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Coffin-Siris syndrome (CSS) is a congenital anomaly syndrome characterized by developmental delay, coarse facial features, and hypoplasia of the fifth digit's nail or phalanges. Herein, we report a case of the 8-year-old female patient who showed developmental delay associated with dysplasia in the macular and large toe area. Comprehensive genomic analysis showed no possible candidate variants, but the subsequent genomic copy number analysis revealed a novel exonic deletion in the coding region of AT-rich interactive domain-containing protein 1B (ARID1B), a gene responsible for CSS. Genomic copy number analysis can aid in diagnosing CSS by confirming undiagnosed exonic deletions in ARID1B. Furthermore, this is the first report of CSS associated with bilateral macular dysplasia.

Isolated complex I deficiency is the most common cause of pediatric mitochondrial disease. Exome sequencing (ES) has revealed many complex I causative genes. However, there are limitations associated with identifying causative genes by ES analysis. In this study, we performed multiomics analysis to reveal the causal variants. We here report two cases with mitochondrial complex I deficiency. In both cases, ES identified a novel c.580G>A (p.Glu194Lys) variant in NDUFV2. One case additionally harbored c.427C>T (p.Arg143*), but no other variants were observed in the other case. RNA sequencing showed aberrant exon splicing of NDUFV2 in the unsolved case. Genome sequencing revealed a novel heterozygous deletion in NDUFV2, which included one exon and resulted in exon skipping. Detailed examination of the breakpoint revealed that an Alu insertion-mediated rearrangement caused the deletion. Our report reveals that combined use of transcriptome sequencing and GS was effective for diagnosing cases that were unresolved by ES.

The level of glycated hemoglobin A1c (HbA1c) is widely used to monitor long-term glycemic control in patients with diabetes mellitus. There are more than 30 methods for measuring HbA1c levels. In recent times, high-performance liquid chromatography (HPLC) has become the most commonly used method in Japan. However, HPLC-based HbA1c level measurements do not accurately reflect glycemic control in the presence of Hb variants. We report the case of a patient with type 2 diabetes mellitus, who was incidentally found to having an extremely rare Hb variant. A 69-year-old Japanese female visited our clinic and was diagnosed with diabetes mellitus. Her HbA1c level, which was measured using HPLC at our clinic, could not be determined. DNA sequencing revealed a heterozygous mutation in the α1 globin gene (HBA1: c.301C > T, p.Leu101Phe). Hb Weesp was detected. Many Hb variants have been reported; however, to the best of our knowledge, this is only the second report about Hb Weesp in the world and the first from Japan. Clinicians should consider the possibility of Hb variants in cases in which abnormal elution patterns are detected during the measurement of HbA1c using HPLC.

Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive disorder characterized by dysmyelination of the central nervous system (CNS). We identified a rare partial duplication of the proteolipid protein 1 gene (PLP1) in a patient with PMD. To assess the underlying effect of this duplication, we examined PLP1 expression in induced pluripotent stem (iPS) cells generated from the patient's fibroblasts. Disease-specific iPS cells were generated from skin fibroblasts obtained from the indicated PMD patient and two other PMD patients having a 637-kb chromosomal duplication including entire PLP1 and a novel missense mutation (W212C) of PLP1, by transfections of OCT3/4, C-MYC, KLF4 and SOX2 using retro-virus vectors. PLP1 expressions in the generated iPS cells were examined by northern blot analysis. Although PLP1 expression was confirmed in iPS cells generated from two patients with the entire PLP1 duplication and the missense mutation of PLP1, iPS cells generated from the patient with the partial PLP1 duplication manifesting a milder form of PMD showed null expression. This indicated that the underlying effect of the partial PLP1 duplication identified in this study was different from other PLP1 alterations including a typical duplication and a missense mutation.

Involvement of the transferrin receptor cycle was noted in erythroblasts from a patient with erythroleukemia (FAB classification M6). The kinetics of transferrin receptor cycle in bone marrow erythroblasts was obtained by pulse-chase experiments before the initiation of therapy. Internalization of transferrin was impaired and resulted in a delayed peak of internalized transferrin, as compared with the kinetics pattern seen in healthy subjects. The subsequent exocytosis of the internalized ligand was also delayed. Thus, transferrin receptor cycle seems to be influenced all along the transferrin pathway, hence transferrin travels more slowly in erythroblasts in erythroleukemia. The altered transferrin receptor cycle led to a diminished iron uptake per surface transferrin receptor (approximately 30% of that in healthy subjects), and the incorporation of iron into heme was greatly reduced. Our observations suggest a possible role for the altered transferrin receptor cycle in the pathogenesis of defective heme synthesis and ineffective erythropoiesis in erythroleukemia.

Evidence from recent epidemiological studies suggests a link between periodontal infections and increased risk of atherosclerosis and related cardiovascular and cerebrovascular events in human subjects. One of the major pathogens of periodontitis, Porphyromonas gingivalis, has the ability to aggregate human platelets in platelet-rich plasma (PRP). Mechanism of P. gingivalis-induced platelet aggregation in PRP was investigated. Proteinase inhibitors toward Arg-gingipain (Rgp) and Lys-gingipain (Kgp) did not suppress P. gingivalis-induced platelet aggregation in PRP, whereas the Rgp inhibitor markedly inhibited P. gingivalis-induced platelet aggregation using washed platelets. Mutant analysis revealed that P. gingivalis-induced platelet aggregation in PRP depended on Rgp-, Kgp- and haemagglutinin A (HagA)-encoding genes that intragenically coded for adhesins such as Hgp44. Hgp44 adhesin on the bacterial cell surface, which was processed by Rgp and Kgp proteinases, was essential for P. gingivalis-induced platelet aggregation in PRP. P. gingivalis cell-reactive IgG in plasma, and FcgammaRIIa receptor and to a lesser extent GPIbalpha receptor on platelets were found to be a prerequisite for P. gingivalis-induced platelet aggregation in PRP. These results reveal a novel mechanism of platelet aggregation by P. gingivalis.

Background. Mutations in the human SLC4A1 (AE1/ band 3) gene are associated with hereditary spherocytic anaemia and with distal renal tubular acidosis (dRTA). The molecular diagnosis of AE1 mutations has been complicated by the absence of highly polymorphic genetic markers, and the pathogenic mechanisms of some dRTA-associated AE1 mutations remain unclear. Here, we characterized a polymorphic dinucleotide repeat close to the human AE1 gene and performed an immunocytochemical study of kidney tissue from a patient with inherited dRTA with a defined AE1 mutation. Methods. One CA repeat region was identified in a phage P1-derived artificial chromosome (PAC) clone containing most of the human AE1 gene and the upstream flanking region. We determined its heterozygosity value in multiple populations by PCR analysis. Genotyping of one family with dominant dRTA identified the AE1 R589H mutation, and family member genotypes were compared with the CA repeat length. AE1 and vH þ -ATPase polypeptides in kidney tissue from an AE1 R589H patient were examined by immunocytochemistry for the first time. Results. This CA repeat, previously reported as D17S1183, is � 90 kb upstream of the AE1 gene and displayed considerable length polymorphism, with small racial differences, and a heterozygosity value of 0.56. The allele-specific length of this repeat confirmed co-segregation of the AE1 R589H mutation with the disease phenotype in a family with dominant dRTA. Immunostaining of the kidney cortex from one affected member with superimposed chronic pyelonephritis revealed vH þ -ATPase-positive intercalated cells in which AE1 was undetectable, and proximal tubular epithelial cells with apparently enhanced apical vH þ ATPase staining. Conclusions. The highly polymorphic dinucleotide repeat adjacent to the human AE1 gene may be useful for future studies of disease association and haplotype analysis. Intercalated cells persist in the endstage kidney of a patient with familial autosomal dominant dRTA associated with the AE1 R589H mutation. The absence of detectable AE1 polypeptide in those intercalated cells supports the genetic prediction that the AE1 R589H mutation indeed causes dominant dRTA.

We describe a 6-year-old girl and her mother with dominant β-thalassemia due to hemoglobin Hradec Kralove (Hb HK). Both patients presented microcytic anemia, jaundice, splenomegaly, cholelithiasis, and recurrent hemolytic bouts. Osmotic resistance tests using saline and coiled planet centrifugation revealed the increased fragility of the red cell membrane. On the other hand, the glycerol lysing time was prolonged, and results of the isopropanol test were weakly positive. Despite mimicking the features of hereditary spherocytosis, the results of the genetic analyses verified the second reported family with Hb HK (codon 115,\(G\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{C} C[Ala] \to G\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{A} C[Asp]\)). Splenectomy was effective for the amelioration of hemolysis. Of 7 reported patients with Hb variants at β-globin codon 115 (Hb Madrid and Hb HK), 5 underwent splenectomy. Because of the variable augmentation of extramedullary hemolysis in dominant β-thalassemias, genotyping is necessary for determining the clinical indication of splenectomy.

Summary. We identified a Japanese family with a β-thalassaemia trait and hereditary elliptocytosis (HE). We studied five members of this family. One was normal, one had only the β-thalassaemia trait, one had heterozygous HE, and two had compound heterozygous β-thalassaemia trait and HE. The last two had already undergone splenectomy. The molecular profile of β-thalassaemia was consistent with that of Hb Gunma: codon 127/128CAGGCT(Gln–Ala) CCT(Pro). Analysis of erythrocyte membrane proteins revealed a partial deficiency of protein 4·1 in all those with HE, whereas the spectrin content was within the normal range. Each heterozygous family member with either the β-thalassaemia trait or HE was asymptomatic, whereas the two with both β-thalassaemia and HE had marked red blood cell deformities and haemolysis. The abnormalities of the red blood cells in patients with the β-thalassaemia trait might be enhanced by association with HE owing to a protein 4·1 deficiency.

We describe two children of nocturnal frontal lobe epilepsy (NFLE) diagnosed using carefully observed nocturnal sleep EEGs and detailed patient histories.Case #1, a 14-year-old boy, showed repeated generalized tonic convulsions and frequent eyes opening seizures during sleep. Conventional EEGs – done with the patient awake or in sleep stage I – showed no abnormalities, while a nocturnal sleep EEG – done during in sleep stage II – revealed the repeated, sharp wave bursts predominantly in the right frontal lobe characteristic of NFLE. During these wave bursts, we noticed the boy's eyes opening, although his parents had not been aware this NFLE symptom.Case #2, a 12-year-old boy, showed one daytime generalized convulsion. He had also been suffering from repeated paroxysmal episodes similar to parasomnia – waking up, sitting, walking, screaming, and speaking – which always followed the same patterns lasting several minutes. During the nocturnal sleep EEG, episodes occurred twice, showing abnormal epileptic discharges predominantly in the frontal lobe. His parents did not mention the episodes to us until questioned, as they had recognized them as parasomnia. The previous conventional EEG showed abnormal slow waves in the frontal lobe, which led us to suspect frontal lobe epilepsy and to take a detailed patient history.The frequency and stereotypy of their symptoms during sleep caused us to perform nocturnal sleep EEGs and led us NFLE diagnosis. Detailed patient histories including sleep habits and carefully observed nocturnal sleep EEGs enabled us to recognize these NFLE clinical features.

Simultaneous presence of hemolytic anemia and bilirubin UDP-glucuronosyltransferase deficiency is a possible cause of misdiagnosis. Seven-year-old and 17-year-old brothers and a 15-year-old sister consecutively suffered from aplastic crises. Although few spherocytes were present, the siblings and their mother had diagnoses of hereditary spherocytosis with flow cytometric analysis of eosin-5'-maleimide-labeled red blood cells in addition to osmotic fragility test. However, inappropriately high values of bilirubin compared with mild hemolysis persisted. Further analysis of UDP-glucuronyltransferase 1A1 revealed all 3 siblings were heterozygous for A(TA)7TAA-P229Q. We report here the importance of careful evaluation of mild hereditary spherocytosis masking UDP-glucuronyltransferase 1A1 deficiency.

We report a case of spherocytosis associated with erythrocyte band 3 deficiency. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of erythrocyte membrane proteins showed that the patient’s band 3 was reduced to about 80% of the control level. Molecular analysis revealed that this quantitative alteration was accompanied by a novel base change at codon 837 (ACG→GCG) of the AE1 gene, resulting in substitution of alanine for threonine. In bone marrow mononuclear cells, both mutant and wild-type mRNA were comparably detected, suggesting that this mutation interfered with band 3 processing or assembly, leading to impaired accumulation of mutant band 3 in the plasma membrane.

Hemolytic properties of human erythrocytes in hereditary spherocytosis (HS) were examined under hydrostatic pressure or hypotonic conditions. In the hypotonic buffer, HS erythrocytes were more fragile than normal erythrocytes, and the osmotic fragility was similarly enhanced if both erythrocytes were treated with 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), an anion transport inhibitor. On the other hand, the hemolysis of HS erythrocytes at 200 MPa was almost the same degree as that of normal cells. Upon DIDS treatment, the hemolysis of normal erythrocytes at 200 MPa was suppressed by about 35%, whereas that of HS erythrocytes was not affected. The absence of a suppressive effect of DIDS on high-pressure-induced hemolysis is likely to be HS-specific and may be a reflection of the underlying defects of band 3-cytoskeleton interactions in HS erythrocytes.

Background Myoclonic-astatic epilepsy (MAE) is an epileptic syndrome characterized by unique myoclonus, myoclonic-astatic, or astatic seizures in childhood. MAE prognosis vary from spontaneous remission to intractable seizures with profound mental retardation. Aim Identifying early risk factors may optimize the treatment of children with MAE. Our hypothesis is early onset age and focal spike discharges on EEG indicate a poor MAE prognosis. Methods Using the medical records of 9 children with MAE, we analyzed their clinical histories, EEG findings, and seizure symptoms. All patients were given follow-up observations/treatments by our department for at least 2 years after MAE onset. Results Five of the patients were given favorable prognoses because their seizures disappeared within 2 years of onset; the other 4 received poor prognoses because their seizures continued more than 2 years. MAE onset in patient with refractory seizures was earlier than that in those with a favorable prognosis (7-24 months vs. 23-38 months). All the patients with refractory seizures showed moderate or severe mental retardation. Among the 5 patients with good prognosis, EEGs showed two with focal spike discharges and three with only generalized spike discharges. In contrast, all cases with a poor prognosis had focal spike discharges. Conclusions MAE onset in patients with refractory seizures occurs earlier than in those with favorable prognosis. Prognosis was excellent when EEG findings show no focal spike discharges. Both early seizure onset and the focal spike discharges associated with MAE are indicators of poor prognosis.

Solid pseudo-papillary tumor (SPT) of the pancreas is a relatively benign tumor that is more frequently reported in females. Most patients usually present with abdominal pain or mass. We experienced the girl who identified SPT with the injury. We diagnosed SPT in a previously healthy 14-year-old Asian girl after abdominal injury. She experienced upper abdominal pain and vomiting after being hit by a basketball. Blood examination revealed a high serum amylase level. Abdominal radiography indicated abnormal bowel gases. Contrast-enhanced computed tomography revealed a smooth, peripheral and unilocular mass approximately 55 mm in diameter in the pancreatic tail. Based on these observations, acute pancreatitis complicated by a pancreatic mass was initially diagnosed. Therapy for acute pancreatitis was instituted, while we simultaneously investigated the mass. Levels of tumor markers were not profoundly elevated in serum. Dynamic contrast-enhanced magnetic resonance imaging (MRI) revealed moderate and gradual increase in contrast-enhanced imaging, consistent with findings of SPT of the pancreas. We thus elected surgical resection for her. Pathological examination of the surgical specimen confirmed our diagnosis of SPT. SPT of the pancreas should be considered as a differential diagnosis of acute abdomen disorders, especially in instances after minor abdominal injuries in young women, and diagnoses must be confirmed with MRIs.

Summary. Human erythrocyte protein 4.2 (band 4.2; pallidin) is a major membrane protein that comprises 5% of the total weight of the human erythrocyte membrane. Deficiencies of this protein have been observed in hereditary spherocytosis with anaemia, suggesting a role of protein 4.2 in erythrocyte stability and integrity. The molecular basis of this disorder remains unknown. As a first step in elucidating the pathogenesis of hereditary spherocytosis associated with protein 4.2 deficiency, we cloned and sequenced the erythrocyte protein 4.2 gene from a normal Japanese person. We prepared sets of oligonucleotide primers for polymerase chain reaction (PCR) and determined nucleotide sequences of exons and exon-intron boundaries of the protein 4.2 gene from three unrelated Japanese patients with hereditary spherocytosis due to a complete defect of protein 4.2, using PCR-related techniques. Two patients were homozygous for a missense mutation in codon 142 with the Ala (GCT) Thr (ACT) amino acid substitution that has been reported previously (protein 4.2NIPPON), whereas one patient was compound heterozygous for the same missense mutation in codon 142 and a guanine-adenine transition in codon 119 that changes the codon for Trp (TGG) to the termination codon (TGA) (protein 4.2Fukuoka). No additional mutation was identified in other exons of the protein 4.2 genes. Dot-blot hybridization with allele-specific oligonucleotide probes showed that homozygosity for the missense mutation in codon 142 and compound heterozygosity for the codon 142 and the codon 119 mutations were related to protein 4.2 deficiency in the families. Although two alleles of missense mutation of the codon 142 were also detected in 100 alleles of healthy Japanese, results obtained in this study indicate that the two mutations described above are closely related to the pathogenesis of hereditary spherocytosis due to protein 4.2 defect.

Summary A case of myelodysplastic syndrome with haemolytic anaemia and a marked elliptocytosis is reported. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of erythrocyte membrane proteins revealed that the patient's band 4.1 was decreased to about 50–70% of that of control and contained abnormal molecule migrating in a faster mobility than normal band 4.1. which was confirmed by immunoblotting. The actin/spectrin ratio of the patient's ghosts diminished to about 70% of that of control ghosts. Flowcytometric analysis showed that the glycophorin C content of the patient's erythrocytes was reduced but maintained the level of about 70% of that of normal, indicating that the glycophorin C-band 4.1 interaction might not be so seriously damaged as to cause elliptocytic shape change. We postulate that the abnormal band 4.1 produced from the abnormal erythroid clone may be the primary molecular defect and result in a dysregulation of spectrinactin interaction to cause erythrocyte shape change and membrane instability.

Band 3-Memphis is the most common variant of erythrocyte band 3 protein, in which a single amino acid substitution (Lys56-->Glu) in a cytoplasmic domain of band 3 has been found. We showed here that the prevalence of the variant was particularly higher in the Japanese population than that in other populations. The calculated gene frequency of the variant in Japanese was 0.156, which was about 4 times higher than that in Caucasian. Although it has been generally accepted that the cytoplasmic domain of band 3 bears no relation to anion transport activity, we found that the transport rate of phosphoenolpyruvate in erythrocytes of homozygotes was decreased to about 80% of that in control cells, and that of heterozygotes was at an intermediate level. The evidence indicates that some structural changes in the cytoplasmic domain of band 3 may have influence on the conformation of anion transport system.

The acquired resistance to various drugs in cancer is mediated by P-glycoprotein (P-gp) which is encoded by the mdr-1 gene. An increased level of mdr-1/P-gp was demonstrated after chemotherapy administered to treat cancer in humans. To clarify the direct effect of anticancer drugs on mdr-1/P-gp expression, we investigated the change in transport of adriamycin (ADR), and the expression of the mdr-1 gene and P-gp in an ADR-treated, multidrug-resistant leukemic cell line (K562/ADR500). The addition of ADR induced the over-expression of mdr-1/P-gp, which led to a transient decrease in the intracellular accumulation of ADR although the difference was not statistically significant. A maximal effect was observed after 4 h incubation, returning to the baseline level after further incubation for 12–24 h. The phosphorylation of P-gp was inversely correlated with the levels of P-gp. These observations suggest that ADR itself modulates both the expression and function of P-gp. Determination of the optimal schedule for administering adriamycin is essential to achieving the optimal effect in treating cancer.

We presented a case, who showed extremely high activity of lactate dehydogenase (LD) and confirmed the presence of the LD linked immunoglobulin in her serum. The maximum activity of LD was 6830 IU/L, and the electrophoretic pattern of LD isozymes showed the broad spectrum from isozyme LD2 to LD5. Analysis by counter immuno electrophoresis revealed that immunogloblin was attached to the M subunit of LD and its subtype was an IgG, lambda-chain. The cause, which produced the complex, might be thought to be the side effects by tiapride administrated for her mild dementia. After discontinuance of this drug, the LD activity in serum had gradually reduced. The serum creatinine also increased gradually after administration of tiapride, and did not reverse to normal level by discontinuance of it. The patient died from acute renal failure, which aggravated from sporadic urinary tract infection. It was suggested that her basal renal dysfunction might be due to the LD IgG complexes. We propose a rapid disruption of suspicious drug for the course of the production of LD linked immunoglobulin, because very high titer of these complexes might suffer irreversible damage to the kidney, which chance to become acute renal failure.

In order to support a faster and more informative clinical practice, we established the criteria for panic (critical) values regarding the blood concentrations of glucose, Na, K, Ca, inorganic phosphate (IP), Hb and number of platelets, and also created a system to report these values directly to the doctors in charge. We initiated this system in September 2003. In order to evaluate the availability of this system, we analyzed the clinical data during a one year period, based on the findings of patients showing panic values, mainly concerning the disease states and the correspondences by the doctors who were directly informed. We also carried out questionnaire surveys about the panic values and the new system for all of the doctors in our hospital (recovery rate: 84.3%). The total number of panic values reported was 113 and the mean percentage of the number of ordered examinations was 0.019%. After the report, 79 cases (69.9%) were examined again or treated, while 34 cases (30.1%) had already been treated or watched carefully at the time of the report. Malignant diseases were the main causes of increased panic values (38 cases), especially in the Na, K and blood glucose of patients. The next disease state, which appeared to demonstrate high rates, was chronic renal failure (16 cases), in the low K, high Ca, and low IP patients. Most of the cases of low Hb were caused from bleeding of the gastro-intestinal tract, with malignancies next. A blood infusion was performed for all of the cases with low Hb except for one. As a result of the questionnaire survey among the staff doctors, we confirmed that this system did indeed work efficiently, and 88% of the doctors who answered the questionnaires, were satisfied with the system. In conclusion, we established a new system, which made it possible for panic values to be directly reported to the doctor in charge and this system was then evaluated for its clinical usefulness.

The effects of the induction of differentiation were investigated on the expression of heat shock proteins (hsps) and thermotolerance. The synthesis of the major hsps in response to heat stress was markedly enhanced in HL-60 human leukemia cells after differentiation. An increased amount of mRNA transcripts for hsp70 was also noted. In addition, induction of differentiation resulted in acquisition of greater resistance to heat, which may be advantageous since cells in the peripheral blood must survive many stresses.

In the present study, we investigated the hsp synthesis in normal human lymphocytes and leukaemic cells in order to detect possible differences

We examined endotoxins and limulus amebocyte lysate-reactive material (LAL-RM) in serum from 87 patients on hemodialysis, using the conventional chromogenic limulus test (CCLT) and a new specific endotoxin assay with factor G-free LAL (endotoxin-specific test: EST). All patients with regenerated cellulose dialyzers had increased CCLT values, whereas the EST values were not higher than in controls. This discrepancy can be explained by the LAL-RM which is cellulose-derived and cross-reacts with LAL via factor G. Using EST for measurements of endotoxin, 6 patients out of 87 had pathological endotoxemia and all of these patients were associated with either cirrhosis, infection, or malignancy. Some patients who had no complications showed fever before or during dialysis, but they did not have high endotoxin levels. EST is useful for the diagnosis of endotoxemia in patients on hemodialysis because of the presence of LAL-RM in serum. Endotoxemia is rare in patients on hemodialysis, if they are not associated with infection, cirrhosis, or other complications.

We have developed three adriamycin (ADR)-resistant K562 sublines with different degrees of resistance. These sublines show a decreased accumulation and an increased efflux of ADR in proportion to the degree of resistance. Two membrane proteins (mol. wt 170,000 and 230,000) reactive with monoclonal antibody against P-glycoprotein were highly expressed in both the K562/ADR200 and the K562/ADR500 subline. Less resistant K562/ADR80 cells contained only small amounts of mol. wt 230,000 protein. Thus, the level of P-glycoprotein expression was not proportionate to the degree of ADR efflux. Verapamil treatment could not completely reverse ADR resistance. No significant change of glutathione-s-transferase activity nor in the level of DNA topoisomerase II was detected in resistant sublines. In our sublines it seems that P-glycoprotein is one of the mechanisms for resistance, but additional mechanisms may be involved.

The majority of a-thalassemia results from the large deletions in a-globin gene cluster, including both or either one of alpha-globin genes (alpha1 and alpha2). Most common a-thalassemia-2 deletions (single gene deletions) are -alpha3.7 and -alpha4.2, and alpha-thalassemia-1 deletions (double gene deletions) are --SEA, --THAI, --FIL, --MED and -(alpha)20.5 Although it is not easy to diagnose these deletions because of the high GC content at this locus and the sequence homology among psi alpha2, psi alpha1, alpha2 and alpha1 genes, these alleles can now be diagnosed by a single tube multiplex gapPCR assay. We showed here two Saudi Arabian patients with a-thalassemia trait who could be determined their gene mutations according to the method of Chong SS et al. (2000). [Case 1: 21-year-old male] GapPCR assay revealed the amplification of only -alpha3.7, whereas PCR of both a-globin genes showed no amplifications. The results indicate case 1 is a homozygote of -alpha3.7(-alpha3.7/-alpha3.7). [Case 2: 31-year-old male] GapPCR assay revealed the amplification of only -alpha3.7, and PCR of both alpha globin genes showed normal amplification. DNA sequencing of the amplified a-globin genes revealed a point mutation in the poly A site of alpha2-globin gene (AATAAA--AATAAG), which is known as alpha(T-Saudi). Thus, case 2 was confirmed to be a compound heterozygote of -alpha3.7 and alpha(T-Saudia) alpha(-alpha3.7 / alpha(T-Saudi) alpha). This gapPCR assay is a rapid, reliable screening test for common alpha-thalassemia deletions and seems to be useful for the diagnosis of thalassemic patients without an increase of Hb A2 and/or an abnormality of beta-globin gene.

Gene analysis in Japanese patients with congenital hemolytic anemia due to red cell membrane disorders, thalassemias, unstable hemoglobinopathies and red cell enzymopathies were summarized. In hereditary spherocytosis, twenty-four mutations of band 3, five mutations of protein 4.2 and twenty mutations of ankyrin have been identified. In beta thalassemia, fourty-seven mutations of beta globin have been found, and ten mutations among them comprise 80% of beta thalassemia patients in Japan. Most common alpha0 and alpha+ thalassemia are--SEA and--alpha3.7, respectively. Fourty glucose-6-phosphate dehydrogenase mutations and twenty-three pyruvate kinase mutations have been identified, allowing a better understanding of the structure-function relationships of these enzymes.

We identified a Japanese family with a beta-thalassaemia trait and hereditary elliptocytosis (HE). We studied five members of this family. One was normal, one had only the beta-thalassaemia trait, one had heterozygous HE, and two had compound heterozygous beta-thalassaemia trait and HE. The last two had already undergone splenectomy. The molecular profile of beta-thalassaemia was consistent with that of Hb Gunma: codon 127/128CAGGCT(Gln-Ala)--CCT(Pro). Analysis of erythrocyte membrane proteins revealed a partial deficiency of protein 4.1 in all those with HE, whereas the spectrin content was within the normal range. Each heterozygous family member with either the beta-thalassaemia trait or HE was asymptomatic, whereas the two with both beta-thalassaemia and HE had marked red blood cell deformities and haemolysis. The abnormalities of the red blood cells in patients with the beta-thalassaemia trait might be enhanced by association with HE owing to a protein 4.1 deficiency.

A case of hereditary thrombocytopenia with large platelets (familial macrothrombocytopenia, FM) is reported. Studies on the platelets from the propositus showed decreased glycosylation of platelet membrane glycoprotein IV, which would distinguish the case from other FM previously described.

The AE1 gene encodes band 3 Cl-/HCO3- exchangers that are expressed both in the erythrocyte and in the acid-secreting, type A intercalated cells of the kidney. Kidney AE1 contributes to urinary acidification by providing the major exit route for HCO3- across the basolateral membrane. Several AE1 mutations cosegregate with dominantly transmitted nonsyndromic renal tubular acidosis (dRTA). However, the modest degree of in vitro hypofunction exhibited by these dRTA-associated mutations fails to explain the disease phenotype in light of the normal urinary acidification associated with the complete loss-of-function exhibited by AE1 mutations linked to dominant spherocytosis. We report here novel AE1 mutations linked to a recessive syndrome of dRTA and hemolytic anemia in which red cell anion transport is normal. Both affected individuals were triply homozygous for two benign mutations M31T and K56E and for the loss-of-function mutation, G701D. AE1 G701D loss-of-function was accompanied by impaired trafficking to the Xenopus oocyte surface. Coexpression with AE1 G701D of the erythroid AE1 chaperonin, glycophorin A, rescued both AE1-mediated Cl- transport and AE1 surface expression in oocytes. The genetic and functional data both suggest that the homozygous AE1 G701D mutation causes recessively transmitted dRTA in this kindred with apparently normal erythroid anion transport.

A 5-year-old male patient with X-linked alpha-thalassemia/mental retardation syndrome is reported. He showed multiple minor anomalies including characteristic facial abnormalities, alpha-thalassemia, severe mental retardation, and hypogonadism. Analysis of his hemoglobin by high performance liquid chromatography using an automated glycated hemoglobin analyzer revealed an abnormal peak. Identification of an abnormal peak by an automated glycated hemoglobin analyzer will aid in the diagnosis of patients with X-linked alpha-thalassemia/mental retardation syndrome.

(1997). β-Thalassemia Mutations in Japanese and Koreans. Hemoglobin: Vol. 21, No. 2, pp. 191-200.

Band 3 is the major membrane protein of erythrocytes, which binds membrane skeletal proteins, glycolytic enzymes, and hemoglobin and transports various kinds of anions. Band 3-Memphis is a variant of Band 3, the amino terminal fragment of which depicts a slow electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel. The frequency of Band 3-Memphis varies among populations, with a higher frequency among the Japanese. We investigated the frequency of Band 3-Memphis in a western Japanese island which is relatively isolated from the main islands, finding that the frequency of Band 3-Memphis of the inhabitants of this island is significantly higher than the frequency of the Japanese based on the survey in Kyushu Island. This indicates that there may be differences of population in the frequency of Band 3-Memphis in Japan and that Band 3-Memphis may be a good marker to genetically differentiate each population.

We investigated a Japanese patient with protein 4.2 deficiency. SDS-PAGE showed a complete deficiency of protein 4.2, while Western blot analysis revealed a marked decrease in the amount of protein 4.2, and the existence of a doublet of 74 and 72 kDa bands. Direct sequencing and dot-blot hybridization with allele-specific oligonucleotide probes indicated that the proband was compound heterozygous for a missense mutation in codon 142 with Ala-->Thr (GCT-->ACT) and a single nucleotide substitution (G-->A) of the first base of intron 6 (G-->A) of the protein 4.2 gene. The former is the commonest mutation observed in cases of protein 4.2 deficiency, whereas the latter is a novel mutation, located within the consensus sequence of the 5' splicing site (AGGU) (Protein 4.2Notame). RT-PCR analysis using total RNA isolated from reticulocytes of the proband revealed that the intron 6 donor site mutation causes an abnormal splicing; exon 6 is spliced out with intron 6. The abnormal mRNA has a premature termination codon, as the result of a frameshift, and this instability may lead to degradation. Thus, there is a close relation between this mutation and the molecular pathogenesis of protein 4.2 deficiency.

The ability to recognize the acquisition of multidrug resistance (MDR) in leukemia patients would improve our ability to predict the responsiveness of patients to chemotherapy. To quantitate the degree of MDR acquisition, we determined the amount of mdr-1 mRNA in leukemic cells from patients by competitive polymerase chain reaction (PCR) analysis. Twenty-one patients including 12 patients prior to treatment and nine relapsed patients with acute myelogenous or lymphoblastic leukemia were examined. The amount of mdr-1 gene expression in K562, K562/ADR500 cells and their mixtures showed a proportional correlation between the ratio of resistant to non-resistant cells and the amount of the mdr-1 gene. Mean mdr-1 gene expression in relapsed patients was greater than that in pretreatment patients. Patients refractory to chemotherapy (NR) showed higher levels of mdr-1 gene expression than the patients who achieved complete remission (CR). Because of the wide variations in values, no statistical differences were observed between pretreatment and relapsed patients, or CR and NR patients. These results suggest that the competitive PCR technique is a reliable method to quantitatively determine mdr-1 gene expression, but it may be difficult to predict responsiveness to chemotherapy by using this technique alone.

We present a patient who developed severe anemia and neutropenia after receiving parenteral nutrition for 2.5 years. The serum levels of copper and ceruloplasmin were low, and the bone marrow showed the presence of ringed sideroblasts and vacuolated immature cells. The administration of copper chloride by bolus injection led to a rapid improvement in anemia and neutropenia. The number of progenitor cells (colony-forming unit-granulocyte-macrophage and erythrocyte) present before the copper supplementation was well preserved. It is therefore suggested that copper enzymes play an important role in the maturation of hematopoietic cells.

We report the first successful use of BMT for the treatment of RBC pyruvate kinase (PK) deficiency in a boy who developed neonatal jaundice and severe transfusion-dependent hemolytic anemia a few months after birth. He received a BMT at the age of 5 from an HLA-identical sister who has normal PK activity after conditioning with busulfan and cyclophosphamide. The post-transplant course was uneventful. At present, 3 years after transplant, he is 8 years old and has a normal hemoglobin level and normal RBC PK activity without evidence of hemolysis. DNA analysis has confirmed full engraftment.

Development of resistance to cytotoxic agents is a common problem in the treatment of acute leukemia. In cell lines having multidrug resistance (MDR) phenotype, a decrease in the intracellular accumulation of drugs has been closely related to the overexpression of P-glycoprotein/mdrl genes. We analyzed the relationship between the cytotoxicity of adriamycin (ADR) in vitro, intracellular accumulation of ADR, and the expression of P-glycoprotein on fresh leukemic cells from 19 patients at their initial presentation and from 9 relapsed patients. Pretreatment patients showed significantly higher ratio of complete remission than relapsed patients, and mean value of IC50 for adriamycin in initial presentation was higher than at relapse. But we found no significant relationship between in vitro cytotoxicity and drug transport. In addition, only 2 of the 5 relapsed patients examined by monoclonal antibody C219 expressed the P-glycoprotein. These results suggest that the acquisition of clinical drug resistance may involve various mechanisms other than the reduction of drug accumulation with P-glycoprotein expression.

The development of alloantibodies to platelets is a major problem in the supportive management of thrombocytopenia in patients with severe aplastic anemia. We report here a case of aplastic anemia refractory to platelet transfusion. An immunosuppressant, cyclosporin A, which was used for the therapy of aplastic anemia, modulated alloimmunization to platelets in this patient, followed by repeated platelet transfusion. The treatment reduced platelet alloantibodies detected by anti-human immunoglobulin lymphocytotoxicity test, with change of the CD4/CD8 ratio in T lymphocytes in peripheral blood. These results suggest the usefulness of cyclosporin A for the prevention of platelet alloimmunization.

Platelet membrane glycoproteins were analyzed in a case of myelodysplastic syndrome with inv(3) (q21q26) presenting with prominent dysmegakaryopoiesis by three different labelling techniques for surface proteins. Markedly decreased level of platelet membrane glycoprotein GPIIb was observed in the patient's platelets by terminal sialic acid labelling method, whereas no significant changes in the levels of glycoproteins including GPIIb could be detected either by penultimate galactose labelling or by tyrosine/histidine labelling. These results indicate a decreased sialylation of GPIIb in the patient's platelets, implying aberrant process in thrombopoiesis in the disease.

Elliptocytosis is reported to occur in at least 1 per 5000 individuals, but most cases are heterozygous and do not show clinical hemolysis. Healthy individuals with silent elliptocytosis, however, may suddenly have an episode of hemolysis [1]. Here we report a woman in the third trimester of pregnancy who suffered from cytomegalovirus (CMV) infection with hemolysis. Scanning electron microscopy showed that half of her red blood cells were oval, and protein analysis revealed a 50% reduction of protein 4.1. We discuss the role of CMV infection and pregnancy in the onset of hemolysis in a patient with otherwise silent elliptocytosis.

A rare hemoglobin variant, Hb J Lome , was identified by chance in a male patient with diabetes mellitus (DM). The patient had no evidence of anemia or hemolysis. However, when his glycated hemoglobin (Hb A 1c ) was examined by high-performance liquid chromatography (HPLC) to assess the state of his DM, an abnormal Hb was unexpectedly detected on the chromatogram. The morphology of the red blood cells was normal. A fast-moving band as well as a normally moving Hb band, of roughly equal intensities, were observed by cellulose acetate membrane electrophoresis. The oxygen equilibrium curve was essentially normal (P 50 = 3.59 kPa). In other words, the ability of the patient's Hb to carry oxygen was nearly the same as that of typical Hb A. The stability of his Hb in isopropanol was normal, and all the functions of his Hb that were tested were essentially normal. The identity of the abnormal Hb was finally determined, by sequencing the globin gene, to be Hb J Lome , which is produced by a point mutation changing AAG to AAC at the 59th codon in exon 2 of the Hb beta chain. As previously reported, replacing the beta 59 lysine with asparagine does not affect the function of Hb or the red blood cells. There have been only five documented cases of Hb J Lome in Japan. Interestingly, all these cases are from Kyushu Island. When an abnormal chromatogram for Hb A 1c is unexpectedly obtained, it is worthwhile searching for an abnormal Hb, even if there are no signs that suggest its existence, such as anemia, hemolysis, erythrocytosis, or cyanosis.

The transport rates of phosphoenolpyruvate in erythrocytes from healthy individuals and from patients with hereditary spherocytosis were examined by incubating the cells with 10 mM phosphoenolpyruvate at 37 degrees C in the citrate buffer (0.10 M Na-citrate, 10 mM NaF, 5 mM glucose; pH 6.1 at 37 degrees C). The transport rate of phosphoenolpyruvate in erythrocytes of hereditary spherocytosis was 0.09 +/- 0.02 mumol/ml cells/min (mean +/- S. D., n = 7), whereas that in normal cells was 0.23 +/- 0.03 mumol/ml cells/min (mean +/- S. D., n = 7). The decreased rate of the transport seemed to be specific to the erythrocytes from patients with hereditary spherocytosis, since the rates in erythrocytes from patients with autoimmune hemolytic anemia, paroxysmal nocturnal hemoglobinuria, iron deficiency anemia and aplastic anemia were almost within normal range. Therefore, the decreased transport rate of phosphoenolpyruvate in erythrocytes of hereditary spherocytosis may be a reflection of some specific abnormality in the erythrocyte membrane. Moreover, the measurement of the transport rate of phosphoenolpyruvate may be used as a new diagnostic method for hereditary spherocytosis.