[Simple and rapid analysis of beta-thalassemia mutation by sequence-specific amplification].

This study was done to detect and diagnose beta-thalassemia (beta-Thal) gene quickly. We applied sequence specific Amplification (SSA) method to the analysis. 13 kinds of beta-Thal and two kinds of hemoglobin variants were able to detect under the same PCR condition. These mutations were found frequently in ten countries of Asian region (the southern part of China, Vietnam, Cambodia, Thailand, Myanmar, Malaysia, Singapore, Indonesia, Pakistan, India), and 15 kinds in total (-28CapA-->G, CD5-CT, CD8/9+-G, CD15G-->A, CD17A-->T, IVSI-1G-->T, CD41/42-4del, CD16-C, CD26G-->A(betaE), IVSI-5G-->C, CD35C-->A, CD71/72 +A, CD6A-->T (betaS), -619del, IVSII-654C-->T). More than 80% of patients are included in these mutations. To make the reagents a kit, the procedure became simple and rapid. DNA was extracted by salting out method. The PCR product was detected by polyacrylamide gel electrophoresis and silver staining. The confirmation of the variant was done by the PCR-direct sequencing method. It took approximately six hours for PCR reaction, electrophoresis and staining. This method made us to detect and diagnose beta-Thal in one day.

[Case of deletion type beta(0)-thalassemia found in an Asian (Malaysian) family living in Japan].

Hb and gene analyses of a Malaysian mother and her two daughters with microcytic anemia living in Japan were performed. Hb analyses of their hemolysates by IEF and DEAE-HPLC revealed high values of Hb A2 and HbF, but abnormal Hbs such as Hb E and Hb Constant Spring, which cause beta- and alpha-thalassemia traits, were not detected. From these data, they were suspected to be beta-thalassemia carriers. The thalassemic mutations commonly found in the Asian area by ARMS and nucleotide sequencing methods were not detected, and the frameworks of the beta-globin gene and the haplotypes of the beta-like globin gene cluster between the mother and daughters were not identical. These results led us to conclude that there was a beta(0)-thalassemia mutation with a large deletion from the beta-globin gene beyond the 3'beta/BamHI polymorphic site 3' downstream to the beta-globin gene. However, the range of the deletion from the beta-like globin gene cluster has not yet been completed in detail. Recently, there have been many foreigners mainly from Asian countries in Japan. We may encounter people with the rare type thalassemic mutation described in the text besides the mutations frequently found in Asian countries.

Hb Constant Spring [alpha 142, Term-->Gln (TAA>CAA in alpha2)] in the alpha-thalassemia of anemic patients in Myanmar.

Hb Constant Spring (Hb CS), the gene (alpha(CS)) of which arises from a point mutation in the termination codon of the alpha2-globin gene, is the most prevalent variety of nondeletional alpha-thalassemia (alpha-thal) in Asian populations. It is a major cause of Hb H disease in compound heterozygotes who have Hb CS combined with a duplicated alpha gene deletion (--/alpha(CS)alpha), and it tends to be more severe than Hb H disease which is caused by a triple alpha gene deletion (--/-alpha). Hb CS is often missed by routine electrophoresis but not by polymerase chain reaction (PCR) methods. During alpha-thal screening and genotyping of 235 patients diagnosed by laboratory tests hemoglobin (Hb), MCV, MCH and Hb H inclusion bodies] using the gap-PCR method, 175 patients were diagnosed to be carriers of an alpha-thal gene, genotypes of which were 133 alpha-thal-2, 34 alpha-thal-1 (including one only by laboratory test) and eight with Hb H disease. Detection of the alpha(CS) gene for the carriers of alpha-thal-1 and Hb H disease was done by the mismatched PCR-RFLP (restriction fragment length polymorphism) method and the alpha(CS) gene was found in the homozygous state in an alpha-thal-1 patient and a single gene form in two Hb H disease patients. These genotypes were characterized by the PCR-sequencing method. These patients clinically presented the aspects of Hb H disease and of a homozygote form of alpha-thal-1. The description of the alpha(CS) gene in Myanmar is of great value in the development of an effective procedure for prenatal diagnosis of Hb Bart's hydrops fetalis syndrome.

High incidence of 3-thalassemia, hemoglobin E, and glucose-6-phosphate dehydrogenase deficiency in populations of malaria-endemic southern Shan State, Myanmar.

Samples from 916 members of various ethnic groups from malaria-endemic southern Shan State, Myanmar, were analyzed for 3-thalassemia (3-thal), 3-thalassemia (3-thal), abnormal hemoglobin variants, and glucose-6-phosphate dehydrogenase (G6PD) deficiency. Of these subjects, 530 (57.9%) were found to have at least one of these red cell genetic disorders. The overall frequencies for the various red cell genetic disorders were as follows: 3-thal, 37.5% (343/916); hemoglobin E (Hb-E), 20.3% (186/916); G6PD-Mahidol, 17.5% (160/916); and 3-thal, 0.3% (3/916). The frequencies of combined disorders were 6.9% (63/ 916) for 3-thal/Hb-E, 5.7% (52/916) for 3-thal/G6PD-Mahidol, 2.8% (26/916) for Hb-E/G6PD-Mahidol, 1.1% (10/916) for 3-thal/Hb-E/G6PD-Mahidol, and 0.1% (1/916) for 3-thal/3-thal/G6PD-Mahidol. Of the various ethnic and non-ethnic groups, the Bamar population showed the highest frequencies of 3-thal (56.9%, 177/311), Hb-E (28.3%, 88/311), and G6PD-Mahidol (21.2%, 66/311) (all duplicated and triplicated cases were included). In addition, 2 new mutations, an 3 gene triplication (/333(anti3.7); 0.2%, 2/916) and Hb-Neapolis (0.1%, 1/916), were detected. Our results showed that race was the dominant factor affecting the frequencies of red cell genetic disorders in malaria-endemic areas of Myanmar.

Hb Nishinomiya [Leu-Gly-inserted between codons 69(E13) and 70(E14) of beta]: a novel unstable hemoglobin with reduced oxygen affinity found in a patient with spherocytic hemolysis.

Substantially decreased oxygen saturation levels were incidentally detected by pulseoxymetry in a patient with spherocytic hemolysis who was undergoing laparoscopic splenectomy. Molecular analysis revealed that he was carrying hemoglobin (Hb) Nishinomiya, a novel Hb variant [Leu-Gly-inserted between codons 69(E13) and 70(E14) of beta]. Amino acid substitutions around positions 70-73(E13-17) of the beta chain are likely to change stability and oxygen affinity, as has been demonstrated in several Hb variants including Hb Seattle. The apparent substitution of the amino acid residues in the heme pocket of the beta chain explains the decreased stability and oxygen affinity of Hb Nishinomiya.

Two children with thalassemia identified during screening for anemia in junior high school.

We present two Japanese students with thalassemia identified during screening for anemia in their junior high school. Blood test results revealed marked hypochromic and microcytic erythrocytosis in one patient and microcytic anemia in the other. Both cases showed a mean corpuscular volume/red blood cell (MCV/RBC) ratio less than 13. Their beta/alpha synthesis ratio was elevated. Deletion of psialpha2, psialpha1, alpha2, alpha1 and theta1 genes in the alpha-globin gene clusters were noted in the first case. This pattern of gene deletion was consistent with heterozygous alpha-thalassemia 1 of the Southeast Asian type. On the other hand, an increased hemoglobin A2 level and reduced beta/alpha synthesis ratio were found in the second case. Direct cloning and DNA sequencing identified a point mutation (guanine to adenine) at position 1 of intervening sequence II in the beta-globin gene (IVS II-1 G-->A). These results suggest that this patient had heterozygous beta0-thalassemia. Diagnosis of thalassemia should be confirmed by molecular analysis in cases with microcytic anemia or hypochromic microcytosis with a MCV/RBC ratio of 13 or less.

A case of hemoglobin Hiroshima (ß146 histidine to aspartic acid) with compensatory erythremia and undetectable HbA1c.

Hemoglobin (Hb) Hiroshima is an Hb variant that travels rapidly on electrophoresis and shows a fourfold increase in oxygen affinity and a decreased Bohr effect. We encountered a 40-year-old male patient with erythremia and an undetectable HbA(1c) level. The presence of an abnormal hemoglobin molecule was suggested by the results of high-performance liquid chromatography analysis. Subsequent gene analysis by direct sequencing confirmed Hb Hiroshima (ß146 histidine → aspartic acid). Caution should be exercised when diagnosing erythremia.

A 6-year-old girl with hemoglobin H disease.

Hemoglobin H (HbH) disease is the severe nonfatal form of α-thalassemia syndrome. It is usually caused by molecular defects of 3 of 4 α-globin genes (--/-α) which cause α-globin expression to be decreased. HbH disease is rare in Japan. Here, we report on a 6-year-old girl with HbH disease who had profound hypochromatic and microcytic anemia. Analysis of the α-globin genes of the patient's family showed that the father, who was Japanese, had an abnormal gene with a 3.7-kb deletion (-α(3.7)/αα), and the mother, who was Filipino, had a deletion removing both α-globin genes of the Filipino type (--(FIL)/αα). Neither parent had anemia. The patient was found to have HbH disease with a heterozygous genetic abnormality (--(FIL)/-α(3.7)). Recently, the number of marriages of Japanese to natives of areas where thalassemia is epidemic has increased. Therefore, the incidence of HbH disease can be expected to increase in Japan. Long-term follow-up will be needed to evaluate the long-term complications and to improve the quality of life of patients with HbH disease.

Method-dependent HbA1c values in a family with hemoglobin Himeji.

BACKGROUND: Hb Himeji is variant hemoglobin associated with increased glycation in a mutated ß chain. We measured HbA1c using various methods in a family with Hb Himeji. METHODS: The proband was a 42-y female. While receiving treatment for Graves' disease, an oral glucose tolerance test showed normal glucose tolerance, but HbA1c by enzymatic assay was abnormally elevated (11.6%). Hemoglobin gene analysis identified Hb Himeji [ß140 (H18) Ala→Asp]. RESULTS: HbA1c values measured by high-performance liquid chromatography (HPLC; HLC-723G8 and HA-8160 instruments), immunoassay, enzymatic assay, affinity method, and electrospray ionization/mass spectrometry were 3.2%, 5.2%, 11.5%, 9.7%, 7.2%, and 9.6%, respectively. Glycation product of the variant hemoglobin measured by HPLC, using HLC-723G8 and HA-8160, was 9.1% and 4.5%, respectively. The proband's father with type 2 diabetes was the first reported case of Hb Himeji. HbA1c by affinity method was markedly elevated (18.0%), but it was 5.3% by HPLC. The proband's two sisters also had Hb Himeji variant and similar method-dependent discrepancies in HbA1c values were observed. CONCLUSIONS: In the patients with Hb Himeji, discrepancies occur between plasma glucose and HbA1c with all measurement methods because of differences in HPLC mobility, increased glycation, and antigenic changes of the variant ß chain.

Hemoglobin variant HbE found in two South Asian diabetic patients.

Glycohemoglobin, also known as hemoglobin (Hb) A(1c), is a marker of long-term glycemic control in patients with diabetes. We present two South-Asian diabetic patients whose HbA(1c) peaks were not measurable using high performance liquid chromatography (HPLC). Further investigations showed that these patients were homozygous for a hemoglobin variant, HbE (beta26 Glu-->Lys). Because of the increasing numbers of immigrants in Japan, area-specific hemoglobinopathies are now encountered more frequently than before. Thus, if discrepant results are found on an HbA(1c) assay or if the HbA(1c) value cannot be measured, such patients should be screened for hemoglobinopathies and alternative measurements for monitoring diabetes should be considered.

Multiple cytokines are involved in the early events leading to the Alzheimer's disease pathology.

It is likely that neuroinflammation begins well before detectable cognitive impairment in Alzheimer's disease (AD) occurs. Clarifying the alterations occurring prior to the clinical manifestation of overt AD dementia may provide valuable insight into the early diagnosis and management of AD. Herein, to address the issue that neuroinflammation precedes development of AD pathology, we analyzed cytokine expression profiles of the brain, with focus on non-demented control patients with increasing AD pathology, referred to as high pathology control (HPC) cases, who provide an intermediate subset between AD and normal control cases referred to as low pathology control (LPC) cases. With a semi-quantitative analysis of cytokine mRNA, among 15 cytokines and their related molecules tested, we found the involvement of eight: interleukin-1(IL-1) receptor antagonist (IL-1ra), IL-1 converting enzyme (ICE), IL-2, IL-6, IL-8, tumor necrosis factor (TNF) α, macrophage-colony stimulating factor (M-CSF) and transforming growth factor (TGF) ß1 during the development from LPC to HPC, while decreases in IL-1ra, IL-8, MCP-1 and TNFα, and an increase in TACE were implicated in the later development from HPC to AD. These findings indicate that neuroinflammation precedes the clinical manifestation of overt dementia, rather than being involved at the later stages of AD.

Hb Sallanches [alpha104(G11)Cys-->Tyr, TGC-->TAC (alpha2)]: an unstable hemoglobin variant found in an Indian child.

We report the fourth observation of Hb Sallanches [alpha104(G11)Cys-->Tyr, TGC-->TAC (alpha2)], an unstable alpha chain variant of intermediate severity in the homozygous state. Heterozygosity occasionally produces mild hypochromia and microcytosis in some patients. A balanced beta/alpha ratio, found in previously reported cases, points to unstable alphabeta dimers formed as a result of the Cys-->Tyr substitution at the alpha1beta1 contact site in this hemoglobin (Hb) variant. Our patient, and the previous two of the three cases reported in patients of Pakistani origin, points to a common population stock, separated by the mass population migration which occurred during the partition of Pakistan and India in 1947.

Common alpha-thalassemia deletions in transfusion-dependent thalassemia patients in the Southeast Asia region of Myanmar.

Screening of 3 common alpha-thalassemia (thal) deletions (-alpha3.7, -alpha4.2 and --SEA) in Southeast Asia was done by polymerase chain reaction in 170 unrelated Myanmar thal patients receiving transfusions. Thal deletions were detected in 27 patients (15.9%) as: (1) alpha-thal-2 (-alpha3.7/alphaalpha) in 12 heterozygous or hemoglobin (Hb) E-beta-thal cases; (2) alpha-thal-1 in 7 patients (2-alpha3.7/-alpha3.7 and 5 --SEA/alphaalpha); and (3) Hb H (-alpha3.7/--SEA) in 8 patients. The latter 15 alpha-thal-1 and Hb H patients had no beta-thal mutations and represented 8.8% of the overall patients seeking transfusion for refractory anemia in Myanmar. This is the first description of alpha-thal in Myanmar from the molecular aspect, and its clinical and racial heterogeneity are described and discussed.

A case of alpha-thalassemia-2 associated with pulmonary infarction.

Pulmonary infarction is an entity of medical significance that develops concurrently in beta-thalassemia but not in alpha-thalassemia. The reason for this difference is yet to be elucidated. We have evaluated a 21-year-old male alpha-thalassemia-2 patient who had profound microcytic anemia and pulmonary infarction. Analysis of the alpha-globin gene revealed -alpha3.7/alpha alpha genotype. His mother also had the same heterozygous gene deletion, though she had neither anemia nor pulmonary infarction. Since the patient had no other predisposition to pulmonary infarction, it is suggested that there is a close etiologic relationship between morphologic abnormality of the erythrocytes caused by alpha-thalassemia-2 and development of pulmonary infarction.

A wider molecular spectrum of beta-thalassaemia in Myanmar.

Two hundred and nine beta-thalassaemia (beta-Thal) alleles of 158 unrelated Myanmar patients (107 HbE-beta-Thal; 51 beta-Thal major) were analysed for beta-globin gene mutations. Amplification refractory mutation system (ARMS) characterized six beta-thal mutations known to Myanmar [betaIVSI-1(G-->T), codon 41/42(-TCTT), betaIVSI-5(G-->C), codon 17(A-->T), betaIVS II-654(C-->T), and -28 Cap (A-->G)] in 166/209 (79.4%) alleles. DNA sequencing of 24 alleles from 43 ARMS-negative samples (20.6%) identified an additional 12 new mutations, to produce a total of 18 different mutations. Nineteen alleles (9.1%) remained for further characterization. The molecular spectrum of Myanmar beta-Thal is wider and more heterogeneous than previously reported.