Enhanced activation of autophagy in β-thalassemia/Hb E erythroblasts during erythropoiesis

Erythropoiesis in β-thalassemia patients is ineffective, primarily because of death of the erythroid progenitor cells at the polychromatic normoblast stage. While it is known that autophagy plays a critical role during erythropoiesis by removing organelles from erythroid cells during terminal differentiation, its role in erythroid cells whose function is impaired remains to be explored. To investigate this, CD34+ erythroid progenitor cells from normal controls and β-thalassemia/Hb E patients were isolated from peripheral blood and cultured under conditions driving differentiation into an erythroid lineage, and levels of autophagy and apoptosis were analyzed both directly and after biochemical manipulation with l-asparagine. A significantly higher level of autophagy was seen in β-thalassemia/Hb E erythroblasts as compared to normal control erythroblasts during erythropoiesis. Interestingly, this activation was mediated in part by the presence of high levels of Ca²⁺ as modulation of Ca²⁺ levels significantly reduced the level of autophagy in these cells. Inhibition of autophagic flux in normal erythroblasts significantly increased apoptosis in normal erythroblasts, but not in thalassemic erythroblasts, although sensitivity to autophagic flux inhibition was restored by reduction of Ca²⁺ levels. These results suggest that high levels of autophagy in β-thalassemia/HbE erythroblasts may contribute to the increased levels of apoptosis that lead to ineffective erythropoiesis in β-thalassemia/Hb E erythroblasts.     

Complex interaction of Hb Q-Thailand and Hb E with α0-thalassemia and hereditary persistence of fetal hemoglobin in a Chinese family

Hemoglobin (Hb) Q-Thailand, Hb E, and other forms of thalassemia are prevalent in Southeast Asia including China. We report a hitherto undescribed condition in which four forms of Hb defects co-segregate. The proband was a 20-year-old Chinese man who presented with moderate hypochromic microcytosis with Hb 73 g/l, hematocrit (Hct) 27.0%, mean corpuscular volume 57.6 fl, mean corpuscular hemoglobin 15.5 pg, and mean corpuscular hemoglobin concentration (MCHC) 268.0 g/l. Both Hb electrophoresis and high-performance liquid chromatography analysis revealed abnormal Hbs. DNA analysis demonstrated that the proband was a double heterozygote of Hb Q-Thailand and Hb E in combination with α⁰-thalassemia and Southeast Asian-type hereditary persistence of fetal hemoglobin (SEA-HPFH). Family study identified that her father was a double heterozygote for Hb Q-Thailand and Hb E, whereas her mother was a heterozygote for SEA-HPFH with α⁰-thalassemia. Moreover, his brother was a classical Hb QH disease patient. The genotype–phenotype relationship observed in this Chinese family with complex thalassemia syndromes is presented. This work will provide some clinical implications for molecular diagnosis for complex hemoglobinopathies.     

Coinheritance of the different copy numbers of α-globin gene modifies severity of β-thalassemia/Hb E disease

β-Thalassemia/Hb E patients show a range of clinical severities, from nearly asymptomatic to transfusion-dependent thalassemia major. This study investigated the clinical heterogeneity and hematologic parameters obtained in the large cohort of 925 Thai β⁰-thalassemia/Hb E patients. Coinheritance of α-thalassemia with β⁰-thalassemia/Hb E produces a milder clinical phenotype in contrast to an interaction of α-globin gene triplication in severe thalassemia. The mean steady-state Hb was also higher, whereas the mean corpuscular volume and the percentage of Hb F were markedly lower in the former group. This finding demonstrates that the genetic combination leading to the more/less degree of α- to non-α-globin chains imbalance is indeed the cause of the severe/mild thalassemia phenotype.     

Analysis of the rs35959442 polymorphism in Hb E/β-thalassemia in Guangxi Province of the Republic of China

Hb E [β26(B8)Glu→Lys]/β-thalassemia (β-thal) is a worldwide inherited disorder. We determined the phenotype of 65 unrelated Hb E/β-thal subjects and 70 healthy individuals in the Guangxi Province of the Republic of China (ROC). Single nucleotide polymorphism (SNP) rs35959442 in HBS1L analysis was performed using the polymerase chain reaction (PCR)/restriction enzyme method. The data suggested that the frequency of the rs35959442 polymorphism was relatively high in patients with Hb E/β-thal in Guangxi Province, ROC, when associated with Hb F augmentation.     

Association of Hb New York with Hb E and α(0)-thalassemia in a Chinese woman identified by Sebia CapillaryS2 system

We describe a Chinese woman who was assumed to be heterozygous for both Hb E [β26(B8)Glu→Lys] and α(0)-thalassemia (α(0)-thal) by a high performance liquid chromatography (HPLC) method, but was later also shown to be a Hb New York [β113(G15)Val→Glu] heterozygote by the Sebia CapillaryS2 system. This study suggested that a single test is never sufficient to allow the correct diagnosis of an abnormal hemoglobin (Hb). We also emphasize the importance of a correct diagnosis of interactions between α- and β-thalassemias.     

Secondary erythrocytosis due to compound homozygosity, but not compound heterozygosity, for Hb Luton and α-thalassemia: a family study

We describe the hematological and clinical features of homozygous Hb Luton (OMIM 141800.0172), a high affinity α-globin variant that has not been previously described in the homozygous state. The proband was found to have a high hemoglobin (Hb) concentration following a routine blood count prior to a planned appendectomy at the age of 16 years. Investigation showed that she was homozygous for both Hb Luton [α89(FG1)His→Leu (CAC>CTC), a high oxygen affinity Hb)] and homozygous for α(+)-thalassemia (α(+)-thal), while her mother, maternal aunt and half-brother were heterozygous for these conditions. Further investigation showed that she also had Gilbert's disease and Raynaud's syndrome. As far as we are aware, this is also the first reported family with a subject homozygous for both Hb Luton and α-thal so that the proband has no nomal α-globin. The parents of the proband are first cousins and originate from Pakistan.     

Hb A2/E levels found in co-inheritance with the α-thalassemia-1 - -(SEA)/type deletion and either Hb E or β-thalassemia

The α-thalassemia-1 (α-thal-1) Southeast Asian (- -(SEA)) type deletion, β-thalassemia (β-thal) and Hb E [β26(B8)Glu→Lys, GAG>AAG] are the most common genetic disorders in Southeast Asian populations. Mean corpuscular volume (MCV) <80.0 fL with normal hemoglobin (Hb) is used for screening α- and β-thal, and a Hb E level of less than 25.0% is used for predicting α-thal-1 in Hb E trait. Thus, levels of Hb, MCV and Hb A(2)/E were reviewed and compared between the SEA type deletion co-inherited with β-thal trait (n = 61), with Hb E trait (n = 102) or homozygous Hb E (n = 13) and β-thal trait (n = 636), Hb E trait (n = 544) or homozygous Hb E (n = 83), respectively. When comparing the values of all three analyzed hematological parameters, only the - -(SEA)/β(E) values were shown to be significantly lower than those of Hb E trait. Furthermore, at a cut-off value of Hb A(2)/E of 21.54%, 95.0% of the - -(SEA)/β(E) had Hb A(2)/E levels lower than this cut-off value, while 94.0% of Hb E trait had Hb A(2)/E at higher levels. Accordingly, the Hb A(2)/E level at 21.54% is the best indicator for predicting co-inheritance of the α-thal-1 - -(SEA)/ deletion and Hb E trait.     

Hydroxyurea in the treatment of major β-thalassemia and importance of genetic screening

Efforts have been undertaken to find an alternative approach to packed red cell transfusion (PRCT) in major -thalassemia. Augmentation of fetal hemoglobin (HbF) by hydroxyurea (HU) has been reported to be less effective in this condition as compared to sickle cell anemia due to molecular heterogeneity of the former disease. HU efficacy and its relation to Xmn1 polymorphism and IVSII-1 mutation was evaluated in major -thalassemics. Forty-five patients, M/F ratio 0.8, aged 6–33 years, received oral HU, 20 mg/kg per day, 4 days per week and daily1 mg folic acid. Thirty-six patients were PRCT dependent (group A) and nine independent (group B). The aim was to stabilize or increase pre-PRCT Hb over 10.0±0.5 g/dl and to reduce the need or cease the PRCT in group A and to increase Hb level and curb the ineffective erythropoesis, e.g., splenomegaly, facial bone deformity, in group B. HU was administered for at least 6 months (mean: 9 months) and discontinued in case of response failure. Screening for Xmn1 polymorphism and IVSII-1 mutation was carried out in most patients. In group A, 25 patients have become PRCT independent for a period of 2.5–7.3 years (mean: 4 years). The mean Hb, pre-HU 10.0 and post-HU 10.7 g/dl (range: 8.8–13.7 g/dl), mean serum ferritin pre- and post-HU was1877 and 525 ng/ml. The PRCT requirement was reduced in one patient, and ten patients did not respond. In group B HU has been given over 3.3 years (range: 2.8–4.8 years), Hb increased from 9.3 to 10.4/dl, and there was no tangible progression of ineffective erythropoesis. Responders in both groups expressed more comfort with this regimen. Xmn1 and IVSII-1 (homo- and/or heterozygosis) are relevant markers in most responding patients. Molecular determination of genetic markers in early childhood will help to identify candidates for pharmacological HbF switching by HU.     

Screening for co-existence of α-thalassemia in β-thalassemia and in HbE heterozygotes via an enzyme-linked immunosorbent assay for Hb Bart’s and embryonic ζ-globin chain

We sought to demonstrate the ability of levels of Hb Bart’s and ζ-globin chain quantified by enzyme-linked immunosorbent assay (ELISA) in detecting α-thalassemia in β-thalassemia and HbE heterozygotes. We developed an in-house sandwich ELISA method using monoclonal antibodies (mAbs) to Hb Bart’s and ζ-globin chain, and quantified levels of Hb Bart’s and ζ-globin chain in 172 and 223 anonymous blood samples of β-thalassemia and HbE heterozygotes, respectively. Genotypes of α-thalassemia 1, β-thalassemia were identified, and HbE allele was confirmed using a newly developed multiplex allele-specific PCR. The in-house sandwich ELISA method detected Hb Bart’s in 6.4% of β-thalassemia heterozygotes, of which 5.2% showed detectable amounts of the ζ-globin chain. 15.2% of individuals heterozygous for HbE showed a detectable amount of Hb Bart’s, and the ζ-globin chain was detected in 11.2% of this cohort. All samples having detectable amounts of Hb Bart’s and the ζ-globin chain were verified to be SEA-type α-thalassemia 1. ELISA-quantified Hb Bart’s and ζ-globin chain levels can be used to detect double heterozygosity of α- and β-thalassemia and of α-thalassemia and HbE. This strategy may be useful in screening for co-existence of α-thalassemia in β-thalassemia and in HbE heterozygotes, particularly in countries where α-, β-thalassemia and HbE are endemic.     

The Effect of Blood Transfusion on Growth of Patients with Hb E/β-Thalassemia

Abstract

A retrospective evaluation of growth in 112 patients (68 males, 44 females) with Hb E (HBB: c.79G>A)/β-thalassemia (β-thal), classified as 88 transfusion-dependent thalassemia (TDT) and 24 non transfusion-dependent thalassemia (NTDT), is reported. Patients with TDT have received regular transfusions of red blood cells (RBCs) 15?mL/kg every 4?weeks to maintain pre transfusion hemoglobin (Hb) levels of at least 9.0?g/dL and were categorized according to age at initiation of regular RBC transfusion as subgroup 1, <4?years; subgroup 2, 4–10?years, and subgroup 3, >10?years. Iron chelation was initiated at the mean age of 7?years. The results revealed that patients in subgroups 1 and 2, receiving RBC transfusions at a young age (2.9 and 6.9?years, respectively), had normal prepubertal growth at enrollment and last follow-up. Patients in subgroup 3, with the lowest initial height Z-score of ?2.10, were able to achieve comparable final adult height as those in subgroups 1 and 2. The mean final height of 21 males and 13 females with TDT at the ages of 18.9 and 18.7?years was 168.1 and 157.7?cm, respectively, which did not significantly differ from their midparental height and those with NTDT. Early initiation of optimal transfusion and iron chelation promoted normal prepubertal growth. However, delayed initiation of transfusion at age 12?years impaired prepubertal growth but they could achieve normal final adult height.     

Allelotyping for loss of heterozygosity on chromosome 18 in gastric cancer

AIM:To investigate the association between loss ofheterozygosity (LOH) on chromosome 18 and sporadicgastric cancer.METHODS:Multiplex PCR was used to screen 14 highlypolymorphic microsatellite markers on chromosome 18 in45 cases of primary gastric cancer.PCR products wereseparated on polyacrylamide gels and the electrophoresismaps were analyzed with Genescan and Genotyper.RESULTS:The LOH frequencies in gastric cancer at all 14markers ranged from 10% to 58%.Eleven markers werefound with over 20% LOH frequencies,in which 9 markerslocated in 18q,and 2 markers in 18p.Two overlapping deletedregions were identified:R1 between D18S61-D18S1161 at18q22 (9cM) with 24% LOH frequency;R2 between D18S462-D18S70 at 18q22-23(6cM) with 32% LOH frequency.CONCLUSION:LOH of chromosome 18 (18q and 18p) maybe involved in gastric tumorigenesis.Two overlapping deletedfragments suggested that there might be unidentified tumorsuppressor genes in those two regions.     

Compound heterozygosity for Hb Korle-Bu (β<Superscript>73</Superscript>; Asp-Asn) and Hb E (β<Superscript>26</Superscript>; Glu-Lys) with a 3.7-kb deletional α-thalassemia in Thai patients

Hemoglobin (Hb) Korle-Bu (beta73; Asp-Asn) is the most frequent of the rare beta-chain variants in the population of West Africa whereas Hb E (beta26; Glu-Lys) is common among the Southeast Asian population. We report a hitherto undescribed condition in which these two beta-chain variants co-segregate. The proband was a 19-year-old Thai pregnant woman in her second trimester of pregnancy who visited our thalassemia screening unit. Cellulose acetate electrophoresis and high-performance liquid chromatography (HPLC) analysis of Hb detected one abnormal Hb in addition to the Hb E. Analysis of DNA sequences revealed a GAT-AAT mutation at codon 73 in trans to a GAG-AAG mutation at codon 26 of the beta-globin gene. Polymerase chain reaction (PCR) analysis of the alpha-globin gene cluster of the patient detected a 3.7-kb deletional alpha-thalassemia 2. Family study identified that her mother had the same genotype and her father was a simple Hb E carrier. The hematological data of these unusual cases of hemoglobinopathy are presented and compared with a simple heterozygote for Hb Korle-Bu found in another unrelated Thai family. beta-Globin gene haplotype linked to the Thai beta(Korle-Bu) and a simple DNA assay based on allele-specific PCR for rapid diagnosis of Hb Korle-Bu are also described.     

Deferasirox effectively decreases iron burden in patients with double heterozygous HbS/β-thalassemia

Iron overload is present in several cases of double heterozygous sickle-cell/beta-thalassemia (HbS/β-thal). Deferasirox is an orally administered iron chelator which is effective on iron overloaded patients with transfusion-dependent anemia. The aim of this study was to investigate the efficacy and safety of deferasirox on HbS/β-thal patients with iron overload. We evaluated 31 adult patients with HbS/β-thal (14M/17F; median age 41 years) who had serum ferritin levels >1,000 ng/mL and who were sporadically transfused. Total iron burden was monitored by measuring serum ferritin levels before and monthly after starting deferasirox, while liver iron concentration and cardiac iron burden were measured by magnetic resonance imaging (MRI) T2 and T2* parameters at baseline and 12 months after deferasirox treatment. Deferasirox managed to reduce the mean serum ferritin levels after 12 months of treatment from 1,989 ± 923 to 1,008 ± 776 ng/mL (P < 0.001). This reduction was accompanied by a significant improvement on MRI T2* of the liver (from 3.9 ± 3.2 to 5.8 ± 3.1 ms; P < 0.01) and by a comparable improvement of biochemical parameters of liver function. Mild nausea and diarrhea of grade 1/2 were reported in 25% of patients within the first month of treatment, but did not re-occur. These data indicate that deferasirox provided effective control of iron levels (mainly of the liver) in minimally transfused patients with HbS/β-thal, without significant adverse events, at similar doses to those studied widely for the treatment of patients with thalassemia syndromes.     

Hb J-Wenchang-Wuming [α11(A9)Lys→Gln (AAG>CAG) (α2 or α1)] compromises neonatal screening for α-thalassemia with the Sebia Capillarys2 electrophoresis system

We describe a Chinese newborn who was assumed to have α(0)-thalassemia (α(0)-thal) by determining the amount of Hb Bart's (γ4) in the cord blood, but was later shown to have only α(+)-thal. Hb J-Wenchang-Wuming [α11(A9)Lys→Gln (AAG>CAG) (α2 or α1)] was mistaken for Hb Bart's as both hemoglobin (Hb) variants have the same mobility.     

Prenatal screening for β-thalassemia major reveals new and rare mutations in the Pakistani population

β-Thalassemia is the most common genetic disorder in Pakistan, where more than 6000 affected children are born annually, and the carrier population is around 10 million. The objective was to study β-globin gene mutations in chorionic villous biopsy samples. Prenatal screening of 383 pregnant women between 2003 and 2010 was carried out using a panel of 13 mutation primers and amplification refractory mutations system (ARMS)-PCR. In addition, DNA sequencing was used to confirm uncharacterized mutations and in some cases fetal disease status was confirmed by linkage analysis. Families enrolled in this study represented major ethnic groups in Pakistan. Of the 13 mutations tested, three mutations accounted 71% of the total, including IVS1-5(G-C)[HBB:c.92+5G>C], codon 8/9(+G) [HBB:c.27_28insG] and del 619[NG_000007.3:g71609-72227del619]. Mutations in four uncharacterized samples were later confirmed by DNA sequencing as −88(C-T)[HBB:c.-138C>G], −90(C-T)[HBB:c.-140C>T] and codon 59(+T)[HBB:c.178_179insT]. To our knowledge, this is the first report of these mutations in Pakistan. Moreover, 19.2% fetal samples were normal and 52.3% heterozygous, whereas 26.4% were affected with thalassemia major. IVS1-5:IVS1-5 was the most common genotype in fetal samples. Prenatal diagnosis of β-thalassemia using ARMS PCR is an efficient approach for reducing the burden of this disease in Pakistan. In addition, rare mutations reported in this study should be incorporated in the diagnostic strategy.     

Allelotyping for loss of heterozygosity on chromosome 18 in gastric cancer

AIM: To investigate the association between loss of heterozygosity (LOH) on chromosome 18 and sporadic gastric cancer. METHODS: Multiplex PCR was used to screen 14 highly polymorphic microsatellite markers on chromosome 18 in 45 cases of primary gastric cancer. PCR products were separated on polyacrylamide gels and the electrophoresis maps were analyzed with Genescan and Genotyper. RESULTS: The LOH frequencies in gastric cancer at all 14 markers ranged from 10% to 58%. Eleven markers were found with over 20% LOH frequencies, in which 9 markers located in 18q, and 2 markers in 18p. Two overlapping deleted regions were identified: R1 between D18S61-D18S1161 at 18q22 (9cM) with 24% LOH frequency; R2 between D18S462-D18S70 at 18q22-23(6cM) with 32% LOH frequency. CONCLUSION: LOH of chromosome 18 (18q and 18p) may be involved in gastric tumorigenesis. Two overlapping deleted fragments suggested that there might be unidentified tumor suppressor genes in those two regions.     

Increased oxidative metabolism is associated with erythroid precursor expansion in β0-thalassaemia/Hb E disease

Erythropoiesis in β0-thalassaemia/Hb E patients, the most common variant form of β-thalassaemia in Southeast Asia, is characterized by accelerated differentiation and over-expansion of erythroid precursor cells. The mechanism driving this accelerated expansion and differentiation remain unknown. To address this issue a proteomic analysis was undertaken to firstly identify proteins differentially expressed during erythroblast differentiation and a second analysis was undertaken to identify proteins differentially expressed between β0-thalassaemia/Hb E erythroblasts and control erythroblasts. The majority of proteins identified as being differentially expressed between β0-thalassaemia/Hb E and control erythroblasts were constituents of the glycolysis/TCA pathway and levels of oxidative stress correlated with the degree of erythroid expansion. A model was constructed linking these observations with previous studies showing increased phosphorylation of ERK1/2 in thalassemic erythroblasts which predicted the increased activation of PKA, PKB and PKC which Western analysis confirmed. Inhibition of PKA or PKC reduced β0-thalassaemia/Hb E erythroblast differentiation and/or expansion. We propose that increased expansion and differentiation of β0-thalassaemia/Hb E erythroblasts occur as a result of feedback loops acting through increased oxidative metabolism.