Short Communication - Hematology and Blood Disorders (2019) Volume 2, Issue 1
Survey of hemoglobinopathies in Central Vietnam: Identification of a novel β-globin gene mutation (HBB:c.-122T>A)
- Corresponding Author:
- Monica Pirastru, PhD
Dip. di Scienze Biomediche Università
di Sassari Via Muroni, 25 - (I) 07100
Sassari (SS) Italy
Tel: +39 079 228610
Fax: +39 079 228636
E-mail: [email protected]
Citation: Pirastru M, Manca L, Nguyen CQ, et al. Survey of hemoglobinopathies in Central Vietnam: Identification of a novel β-globin gene mutation (HBB:c.-122T>A). Hematol Blood Disord 2019;2(1):13-15.
The thalassemias, together with sickle cell disease (HBB: c. 20A>T), are the world ? s most common form of inherited anemia. The myriad manifestations of thalassemia result from the imbalanced synthesis of ?-like and non-?-like globin chains and from the accumulation of unpaired counterpart. Unpaired globin chains are unstable: they form intracellular aggregates which are insoluble and precipitate causing decreased deformability, membrane damage and selective removal of the damaged cell. Ineffective erythropoiesis and shortened red cell survival will lead to chronic anemia.
The thalassemias, together with sickle cell disease (HBB: c. 20A>T), are the world ’ s most common form of inherited anemia. The myriad manifestations of thalassemia result from the imbalanced synthesis of α-like and non-α-like globin chains and from the accumulation of unpaired counterpart.
Unpaired globin chains are unstable: they form intracellular aggregates which are insoluble and precipitate causing decreased deformability, membrane damage and selective removal of the damaged cell. Ineffective erythropoiesis and shortened red cell survival will lead to chronic anemia.
The β-thalassemias are characterized by a quantitative deficiency of β-globin chains underlaid by a striking heterogeneity of molecular defects. Mutations that completely inactivate the β gene resulting in no β-globin production cause β0-thalassemia. Other mutations allow the production of some β globin but in markedly decreased amounts, and are classified as β+-thalassemias.
According to the severity of the phenotype, β-thalassemias can be subclassified in three different groups: β-thalassemia minor (Tm), β-thalassemia intermedia (TI) and β-thalassemia major (TM). Carriers for Tm, which are heterozygotes for a defective β-gene, present with microcytic hypochromic parameters with or without anemia. Inheritance of two β0-thalassaemia alleles, homozygous or compound heterozygous states, results in TM with severe transfusion-dependent hemolytic anemia associated with marked ineffective erythropoiesis. Homozygotes β+β+ or compound heterozygotes β+β0 are usually affected with TI and show a moderate to severe, partially compensated, hemolytic anemia that usually does not require chronic transfusion therapy to maintain a satisfactory circulating Hb level in the affected patient, although occasional transfusions may be required if the anemia worsens because of associated complications .
After 60 years of significant progress, the management of thalassemia patients still depends on supportive care, regular lifelong blood transfusions and iron chelation. Cure is possible only in the limited case of a patient having an HLA-identical donor. Approximately 80% of the annual births of babies with severe conditions occur in developing and low-income countries, many of which have extremely limited facilities for their control and management. It is therefore important to accurately identify carriers of these disorders and offer the option of preventive measures by prenatal diagnosis to couples at risk of having a child with severe disease.
Routine diagnosis of β-thalassemia trait is based on microcytic parameters and/or elevated levels of Hb A2 (≥ 3.5). However, a reliable diagnosis can only be achieved by DNA analysis.
The ability to perform DNA analysis has become an increasingly important requirement and the acquisition of such skills requires the development of suitable training programs involving hematologists, pediatricians, biologists and technical staff. A start has been made in developing and low-income countries and there is already considerable evidence that much more can still be done, including the further development of partnerships between countries where expertise in this field has been developed and countries where no such expertise exists.
Here we report data obtained during a screening program for hemoglobinopathies (Hbpathies) carried out as part of the ongoing cooperation between the Universities of Hue, Vietnam, and Sassari, Italy.
In Vietnam, both β-thalassemia and Hb E (HBB: c.79G>A) are prevalent and represent an important cause of childhood chronic disease . The first study for β-thalassemia was performed in the North of Vietnam . Later, the spectrum of β-globin mutations has been investigated in Ho Chi Minh City, South Vietnam [2,4]. Data concerning the central area of the country were recently published [5-7]. Nonetheless, epidemiological data is still insufficient and fragmented.
To date, eight β-thalassemia mutations have been identified in the Vietnamese population, with distinct incidence in the different areas: six are β0 mutations and two belong to the β+ type [2-8]. The most commons are the frameshift mutation at codons 41/42 (HBB:c.126_129delCTTT), the nonsense mutation at codon 17 (HBB:c.52A>T) and the frameshift mutation at codon 95 (HBB:c.287_288insA), which is known as the “Vietnamese” mutation. The other mutations are less frequent: the β+-28 (HBB: c.78A>G), the codon 26 (HBB: c. 79G>T), the IVS-I-1 (HBB:c.92+1G>T), the frameshift mutation at codons 71/72 (HBB:c.216_217insA) and the β+ IVS-II-654 (HBB:c.316-197C>T). The latter, despite it being a β+ mutation, leads to a transfusion-dependent phenotype in homozygous or compound heterozygous states.
The missense mutation GAG>AAG (Glu>Lys) at codon 26 of the Hb E variant also results in the activation of a cryptic, alternative, splice site and leads to a β+-thal phenotype due to a 25.0%-30.0% reduction of βE-globin synthesis. The Hb E heterozygote is mildly affected and the Hb E homozygosity is a benign disorder with a mild β-globin chain deficit which is comparable to that seen in a β0-thalassemia heterozygote. However, compound heterozygotes βEβ0 are often severely affected.
Our study included nearly 200 individuals referred to Hue Medicine and Pharmacy College and PhuVang District Hospitals for hematological and clinical evaluation. Samples have undergone to Hbpathies screening on whole blood lysates performed by cation-exchange high-performance liquid-chromatography (CE-HPLC) and by isoelectric focusing . Any qualitative or quantitative alteration observed in Hb profiles was further investigated by DNA analysis which included: Multiplex Ligation-Dependent Probe Amplification (MLPA) analysis of HBB and HBA cluster, PCR and Sequencing Analysis of the β-globin gene, K562 cell-based luciferase reporter assay on β-globin gene promoter .
A total of three known β-globin gene mutations and a novel one were observed. The three previously described were: the Hb E (HBB: c.79G>A), the mutation at codons 41/42 (HBB:c. 126_129delCTTT) and the mutation at codon 17 (HBB:c. 52A>T). These have been found in heterozygousity or in compound heterozygous βEβ0 state. Wide phenotypic variability in βEβ0 patients is described: approximately one half of them are phenotypically similar to patients with TM who require regular transfusion therapy, and the other half resembles TI. Notable are variations in anemia, growth, development, hepatosplenomegaly, and transfusion requirements .
The cause of the striking variability remains largely unknown. Despite seemingly identical genotypes, patients from the same family may show significant differences in clinical severity. Hb F level is the strongest predictor of morbidity. However, the basis of increased Hb F is usually unknown. In a review of 378 patients with Hb E-β0-thalassemia from Thailand, the Hb concentrations ranged from 3 to 13 g/dl, with an average of 7.7 g/dl .
In our study, the mean value of Hb concentration was 8.13 g/dl, with Hb F markedly elevated. None of the βEβ0 patients undergoes to a regular transfusion program. They have occasionally received transfusions and the level of persistent Hb F synthesis appears to be the most important factor in their clinical mildness.
A novel mutation at position -72 HBB:c.-122T>A) in the CCAAT box of the β promoter region was found in a 5-yearold child. The proband presented with a mild microcytic anemia as both his father and paternal grandfather. None of them had ever required transfusions. The other family members showed normal hematological parameters and Hb components. Sequencing revealed that the same mutation identified in the proband's β-globin gene was also present in both father and grandfather. No alteration was observed in the HBA cluster of the proband and his grandfather, whereas a triplicated HBA cluster, that seems to have no effect on the phenotype, was pointed out in the proband's father. The presence of Hb Constant Spring was excluded in all carriers .
To date, one mutant at β-promoter, the -28 (HBB: c.78A>G), has been identified in Vietnam [2,4]. This variant occurs in the TATA box and has been introduced into the HbVar database as β+-thalassemic allele. Indeed, the mild thalassemic phenotype observed in compound heterozygotes βEβ-28 from Thai population may indirectly indicate that the -28 mutation is a mild thalassemic allele .
The functional effect of the -72 mutation, identified in our study in a heterozygous state, was evaluated by luciferase reporter assays.
Our in vitro experiments shows that the transcriptional activity of the mutated promoter is roughly half that of the wild type promoter . This finding suggests that the -72 mutation can be classified as a β+-thalassemic allele. However, the absence of homozygous or compound heterozygous states does not allow us to precisely predict its clinical impact and, consequently, its relevance in management programs. Indeed, association of β+-thalassemia with β0 or βE mutations results in a markedly heterogeneous hematological picture, ranging in severity from that of the β-thalassemia carrier state to that of transfusion-dependent state .
Furthermore, it has been noted that even in the case of mild disease, β-TI patients may still suffer from many complications including a hypercoagulable state and subsequent thrombotic events .
The ability to predict phenotype from genotype has important implications for the screening of β-thalassemia carriers, for genetic counseling and prenatal diagnosis and for planning the appropriate treatment regimen. This ability requires in turn a comprehensive knowledge of the spectrum of β-thalassemia mutations, more so in countries, like Vietnam, where the extraordinary phenotypic diversity involves particular management problems.
Our results further underline the importance of identifying and characterizing new or rare β-thalassemic alleles, even when mild, in carrier screening and prenatal diagnosis in order to reduce the burden of thalassemias, avoid unnecessary transfusions in TI and start early transfusions in TM patients.
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