the major transmembrane protein band 3, the anion channel protein. Additional attachment sites are those of protein 4.1 with several glycoproteins, namely glycophorin A and C, as well as direct interactions between spectrin, protein 4.1 and the negatively charged lipids of the inner membrane lipid bilayer. Hereditary spherocytosis, elliptocytosis and pyropoikilocytosis represent a group of disorders that are due to deficiency or dysfunction of one of the membrane skeletal proteins (Fig. 1). Known deficiency states include that of spectrin, ankyrin and protein 4.1. Severe spectrin and ankyrin deficiencies (with decrease in spectrin and ankyrin contents to about 50% of the normal amount) are both rare disorders associated with severe autosomal recessive hereditary spherocytosis. On the other hand, mild spectrin deficiency is found in the majority of patients with autosomal dominant spherocytosis in which the degree of spectrin deficiency correlates with the clinical severity of the disease. Protein 4.1 deficiency, in contrast, is associated with hereditary elliptocytosis, which in certain populations constitutes about 20% of all such patients. Known skeletal protein dysfunctions include mutants of both alpha and beta spectrin that involve the spectrin heterodimer self-association site. These are clinically expressed as hereditary elliptocytosis (HE) and a closely related disorder, hereditary pyropoikilocytosis (HPP). At the level of protein function, this defect can be detected by analysis of the content of spectrin dimers and tetramers in 0 degrees C low ionic strength extracts of red cell membranes. Their structural identification is accomplished by limited proteolytic digestion of spectrin followed by two-dimensional tryptic peptide mapping.(ABSTRACT TRUNCATED AT 400 WORDS)

Fine structures of the spleen in hereditary elliptocytosis.

Light and electron microscopic studies of the spleen and liver from a patient with hereditary elliptocytosis have been reported. Characteristic findings are congestion of the cordal space, diminished variation of the erythrocyte shape in the cord, relatively empty sinuses and erythrophagocytosis by the cordal macrophages. The same basic defect of the red cell membrane as has been documented in hereditary sperocytosis might be a possible explanation on the pathogenesis of this rare hemolytic anemia.

Red blood cell abnormalities in cardiac valvular disease.

Nineteen patients with morphological abnormalities of the red blood cells are described, and these formed approximately 3% of the total cases of cardiac valvular disease. In two patients the abnormal blood film developed after the insertion of an aortic and mitral valve prosthesis respectively, but in another two patients the abnormal blood film was corrected by aortic valve surgery. Anisopoikilocytosis may have been associated with microangiopathic haemolytic anaemia in one patient, but in the others the cardiac valvular disease was severe and other mechanical factors were not present. The mitral valve was involved in 16 patients and the aortic valve in eight. Elliptocytosis was the only abnormality in 11 blood films, schistocytes and burr cells were present in seven, and in three there were a few microspherocytes. Family studies in seven patients produced evidence of hereditary elliptocytosis in three. Anaemia was present in only two patients. One of these had infective endocarditis, and the other developed overt haemolytic anaemia following the replacement of a diseased mitral valve by a Starr-Edwards prosthesis. In this latter case there was a transiently positive direct antiglobulin test, and the anaemia and abnormal blood picture were corrected without further surgical treatment. Haemolytic anaemia did not develop in 23 patients after the insertion of an aortic valve prosthesis or homograft. Indirect evidence of haemolysis was obtained in some patients who were not anaemic. There was a reticulocytosis in one third and serum haptoglobins were decreased or absent in over half of the patients tested.