Interaction of heterozygous βo-thalassemia with single functional α-globin gene

In this study, we analyzed the phenotypic manifestations resulting from the interaction of heterozygous β^o-thalassemia(β^o-39 nonsense mutation) with the functional loss of three α-globin structural genes in six subjects, of whom four had the [-αl–] α-globin genotype and two the [–/αThα] α-globin genotype. The β-thalassemia defect was in all cases the nonsense mutation at codon 39. The nondeletion α-thalassemia α^th was the initiation codon mutation (AUG→GUG) of the α-2 gene. In all these subjects hypochromia and microcytosis were more marked than in βα -thalassemia heterozygotes with a full complement of four α-globin genes. All but one had moderate anemia. The α:β globin chain synthesis ratios were consistently decreased. No cases had Hb H on electrophoresis. Subjects with [–/αThα] α-globin genotype had more severe thalassemia-like manifestations than those with [–/-α] α-globin genotype.     

The Different Types of α-Thalassemia: Practical and Genetic Aspects

From May 1985 to October 1987, 1,564 Southeast Asians living in Hawaii were screened for hereditary anemias. Microcytosis was determined by electronic red cell indices and morphology; iron deficiency was ruled out by normal red cell distribution width and normal protoporphyrin levels; Hb E was determined by electrophoresis; β-thalassemia (thal) heterozygotes were identified by raised Hb A₂ on column chromatography. α-Thalassemia heterozygotes were diagnosed by exclusion. Family studies helped identify or confirm diagnoses, especially for the α-thal-2 heterozygotes (-α/αα) and homozygotes (-α/-α). Provisional diagnoses are being checked by DNA analyses. Iron deficiency prevented detection of possibly coexisting α-thalassemias in 97 individuals.

Technical problems included the obscuring of standard criteria for recognizing the α-thal variants by the presence of Hb E or β-thal. In such cases, α-thal could only be detected by family studies or DNA analyses. Problems with hemoglobin (Hb) electrophoresis included Hb H migrating beyond the edge of the strip if incubation was not closely monitored, and difficulty in detecting the small amounts of unstable Hb Constant Spring. DNA analyses also had limitations, since the nondeletion α-thalassemias would not be detected by routine Southern blotting.     

MOLECULAR CHARACTERIZATION OF Hb D-PUNJAB [β121(GH4)Glu→Gln] IN THAILAND

We describe the hematological and DNA characterization of Hb D-Punjab [β121(GH4)Glu→Gln] in Thailand. Nine patients from five unrelated families were studied; four patients were simple carriers of Hb D-Punjab, two were compound heterozygotes for Hb D-Punjab/β⁺-thalassemia;another two patients were double heterozygotes for Hb D-Punjab and α-thalassemia-2, and one patient was a compound heterozygote for Hb D-Punjab and Hb E [β26(B8)Glu→Lys]. Typical thalassemic indices with hypochromic microcytosis were observed in compound Hb D-Punjab/β⁺-thalassemia and Hb D-Punjab/Hb E but normal hematological profiles were observed in the remaining cases. DNA sequencing of the β-globin gene identified the G AA→C AA substitution at codon 121 causing Hb D-Punjab in all cases, and the ?28 (A→G) mutation for the β⁺-thalassemia alleles. β-Globin gene haplotype analysis demonstrated, for the first time, that all these Asian β^D-Punjab globin genes were associated with haplotype [?++?+++], previously undescribed in other populations. The finding of Hb D-Punjab in Thailand is compatible either with an independent origin of this abnormal hemoglobin or a spread of the Hb D-Punjab gene with a single origin among Asians.     

First Description in Tunisia of a Point Mutation at Codon 119 (CCT→TCT) in the α1-Globin Gene: Hb Groene Hart in Association with the − α3.7 Deletion

Herein we describe the case of a Tunisian girl who presented with 3% Hb Bart's (γ₄) at birth. At the age of 3 years, she showed microcytosis and hypochromia in the absence of iron deficiency. The first step of molecular analysis was to test for the common Mediterranean mutations and the classical ? α^3.7 deletion was found in the heterozygous state. Since this finding could not explain the level of Hb Bart's at birth, or the hypochromia and microcytosis, all the α-globin genes were sequenced. This revealed a rare point mutation at codon 119 (CCT→TCT) in the α1-globin gene, identified for the first time in Tunisia, and which has previously been described as an unstable hemoglobin (Hb) variant named Hb Groene Hart [α119(H2)Pro→Ser (α1)]. Here the ? α^3.7/αα^{119(CCT→TCT)} genotype is responsible for the α-thalassemia (thal) trait phenotype.     

Clinical,hematologic and biosynthetic studies in sickle cell-β°-thalassemia: A comparison with sickle cell anemia

The diseases commonly confused with sickle cell anemia include sickle cellβ-thalassemia in which synthesis of β^A-chains are completely suppressed (HbS-β^O-thalassemia). We obtained hematologic measurements and studied globin biosynthesis in five patients with this disorder and compared the results with those obtained in five patients with “mild” sickle cell anemia and seven individuals with sickle cell-β-thalassemia having hemoglobin A levels of 20–30% (HbS-β⁺-thalassemia). A distinction between HbS-β^O-thalassemia and sickle cell anemia was not always possible on clinical, hematologic, or electrophoretic grounds. Thalassemia heterozygotes had hypochromia and microcytosis, not generally a feature of sickle cell anemia, although overlap of values did exist. The ratio of β to non-β, or β to β^S-chains in sickle cell anemia approximated unity, whereas patients with HbS-β^O-thalassemia had a deficit of β-chain production relative to that of the β-chain. The differentiation of HbS-β^O-thalassemia and sickle cell anemia can be best made on the basis of family or biosynthetic study. We estimated the regional prevalence of HbS-β^O-thalassemia to be 1:23,000 of the black population.     

Investigation of microcytosis: a comprehensive approach

Abstract: Microcytosis is a highly prevalent finding during blood examination. This study investigates the causes of microcytosis (defined as mean corpuscular volume (MCV)<82 fl) in 466 patients referred to our laboratory for suspected hemoglobinopathy. The following data were obtained: Hb, MCV, serum iron, transferrin, ferritin, HbA2, HbF, isoelectric focusing of the Hb, gene mapping of chromosome 16 with Xba I and Bgl II and hybridization with an α- and a ζ-probe, inflammatory status. Results show that iron deficiency remains the first cause of microcytosis (35.2% of our patients), even in a selected population such as ours. Deletional α-thalassemia, probably the most frequent hemoglobinopathy throughout the world, represents the second most frequent cause of microcytosis (31.1%), followed by β-thalassemia heterozygous state (18.9%). Of our patients, 1.3% had microcytosis due to the presence of an abnormal hemoglobin (HbC, Hb S/C, HbE). Three cases (0.6%) had other possible causes of microcytosis. Of the remaining 60 cases, 28 had an inflammatory state. Finally, 32 cases (6.9%) remain unexplained; taking into consideration the origin of these cases, their hematological parameters and their family history, we postulate that these cases are at high risk for non-deletional α-thalassemia.     

Variable and Often Severe Phenotypic Expression in Patients with the α-Thalassemic Variant Hb Agrinio [α29(B10)Leu→Pro (α2)]

Hb Agrinio [α29(B10)Leu→Pro] is a highly unstable variant, classified as a nondeletional α-thalassemia (α-thal) mutation. To date it has only been described in individuals of Greek and Cypriot origin. Evaluation of the phenotypic presentation of 12 Hb Agrinio homozygotes or compound heterozygotes, diagnosed in a single center in Greece during a 15-year period, found a wide clinical expression, ranging from thalassemia intermedia (with or without transfusion requirement) to Hb H hydrops fetalis, with some phenotype-to-genotype correlation. The often severe clinical presentation of Hb Agrinio homozygotes or Hb Agrinio compound heterozygotes, coinheriting severe α-thal determinants, indicates that molecular identification of carriers of the Hb Agrinio mutation should be considered within the context of screening programs involving individuals of Greek and Cypriot origin. Selective molecular investigation of candidate carriers is facilitated by the observation that all heterozygotes for the Hb Agrinio mutation present with at least one hematological parameter implicating an α-thal carrier state.     

The association of sickle cell anemia with heterozygous and homozygous α-thalassemia-2: In vitro HB chain synthesis

The in vitro synthesis of hemoglobin chains was investigated in 34 scikle cell anemia (SS) patients and five patients with Hb S-β^o-thalassemia. Incubations were made for 30 minutes and for 120 minutes. Hematological and family data were also obtained. Although the 30-minute α/non-α total activity ratios were more widely distributed than the ratios at 120 minutes, a distinct classification of SS patients into groups without an α-thalassemia, with a heterozygous α-thal-2, or with a homozygous α-thal-2 could not be made. Family studies indicated that four patients who had 30-minute α/non-α ratios below 0.82 and mean corpuscular volume (MCV) values below 70 fl had a homozygosity for both α-thal-2 and Hb S. They had mild hematological features of SS disease. Many SS patients with 30-minute α/non-α ratios between 0.8 and 1.0 and MCV values above 70 fl had an associated α-thal-2 heterozygosity. Their hematological features were similar to those of SS patients with four active α chain genes. It appears that an α-thal-2 heterozygosity (−α/αα; β^S/β^S) does not alter the hematological expression of SS disease. An α-thal-2 homozygosity −α/−α;β^S/β^S results in a microcytosis similar to that seen in Hb S-β^o-thalassemia patients. The diagnosis of these α chain deficiencies in association with SS disease (by in vitro chain synthesis analyses) leaves several uncertainties.     

Different hematological phenotypes caused by the interaction of triplicated α-globin genes and heterozygous β-thalassemia

The pathophysiology and clinical severity of β-thalassemia are related to the degree of α/non-α-chain imbalance. A triplicated α-globin gene locus can exacerbate effects of excess α-chains caused by a defective β-globin gene, although this is not observed in all cases. Extensive studies on this condition are lacking. We report a group of 17 patients who are heterozygous for both the ααα^anti-3.7 allele and a mutation in the β-globin gene cluster. Their clinical phenotypes varied: six had mild anemia with microcytosis and hypochromia, while 11 had more severe anemia with splenomegaly requiring splenectomy (three cases) and blood transfusions (four cases). Different phenotypes were also evident in the presence of the same β-thalassemia mutation: in one family, two individuals had the same α- and β-globin genotypes but presented with different hematologic phenotypes. In addition, the complex interaction involving a triplicated α-globin gene, β³⁹- and δ⁺²⁷-thalassemia mutations is studied in a family with two siblings presenting with hemolytic anemia, normal Hb A₂ and increased Hb F. Analysis of this series of patients suggests that additional genetic determinants play a role in modulating phenotypic expression in individuals with identical α- and β-globin genotypes. Interaction with a triplicated α-gene can play a role in the clinical presentation of patients with defective β-globin gene expression and should be considered in the diagnosis of atypical cases. Am. J. Hematol. 55:83-88, 1997. © 1997 Wiley-Liss, Inc.     

A Novel α2-Globin Gene Mutation: Hb Debao [α31(B12)Arg→Trp; HBA2: c.94A>T]

We report a novel mutation on the α2-globin gene, Hb Debao [α31(B12)Arg→Trp; HBA2: c.94A>T] detected in a Chinese family. This mutation gives rise to a previously undescribed hemoglobin (Hb) variant that was undetectable by electrophoretic or chromatographic methods. Hb Debao was associated with an α⁺-thalassemia (α⁺-thal) deletion [?α^3.7 (rightward)] producing a mild phenotype with significant microcytosis and hypochromia, while the combination of this mutation with an α⁰-thal deletion (??^SEA) resulting in a severe form of Hb H (β4) disease, which is consistent with a thalassemic phenotype associated with the novel mutation.     

Molecular Characterization of α-Thalassemia in the Dohuk Region of Iraq

The molecular basis of α-thalassemia (α-thal) has been addressed by several studies from the eastern Mediterranean region, but not from Iraq. To address this issue, we studied 51 individuals with unexplained hypochromia and/or microcytosis, as well as nine patients with documented Hb H disease from the Dohuk region in northern Iraq. We used multiplex gap-polymerase chain reaction (gap-PCR), reverse hybridization, and sequencing for this purpose. It was found that the most common genotypes in those with unexplained hypochromia and/or microcytosis were ?α^3.7/αα, followed by ? ?^MED-I/αα, then ?α^3.7/?α ^3.7, respectively, detected in 84.3% of the above individuals. Other genotypes identified sporadically were ?α^4.2/αα, α^{poly A1}α/αα (AATAAA>AATAAG), α^Adanaα/αα [Hb Adana, codon 59 (Gly→Asp) or HBA1:c.179G>A], and α^Evanstonα/αα [Hb Evanston, codon 14 (Trp→Arg) or HBA1:c.43 T>C]. Three cases (5.88%) remained uncharacterized even after sequencing. All nine Hb H cases carried the ?α^3.7/? ?^MED-I genotype. Such findings are rather different from those in other eastern Mediterranean populations, particularly with relevance to an Hb H molecular basis.     

A comprehensive screening program for β-thalassemia and other hemoglobinopathies in the Hooghly District of West Bengal,India, dealing with 21 137 cases

We here present a report of population screening programs (January 2012–December 2015) conducted by the Thalassemia Control Unit, Imambara Sadar Hospital, Chinsurah, Hooghly in the Hooghly District of West Bengal, India for prevention of thalassemia. We screened β-thalassemia (β-thal) heterozygotes and homozygotes, and Hb E (HBB: c.79G?>?A)-β-thal compound heterozygotes. Among 21?137 cases, we found 1968 heterozygotes and 192 homozygotes or compound heterozygotes. Results were evaluated with standard hematological analyses including red cell indices, hemoglobin (Hb) typing and quantification. The participants of the screening program were divided into six groups (children, pre-marriage cases, post-marital cases, family members of affected individuals, family members of carriers and pregnant women). While considering the average frequency of carriers, many reports recorded both related individuals (family members of trait and affected individuals) as well as unrelated individuals such as school children and pregnant women. These would have to be considered separately and only the unrelated individuals taken to estimate carrier frequencies in this article that would give more realistic data on carrier frequency of unrelated individuals.     

PRENATAL DIAGNOSIS OF β-THALASSEMIA MAJOR BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY ANALYSIS OF HEMOGLOBINS IN FETAL BLOOD SAMPLES

In Thailand and adjacent countries, most of the β-thalassemia genes are β⁰-thalassemia mutations that prevent the production of Hb A. We propose the quantitation of the Hb A fraction in fetal blood in the mid-trimester of pregnancy by automated high performance liquid chromatography as a reasonable prenatal diagnostic method to be applied in areas with limited laboratory facilities. Forty pregnant women at risk of delivering a child with β-thalassemia major were identified using an erythrocyte osmotic fragility test and quantitation of Hb A₂. Cordocentesis was performed at the gestational age of 18–22 weeks and fetal blood was analyzed for hemoglobin fractions by automated high performance liquid chromatography. The β-globin gene mutations were characterized by β-globin gene sequencing. The 4 bp deletion at codons 41/42 (?TTCT) was the most frequent of the 40 β-thalassemia mutations observed (20/40 = 50%), followed by the splice site mutation IVS-I-1 (G → T) (7/40 = 17.5%), the nonsense mutation at codon 17 (A → T) (7/40 = 17.5%), the nonsense mutation at codon 35 (C → A) (3/40 = 7.5%), and the β⁺ -thalassemia promoter mutation at ?28 (A → G) (3/40 = 7.5%). High performance liquid chromatography revealed nine fetuses which had only Hb F and no Hb A. All were homozygotes or compound heterozygotes for β⁰-thalassemia mutations. In the remaining 31 fetuses, a Hb A peak was present in the chromatograms. One fetus with 0.5% Hb A was a compound heterozygote for the ?28 (A → G) and codons 41/42 (?TTCT) mutations. In the remaining 30 fetuses, the Hb A values ranged between 0.8 and 7.4%. Twenty of these, with a Hb A concentration of 1.82 ± 0.49% (range 0.8–2.8%), were β-thalassemia heterozygotes. The remaining 10 fetuses had Hb A values of 4.89 ± 1.47% (range 2.9–7.4%) and normal β-globin genes. The absence of Hb A in homozygotes or compound heterozygotes for β⁰-thalassemia mutations and the presence of measurable amounts of Hb A in heterozygotes and normal homozygotes, permits the diagnosis of fetuses expected to develop postnatal β-thalassemia major.     

Studies on the Proportion and Synthesis of Haemoglobin G Philadelphia in Red Cells of Heterozygotes, a Homozygote, and a Heterozygote for both Haemoglobin G and α Thalassaemia

SUMMARY. The proportion of Hb G Philadelphia (α68-Asnalys) in heterozygotes has been found to have a well-defined bimodal distribution around means of 33% and 46% Hb G. Microcytosis and hypochromia are consistently associated with the latter group, who also have a decreased ratio of α/β-chain synthesis in the peripheral blood, but these characters are not linked to the Hb-Gα gene, because a parent with microcytosis and 46% Hb Gα may have offspring with 33% Hb G without significant microcytosis. In one family a subject with Hb G and Hb G₂ but no Hb A or Hb A₂ is presumably a homozygote for α^G. This subject has microcytosis and a decreased ratio of α/β chain synthesis. In another family a subject with Hbs H, G and G₂ but without Hbs A or A₂ is heterozygous for both Hb G and α thalassaemia 1. These findings are compatible with the hypothesis that the α^G mutation occurs on a chromosome with only a single αchain locus and that the expression in heterozygotes as 46% or 33% Hb G is determined by the homologous chromosome in trans having either one or two normal α^A genes respectively. The significance of this polymorphism for chromosomes carrying αchain genes is discussed.     

A New Gγ Chain Variant: Hb F-Bron [γ20(B2)Val→Ala]

A new ^Gγ hemoglobin (Hb) variant, Hb F-Bron [γ20(B2)Val→Ala] on the first exon of the ^Gγ-globin gene is described. The variant was characterized by DNA sequencing and mass spectrometry (MS). Hematological abnormalities included hypochromia and microcytosis and were probably caused by an interaction with an α-thalassemia (thal) (3.7 kb) deletion in the heterozygous state.     

Haemoglobin E variants: a clinical, haematological and biosynthetic study of 4 South African families

The clinical, haematological and biosynthetic features of subjects with Hb E variants are described. An association with red cell hypochromia and microcytosis was confirmed, although this was not invariable in Hb E trait. Imbalanced globin chain synthesis was found in the majority of Hb E carriers. A patient doubly heterozygous for Hb E and Hb S, a condition we have not previously seen reported, had a benign clinical course with minor haematological changes, despite a relatively large amount of Hb S (67%).     

Population Screening and Prevention Strategies for Thalassemias and other Hemoglobinopathies of Eastern India: Experience of 18,166 cases

Abstract

We evaluated population screening programs (1999–2011), conducted by the Thalassaemia Foundation, Kolkata, India, for the first time in Eastern India in different districts of West Bengal, for prevention of thalassemia comprising screening of heterozygotes and β-thalassemia intermedia (β-TI) cases [β⁺, β⁺⁺, β⁰/β⁺, β^E/β^E (codon 26 or HBB: c.79G?>?A), Hb-E-β-thalassemia (Hb E-β-thal)]. Among 18,166 cases, we found 2092 heterozygotes and 2245 β-TI individuals (who had no information about their disorders). Results were evaluated with standard hematological analyses including erythrocyte indices, hemoglobin (Hb) typing and quantification. Participants were divided into five groups (children, pre-marriage cases, pre-pregnancy cases, affected family members, pregnant women). The objectives of this evaluation were to fix cut-off values of red blood cells (RBCs), mean corpuscular volume (MCV), mean corpuscular Hb (MCH), red blood cell distribution width (RDW) and Hb A₂, as the standard World Health Organization (WHO) guidelines were not strictly followed in mass-scale screening programs. We have observed many dilemmas in considering the status of the thalassemia subject, due to presence of some other clinical conditions such as iron deficiency anemia, α-thalassemia (α-thal), δ-thalassemia (δ-thal), clinically silent Hb variants, and some cases of non hemoglobinopathies (such as pregnancy) along with thalassemia. The MCV values varied greatly in different conditions of hemoglobinopathies, whereas MCH provided a more stable measurement. We found an MCH value of <27.0?pg is a suitable cut-off point for screening in this population. Participants with an MCH of <27.0?pg should be investigated further to confirm or exclude a diagnosis of β-thal trait.     

Clinical and Molecular Characteristics of Non-Transfusion-Dependent Thalassemia in Kuwait

Abstract

Although not regularly transfused, patients with non-transfusion-dependent thalassemia (NTDT) are prone to iron overload and its complications. Their molecular, phenotypical and laboratory characteristics vary in different populations and there is a need to document local prevailing patterns. We have reviewed the records of our patients with NTDT in Kuwait and documented their clinical and molecular characteristics in addition to iron status [serum ferritin and liver magnetic resonance imaging (MRI) T2*], management and complications. There were 41 patients, made up of 20 with β-thalassemia intermedia (β-TI), 18 with Hb H (β4) disease and three with Hb E (HBB: c.79G?>?A)-β-thalassemia (Hb E-β-thal); their ages ranged from 3 to 36 years (mean 12.5?±?7.7). While 18 (43.9%) had been transfused at least once, only three (7.3%) had been transfused on multiple occasions. Three patients had serum ferritin >500?ng/mL; while four of 38 had mild or moderate liver iron overload. Seven (35.0%) of the β-TI patients were managed with hydroxyurea (HU) with good response. Other complications included five patients with gallstones and one each of hypothyroidism and moyamoya. The most common mutations among the β-TI patients were IVS-II-1 (G?>?A) and IVS-I-6 (T?>?C), while among the Hb H patients, the Saudi α2-globin gene polyadenylation (polyA) (AATAAA?>?AATAAG) mutation was responsible for all cases either as homozygotes (61.1%) or compound heterozygotes with the α-thal-2 (–α^3.7) allele (33.3%). Although the pattern of NTDT in Kuwaiti patients is generally mild, there is a need to follow them to adulthood as the complications are cumulative and more prevalent in this group.