A Iolascon

Erythrocyte membrane protein alterations underlying clinical heterogeneity in hereditary spherocytosis

Summary. Hereditary spherocytosis (HS) is a very heterogenous condition both at clinical and biochemical level. To establish the relationship between these aspects we performed a clinical and biochemical study in 87 Italian HS subjcts. Patients were divided into three groups based on clinical severity (mild, typical and severe) and into five subgroups based on specific membrane abnormalities identified by polyacrylamide gel electrophoresis (isolated spectrin deficiency, spectrin deficiency combined with mild ankyrin reduction, spectin deficiency combined with severe ankyrin reduction, band 3 reduction and isolated protein 4.2 reduction). We were not able to assess any alteration in six HS patients. A good correlation between clinical HS forms and memberane protein defects is shown. We conclude that erythrocyte memberane analysis should be carried out after diagnosis of HS in order to predict the clinical course of the disease.

Bone marrow transplantation in a case of severe, type II congenital dyserythropoietic anaemia (CDA II)

Type II congenital dyserythropoietic anaemia (CDA-II or HEMPAS) is an autosomal recessive disorder, representing the most frequent form of congenital dyserythropoiesis. It is characterised by normocytic anaemia, variable jaundice and hepato-splenomegaly. Gallbladder disease and secondary haemochromatosis are frequent complications. We report a case characterised by severe transfusion-dependent anaemia. The proband inherited CDA-II in association with beta-thalassaemia trait. Splenectomy did not abolish the transfusion dependence and this, in association with poor compliance to iron-chelation therapy, prompted us to consider bone marrow transplantation (BMT) from his HLA-identical sibling. The preparative regimen included busulfan, thiotepa and fludarabine, and graft-versus-host disease prophylaxis consisted of cyclosporin A and short-term methotrexate. Engraftment of donor cells was prompt and the post-transplant course uncomplicated. The patient is alive and transfusion-independent 36 months after allograft. This is the first case of severe CDA-II to undergo BMT. Analysis of this pedigree suggests that interaction with beta-thalassaemia enhanced the clinical severity of CDA-II, making BMT an attractive therapy for patients with transfusion dependence. Bone Marrow Transplantation (2001) 27, 213–215.

Gilbert's syndrome accounts for the phenotypic variability of congenital dyserythropoietic anemia type II (CDA-II)

The molecular basis for the considerable variation of serum bilirubin levels and the incidence of gallstone formation in patients with congenital dyserythropoietic anemia (CDA) type II are unknown. We show that the combined effect of an increased bilirubin load caused by dyserythropoiesis in CDA II and decreased bilirubin conjugation caused by reduced expression of uridine diphosphate glucuronosyl transferase (UGT1A) would increase the risk of hyperbilirubinemia (P <.005) and gallstone formation (chi(2): P <. 001). The rate of gallstone formation in patients with CDA II is 4. 75-fold the rate of patients without Gilberts syndrome, and gallstone diagnosis occurs at a younger age (P < 0.01). These findings should be considered during the follow-up of patients with CDA II.

Novel band 3 variants (bands 3 Foggia, Napoli I and Napoli II) associated with hereditary spherocytosis and band 3 deficiency: status of the D38A polymorphism within the EPB3 locus

We report three novel variants of band 3 associated with hereditary spherocytosis: band 3 Foggia (311delC; ACCCAC → ACCAC), band 3 Napoli I (447insT; TCT → TTCT) and band 3 Napoli II (I783N; ATC → AAC). The first two mutations resulted in premature termination of translation, making one haploid set of band 3 mRNA unavailable. Since it affected a highly conserved position at the terminal end of transmembrane domain 11, the third mutation prevented one haploid set of band 3 from becoming incorporated or stabilized into the membrane. These three mutations resulted in a reduction of the band 3 level in the red cell membrane (by 20–25%) and were dominantly transmitted. The D38A substitution (GAC → GCC) is a low frequency change of band 3. In one compound heterozygote D38A/Napoli II, a markedly aggravated picture required early splenectomy. In contrast, the D38A change was not associated with deterioration in another compound heterozygote, carrying in trans, the previously recorded R760W mutation (CGG → TGG). In the aggravated case, SSCP analysis did not exhibit any additional change in the two EPB3 alleles. Nor did it show any alteration in the exons of the two ANK1 alleles, and the aggravating factor remained elusive. The D38A alteration should be regarded as an innocuous polymorphism.

Ankyrin Napoli: a de novo deletional frameshift mutation in exon 16 of ankyrin gene (ANK1) associated with spherocytosis.

We report a case of apparently recessive hereditary spherocytosis in an Italian child. The proband exhibited a reduction of overall ankyrin in the red cell membrane. The parents were free of any haematological manifestations. The VNDR associated with the ankyrin gene (ANK1) were consistent with the following diplotypes: AC11/AC14 (father), AC14/AC14 (mother) and AC11/AC14 (child). The cDNA of the patient disclosed the expression of the AC11 allele only. As a consequence, we put forward the hypothesis of a de novo inactivation affecting the ankyrin allele of maternal origin (AC14) and accounting for the disease. PCR amplification of exons, SSCP analysis and nucleotide sequencing disclosed a polymorphism: GAC → AAC; Asp → Asn in codon 328 of exon 10, and a one‐nucleotide deletion : CTG → CG in codon 573 of the exon 16. This frameshift mutation placed in phase the TGA triplet that normally overlaps codons 636 and 637. Termination of translation near the middle of ankyrin mRNA coding sequence resulted, presumably, in its premature degradation. The present allele has been designated allele Napoli.

Frequent de novo mutations of the ANK1 gene mimic a recessive mode of transmission in hereditary spherocytosis: three new ANK1 variants: ankyrins Bari, Napoli II and Anzio

A subset of spherocytosis cases associated with mutations of the ANK1 gene present an apparently recessive inheritance pattern on a clinical and haematological basis. We identified three novel out‐of‐frame deletions in the ANK1 gene: allele Bari (1361delG), Napoli II (2883delC) and Anzio (3032delCA) in three Italian patients, two of whom have been splenectomized. Analysis of the cDNA showed small or trace amounts of ankyrin mRNAs in Bari, Napoli II and Anzio. The parents were normal clinically and haematologically and did not carry the mutations exhibited by their children. We confirmed the de novo character of the HS mutations based on paternity testing. Recessive HS associated with the ANK1 gene is probably rarer than initially thought, and spherocytosis may often be due to de novo mutations.

Severe poikilocytosis associated with a de novo α28 Arg→Cys mutation in spectrin

Summary Severe poikilocytosis was observed in an Italian child. The mutation responsible was a de novoα28 Arg→Cys substitution (CGT→TGT) in spectrin, a mutation known to cause hereditary elliptocytosis or hereditary pyropoikilocytosis. In this particular case the severity of the manifestations were accounted for by the occurrence, in trans to the α28 mutation, of the αv/41 polymorphism. The latter has been shown previously to be associated with structural abnormalities at the αIV‐αV domain junction and with a low expression level. The pronounced alteration of the dimer self association process was also explained by the location of the α28 mutation. This mutation occurs in helix 3 of repeating segment α1, e.g. precisely in the head‐to‐head contact between the spectrin α and β chains. The present phenotype was compared to that yielded by another α28 mutation (Arg→His) also combined, in trans, with the αv/41 polymorphism. The pictures were very much alike, stressing the functional importance of residue α28. The de novo character of the present mutation strengthens the view that codon α28 is a ‘hot spot’ for mutations.

Reversible erythrocyte skeleton destabilization is modulated by beta-spectrin phosphorylation in childhood leukemia

The erythrocyte skeleton plays an essential role in determining the shape and deformability of the red cell. Disruption of the interaction between components of the red cell membrane skeleton may cause loss of structural and functional integrity of the membrane. Several observations based on studies in vitro strongly suggest that phosphorylation may modify interactions between proteins, leading to a reduced affinity. In particular, increased phosphorylation of β-spectrin decreases membrane mechanical stability. In order to investigate the presence of membrane protein defects we investigated the erythrocyte membrane protein composition and phosphorylation in 22 children with leukemia at diagnosis and during the remission phase. Sixteen children had acute lymphoblastic leukemia (ALL), three had chronic myeloid leukemia (CML) and three had acute myeloid leukemia (AML). Ten patients (eight ALL and two CML) displayed elliptocytosis and poikilocytosis, an increase of spectrin dimers (41.8 ± 15.6) and an enhanced phosphorylation of β-spectrin (108 ± 15%) at diagnosis. These alterations disappeared during the remission phase. This is the first demonstration of a reversible erythrocyte membrane alteration in leukemia. Since the β-spectrin phosphate sites are located near the C-terminal region and close to the head of the β-chain that is involved in dimer-dimer interaction, we supposed that the β-chain phosphorylation has an effect upon the interactions between spectrin dimers, ie the tetramerization process. The weakening of this process should be responsible for the presence of elliptocytes and poikilocytes as reported in hereditary elliptocytosis and pyropoikilocytosis.

Cell Division Cycle Alterations and Human Tumors

A large series of evidence has conclusively demonstrated that the development and progression of a cancer are due to the accumulation of a number of genetic alterations which finally result in a full malignant phenotype. This complex phenomenon is clearly illustrated by colorectal tumors, which often require more than a decade to be clinically evident and at least seven genetic events for completion.1

A DELETIONAL FRAMESHIFT MUTATION IN SPECTRIN BETA -GENE ASSOCIATED WITH HEREDITARY ELLIPTOCYTOSIS IN SPECTRIN NAPOLI

Summary. We studied a clinically manifest. dominantly transmitted elliptocytosis in an Italian family. We found a new spectrin variant, designated spectrin Napoli. Its β‐chain was truncated in its C‐terminal region (apparent MW 216kD. It its C‐terminal region (apparent MW 216kD. It displayed a low expression level (15%). There was a 8 nt deletion; CTTTTGAGAAGT → CTGT (nt 62556262), starting after condon 2053. This deletion was followed by a 54nt (18 amino acids missense sequence and terminated by the TGA triplet which normally overlaps codons 2074 and 2075 (CTTGAG0. The overall length of the mutrated β‐chain was comparable to that found in spectrin Nice, spectrin Tokyo and spectrin Tandil, which are other variants with truncated β‐chains; however, a distinct nonsense codon was used in spectrin Napoli.