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Case 1 presented with severe anemia and received an intrauterine blood cell transfusion at 33 weeks of gestation. The anemia spontaneously improved in early infancy. Case 2, the father of Case 1, had an uneventful birth with no evidence of anemia, though microcytic anemia was observed during childhood. The genetic analysis of the ß-globin gene cluster identified a novel heterozygous deletion of DNA extending from the Gγ-globin gene downstream to the ß-globin gene, confirming a diagnosis of (G γA γδß)0 -thalassemia. In cases where thalassemia is suspected based on blood tests, a genetic diagnosis should be performed for the sake of the offspring.

A Case of Hb Aalborg (HBB: c.223G>C) with Chronic Obstructive Pulmonary Disease: A First Familial Presentation in Japan.

The proband was a male in his seventies who came to our facility because of shortness of breath. He was not anemic but presented dissociation between oxygen saturation (SpO2) and partial pressure of oxygen (PaO2) by blood gas analysis, and also demonstrated hemoglobinopathy after measurement of Hb A1c using high performance liquid chromatography (HPLC). Twenty-three percent of unknown hemoglobin (Hb) bands were detected. After sequencing the ß-globin gene, we noted a missense mutation at codon 74 (GGC>CGC) (Gly→Arg) of the ß-globin chain and he was diagnosed with Hb Aalborg (HBB: c.223G>C). One of the proband's siblings was diagnosed to have a low SpO2 level and also diagnosed to carry Hb Aalborg; she was also mildly anemic. This is the first known familial case of Hb Aalborg in Japan. In addition to Hb Aalborg, our case had underlying chronic obstructive pulmonary disease (COPD). Herein we present this case as a rare addition to the hematological literature.

A Japanese Family with the Unstable Hb Sydney (HBB: c.203T>C) Variant and Persistent Low Hemoglobin Oxygen Saturation.

Patients with unstable hemoglobin (Hb), caused by a qualitative abnormality in α- and ß-globin genes, are often asymptomatic or mildly symptomatic. It is often difficult to diagnose unstable Hb patients with only mild hemolysis or low oxygen saturation. We herein report a case of a family with an unstable Hb, specifically, Hb Sydney (HBB: c.203T>C), an abnormal ß-globin chain. A 5-year-old boy was referred to our hospital for low percutaneous oxygen saturation (SpO2) in the setting of bronchitis. During hospitalization, low SpO2 persisted despite the improvement in respiratory distress symptoms. As he had mild hemolysis and splenomegaly, his disease was diagnosed to carry Hb Sydney based on gene analysis. His mother and brother also carried Hb Sydney. In this case, bronchial asthma had been treated, but unstable Hb was not assessed. Low SpO2 may be tolerated and overlooked in cases of asthma and it took time to diagnose this patient. The present case suggests that unstable Hb should be considered in patients with bronchial asthma and prolonged low SpO2.

A Coincidental Discovery of a New Stable Variant (Hb Hachioji or HBB: c.187C>T) in a Patient with Chronic Hemolytic Anemia of Unexplained Origin.

We report a new hemoglobin (Hb) variant, Hb Hachioji (HBB: c.187C>T), which was detected in a 32-year-old male with hemolytic anemia. The proband had undergone splenectomy in his childhood after being diagnosed with hereditary spherocytosis (HS) with no clinical improvement. A recent study showed that Heinz bodies were frequently observed in his red cells, however, no abnormal band was separated by isoelectric focusing (IEF), and the isopropanol (instability) test was negative. Direct sequencing revealed that the proband was a heterozygous carrier of a novel mutation (GCT>GTT) at codon 62 of the ß-globin gene, leading to an alanine to valine substitution. This variant was named Hb Hachioji. Characterization at the mRNA level by cDNA sequencing detected ßHachioji mRNA, as well as ßA mRNA. Subsequently, study of the proband's family indicated that his father was a carrier of this Hb variant, although unexpectedly, the father was asymptomatic and clinically healthy. Oxygen affinity measurement of total Hb showed no alteration in the P50 and oxygen equilibrium curve. The presence of Hb Hachioji was confirmed by mass spectrometry (MS). Hb Hachioji comprised approximately 50.0% of the total Hb and was a stable variant. The phenotypic discrepancy between these two carriers suggests that Hb Hachioji may not be associated with the hemolytic involvement in the proband. P4.2Nippon, which is the primary cause of most cases of Japanese HS, was absent in the proband's parents. The coexistence of glucose-6-phosphate dehydrogenase (G6PD) deficiency was ruled out. Thus, the cause of hemolytic involvement in this patient remains unclear.

A new Krüppel-like factor 1 mutation (c.947G > A or p.C316Y) in humans causes ß-thalassemia minor.

Here we describe a Japanese patient with mild ß-thalassemia (ß-thal) with an intact ß-globin gene but a new missense mutation of c.947G > A or p.C316Y in the erythroid Krüppel-Like Factor (KLF1) gene which is strongly associated with the expression of the ß-globin gene. The association of the KLF1 mutation with ß-thal, is here described. The p.C316Y mutation occurred at one of the cysteines that constitute the second zinc finger motif of KLF1, and would have changed the zinc finger conformation to impair the DNA binding properties or the promoter function of the ß-globin gene. Our expression study found that the mutant KLF1 gene had a markedly negative effect on the ß-globin gene expression, or 7.0% of that of its normal counterpart. A presumed heterozygous state, or equimolar presence of the mutant and normal KLF1s reduced the expression rate to 70.0% of the normal alone. This degree of the decrease may explain the very mild phenotype of the patient's ß-thal. Furthermore, the patient's whole-exome analysis using next-generation sequencing revealed that the ß-thal defect is caused by only this KLF1 gene mutation. The Hb A2 and Hb F levels that are frequently elevated in KLF1 mutations were elevated by 4.1 and 1.3%, respectively, in this case. The contribution to their elevation by KLF1: p.C316Y is uncertain.

[Testicular Tumor in a Patient with Delta Thalassemia Complicated with Hereditary Persistence of Fetal Hemoglobin].

A 30s male was diagnosed as having the left testicular tumor in 2010. He received the anti-neoplastic chemotherapy, and could achieve the complete remission. But, he relapsed with solitary retroperitoneal lymph node swelling in 2012, and he was referred to our hospital. Laboratory examination on his admission showed the significant increase of fetal hemoglobin (HbF) up to 16.4%. But, neither anemia nor hemolysis was found at that time. Coexistence of therapy-related myeloid neoplasm or HbF production by metastatic lesion was not definite. Isoelectrofocusing of his hemolysate showed the faint HbA2 in addition to dense HbF band. Molecular analysis of his Hb gene revealed the homozygous (G)gamma-158 (C-T) together with homozygous delta-77(T-C). From these findings, he was diagnosed as having hereditary persistence of HbF (HPFH) and homozygous delta thalassemia. The precise incidence of such combined genetic variation has been unknown because the majority of such cases seem to show no significant clinical symptoms as our case. Whereas, it seems necessary to remind the possibility of such genetic variation when adult patients with various acquired diseases such as testicular tumor or hematologic malignancies show the elevated HbF level.

[Hemoglobinopathies in Japan: characteristics and comparison with those of other ethnic groups].

Abnormalities of hemoglobin (Hb), or hemoglobinopathies, are classified into Hb abnormalities that arise from altered quality induced mainly by amino acid substitution. Furthermore, thalassemia is a quantitative abnormality of normal Hb. Most hemoglobinopathies are inherited disorders. The abnormal Hb known to date comprise more than 210 types in the Japanese population. The rate of thalassemia in Japan is less than that in endemic regions, but the frequencies of ß- and α- thalassemias are 1/1,000 and 1/3,500 in the general population, respectively, so not particularly rare. The mutation spectrum is different from that of endemic regions, probably because Japanese have been historically isolated islanders. Japanese hemoglobinopathy generally has minor symptoms, which are different from those in endemic areas where thalassemia exhibits major clinical manifestations. This might be why useful knowledge is obtained in our laboratory which concentrates on detailed observation of clinical data in addition to genetic analysis. We have, in fact, discovered new clinical characteristics and the significance of hemoglobinopathy, especially of minor or intermediate thalassemia. This approach is quite different from that in other countries coping with only the major type. By focusing on this novel approach, we aim to contribute to improving diagnostic technology for patients.

Oxidation status of ß-thalassemia minor and Hb H disease, and its association with glycerol lysis time (GLT50).

ß-Thalassemia (ß-thal), especially ß-thalassemia major (ß-TM), is reported to be related to reactive oxygen species (ROS) and enhanced oxidation status. It is reflected by increased malondialdehyde (MDA), by membrane lipid peroxidation and decreased by the newly developed total antioxidant capacity (TAC). However, there is less evidence for ß-thal minor and Hb H (ß4) disease on its association with oxidation status. On the other hand, hemolysis by glycerol lysis time (GLT50) is invariably prolonged in thalassemia. The reason for the prolongation of GLT50 is not well understood. The aim of this study was to investigate the oxidation state in ß-thal minor and Hb H disease and to find out the association of the oxidation with the prolongation of GLT50. Blood samples from 39 subjects (33 with ß-thal minor, six with Hb H disease) were collected from individuals living in Japan. The clinical screening tests and molecular identification of the thalassemias were performed. Malondialdehyde and TAC were measured using spectrophotometric analyses. In ß-thal minor and Hb H disease, the plasma MDA level was significantly elevated and the TAC reduced. A highly reversed correlation between MDA and TAC was noted. Their GLT50 levels were evidently prolonged, and the GLT50 has significant correlations with MDA and TAC. ß-Thalassemia minor and mild Hb H disease are evidently in a milieu of reduced redox state, and GLT50 prolongation in ß-thal minor and Hb H disease has a close correlation with the oxidation state, possibly by oxidative impairment of the membrane protein of the red cell.

A new ß(0)-thalassemia mutation (codon 102, AAC>ATCAC) in coexistence with a heterozygous P4.2 Nippon gene.

A new ß-thalassemia (ß-thal) frameshift mutation was found at codon 102 (AAC>ATCAC) in a 17-year-old Japanese male and his 14-year-old sister. Both demonstrated a more severe phenotype than the usual ß-thal minor with mild hemolytic involvement. No mRNA derived from the thalassemic allele, or ß(T)mRNA, was detected in the sequencing analysis of the whole mRNA (cDNA). However, the ß(T)mRNA from the whole ßmRNA was specifically amplified by amplification refractory mutation system (ARMS), and was actually found to be present. Furthermore, quantitative polymerase chain reaction (q-PCR) demonstrated a negligible amount of ß(T)mRNA. Thus, their more severe phenotype was not caused by the "dominant type" ß-thal in which a considerable amount of the ß(T)mRNA would be expected. In fact, our proband had a total ßmRNA level that was mostly normal. Thus, the cause of a ß-thal phenotype by the frameshift mutation was ascribed to the reduced amount of mRNA. We further searched for the cause of their severe phenotype. However, factors that exacerbated the phenotype of ß-thal, such as α-globin gene triplication, coexisting iron deficiency and infection were not found. Finally, we noticed that the red cell morphology revealed ovalocytosis and small numbers of stomatocytes that were seen in the hereditary spherocytosis (HS), especially by P4.2 mutations. The sequence of the P4.2 gene disclosed heterozygous P4.2 Nippon, or missense mutation at codon 142 (GCT>ACT) on exon 3, the most common mutation of Japanese HS. Frequent mutations of other membrane proteins, Band 3 and ankyrin that are common cause of HS in the Japanese population, other than P4.2, were not detected. When HS by P4.2 Nippon develops it is homozygous, and no P4.2 protein is observed in sodium dodecilsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), while in our case the amount of the P4.2 was almost normal in the SDS-PAGE. However, there are several reports that revealed more severe phenotypes of ß-thal by the coexisting abnormality of membrane protein. It is uncertain, but the presence of heterozygous P4.2 Nippon may be associated with the exacerbation of the phenotype of ß-thal minor.

Identification of a novel mutation in the ß-globin gene 3' untranslated region [+1,506 (A>C)] in a Japanese male with a heterozygous ß-thalassemia phenotype.

ß-Thalassemia (ß-thal) is characterized by the absent or reduced production of ß-globin chains. The precise molecular lesion that causes decreased ß-globin synthesis in ß(+)-thal is difficult to predict when mutations occur in the locus control region (LCR), the promoter, the introns or 3' untranslated regions (3'UTRs). Among them, the role of the 3'UTR of ß-globin gene in mRNA stability is poorly understood, mainly due to very few cases that have mutations in this region. So far, only three mutations have been reported in the 3'UTR of ß-globin gene. Although, it is speculated that some of these reported mutations could be associated with mRNA stability, the precise molecular basis still remains unclear. We report here a novel mutation in the ß-globin gene 3'UTR [+1,506 (A>C)] in a 31-year-old Japanese male with hematological parameters suggestive of heterozygous ß-thal. Further functional studies on this novel mutation reported here, may help in understanding of the regulation and expression of the ß-globin gene and its products.

The +1,506 (A>C) mutation in the 3' untranslated region affects ß-globin expression.

The 3' untranslated region (3'UTR) is known to be important to mRNA stability but the stabilization mechanism on the ß-globin gene is not fully elucidated. We speculated in our previous report that +1,506 (A>C) mutation (HGVS nomenclature: *32A>C) on the ß-globin 3'UTR causes ß-thalassemia (ß-thal) in order to destabilize the mRNA. To investigate further, we studied the expression efficiency for the mutation with a luciferase assay. We made recombinant pGL4.74 vectors in which the luciferase 3'UTR was replaced with the wild-type and mutant 3'UTR of the ß-globin gene. For a comparison experiment, recombinant vectors were made not only for this mutation but also six other mutations in the ß-globin 3'UTR which bring about ß-thal or affect mRNA stability. The +1,506 mutation led to a 30.0% lower protein expression than normal in this assay. We concluded that this mutation destabilizes mRNA and consequently decreases the ß-globin amount to finally cause ß-thal. Our study highlights the crucial area of ß-globin 3'UTR for protein expression.

A 25-year observation of a Japanese female patient with Hb Nottingham who has two children with the same disorder.

We undertook a 25-year observation of a female patient with an unstable variant, Hb Nottingham or ß98(FG5)Val→Gly, GTG>GGG. The proband was diagnosed with Hb Nottingham at the age of 9 years. Splenectomy was performed in order to successfully aid her height growth due to chronic anemia at the age of 11, although anemia improvement was transient. She experienced pregnancy/delivery twice, at age 23 and 26, respectively. During both pregnancies, a large number of nucleated red blood cells (NRBCs) appeared in her peripheral blood. No developmental delay of the fetus was noted in either pregnancy, and she gave birth without any maternal complications or perinatal problems. Both babies were diagnosed with Hb Nottingham. To the best of our knowledge, this is the first report of a long-term observation of a proband with Hb Nottingham, including her pregnancy/delivery and the neonatal course of her children with the same disorder.

[Establishment of multiplex hybri-probe method--analysis using the polymorphism of UGT1A1 gene].

There are a number of methods for gene analysis of a point mutation and deletion/insertion of several nucleotides. In 2011, we reported an improved hybridization probe methods (Hybri-Probe method) that are highly sensitive and accurate, and excellent in cost and time effectiveness. Here, we have developed the Multiplex Hybri Probe method for several types of mutations or polymorphisms including the microsatellite polymorphisms, especially of palindromic sequence such as (TA)n and (GC)n. In addition, several mutations are analyzed at a time. In this research we focused on the three types of polymorphism on the Uridine diphosphate glucuronyltransferase (UGT) gene. Design of the probes for the detection of UGT1A1*6(211 G --> A G71R) and UGT1A1*27 (686 C --> A P229Q) was not difficult because the mutations were a single base substitution. However, UGT1A1*28 (A (TA)6TAA --> A (TA)7TAA) has tandem and palindromic sequence. Since the probes to detect the mutation in such sequence resulted in failure, we made several mismatches, i.e., TATATATATATA --> TATGTGTATATA. As a result, the probes designed for the three polymorphisms above did not overlap in the Tm and were separated by approximately 10 degree intervals between 63.2 degrees C and 37.5 degrees C. In this Multiplex Hybri-Probe method, the three kinds of probe are added tube into the PCR product in the same tube and followed by the measurement of the Tm using the LightCycler. Thus, it is very simple, and performed by one step for any mutation including the microsatellite polymorphisms, and it also has good cost performance and favorable time efficiency. It takes two hours including the running time of PCR for completion of the analysis. It may also be available to the detection of other gene abnormalities.

A case of a novel hemoglobin variant, Hb A2-Karatsu, discovered following a falsely elevated HbA1c value.

The patient, a man in his thirties, presented to our hospital for a secondary examination after a 2020 medical check-up found a high hemoglobin A1c (HbA1c) level on high-performance liquid chromatography (HPLC). The HbA1c level determined by HPLC (HA-8180V, fast mode) was elevated at 6.8%, but a 75-g glucose tolerance test showed normal glucose tolerance. The glycated albumin level was within the reference range at 14.6%. The continuous glucose monitoring-derived mean blood glucose and the percentage of time in range were 99 mg/dL and 98%, respectively. The HbA1c levels determined by HPLC (G9, fast mode), enzymatic assay, and immunoassay were all 5.3%. An isoelectric focusing analysis showed an abnormal band on the anode side of HbA2, and a globin gene analysis detected a heterozygous mutation at codon 144 [AAG (Lys) → TAG (stop codon)] in the δ-chain. Since this mutation is a novel δ-chain hemoglobin variant, it was given the name 'Hb A2-Karatsu'.

Internet-based approach to population screening for common hemoglobinopathies in United Arab Emirates.

This article reports on efforts to overcome common hurdles that were faced during population-based screening for common hemoglobinopathies in the United Arab Emirates. An Internet-based approach was designed and implemented to increase the acceptance of the screening program. The process involved: an awareness campaign, a simple bilingual (Arabic/English) online consent form and registration process, the use of a barcode for sample labeling, an equipment upgrade, electronic communication of a successful registration process, test results, and a counseling process. Before the implementation of the Internet-based system, great concern was noted among the clients in terms of the availability of accurate and timely test results, the need for pretest and post-test counseling, and the way that their personal health information was handled. Lapses in information exchange between the clients who participated in the screening program for the carrier state of inherited disorders and the screening laboratory posed significant challenges. The emphasis on confidentiality and the ease of access to services was instrumental in increasing the level of acceptance of these services in our community. Based on an analysis of > 10,000 samples, we conclude that Internet-based reporting holds much promise for improving the quality of care that clients receive.

[Improved hybridization probe method for genetic analysis of beta-thalassemia].

There are a number of methods for genetic diagnosis of a point mutation and/or deletion/insertion of several nucleotides. However, none of these methods are satisfactory with respect to sensitivity, accuracy and cost effectiveness. In 2003, the Hybridization Probe method was first reported, but it was unsatisfactory. We attempted to improve this method by separating the step of using real-time PCR with melting curve analysis. In our improved method, or Hybri Probe method, the sensitivity and accuracy of Tm were remarkably improved by employing H2O and Hi-Di Formamide. In order to detect several beta-thalassemia mutations, relevant probes of basically wild sequence were prepared, and optimal conditions for detecting mutations were studied. This method yielded highly sensitive and accurate results. This Hybri Probe method is available for SNPs and microsatellite polymorphisms, and it may also be useful for the detection of gene abnormalities other than those in beta-thalassemia.

A Newly Characterized Hemoglobin Variant with a High Oxygen Affinity, Hb Fuchu-II, Presenting with Acute Myocardial Infarction.

A 65-year-old Japanese man presented with acute myocardial infarction (AMI) and polycythemia. Biochemical studies of the patient's hemoglobin (Hb) and the sequencing of his globin genes revealed that the polycythemia was secondary to a high oxygen affinity Hb variant, Hb Fuchu-II. Hb variants with high oxygen affinity can be an additional thrombotic risk factor in older patients and/or those with other risk factors. The patient was diagnosed with hemoglobinopathy after the development of AMI and exemplifies the importance of recognizing such conditions and of taking appropriate prophylactic interventions.

Nucleotide sequence of complementary DNA encoding for quaking protein of cow, horse and pig.

Complementary DNA (cDNA) for bovine quaking gene (Bqk), equine quaking gene (Eqk) and porcine quaking gene (Pqk), which are homologous to mouse quaking gene (qkI), were isolated, and their nucleotide sequences were determined. cDNA sequences of Bqk, Eqk and Pqk showed very high homology to that of qkI at nucleotide level; 94.2, 95.7 and 95.6%, respectively. Deduced amino acid sequences for Bqk, Eqk and Pqk perfectly matched to that of qkI. These findings suggest that the quaking gene family is highly conserved during mammalian evolution, and that Bqk, Eqk and Pqk are likely to have important biological functions also in cow, horse and pig.