Akio Kanzaki

Total absence of protein 4.2 and partial deficiency of band 3 in hereditary spherocytosis

Unlike previously reported cases with total protein 4.2 deficiency due to mutations in the EPB42 gene, we describe a total deficiency in protein 4.2 with normal EPB42 alleles. Hereditary spherocytosis (HS) was observed in a Japanese woman (unsplenectomized) and her daughter (splenectomized). The mother showed a partial deficiency in band 3 and a proportional reduction in protein 4.2. She was heterozygous for a novel allele of the EPB3 gene, allele Okinawa, which contains the two mutations that define the Memphis II polymorphism (K56E, AAG → GAG, and P854L, CCG → CTG) and, additionally, the mutation: G714R, GGG → AGG, located in a highly conserved position of transmembrane segment 9. The latter change was responsible for HS. In trans to allele Okinawa, the daughter displayed allele Fukuoka: G130R, GGA → AGA, an allele known to alter the binding of protein 4.2 to band 3. The daughter presented with a more pronounced decrease of band 3, and lacked protein 4.2, resulting in aggravated haemolytic features. Although the father was not available for study, heterozygosity for allele Fukuoka has been documented in another individual who showed no clinical or haematological signs, and a normal content of band 3. We suggest that band 3 Okinawa binds virtually all the protein 4.2 in red cell precursors, band 3 Fukuoka being unable to do so, and that the impossibility of band 3 Okinawa incorporation into the membrane leads to degradation of the band 3 Okinawa protein 4.2 complex. In contrast, band 3 Fukuoka, free of bound protein 4.2, could then incorporate normally into the bilayer. Thus, protein 4.2 would not appear in the daughters red cell membrane.

Ankyrin gene mutations in Japanese patients with hereditary spherocytosis

We studied mutations of theankyrin-1 (ANK-1) gene of genomic DNA from Japanese patients with hereditary spherocytosis (HS). Forty-nine patients from 46 unrelated families were included in this study. Of these patients, 19 cases from 16 unrelated families had HS of autosomal-dominant inheritance, and 30 patients had non-autosomal-dominant HS. Fifteen mutations of theANK-1 gene pathognomonic for HS were identified: 4 nonsense mutations, 7 frameshift mutations, and 4 abnormal splicing mutations. These 15 mutations have not been previously reported. The frameshift mutations were found from exon 1 to exon 26, corresponding particularly to the band 3-binding domain of ankyrin. The nonsense mutations, on the contrary, were present mostly at the 3′-terminal side, especially in the spectrin-binding domain and the regulatory domain.The patients with ankyrin gene mutations tended to be more anemic with a higher level of reticulocytosis than those without these mutations. Fifteen silent mutations of theANK-1 gene, most of which have previously been detected in HS patients in Western populations, were also found. The allele frequency of these silent mutations in the HS patients was nearly identical to that in normal subjects. There was no difference between the Japanese and Western populations in the allele frequency of these gene polymorphisms in healthy subjects or HS patients.

Late expression of red cell membrane protein 4.2 in normal human erythroid maturation with seven isoforms of the protein 4.2 gene

The expression of protein 4.2 in normal human erythroid cells was studied utilizing erythroblasts from bone marrow and erythroid cells cultured by the two-phase liquid culture method from burst-forming unit erythroid (BFU-E) in peripheral blood. As opposed to spectrin, which was expressed in erythroid progenitors or very early erythroblasts, protein 4.2 was first detected in late erythroblasts with a morphology nearly identical to orthochromatic erythroblasts. Among the various major membrane proteins, the expression of protein 4.2 was the latest. At the gene level, protein 4.2 gene mRNA was expressed in early erythroblasts. During normal erythroid maturation, the expression of seven different protein 4.2 gene products was observed by Southern blot analysis. These seven gene products appeared to be derived from protein 4.2 gene in the presence or absence of skipping of the 90 bp in exon 1, exon 3, and/or exon 5, as judged by deduction from the protein 4.2 sequence. Therefore, it can be speculated that protein 4.2 is expressed after the cytoskeletal network has been constructed and assembled with integral proteins in the membrane lipid bilayer.

Ethnic distribution of allele aLELY, a low-expression allele of red-cell spectrin a-gene

Summary. AlleleαLELY low‐expression allele of erythroid spectrin a‐chain. It carries mutations both in exon 40 and intron 45 and is associated with partial skipping of exon 46. Allele αLELY remains asymptomatic by itself. In contrast, it enhances the expression level of deleterious α‐alleles occurring in trans, and as such has clinical importance. The aim of this study was to evaluate the incidence of allele QLBLY mvarjous ethnic groups, i.e. Caucasians, African Blacks, Japanese and Chinese. Allele QLELY occurred in all groups investigated with a fairly uniform frequency: 31%, 21%, 20% and 22%, respectively. Mutations in exon 40 and intron 45 appeared linked to one another without exception. Partial skipping of exon 46 or the low‐expression feature, whenever they could be assessed, were invariably observed. Allele αLELY appears to be an ancient and stable allele.

Immunohistochemical identification of erythroid precursors in paraffin embedded bone marrow sections: spectrin is a superior marker to glycophorin.

AIM: To investigate whether spectrin can be used as an immunohistochemical marker for erythroid precursors in routinely processed paraffin embedded bone marrow sections. METHODS: Bone marrow biopsies and clot sections were stained with rabbit antihuman erythrocyte spectrin antibodies, specific for erythroid cells as shown by western blotting and bone marrow smears, and compared to sections stained with antiglycophorin monoclonal antibodies (JC159 and Ret49f). RESULTS: Antispectrin antibodies resulted in diffuse cytoplasmic staining of early erythroblasts and membranous staining of late erythroblasts as well as erythrocytes. In haematopathological samples, immature erythroid cell clusters were clearly identified. In contrast, antiglycophorin monoclonal antibodies resulted in only membranous staining of late erythroblasts, and faint staining of early erythroblasts. CONCLUSIONS: Spectrin may be a superior marker to glycophorin for the identification of erythroid precursors in paraffin embedded sections.

Homozygous missense mutation (band 3 Fukuoka: G130R): a mild form of hereditary spherocytosis with near-normal band 3 content and minimal changes of membrane ultrastructure despite moderate protein 4.2 deficiency

The characteristics of phenotypic expression were studied in a Japanese family with hereditary spherocytosis and an extremely rare homozygous missense mutation of the band 3 gene (band 3 Fukuoka: G130R). The homozygous unsplenectomized proband was a 29‐year‐old male with compensated haemolytic anaemia (red cell count 4.21 × 1012/l, reticulocytes 278 × 109/l, and indirect bilirubin 44 μmol/l). His red cell band 3 (B3) protein demonstrated a 9.3% reduction and his protein 4.2 (P4.2) level was substantially reduced (45.0%), compared to normal subjects. P4.2 protein was composed mostly of a wild type (72 kD) with a trace of 68 kD peptide. The binding properties of the mutated B3 to normal P4.2 were significantly impaired, which probably resulted in the substantial reduction of P4.2 in this proband, since no abnormalities were detected on the P4.2 gene. Electron microscopy (EM) using the freeze‐fracture method demonstrated a mild decrease in intramembrane particles (IMPs) of near‐normal size (8 nm in diameter) with no substantial increases in their oligomerization. Their distribution on the membrane P face was almost normal, although most of the IMPs could represent the homozygously mutated B3 protein. EM (quick‐freeze deep‐etching method) disclosed a skeletal network of near‐normal size and size distribution of the skeletal units, suggesting that the mutated B3 protein itself did not have much effect on the skeletal network in situ. Therefore the reduced P4.2 content (45% of that of normal subjects), which remained on the red cell membrane of this proband, appeared to be nearly sufficient for maintaining the normal structure of the skeletal network and IMPs in situ, contrary to the marked abnormalities in both IMPs and the skeletal network in complete P4.2 deficiencies.

Granulocyte‐colony stimulating factor‐induced proliferation of primary adult T‐cell leukaemia cells

Granulocyte‐colony stimulating factor (G‐CSF) is known to induce proliferation and differentiation of granulocyte progenitors, and is widely used to treat neutropenia induced by intensive chemotherapy for malignant lymphoma or adult T‐cell leukaemia/lymphoma (ATL). G‐CSF is thought not to stimulate malignant lymphoid cells. In the present study we examined the ability of G‐CSF to induce in vitro growth of primary ATL cells from 14 patients (nine acute‐type, two chronic‐type and three lymphoma‐type), and we analysed the in vivo counts of ATL cells in patients who received G‐CSF for neutropenia. FACS analysis using phycoerythrin‐labelled recombinant G‐CSF demonstrated that ATL cells from 11/14 patients express some G‐CSF receptor (G‐CSFR), with a range between 5.4% and 87.3%. Cells expressing G‐CSFR also expressed CD4. Reverse polymerase chain reaction (PCR) analysis demonstrated expression of G‐CSFR messenger RNA in G‐CSFR expressing cells. Leukaemic cells derived from seven (four acute‐type, one chronic‐type and two lymphoma‐type) of the 14 patients proliferated in vitro in response to G‐CSF, as measured by [3H]thymidine incorporation; maximum responses were at G‐CSF concentrations of 10–100 ng/ml. Nine of 14 patients receiving rG‐CSF for neutropenia were analysed retrospectively for ATL cell numbers. Four patients whose primary tumour cells proliferated in response to rG‐CSF in vitro showed a significant increase in ATL cell count after administration of rG‐CSF (P =0.038), whereas five patients whose leukaemic cells did not proliferate in vitro showed no significant increase in ATL cell count. G‐CSF can stimulate proliferation of ATL cells which may complicate therapy for this disease.

Band 4.2 Shiga: 317 CGC → TGC in compound heterozygotes with 142 GCT → ACT results in band 4.2 deficiency and microspherocytosis

Summary. A novel compound heterozygous mutation of 317 CGC → TGC with 142 GCT → ACT in human red cell band 4.2 deficiency is described. A proband and his son suffered from compensated haemolysis with nearly complete deficiency of red cell band 4.2. Their red cell morphology exhibited microspherocytosis resembling classic hereditary spherocytosis (HS). Sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) showed band 4.2 to be nearly missing (< 1% of normal controls) with the presence of 74 kU and 72 kD isoforms in trace amounts. Other family members (daughters older and younger than the son) exhibited nearly normal amounts of 72 kD as a wild form of band 4.2 on SDS‐PAGE with the presence of the 74 kD isoform in a trace amount. The proband and his son demonstrated two compound heterozygous mutations in trans: i.e. nucleotide (nt) 949 CGC → TGC (codon 317 Arg → Cys) in exon 7 and nt 424 GCT → ACT (codon 142 Ala → Thr) in exon 3 of the band 4.2 gene. The two daughters demonstrated only the mutation of nt 949 CGC → TGC in exon 7 in heterozygous states, but no 142 mutation. Therefore the proband and his son were compound heterozygotes of these two mutations in trans. It is interesting to note that the 74 kD isoform of band 4.2 protein existed in a trace amount in the two daughters in spite of the absence of the 142 Ala → Thr mutation. In addition, even in the presence of the 142 mutation in one allele in the proband and his son, their red cell morphology demonstrated classic HS with microspherocytosis, although a homozygous state of the 142 mutation known as the Nippon type of band 4.2 deficiency exhibits ovalostomato‐cytosis.

Multiple cerebral infarctions in a patient with refractory idiopathic thrombocytopenic purpura

Multiple cerebral infarctions were observed in a patient with refractory idiopathic thrombocytopenic purpura who was positive for lupus anticoagulant (LAC) when her platelet counts were 2000 μL−1. It is suspected that LAC may have played an important role in the pathogenesis of this patients cerebral infarctions, although she had severe thrombocytopenia.

Germline mosaicism of MPZ Gene in Dejerine-Sottas syndrome (HMSN III) associated with hereditary stomatocytosis

We report on two sisters with Dejerine-Sottas syndrome (DSS) who had a heterozygous Gly 167 Arg mutation in the myelin protein zero (MPZ) gene and hereditary stomatocytosis (HSt). Genetic haplotype analysis suggested that the allele with the MPZ gene mutation originated from maternal lineage. However, the parents, who were normal clinically and electrophysiologically, had no mutation in the MPZ gene. Therefore, the MPZ gene mutation in these sisters was due to germline mosaicism of the MPZ gene in their mother. Stomatocytosis was detected in their mother and a sister who had no neurological symptoms, and therefore autosomal dominant HSt was suspected in this family. As stomatocytosis is very severe in our patients with DDS, we speculate that the association of DSS with stomatocytosis is coincidental but may have additively affected erythrocyte morphology. To our knowledge, these are the first familial cases of DSS with a mutation due to germline mosaicism of the MPZ gene to be reported.