Diagnostic challenges posed by a rare alpha globin chain variant Hb Fontainebleau in a pregnant female and its potential effects in her children in view of multiple globin gene defects in her husband

Alpha globin chain variants per se do not cause severe morbidity and mortality but can modify – usually ameliorate – the clinical manifestations of beta globin chain variants when co-inherited with the latter. They also pose challenges in interpretation of high-performance liquid chromatography histograms and require molecular analysis for proper characterization. Hemoglobin (Hb) Fontainebleau is a rare alpha globin chain variant [alpha 21(B2) Ala?Pro], of which only three families have been reported from India in the past. Here, we describe a case of Hb fontainebleau detected in heterozygous condition in a 19-year-old primigravida. Her husband was found to have a double heterozygous state for HbQ India and beta-thalassemia trait. This opens up the possibility of multiple combinations of hemoglobinopathies in the offspring.

Analysis of clinical phenotype of a α2-globin gene mutation -IVS-Ⅱ-55 (T→G) / 临床检验杂志

Objective@#To identify a α-globin gene mutation-IVS-Ⅱ-55 (T→A) and analyze hematological characteristics of IVS-Ⅱ-55 (T→G) carriers. @*Methods@#The peripheral blood samples were collected from the members of five family and three sporadic IVS-Ⅱ-55(T→G) carriers for the analysis of RBC parameters and hemoglobin electrophoresis. Gap-PCR, PCR-RDB (reverse dot blot) and DNA sequencing were carried out for the identification of gene deletion and mutation of α-globin and β-globin. @*Results@#The results of RBC parameters of five infant probands which presented with microcytic hypochromic anemia were below the normal reference interval. One of the adult carriers of IVS-Ⅱ-55 (T→G) heterozygote alone presented with microcytic hypochromic anemia, and the others showed normal RBC parameters. The hematological phenotype index (MCV, MCH and HbA 2 ) of one adult carrying a compound heterozygote for IVS-Ⅱ-55 (T→G) and βCD27-28M/N were 65.0 fL, 20.3 pg and 5.8% respectively. The hematological phenotype index (MCV, MCH, HbA 2 and HbF) of one adult carrying a compound heterozygote for IVS-Ⅱ-55 (T→G) and SEA-HPFH were 81.9 fL, 26.5 pg, 3.0% and 29.0% respectively. The HbA 2 levels of all carriers of IVS-Ⅱ-55 (T→G) heterozygote alone were in normal range. No abnormal hemoglobin band was detectable on hemoglobin electrophoresis for all the carries. @*Conclusion@#The carriers of IVS-Ⅱ-55(T→G) heterozygote alone were asymptomatic. The phenotype of compound heterozygote for β-thalassemia was similar to that of β-thalassemia alone.

Hemoglobin Fontainebleau [a21(B2)Ala>Pro]: The second report from India.

Structural hemoglobin (Hb) variants are mainly due to point mutations in the globin genes resulting in single amino acid substitutions. Until date, about 200 alpha chain variants have been identified and they are usually detected during the hemoglobinopathy screening programs. Under a community control program for hemoglobinopathies, which involved screening of antenatal cases followed by prenatal diagnosis if indicated. Here, we report a rare alpha globin gene variant Hb Fontainebleau [a21(B2)Ala>Pro] detected in the heterozygous condition in a 35‑year‑old pregnant lady screened during this program. This is the second report of this alpha globin variant from India. Unlike the earlier case from India where Hb Fontainebleau was reported in a neonate who was also a carrier of Hb Sickle and had no clinical problems, this case presented with a bad obstetric history associated with the secondary infertility. However, the presence of the variant and the obstetric complications may be unrelated.

Mechanisms of sirolimus inducing expression of γ-globin gene in K562 cells / 中国药学杂志

OBJECTIVE: To investigate the mechanisms of sirolimus inducing γ-globin gene expression in K562 cells. METHODS: K562 cells were cultured in the presence of 10 nmol · L-1 sirolimus, butylate, or DMSO for 3 d. Western blot and real time PCR-based chromatin immunoprecipitation was employed to measure the levels of p38MAPK and acetylated histone H3 (acH3) at γ-globin gene promoter regions, respectively. RESULTS: In K562 cells with 10 nmol · L-1 sirolimus treatment, phospholylated p38MAPK (p-p38MAPK) was 2.8-fold greater and acH3 was 9.8-fold greater than that in untreated K562 cells, and there was a 2.9-fold in p-p38MAPK and a 9.1-fold in acH3 increase comparing with K562 cells treated with DMSO, no significant difference in p-p38MAPK and acH3 level was found between cells treated with sirolimus and with butylate. SB203580 completely abolished induction of p38MAPK activation and acH3 increase by sirolimus. CONCLUSION: Our results indicate that sirolimus actives p38MAPK signal and increases acetylation of H3 at γ-globin gene promoter regions, which may be the mechanisms of induced expression of γ-globin genes by sirolimus in K562 cells.

Detection of β-globin Gene Mutations Among β-thalassaemia Carriers and Patients in Malaysia: Application of Multiplex Amplification Refractory Mutation System– Polymerase Chain Reaction

Background: β-thalassaemia is one of the most common single-gene disorders worldwide. Each ethnic population has its own common mutations, accounting for the majority of cases, with a small number of mutations for the rarer alleles. Due to the heterogeneity of β-thalassaemia and the multi-ethnicity of Malaysians, molecular diagnostics may be expensive and time consuming. Methods: A simple polymerase chain reaction (PCR) approach involving a multiplex amplification refractory mutation system (MARMS) and one amplification refractory mutation system (ARMS), consisting of 20 β-globin gene mutations, were designed and employed to investigate β-thalassaemia patients and carriers. Results: Out of 169 carriers tested with the MARMS, Cd 41/42 (–TTCT), Cd 26 (A–G) HbE, IVS 1–1 (G–T), and IVS 1–5 (G–C) were the most common mutations, accounting for 78.1%. Among the Malays, Cd 26 (A–G) HbE, Cd 41/42 (–TTCT), IVS 1–1 (G–T), and IVS 1–5 (G–C) were the most common mutations, accounting for 81.4%, whereas Cd 41/42 (–TTCT) and IVS 2–654 (C–T) were most common among the Chinese (79.1%). Conclusion: We propose the use of this cheap, easy to interpret, and simple system for the molecular diagnostics of β-thalassaemia among Malaysians at the Institute for Medical Research (IMR)

Comparative evolutionary analyses of beta globin gene in eutherian, dinosaurian and neopterygii taxa.

Background & objectives: Comparative genomics and evolutionary analyses of conserved genes have enabled us to understand the complexity of genomes of closely related species. For example: -globin gene present in human hemoglobin is one such gene that has experienced many genetic changes in many related taxa and produced more than 600 variants. One of the variant, HBS causes sickle-cell anemia in humans but offers protection against severe malaria due to Plasmodium falciparum. In the present study, we characterized and performed evolutionary comparative analyses of the -globin gene in different related and unrelated taxa to have a comprehensive view of its evolution. Methods: DNA and protein sequences of -globin gene were downloaded from NCBI and characterized in detail in nine eutherian (Homo sapiens, Pan troglodytes, Macaca mulatta, Mus musculus, Rattus norvegicus, Bos taurus, Canis familiaris, Equus caballus, Oryctolagus cuniculus), a dinosaurian (Gallus gallus) and a neopterygii (Danio rerio) taxa. Three more eutherian (Papio anubis, Ovis aries and Sus scrofa) taxa were included for an analysis at the protein level but not included at the gene level owing to lack of genomic information. Computational and phylogenetic analyses were performed using evolutionary comparative approach. Results: Results of comparative and phylogenetic analyses revealed less conservation of genetic architecture of -globin compared to its protein architecture in all eutherian taxa. Both dinosaurian and neopterygii taxa served as outgroups and varied at gene and protein levels. Interpretation & conclusion: Most remarkably, all primates from eutherian taxa including P. anubis showed only nine codon position differences and an absolute similarity between H. sapiens and P. troglodytes. Absolute conservation of coding region in Equus caballus (horse) was observed. The results were discussed with an inference on the role of evolutionary forces in maintaining such close similarities and variations across closely related taxa. Further, the need to utilize more comparative approaches in understanding the disease causing genes’ evolution in closely related taxa is hoped for.

Anemia hemolítica causada por hemoglobina evans en una familia argentina / Hemolytic anemia due to hemoglobin evans in an Argentinean family

Las hemoglobinas inestables son variantes estructurales de lamolécula de hemoglobina originadas, en su mayoría, por sustitucionespuntuales de aminoácidos en alguna de las cadenas de globina. Estos cambios afectan la estabilidad de la molécula,causan pérdida de la solubilidad y precipitación dentro del eritrocito, lo cual provoca su destrucción acelerada. Desde el punto de vista clínico, las hemoglobinas inestables puedenpresentar anemia hemolítica crónica de gravedad variable.La hemoglobina Evans es una hemoglobina inestable causadapor la sustitución de valina por metionina en la posición 62 de la cadena de alfa globina. Hemos identificado esta variante en una niña con crisis hemolítica aguda asociada a faringitis y en dos miembros de su grupo familiar. Éste es el tercer caso de anemia hemolítica congénita causada por hemoglobina Evans comunicado en la bibliografía mundial.

Unstable hemoglobins are structural variants of the hemoglobin molecule, mostly originated by single amino-acid replacement in some globin chains. These changes affect molecule stability, leading to loss of solubility, precipitation, and cellular lysis. Patients carrying these unstable hemoglobins may present mild to severe chronic hemolytic anemia. Hemoglobin Evans is an unstable variant originated by replacement of valine with methionine at position 62 of the αa-globin chain. We have identified this variant in a girl with an acute hemolytic crisis associated to pharyngitis, as well as in two of her family members. This is the third case of hemolytic anemia due to hemoglobin Evans reported in the literature.

Familial congenital cyanosis caused by Hb-M Yantai (alfa-76 GAC - TAC, Asp - Tyr)

Methemoglobin (Hb-M) is a rare hemoglobinopathy in China. We hereby report on a family living in Yantai, East China, with congenital cyanosis due to Hb-M mutation. The proband, a 65-year-old female, presented 63 percent oxygen saturation. Both Hb-M concentration and arterial oxygen saturation remained unchanged, even following intravenous treatment with methylene blue. There was also no change in blood-color (chocolate-brown) after adding 0.1 percent KCN. A fast-moving band (Hb-X) in hemolysates was found by cellulose acetate electrophoresis, the Hb-X/Hb-A ratio exceeding 10 percent. GT transition at 131nt of exon 2, although present in one of the alfa2-globin alleles, was not found in alfa1-globin alleles as a whole. This mutation leads to the aspartic acid to tyrosine substitution (Asp76Tyr). In this family, the novel mutation in the alfa2-globin gene resulted in a rare form of congenital cyanosis due to Hb-M. This hemoglobin was named Hb-M Yantai.

Sequence change in the HS2-LCR and Gg-globin gene promoter region of sickle cell anemia patients

The fetal hemoglobin (HbF) levels and betaS-globin gene haplotypes of 125 sickle cell anemia patients from Brazil were investigated. We sequenced the Gg- and Ag-globin gene promoters and the DNase I-2 hypersensitive sites in the locus control regions (HS2-LCR) of patients with HbF level disparities as compared to their ßS haplotypes. Sixty-four (51.2 percent) patients had CAR/Ben genotype; 36 (28.8 percent) Ben/Ben; 18 (14.4 percent) CAR/CAR; 2 (1.6 percent) CAR/Atypical; 2 (1.6 percent) Ben/Cam; 1 (0.8 percent) CAR/Cam; 1 (0.8 percent) CAR/Arab-Indian, and 1 (0.8 percent) Sen/Atypical. The HS2-LCR sequence analyses demonstrated a c.-10.677G>A change in patients with the Ben haplotype and high HbF levels. The Gg gene promoter sequence analyses showed a c.-157T>C substitution shared by all patients, and a c.-222_-225del related to the Cam haplotype. These results identify new polymorphisms in the HS2-LCR and Gg-globin gene promoter. Further studies are required to determine the correlation between HbF synthesis and the clinical profile of sickle cell anemia patients.

Clinical and molecular characteristics of sickle cell anemia in the northeast of Brazil

Beta S-globin gene (âS-globin) haplotypes, markers for severe sickle cell anemia (SCA), and the alpha-thalassemia 2 gene 3.7 kb deletion (-á2(3.7 kb) thal) along with demographic and clinical data were investigated in SCA outpatients (n = 125, 63 female and 62 male) in the Brazilian state of Bahia, which has a high prevalence SCA. PCR-RFLP showed that the Central African Republic/Benin (CAR/BEN, 51.2 percent) haplotype was most frequent, followed by the Benin/Benin (Ben/Ben, 28.8 percent). At least one CAR haplotype was present in every outpatient with a history of cerebrovascular accident. The Cameroon (Cam), Senegal (Sen) and Arab-India haplotypes occurred in small numbers, as did atypical haplotypes. Fetal hemoglobin (HbF, percent) was unevenly distributed. Compared to those > 18 y, those aged < 18 y had had fewer erythrocyte transfusions and high HbF levels (12.3 percent ± 7.01 to 7.9 percent ± 4.36) but a higher frequency of spleen sequestration and pneumonia. Compared with normal á - genes carriers values, the outpatients with -á2(3.7 kb) thal (determined by PCR analysis) had significantly higher mean hemoglobin concentration (Hb) (8.3 ± 1.34 g/dL, p = 0.018) and packed cell volume (PCV = 27.1 percent ± 4.26, p = 0.019) but low mean corpuscular volume (MCV = 86.1 fL = 10-15 L ± 9.56, p = 0.0004) and mean corpuscular hemoglobin (MCH = 26.6 percent ± 4.60, p = 0.039).

Adeno-associated Virus Serotype 2 Mediated Transduction and Expression of The Human β-Globin Gene in Human Early Fetal Liver Hematopoietic Cells / 生物化学与生物物理进展

β-Thalassemia is an inheritance anaemia disease due to the defect in β- globin gene. Gene therapy is considered to be the only method which could cure this disease. Adeno-associated virus type 2 (AAV2) has benn gaining more attention as a vector in human gene therapy for its non pathogenic character and broad host range. Although, the efficacy of recombinant AAV2 (rAAV2) in transducing human hematopoietic stem cells has been investigated by researchers, the results were varied from different laboratory. The view was proposed recently that it may be resulted from helper virus in their packaging system. Respecting this, the packaging system without helper virus was used to produce rAAV2. Human early fetal liver hematopoietic cells not only possess many superior peculiarity compared to hematopoietic cells of bone marrow or cord blood, but also the inherent β-globin gene in the cells is not expressed. Studies on the AAV2 transduction of human fetal liver hematopoietic cells and mediated expression of β- globin gene in vivo were performed and the potential role of AAV2 in β-thalassemia gene therapy was analyzed. The rAAV2 containing a normal human β-globin gene (rAAV2-β-globin) without helper virus contamination were produced. The viral titer, purity and the ability of mediating expression of β-globin gene were detected in vitro. Then, human early fetal liver hematopoietic cells were isolated and were further transducted with the rAAV2-β-globin, followed by transplantation into sublethally irradiated BALB/C nude mice to analyze the β-globin gene expression. The results showed that the high titer and purity of rAAV2-β-globin had the ability of mediating β-globin gene expression in vitro. In 8 recipient BALB/C nude mice, the β-globin gene expression were detected in the 2 mice marrow by RT-PCR. The results suggested that rAAV2 could transduce human fetal liver hematopoietic cells and mediate the β-globin gene expression in BALB/C nude mice, meanwhile the expression level of the gene was still rather low. It is necessary to perform further research on AAV2 biology before applying in β-thalassemia gene therapy.

Amplification of?-globin gene to assess the quality of genitourinary tract samples for PCR detection / 临床检验杂志

Objective To investigate whether amplification of?-globin gene can be used to monitor the quality of samples for PCR detection of DNA of Chlamydia trachomatis(CT).Methods First-void urines(FVU),endocervical swabs(ECS),and urethral swabs (UTS) were collected for PCR detection of CT-DNA and cellular?-globin gene.Samples negative for?-globin gene were retested after 10-fold dilution.Results The positive rates of?-globin gene in FVU and ECS were 95.6%(255/264),and 91.7%(413/450) respectively, both higher than that(77.8%,172/221) in UTS(P

Adeno-associated Virus Serotype 2 Mediated Transduction and Expression of The Human ?-Globin Gene in Human Early Fetal Liver Hematopoietic Cells / 生物化学与生物物理进展

?-Thalassemia is an inheritance anaemia disease due to the defect in?- globin gene. Gene therapy is considered to be the only method which could cure this disease. Adeno-associated virus type 2 (AAV2) has benn gaining more attention as a vector in human gene therapy for its non pathogenic character and broad host range. Although, the efficacy of recombinant AAV2 (rAAV2) in transducing human hematopoietic stem cells has been investigated by researchers, the results were varied from different laboratory. The view was proposed recently that it may be resulted from helper virus in their packaging system. Respecting this, the packaging system without helper virus was used to produce rAAV2. Human early fetal liver hematopoietic cells not only possess many superior peculiarity compared to hematopoietic cells of bone marrow or cord blood, but also the inherent?-globin gene in the cells is not expressed. Studies on the AAV2 transduction of human fetal liver hematopoietic cells and mediated expression of ?- globin gene in vivo were performed and the potential role of AAV2 in?-thalassemia gene therapy was analyzed. The rAAV2 containing a normal human?-globin gene (rAAV2-?-globin) without helper virus contamination were produced. The viral titer, purity and the ability of mediating expression of?-globin gene were detected in vitro. Then, human early fetal liver hematopoietic cells were isolated and were further transducted with the rAAV2-?-globin, followed by transplantation into sublethally irradiated BALB/C nude mice to analyze the?-globin gene expression. The results showed that the high titer and purity of rAAV2-?-globin had the ability of mediating ?-globin gene expression in vitro. In 8 recipient BALB/C nude mice, the ?-globin gene expression were detected in the 2 mice marrow by RT-PCR. The results suggested that rAAV2 could transduce human fetal liver hematopoietic cells and mediate the ?-globin gene expression in BALB/C nude mice, meanwhile the expression level of the gene was still rather low. It is necessary to perform further research on AAV2 biology before applying in ?-thalassemia gene therapy.

Analysis for single nucleotide polymorphisms of beta-globin gene in beta-thalassaemia carriers / 临床检验杂志

Objective To investigate the single nucleotide polymorphisms(SNP) of beta-globin gene in beta-thalassaemia carrier.Methods The target fragment of beta-globin gene in beta-thalassaemia carriers was first amplified using PCR,the single nucleotide polymorphisms were determined by the automatic DNA sequencing.Results Three single nucleotide polymorphisms were found in the amplified fragment of beta-globin gene from beta-thalassaemia carriers.They were respectively the T/C polymorphism at nucleotide 59 in exon 1,the G/C polymorphism at nucleotide -16 in intron 2,and the T/G polymorphism at nucleotide-74 in intron 2.Conclusion There are some differences in the single nucleotide polymorphisms of the beta-globin gene between beta-thalassaemia carriers and normal subjects.The number of single nucleotide polymorphisms among beta-thalassaemia carriers is lesser.The frequency of bases is also different between beta-thalassaemia carriers and normal subjects.

Effect of Sodium Butyrate on Phosphorylation of Histone at ?-Globin Gene Promoter Regions in K562 Cells / 实用儿科临床杂志

Objective To explore the effect of sodium butyrate(NaB) on phosphorylation/ acetylation of histone H3(ph/acH3) at G?-globin gene and A?-globin gene promoter regions in K562 cells.Methods K562 cells were devided into 2 groups:K562 cells were grown in the presence or absence of 0.5 mmol?L-1NaB for 48 h [K562(NaB) group] and untreated K562 cells group(K562 group).Semi-quantitative RT-PCR was employed to measure the levels of G?-globin mRNA and A?-globin mRNA.The real time PCR-based chromatin immunoprecipitation(ChIP) was used to detect the levels of ph/acH3 at G?-globin gene and A?-globin gene promoter regions.Results Compared with the K562 group,there was a 1.4-fold(t=-149.022,P=0.000) and 1.2-fold(t=-13.363,P=0.000) increase in G?-globin mRNA and A?-globin mRNA,respectively,in K562(NaB) group.The level of ph/acH3 at G?-globin gene and A?-globin gene promoter region increased by 2.9-fold(t=-12.833,P=0.006) and 3.2-fold(t=-10.484,P=0.000),respectively,in K562(NaB) group,compared with the K562 group.The %Input value of G?-globin and A?-globin promoter fragment was 10.0-fold(P=0.000) and 9.5-fold(P=0.000) higher than that value of Necdin gene promoter fragment in the K562(NaB) group,while the %Input value of G?-globin and A?-globin promoter fragment was 3.2-fold(P=0.000) and 2.7-fold(P=0.000) higher than that value of necdin gene promoter fragment in K562 group.Conclusions NaB improves the phosphorylation and acetylation of H3 at ?-globin gene promoter regions,and this may be one of the mechanisms of expression of ?-globin genes induced by NaB.

Influence of Astragalus Polysaccharides on Fetal Hemoglobin Synthesis and Cell Proliferation in K562 Cells / 实用儿科临床杂志

Objective To explore the effects of astragalus polysaccharides(APS) on fetal hemoglobin(HbF) synthesis and cell proli-feration in K562 cells.Methods K562 cells were chosen as the cell model and cells treated with Na-butyrate(NaB) were taken as the po-sitive control.Western blot was applied to study the level of HbF expression in K562 cells and Trypan blue dye exclusion test was employed to analyze the influence of APS(150 mg/L,300 mg/L,450 mg/L)on K562 cells proliferation.Results 1.Dosage effect:when compared with untreated K562 cells,the HbF expression level increased to(1.56?0.03),(1.78?0.04) and(1.51?0.32) fold,respectively after 48 h treated with different concentrations of APS(150 mg/L,300 mg/L,450 mg/L,F=310.476 P=0).The best inducing concentration was 300 mg/L(P=0.005).2.Time course: HbF levels raised up gradually and the maximum was(2.88?0.27) fold over baseline(P=0) at 48-60 h in the presence of 300 mg/L APS.Then it went to decline.There was statistical significance of HbF expression between K562 cells treated with 300 mg/L APS or NaB [(2.88?0.27) folds,P=0].3.Effects of APS on K562 cells proliferation:the highest reduction of the cell proliferative was obtained in K562 cells cultured in the presence of 0.5 mmol/L NaB.As detected by Trypan blue exclusion met-hod,growth rate of cells stimulated by APS was affect in a dose dependent manner,and significantly higher than NaB.For example,the inhibition rate at 48 hours was 20.45% for 300 mg/L APS but 79.55% for 0.5 mmol/L NaB(P=0).Conclusion APS has ability to induce HbF synthesis in K562 cells and revealed less cells reduction than that of NaB.

RFLP Haplotypes of beta-Globin Gene Complex of beta-Thalassemic Chromosomes in Koreans

Korea is in the low-prevalence area of beta-thalassemia and the Korean population has relatively homogenous racial characteristics. Recently, we identified some causative mutations of the Korean beta-thalassemia patients. In order to elucidate the genetic background of beta-thalassemia alleles in Koreans, we determined the restriction fragment length polymorphism (RFLP)-haplotype and framework (FW) in nine beta-thalassemia chromosomes of five different causative mutations by PCR-based method and family linkage study. The result that the haplotype and the framework linked to the initiation codon ATG-->AGG mutation were -+-++-+ and FW3A, respectively, in all of three families in this study suggests a common origin of this mutation at least in Koreans. A novel beta-thalassemia mutation, codons 89/90 -TG, showed discrepancy between -++--++- and FW1, which could be explained by gene conversion. A case of codons 8/9 +G frameshift mutation had +----++ and FW1. The linkage of the two beta-thalassemia mutations, codon 17 AAG-->TAG and codons 41/42 -TTCT, with specific haplotypes and frameworks common to the Koreans and the neighboring countries suggests that those mutations are influenced by the genetic flow from the south China.

A Korean Family with Thalassemia Intermedia due to Co-inheritance of Triplicated alpha-Globin Genes (alphaalpha/alphaalphaalphaanti3.7) and beta-Thalassemia Trait (IVSII-1 G -> A) / 대한혈액학회지

We report a Korean family in which the interaction of a triplicated alpha-globin locus and a heterozygous beta-thalassemia gives rise to a clinical phenotype of thalassemia intermedia. The propositus, a 36year-old woman, was evaluated because of moderately severe chronic anemia. Molecular analysis revealed heterozygosity for a single beta-thalassemia mutation, IVSII-1 (G->A). Additionally, she was found to have co-inherited a triplicated alpha-globin gene (alphaalpha/alphaalphaalphaanti3.7). In contrast, her brother heterozygous for the same triplicated alpha-locus and beta-thalassemia was clinically normal, suggesting that the delicate balance between alpha- and beta-chains is controlled by other currently not identified factors. Thalassemia intermedia due to co-inheritance of alphaalpha/alphaalphaalphaanti3.7 and IVSII-1 (G->A) was rare, and in Korea, this patient is the first case of thalassemia intermedia attributable to this combined abnormalities.

DNA-Protein Interaction of gamma-Globin Gene Promoter by Differentiation Inducers / 대한혈액학회지

BACKGROUND: The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic cells. There are numerous similarities between the erythroid or megakaryocytic lineages. In this study, we examined role of the region -269~-240 of gamma-globin gene promoter in fetal hemoglobin expression during either erythroid or megakaryocytic differentiation. METHODS: K562 cells were cultured and treated with differentiation inducers. Hemoglobin content was scored by benzidine staining, and hemoglobin F was stained by acid elution technique. To determine whether transcription factor binding to the gamma-globin gene promoter is critical to lineage determination, DNA-protein interaction of gamma-globin gene promoter was examined under both uninduced and induced conditions of K562 cells using gel mobility shift assay and southwestern blot analysis. RESULTS: Phorbol 12-myristate 13-acetate (PMA) induced a megakaryocytic differentiation, but suppressed erythroid differentiation. On the other hand, hydroxyurea (HU), hemin, n-butanol, and sodium butyrate (NaB) induced the expression of erythroid phenotypes. Parallel to hemoglobinization, increase in gamma-globin mRNA was observed in HU- and hemin-treated K562 cells. Gel mobility shift assay and southwestern blot analysis revealed that binding of a erythroid-specific protein (p120) to the region -269~-240 of gamma-globin gene promoter occurred with treatment of erythroid differentiation inducers and did not occur with treatment of PMA. CONCLUSION: These results suggest that erythroid differentiation inducers may act via DNA- protein interaction at the gamma-globin gene promoter region to induce erythroid differentiation.

Role of Gamma Globin Promoter Region -269~-240 in Hydroxyurea Treated Erythroid Progenitor Cells / 대한임상병리학회지

BACKGROUND: Recently, a great deal of interest has been focused on the use of hydroxyurea and hemin that may augment Hb F levels in patients with hemoglobinopathies and thalassemia, although the molecular mechanism of those chemicals remains unclear. In this study, we examined the effects of hydroxyurea and hemin on human adult peripheral and cord blood erythroid cells grown in a two-phase liquid culture system. METHODS: Four adult peripheral and four cord blood cells were cultured in two-phase liquid culture, and were treated with hydroxyurea or hemin. We counted isolated erythroid cells by acid benzidine and glycophorin A stains. To determine whether transcription factor binding to the promoter is critical, we also examined the promoter region of gamma globin gene both under uninduced and hydroxyurea or hemin induced conditions using gel mobility shift assay and southwestern blot analysis. RESULTS: When added together with erythropoietin, hydroxyurea led to significant increase in the percentage of erythroid cells in cord blood. In contrast, hemin greatly accelerated hemoglobin accumulation in adult erythroid progenitor cells. At -230 and -264 regions of gamma globin gene promoter, different protein binding patterns were observed in uninduced and hydroxyurea or hemin induced conditions between adult and cord blood. CONCLUSIONS: These results suggest that hydroxyurea and hemin may act via alteration in DNA-protein interactions to induce gamma globin gene expression. In addition, we can conclude that different transcription factors may be involved in the gamma globin induction process between the adult and cord blood erythroid cells.