SEARCH

SEARCH BY CITATION

Keywords:

  • homozygous missense mutation;
  • protein 4.2 deficiency;
  • band 3 Fukuoka (G130R);
  • intramembrane particles;
  • skeletal network

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

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.

Many B3 mutations have been reported in patients with hereditary spherocytosis (HS) ( Gallagher et al, 1998 ). Among these mutations, frameshift mutations and nonsense mutations have been found mostly in HS patients with autosomal dominant (AD) inheritance. Missense mutations, on the other hand, have been reported in HS patients with autosomal recessive (AR) transmission or with so-called ‘sporadic’ occurrence, in which the mode of inheritance has not been clarified. Therefore evaluation of such missense mutations would appear to be difficult with regard to the pathogenesis of HS itself, especially in a heterozygous state, in which an unaffected allele is clearly present in the patient in addition to a mutated allele. Therefore the homozygous state is crucial to clarify the significance of such missense mutations. Such a pathological condition is extremely rare, however. At the present time, only two definite cases have been reported; (1) band 3 Coimbra, which has a mutation of V488M in the membrane domain of the band 3 gene ( Ribeiro et al, 1997 ), and (2) the homozygous HS patient with a missense mutation in a cytoplasmic domain of the band 3 gene (codon 130 GGA  [RIGHTWARDS ARROW]  AGA:Gly  [RIGHTWARDS ARROW]  Arg in exon 6: B3 Fukuoka), who is reported here.

Therefore, the phenotypic expressions in this homozygous proband were studied to evaluate the significant contribution of this missense mutation to the pathogenesis of HS.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Subjects

The proband (a 29-year-old Japanese male) has suffered from compensated haemolysis (red cell count 4.21 × 1012/l, haemoglobin 13.6 g/dl, haematocrit 40.2%, MCV 95.5 fl, MCH 32.3 pg, MCHC 33.8 g/dl, increased indirect bilirubin 44 μmol/l, reticulocytosis 278 × 109/l, and increased osmotic fragility) since birth. The red cell morphology with microspherocytosis was compatible with hereditary spherocytosis. This patient has not been splenectomized, and blood transfusion has not been required. His parents demonstrated normal clinical haematological findings with normal red cell morphology. The patient has no brothers or sisters.

Analysis of the red cell membrane proteins

Membrane proteins were studied using sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) by the methods of Fairbanks et al (1971 ) and Laemmli (1970) with Coomassie blue staining, and were scanned by Protein+DNA Image Ware Systems (PDI-Toyobo Co., Tokyo, Japan). Western blotting was performed based on anti-human P4.2 rabbit polyclonal IgG antibody ( Kanzaki et al, 1997a ). The contents of each membrane protein fraction were determined on SDS-PAGE gels densitometrically and were expressed as relative amounts of the areas based on the O.D. values and the band widths. The content of the membrane proteins was expressed as the ratio of each membrane protein to that of total membrane proteins (TMP) or the TMP except for protein 4.2 (TMP-P4.2). Band 3 was also quantitated in individual red cells by cytofluorometry of red cells labelled with eosin-5-maleimide (E5M; Molecular Probes, Eugene, Ore.), as described previously ( Jennings et al, 1985 ).

Separation of band 3 dimers and tetramers

Separation of band 3 dimers and tetramers (and higher order oligomers) was performed using size-exclusion high-power liquid chromatography (HPLC) essentially according to the method of Casey & Reithmeier (1991).

Binding assay of the proband's inside-out vesicles (IOVs) to normal protein 4.2

Binding of pH 11-stripped inside-out vesicles (IOVs) to 125I-labelled protein 4.2 was measured according to the method of Korsgren & Cohen (1986).

Electron microscopy (EM) for the intramembrane particles (IMPs) and for the skeletal network

The intact red cells were examined for IMPs by EM using the freeze fracture method as described previously ( Yawata et al, 1996 ). Freeze fracture replicas were prepared in a Balzers BAF 301 apparatus (Balzers, Liechtenstein), and were examined with an electron microscope (JEM-2000 EXII, JEOL, Tokyo, Japan).

EM studies with the quick-freeze deep-etching (QFDE) method were performed by utilizing the quickly frozen red cell ghosts with Balzers BAF 301 (Balzers, Liechtenstein), as described previously ( Yawata et al, 1994 , 1997). The replicas were examined as described above.

Studies on nucleic acids: cDNA analysis

Total reticulocyte RNA was prepared from peripheral blood as a polysomal precipitate and extracted with phenol–chloroform–isoamylalcohol. Reverse transcription (RT) was carried out using random hexamers. Band 3 cDNA was amplified by the polymerase chain reaction (PCR) essentially as described by Kanzaki et al (1997a , b). Five PCR pairs of primers were used to cover the entire band 3 cDNA coding sequence (from nucleotide (nt) −148 upstream from the ATG initiation codon to nt 2815). The PCR products were digested with appropriate restriction enzymes to generate subfragments suitable for single-strand conformational polymorphism (SSCP) analysis as described by Spinardi et al (1991 ). The primers used are shown in Fig 1. Determination of the entire coding sequence of cloned band 3 cDNA (nt −128 to 2795) in the proband was performed by cloning PCR fragments into pCRTM II vector (TA cloning TMKit, Invitrogen, San Diego, Calif.). The clones were screened in order to distinguish the two types of cloned cDNA prior to sequencing, and then the PCR was performed. PCR products were analysed using PstI and MscI digestion of fragment C2 and SSCP analysis was carried out. Finally, nucleotide sequencing of protein 4.2 cDNA (the coding protein and the 180 nt sequence upstream from the ATG initiation codon) was also carried out, as previously described ( Kanzaki et al, 1995a , [12]b).

image

Figure 1. 30 is indicated by an arrow in fragment C-2.

Download figure to PowerPoint

Genomic DNA analysis

Total genomic DNA from the proband and his parents was extracted from peripheral blood and subjected to Bam HI digestion. The exon was examined by SSCP ( Spinardi et al, 1991 ). In these specimens, exon 6 was studied by asymmetric PCR using a 100-fold excess of the 5′-primer with regard to the 3′-primer. The corresponding 3′-primer was used for direct nucleotide sequencing. In the proband they were further cloned into the pCRTM II vector (TA cloning TMKit), PCR-amplified using the same primers, screened by SSCP ( Spinardi et al, 1991 ), and sequenced using universal primers.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Protein chemistry

Band 3 in the proband demonstrated a 9.3% reduction, compared with normal subjects, as shown in Fig 2A. Quantitation of band 3 by cytofluorometry of red cells labelled with eosin-5-maleimide confirmed this result. The ratio of band 3 tetramer to band 3 dimer was 25.6% in the proband compared with 24.4% in a normal subject, when high-performance liquid chromatography was performed. The protein content of P4.2 was substantially reduced in the proband (45.0% of that of normal subjects), as shown in Fig 2A. It is also noteworthy that, in addition to the 72 kD peptide (a wild type of P4.2), a trace amount of the 68 kD peptide was detected in the proband by Western blotting with anti-human P4.2 rabbit polyclonal antibody, when an excessive amount (50 μg per line) of membrane proteins was loaded, as shown in Fig 2B. The 74 kD peptide was not detected in the proband.

image

Figure 2. antibodies. Apparent molecular weights are expressed as kD. C: normal control; P: proband; F: father; M: mother.

Download figure to PowerPoint

In the proband, membrane proteins other than band 3 and P4.2 showed no abnormalities. No significant abnormalities were observed in his parents.

cDNA sequencing

SSCP analysis of the entire coding region of band 3 cDNA (from the nucleotide (nt) −148 to nt 2815) was performed. One point mutation with a G [RIGHTWARDS ARROW] A transition (GGA [RIGHTWARDS ARROW] AGA) at nt 388 introducing arginine (Arg) in place of glycine (Gly) at codon 130 in exon 6 was found, as shown in Fig 3A. This was previously designated as allele band 3 Fukuoka ( Ideguchi et al, 1994 ). The proband was homozygote for this mutation, as shown in Fig 3B, whereas his parents were heterozygotes.

image

Figure 3. 0R, and father and mother were heterozygous for this mutation.

Download figure to PowerPoint

Binding studies of the proband's IOVs to normal protein 4.2

pH 11-stripped IOVs were prepared from normal red cells and those of the proband. A rebinding assay of pH 11-stripped vesicles was reproducibly performed with normal protein 4.2 on three independent occasions. Representative results are shown in Fig 4. The extent of the rebinding of the proband's IOVs to the normal protein 4.2 was markedly reduced, compared with that of normal subjects. Scatchard plots indicated the average rebinding capacity in the proband was 207 μg of P4.2 per mg of vesicle proteins versus 295 μg in a normal subject. Therefore the rebinding capacity of the mutated band 3 Fukuoka to normal protein 4.2 appeared to be reduced to approximately 70% of the normal band 3.

image

Figure 4. .2 was measured according to Korsgren & Cohen (1986). (A) Representative results are shown by open circles (a normal subject) and closed circles (the proband). (B) Scatchard plots.

Download figure to PowerPoint

IMPs examined by EM using the freeze fracture method

Intact red cells were subjected to electron microscopy (EM) using the freeze fracture method. Representative results are shown in Fig 5. The number of IMPs was 4740 ± 125 per μm2 in the proband (n = 1, observed 30), compared with 5275 ± 329 in normal subjects (n = 24, observed 142). The size distribution of the IMPs in the proband was 67 ± 8% of small size (4–8 nm; normal 71 ± 8%), 31 ± 4% of medium size (9–20 nm; normal 27 ± 3%) and 2 ± 1% of large size (>21 nm; normal 2 ± 1%). Therefore no significant changes in IMPs were observed in the proband except for a slight reduction in number (89.8% of those in normal subjects).

image

Figure 5. Fig 5. Electron micrographs of red cell membrane studied by the freeze fracture method. Representative results are shown in a normal subject (A) and the proband (B). Intramembrane particles (IMPs) were detected on the inner (so-called ‘P’) face. The number of IMPs was slightly diminished (−10.2%) with normal size distribution in the proband.

Download figure to PowerPoint

Skeletal network examined by EM using the QFDE method

Red cell membrane ghosts were subjected to EM using the QFDE method, and representative results are shown in Fig 6. In normal subjects a fairly uniform distribution of filamentous structures and also uniformity of apparent branchpoints of the filamentous elements in an essentially orderly fashion were observed. The skeletal network in the normal subjects showed numerous basic units, resembling ‘cages’, the number of which was 548 ± 39 per μm2. In the proband the number of basic skeletal units was 484 ± 10 per μm2 (88.3 ± 1.8% of normal) with 68 ± 12% of those of basic small size (20–44 nm as determined by the interdistance of the longer axis of each structure; normal 70 ± 10%), 27 ± 6% of medium size (45–68 nm; normal 25 ± 6%), and 5 ± 3% of large size (69–92 nm; normal 5 ± 1%). Therefore the skeletal network in situ was almost normally maintained in the proband.

image

Figure 6. Fig 6. Electron micrographs of red cell membrane studied by the quick-freeze deep-etching method. Representative results are shown in a normal subject (A) and the proband (B). The number of the basic units of the skeletal network was only minimally reduced (−11.7%) with normal size distribution in the proband.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

We report on a homozygous patient with HS associated with a missense mutation (codon 130 GGA [RIGHTWARDS ARROW] AGA: Gly  [RIGHTWARDS ARROW] Arg in exon 6: band 3 Fukuoka) who demonstrated compensated haemolysis with disproportionally decreased (−55%) protein 4.2, compared with a mild reduction (−9.3%) of band 3. The pathogenesis is most probably due to an impaired binding capacity of the mutated band 3 protein to normal protein 4.2 as shown in band 3 Tuscaloosa ( Jarolim et al, 1992 ) and band 3 Montefiore ( Rybicki et al, 1993 ), in which the inheritance pattern appears to be autosomal recessive. Patients are expected to demonstrate clinical hazards only in the homozygous state, which has not been well documented. In the present proband, codon 130 appears to be one of the hot spots as a binding site to protein 4.2. Regarding the mutation (G130R) found in this proband, highly conserved glycine at segment 7 was replaced by arginine, which is positively charged. Therefore this mutation is truly pathognomonic in a homozygous state, but probably not in a heterozygous state, because normal haematology and protein chemistry were demonstrated in his heterozygous parents.

Although the exact role of P4.2 in the red cells has not been elucidated, many reports have recently been published regarding complete P4.2 deficiencies. The most striking phenotypic feature of the complete P4.2 deficiencies is the tremendous abnormalities in IMPs; that is, the decreased number, but markedly increased sizes, of IMPs ( Yawata, 1994a, b; Inoueet al, 1994 ; Kanzaki et al, 1995b ; Yawata et al, 1996 ). In addition, a marked disruption of the skeletal network in situ has been observed in complete P4.2 deficiencies ( Yawata, 1994a, b; Inoueet al, 1994 ; Kanzaki et al, 1995b ; Yawata et al, 1996 ; Golan et al, 1996 ). These findings appear to indicate that P4.2 controls the biophysical state of both band 3 and the skeletal network, which should be tightly linked to IMPs. Therefore, in the total absence of P4.2, serious derangements of red cell membranes in situ have been observed, not only in the integral proteins vertically, but also in the skeletal proteins horizontally.

The question then arises as to what extent protein 4.2 deficiency may cause serious damage to the red cell membrane structure. To try to answer that question, we investigated the homozygous proband with a substantial (−55%) deficiency of P4.2, which resulted from a band 3 mutation (G130R: GGA  [RIGHTWARDS ARROW]  AGA). We analysed the IMPs by EM using the freeze fracture method and the skeletal network in situ by EM with the QFDE method.

First of all, we found that the number of IMPs was only minimally diminished (roughly −10%), and that the size distribution was almost identical to that of normal subjects. The distribution of IMPs on the membrane plane was also nearly normally maintained. On SDS-PAGE gels the amount of band 3, which is a major component (approximately 80%) of IMPs, was only decreased by approximately 10%, even though the patient was homozygous for the band 3 mutation (G130R). On EM the mutated band 3 protein itself appeared to behave almost normally on the P face of the red cell membrane.

The skeletal network was also examined by EM to determine whether the network might be deranged by the decreased protein 4.2 content. The number of the basic skeletal units, however, was near-normal (484 ± 10/μm2; normal 548 ± 39). The size distribution of these skeletal units was also unaffected, and consisted mostly of units of small size (68 ± 12%, normal 70 ± 10%). Therefore even decreased (to approximately 45% of normal subjects) protein 4.2 appeared to be sufficient to maintain the normal structure of the skeletal network.

Although this patient was a homozygote of the G130R mutation of the band 3 gene, increased haemolysis was reasonably compensated with normal red cell counts and minimal changes in red cell morphology. Neither blood transfusion nor splenectomy was required, contrary to the serious clinical pictures and striking abnormalities in red cell membrane structure in the total absence of P4.2.

In summary, a partial deficiency of P4.2, if the extent of the deficiency was within approximately 45% of normal, appeared to not be critical for maintaining the normal integrity of red cell membrane structure in situ, both in the integral membrane components and also in the skeletal network. Therefore the state of P4.2 appears to play a critical role in determining the whole picture of red cell membrane abnormalities.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

We thank Professors J. Delaunay, W. Doerfler and S. Eber for their useful discussion. This work was supported by Grants-in-Aid for Scientific Research (07457236, 07670180, 09470235 and 09670164) and by the International Scientific Research Program: Joint Research (06044212, 08044328, 09044346 and 10044329) from the Ministry of Education, Science, Sports and Culture of the Japanese Government, by the Japanese (JSPS)–German (DFG) Cooperative Science Promotion Programme from the Japan Society for the Promotion of Science (JSPS), a research grant for Idiopathic Disorders of Haematopoietic Organs from the Japanese Ministry of Health and Welfare, a research grant from the Uehara Memorial Foundation, and research grants from Kawasaki Medical School (9-109, 9-809 and 10-111).

References

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References
  • 1
    Casey, J.R. & Reithmeier, R.A.F. (1991) Analysis of the oligomeric state of band 3, the anion transport protein of the human erythrocyte membrane, by size exclusion high performance chromatography: oligomeric stability and origin of heterogeneity. Journal of Biological Chemistry, 266, 15726 15737.
  • 2
    Fairbanks, G., Steck, T.L., Wallach, D.F.H. (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry, 10, 2606 2617.
  • 3
    Gallagher, P.G., Forget, B.G., Lux, S.E. (1998) Disorders of the erythrocyte membrane. Hematology of Infancy and Childhood (ed. by D. G. Nathan and S. H. Orkin), pp. 544 664. Saunders, Philadelphia.
  • 4
    Golan, D.E., Corbett, J.D., Korsgren, C., Thatte, H.S., Hayette, S., Yawata, Y., Cohen, C.M. (1996) Control of band 3 lateral and rotational mobility by band 4.2 in intact erythrocytes: release of band 3 oligomers from low-affinity binding sites. Biophysical Journal, 70, 1534 1542.
  • 5
    Ideguchi, H., Okubo, K., Nishimura, J., Eto, T., Fukumaki, Y. (1994) Abnormal band 3 protein (band 3 Fukuoka) found in two cases of erythrocyte band 4.2 deficiency. (Abstract). International Journal of Hematology, 59, 255.
  • 6
    Inoue, T., Kanzaki, A., Yawata, A., Tsuji, A., Ata, K., Okamoto, N., Wada, H., Higo, I., Sugihara, T., Yamada, O., Yawata, Y. (1994) Electron microscopic and physicobiochemical studies on disorganization of cytoskeletal network and integral protein (band 3) in red cells of band 4.2 deficiency with a mutation (codon 142 GCT [RIGHTWARDS ARROW] ACT). International Journal of Hematology, 59, 157 175.
  • 7
    Jarolim, P., Palek, J., Rubin, H.L., Prchal, J.T., Korsgren, C., Cohen, C.M. (1992) Band 3 Tuscaloosa: Pro327 [RIGHTWARDS ARROW] Arg327 substitution in the cytoplasmic domain of erythrocyte band 3 protein associated with spherocytic hemolytic anemia and partial deficiency of protein 4.2. Blood, 80, 523 529.
  • 8
    Jennings, L.K., Brown, L.K., Dockter, M.E. (1985) Quantitation of protein 3 content of circulating erythrocytes at the single-cell level. Blood, 65, 1256 1262.
  • 9
    Kanzaki, A., Hayette, S., Morlé, L., Inoue, F., Matsuyama, R., Inoue, T., Yawata, A., Wada, H., Vallier, A., Alloisio, N., Yawata, Y., Delaunay, J. (1997a) Total absence of protein 4.2 and partial deficiency of band 3 in hereditary spherocytosis. British Journal of Haematology, 99, 522 530.
  • 10
    Kanzaki, A., Takezono, M., Kaku, M., Yawata, A., Özcan, R., Kugler, W., Eber, S.W., Yawata, Y. (1997b) Molecular and genetic characteristics in Japanese patients with hereditary spherocytosis: frequent band 3 mutations and rarer ankyrin mutations. (Abstract). Blood, 90, (Suppl. 1), 6b 7b.
  • 11
    Kanzaki, A., Yasunaga, M., Okamoto, N., Inoue, T., Yawata, A., Wada, H., Andoh, A., Hodohara, K., Fujiyama, Y., Bamba, T., Harano, T., Harano, K., Yawata, Y. (1995a) Band 4.2 Shiga: 317CGC [RIGHTWARDS ARROW] TGC in compound heterozygotes with 142 GCT [RIGHTWARDS ARROW] ACT results in band 4.2 deficiency and microspherocytosis. British Journal of Haematology, 91, 333 340.
  • 12
    Kanzaki, A., Yawata, Y., Yawata, A., Inoue, T., Okamoto, N., Wada, H., Harano, T., Harano, K., Wilmotte, R., Hayette, S., Nakamura, Y., Niki, T., Kawamura, Y., Nakamura, S., Matsuda, T. (1995b) Band 4.2 Komatsu: 523 GAT [RIGHTWARDS ARROW] TAT (175 Asp [RIGHTWARDS ARROW] Tyr) in exon 4 of the band 4.2 gene associated with total deficiency of band 4.2, hemolytic anemia with ovalostomatocytosis and marked disruption of the cytoskeletal network. International Journal of Hematology, 61, 165 178.
  • 13
    Korsgren, C. & Cohen, C.M. (1986) Purification and properties of human erythrocyte band 4.2: association with the cytoplasmic domain of band 3. Journal of Biological Chemistry, 261, 5536 5543.
  • 14
    Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680 685.
  • 15
    Ribeiro, M.L., Alloisio, N., Almeida, H., Texier, P., Lemos, C., Mimoso, C., Morlé, L., Bey-Cabet, F., Rudigoz, R.-C., Delaunay, J., Tamagnini, G. (1997) Hereditary spherocytosis with total absence of band 3 in a baby with mutation Coimbra (V488M) in the homozygous state. (Abstract). Blood, 90, (Suppl. 1), 265a.
  • 16
    Rybicki, A.C., Qiu, J.J.H., Musto, S., Rosen, N.L., Nagel, R.L., Schwartz, R.S. (1993) Human erythrocyte protein 4.2 deficiency associated with hemolytic anemia and a homozygous 40Glutamic acid [RIGHTWARDS ARROW] Lysine substitution in the cytoplasmic domain of band 3 (band 3Montefiore) . Blood, 81, 2155 2165.
  • 17
    Spinardi, L., Mazars, R., Theillet, C. (1991) Protocols for an improved detection of point mutations by SSCP. Nucleic Acids Research, 19, 4009.
  • 18
    Yawata, A., Kanzaki, A., Gilsanz, F., Delaunay, J., Yawata, A. (1997) A markedly disrupted skeletal network with abnormally distributed intramembrane particles in complete protein 4.1-deficient red blood cells (allele 4.1 Madrid): implications regarding a critical role of protein 4.1 in maintenance of the integrity of the red blood cell membrane. Blood, 90, 2471 2481.
  • 19
    Yawata, A., Kanzaki, A., Uehira, K., Yawata, Y. (1994) A surface replica method: a useful tool for studies of the cytoskeletal network in red cell membranes of normal subjects and patients with a β-spectrin mutant (Spectrin Le Puy: β220/214) . Virchows Archiv, 425, 297 304.
  • 20
    Yawata, Y. (1994a) Red cell membrane protein band 4.2: phenotypic, genetic and electron microscopic aspects. Biochimica et Biophysica Acta, 1204, 131 148.
  • 21
    Yawata, Y. (1994b) Band 4.2 abnormalities in human red cells. American Journal of the Medical Sciences, 307, 190 203.
  • 22
    Yawata, Y., Yawata, A., Kanzaki, A., Inoue, T., Okamoto, N., Uehira, K., Yasunaga, M., Nakamura, Y. (1996) Electron microscopic evidence of impaired intramembrane particles and instability of the cytoskeletal network in band 4.2 deficiency in human red cells. Cell Motility and the Cytoskeleton, 33, 95 105.