Nondeletional Hb Queens Park [α32(B13)Met→Lys]/Hb H (β4) disease

A rare nondeletional α-thalassemia-2 (α-thal-2) allele was identified in a Thai boy with Hb H (β4) disease. The proband has α-thal-1 (- -(SEA) type) together with a non productive Hb Queens Park (HBA1:c.98T>A) [α32(B13)Met→Lys] α1-globin variant. No abnormal hemoglobin (Hb) fraction was detected by high performance liquid chromatography (HPLC). The clinical effect of this mutation in the proband was comparable to that of deletional α-thal-2 present in Hb H disease.     

Five Variants of the β-Globin Gene without Clinical or Hematological Effects: Hb Maryland [β47(CD6)Asp→His], Hb Kent [β37(C3)Trp→Cys], Hb Visayan [β136(H14)Gly→Cys], Hb Cutlerville [β138(H16)Ala→Val] and Hb Hornchurch [β43(CD2)Glu→Lys]

We report on five hemoglobin (Hb) β chain variants that were initially identified either by electrophoretic, chromatographic or isoelectric focusing (IEF) methods. These variants do not appear to be associated with clinical or hematological abnormalities. All variants were confirmed by DNA sequence analysis.     

Coinheritance of High Oxygen Affinity Hb Helsinki [HBB: c.248A>T; β82(EF6)Lys→Met] with Hb H Disease

Hb Helsinki [HBB: c.248A>T; β82(EF6)Lys→Met] is a high oxygen affinity hemoglobin (Hb) causing polycythemia, whereas Hb H (β4) disease causes mild to severe chronic hemolytic anemia. The clinical characteristics, gel electrophoresis, capillary electrophoresis (CE) and molecular genotyping of a case of Hb Helsinki coinherited with Hb H disease in an ethnic Malay is described, illustrating the interaction between the β-globin variant and coinheritance of three α gene deletions. The proband was asymptomatic, exhibited microcytosis and a normal with Hb value.     

Hb H (β4) Disease in Çukurova,Southern Turkey

In this study, 32 patients with Hb H (β₄) disease have been identified. Three different α-thalassemia-1 (thal) determinants; nine with the ?17.4 kb (MED I) type, 12 with the ?20.5 kb type and 10 with the ?26.5 kb (MED II) type were characterized. Of the 32 patients, 19 had the 3.7 kb deletion and one had the 4.2 kb deletion in trans to α-thal-1 determinants. Only one patient, homozygous for the polyadenylation signal (poly A) site (PA 1) mutation, was identified to be associated with Hb H disease. The other patient had the poly A (PA 1) mutation in trans to the MED I (?17.4 kb) determinant. The 5 nt (nucleotide) deletion was present in three patients, two of them in the same family; this mutation was found in association with the MED II (26.5 kb deletion). The other patient had the ?5 nt mutation in trans to the MED I (?17.4 kb) determinant. An unstable hemoglobin (Hb) variant [Hb Adana, codon 59 (C→A)] was present in association with the α‐thal-1 deletion (20.5 kb) in two adults and caused a severe type of Hb H disease. Five patients with Hb H disease had the genotype – –^{MED II}/α^{PA 2}α one had a Hb S heterozygosity (– –^{MED II}/α^{PA 2}α + Hb AS). A patient with Hb H disease (– –^{MED I}/?α^3.7) also had Hb S trait.     

Description of Two New α Variants: Hb Canuts [α85(F6)Asp→His (α1)] and Hb Ambroise Pare [α117(GH5)Phe→Ile (α2)]; Two New β Variants: Hb Beaujolais [β84(EF8)Thr→Asn] and Hb Monplaisir [β147 (Tyr-Lys-Leu-Ala-Phe-Phe-Leu-Leu-Ser-Asn-Phe-Tyr-158-COOH)] and One New δ Variant: Hb A2-North Africa [δ59(E3)Lys→Met]

We present here five new hemoglobin (Hb) variants which have been identified during routine Hb analysis before their genotypic characterization. Four of these result from a classical missense mutation: Hb Canuts [α85(F6)Asp→His (α1)], Hb Ambroise Pare [α117(GH5)Phe→Ile (α2)], Hb Beaujolais [β84(EF8)Thr→Asn] and HbA₂-North Africa [δ59(E3)Lys→Met]. The last one, Hb Monplaisir [β147 (Tyr-Lys-Leu-Ala-Phe-Phe-Leu-Leu-Ser-Asn-Phe-Tyr-158-COOH)], results from a frameshift mutation at the stop codon of the β-globin gene which leads to a modified C-terminal sequence in the β-globin chain. None of these variants seem to have a particular clinical expression in the heterozygous state. The circumstances of the discovery of these five new Hb variants emphasize the fact that an association of techniques is necessary for a complete screening of Hb variants during routine Hb analysis. Globin chain separation by reversed phase liquid chromatography (RP‐LC) appears to be the most relevant method.     

Complex Interaction of Hb E [β26(B8)Glu→Lys], Hb Korle-Bu [β73(E17)Asp→Asn] and a Deletional α-thalassemia-1 in Pregnancy

A pregnant Thai woman with mild hypochromic microcytic anemia caused by α- and β- globin defects is described. The proband was a 26-year-old pregnant woman discovered through our ongoing thalassemia screening program. Initial hemoglobin (Hb) high performance liquid chromatography (HPLC) analysis revealed a homozygosity for an unknown variant at the D window, inconsistent with results of family analyses. Further Hb analysis using automated capillary zone electrophoresis identified that the proband was in fact a compound heterozygote for Hb E [β26(B8)Glu→Lys, GAG>AAG] and another β chain variant. DNA analysis demonstrated that she carried the Hb Korle-Bu mutation [β73(E17)Asp→Asn (GAT>AAT)] in trans to the Hb E and an α-thalassemia-1 (α-thal-1) with the Southeast Asian (? ?^SEA) deletion. Family studies identified that her father and sister were double heterozygotes for Hb Korle-Bu and α-thal-1, whereas her mother was a double heterozygote for Hb E/Hb Constant Spring [Hb CS; α142, Term→Gln (TAA>CAA in α2)]. The genotype-phenotype relationship observed in this Thai family with complex hemoglobinopathies and methods for characterization are presented.     

Hb Koln [β98(FG5) [GTG → ATG,Val → Met]: The first report from India

Background

The group of unstable hemoglobins are associated with congenital non-spherocytic hemolytic anemia due to instability of the hemoglobin molecule. They often lead to formation of the characteristic inclusion bodies or Heinz bodies.

Aim

To identity the cause of mild anemia, reticulocytosis, and hepatosplenomegly in a case of non-spherocytic hemolytic anemia.

Materials and methods

A 34-year-old female patient originating from Maharashtra, western India presented with mild anemia and jaundice which had persisted since childhood. Investigations included a complete blood count, screening for red cell membrane protein defects, Hb analysis by high-performance liquid chromatography (HPLC) and cellulose acetate electrophoresis (pH 8.9), heat instability test and DNA sequencing.

Results

Hemoglobin analysis by HPLC showed an abnormal peak in the Hb C window (9.8%) with a retention time of 4.90 minutes. Cellulose acetate electrophoresis (pH 8.9) showed a slow moving band (6.15%) between Hb A₂ and Hb S. The heat instability test was positive. DNA analysis of α globin genes showed absence of both deletional and non- deletional α thalassemia. DNA sequencing of the β globin gene revealed heterozygosity for a mutation at codon 98 [GTG → ATG, Val → Met], which gives rise to Hb-Koln.

Conclusion

Hb Koln is the commonest unstable Hb variant reported from many populations in the world. However, this is the first report of this unstable Hb variant from India.     

Characterization of a new hemoglobin variant: Hb Badalona (β31[B13]Leu→Val)

Hemoglobin (Hb) Badalona was identified in a 35-year-old Spanish female and two other family members. All affected subjects presented erythrocytosis and increased oxygen affinity (P(50): 21 mmHg). Hemoglobinopathy was not detected with electrophoretic methods. It was, however, separated and quantified by cation exchange and reverse-phase high-performance liquid chromatography. Hb Badalona accounted for 35% of the total Hb. No significant clinical symptoms were found to be related to this hemoglobinopathy. This is the first case of a Leu-->Val replacement at position beta31(B13) reported in the world literature.     

Hb I-High Wycombe [β59(E3)Lys→Glu]: The First Instance in Japan

15,661 cord bloods from Jamaican infants were examined for abnormal hemoglobins wine alkaline cellulose acetate electro-phoresis for the initial screening, supplemented by acid agar gel electrophoresis for samples exhibiting abnormal hemoglobin bands. Of the 16 electrophoretic variants which were detected, six were fully characterized and found to be: four Hb F Port Royal (α₂^Gγ₂125 Glu→Ala) and two Hb F Victoria Jubilee (α₂^Aγ₂80 Asp→Tyr). The Hb F Port Royal samples each constituted about one eighth of the total Hb F as did seven additional samples presumed to be Hb F Port Royal. The infants with this variant exhibited no special hematological characteristics or other consistent associations. Both Hb F Victoria Jubilee samples occurred in somewhat lower proportions of the total Hb F compared with Hb F Port Royal and exhibited an apparent increase of free alpha chains in the whole hemolysate. The data available on detectable gamma chain variants suggest that a specific point mutation may occur in either a Hb_Gγ or a Hb_Aγ locus.     

Complex interaction of hemoglobin (Hb) Nakhon Ratchasima [α63(E12)Ala→Val], a novel α2-globin chain variant with Hb E [β26(B8)Glu→Lys] and a deletional α(+)-thalassemia

Objectives: To describe the hematological and molecular features as well as diagnostic aspects of a complex hemoglobinopathy caused by interaction of a novel α2‐globin chain variant with hemoglobin (Hb) E and α⁺‐thalassemia. Methods: Blood specimen of a 41‐yr‐old Thai man was transferred to our center for the analysis of unknown Hb variant. Hb analysis was carried out using automated high‐performance liquid chromatography (HPLC) and capillary electrophoresis system. Mutation was identified by PCR and related techniques. Results: RBC analysis revealed a mild anemia but blood indices were within normal ranges. Hb‐HPLC analysis demonstrated, in addition to the Hb E and Hb A, two abnormal peaks not fully separated from Hb A and Hb E, but capillary electrophoresis showed a pattern of Hb E heterozygote with 4.0% Hb A₂. DNA analysis of the α2 globin gene identified a novel mutation (namely Hb Nakhon Ratchasima), GCC (Ala)→GTC (Val) at codon 63 in trans to the α⁺‐thalassemia (3.7‐kb deletion). Association of this novel α‐chain variant with β^E globin chain leads to the formation of another novel Hb derivative with different HPLC characteristics. Conclusion: Although Hb Nakhon Ratchasima might be clinically innocuous, differential diagnosis from other clinically relevant hemoglobinopathies is essential in routine setting. This could be made by using a simple PCR–restriction fragment length polymorphism assay or allele‐specific PCR assay developed in this study.     

IDENTIFICATION OF Hb VILLEJUIF [β123(H1)Thr→Ile] IN SOUTHERN ITALY

Hb Villejuif [β123(H1)Thr→Ile] is a silent and asymptomatic variant described in 1989 in an 87-year-old woman of French origin suffering from coincidental polycythemia vera. This paper reports the second observation of Hb Villejuif in three related subjects from Montesarchio, Southern Italy. All routine techniques for hemoglobin analysis yielded normal results with the exception of a slight increase in the Hb A₂ value. The occurrence of a variant β-globin was rapidly assessed by liquid chromatography mass spectrometric analysis and the abnormal chain purified by high performance liquid chromatography. The amino acid replacement Thr→Ile at β123 was determined by tandem electrospray mass spectrometric analysis of the tryptic digest of the variant β chain. The corresponding DNA mutation was established as C→T at the second position of codon 123 (ACC → ATC) by polymerase chain reaction amplification techniques.     

Compound Heterozygosity for Hb S [β6(A3)Glu→Val,GAG→GTG] and a New Thalassemic Mutation [β132(H10)Lys→Term,AAA→TAA] Detected in a Family from West Africa

We describe a Hb S/β-thalassemia (β-thal) mutation involving an A→T transition at codon 132 of the β-globin gene. The mutation, in the heterozygous state, unlike several other mutations in exon 3, shows no signs of dominant thalassemia but those of a typical β⁰ carrier. Compound heterozygosity with Hb S [β6(A3)Glu→Val, GAG→GTG] showed a severe clinical picture.     

Hb Extremadura [β64(E8)Gly→Ser;β133(H11)Val→Leu]: a new molecular analysis

Unstable hemoglobin (Hb) variants account for 9.5% of structural hemoglobinopathies. The majority of these unstable variants are the result of gene point mutations resulting in the substitution of a single amino acid by another. The presence of two mutations in the same allele is infrequent: of the 781 variants of the β-globin cluster described, only 32 are due to two point mutations (4.1%). Hb Extremadura is a structural variant that is included within the so-called unstable Hb anomalies. It was first described in 1989, employing the most up-to-date techniques available at that time, reversed phase high performance liquid chromatography (HPLC) to separate the abnormal chain (β(X)) digesting it with trypsin and analysis of the fragments with an automatic analyzer.     

Low oxygen saturation and severe anemia in compound heterozygous Hb Louisville [β42(CD1)Phe→Leu] and Hb La Desirade [β129(H7)Ala→Val]

Objective: To investigate the cause(s) of a Thai male proband presenting low oxygen saturation by pulse oximetry (SpO₂) and severe anemia.

Methods: As Hb variant was suspected, Hb typing was determined by high-performance liquid chromatography and capillary electrophoresis, and subsequently Hb variant was identified by DNA sequencing. Complete blood counts were performed using automated blood cell counter and oxygen saturation was measured by pulse oximetry.

Results: Proband was compound heterozygous for Hb Louisville [β42(CD1)Phe→Leu] and Hb La Desirade [β129(H7)Ala→Val]. Of the proband’s two sons, one was compound heterozygous for Hb Louisville and Hb E and the other for Hb La Desirade and Hb E. The former son had similar clinical features and laboratory findings with those of the proband while the latter showed had no abnormal clinical manifestations.

Conclusion: This the first report of compound heterozygosity of Hb Louisville and Hb La Desirade in an individual of Southeast Asian ethnicity. Hb variant identification is crucial for genetic counseling and appropriate treatment in regions where hemoglobinopathies are common.     

Hb Sarrebourg [β131(H9)Gln→Arg, CAG>CGG] in Turkey

We describe Hb Sarrebourg [β131(H9)Gln→Arg, CAG>CGG] in four heterozygous members of a Turkish family. It was associated with iron deficiency in the proband.     

Hb Taradale [β82(EF6)Lys→Arg]: A Novel Mutation at a 2,3-Diphosphoglycerate Binding Site

Hb Taradale [β82(EF6)Lys→Arg] was initially detected as a split Hb A₀ peak on Hb A_1c monitoring. Red cell parameters, hemoglobin (Hb) electrophoresis and stability tests were normal. Mass spectrometry (ms) clearly identified a variant β chain with a mass increase of 28 Da and peptide mapping located the mutation site to peptide βT-9. DNA sequencing confirmed the presence of a novel β82(EF6)Lys→Arg mutation. This conservative substitution at a 2,3-diphosphoglycerate (2,3-DPG) binding site did not, however, appear to affect the P₅₀ for oxygen binding.     

A new α1-globin mutation, Hb Brugg [α20(B1)His→Gln

A 2?-year-old male child and a 23-year-old woman with no clinical symptoms were investigated during routine consultations. Cation exchange high performance liquid chromatography (HPLC) revealed an additional peak eluting before Hb A. DNA sequencing showed a novel heterozygous mutation at codon 20 of the α1-globin gene. The hemoglobin (Hb) variant was named Hb Brugg. Analysis of oxygen affinity Hb and Hb stability did not show any changes compared to normal Hb constellation.     

Hb Clinico-Madrid [α90(FG2)Lys→Alg]: a New Hemoglobin Mutation in the α2-Globin Gene

High performance liquid chromatography was compared with carboxymethyl cellulose column chromatography data for analysis of globin chain composition and biosynthetic ratios in fetal blood. A formula was derived for mathematical correction for losses of small amounts of globin protein on the high performance liquid chromatography column. Radioactive globin chain ratios by high performance liquid chromatography are equivalent to those by carboxymethyl cellulose column chromatography, but only when carrier Hb A is added. We conclude that high performance liquid chromatography provides a suitable alternative to carboxymethyl cellulose columns, and has several advantages.     

The Mutation of Hb Turriff [α99(G6)Lys→Glu (AAG→GAG)] Is Carried by the α1‐Globin Gene in a Japanese (Hb Turriff‐I)

The forms and severity of cardiac complications were investigated in patients with asymptomatic thalassemia intermedia and thalassemia major by M‐mode, bi‐dimensional echocardiography (ECHO) and echo‐Doppler. Twenty‐eight patients of both sexes with β‐thalassemia intermedia (β‐TI), mean age 23.2 ± 6.3 years, untransfused or minimally transfused, were compared to 42 age‐ and sex‐matched subjects with thalassemia major, who were regularly treated with hemotransfusive therapy [pre‐transfusion hemoglobin (Hb) values 9.5 ± 0.9 g/dL] and iron chelation. All patients were splenectomized. Age and sex matched healthy control subjects were randomly selected. β‐Thalassemia major (β‐TM) patients showed a marked reduction in contractile state and a milder left ventricular (LV) enlargement than β‐TI patients. Cardiac output (CO) and cardiac index (CI) were increased in both groups of patients but appeared significantly higher in β‐TI patients with consequent altered LV diastolic function indices. In addition, β‐TI patients had reduced indices of pulmonary artery flow related to long‐term chronic anemia rather than iron overload. The progressive rise in CO and CI casts doubts on the current management of β‐TI syndromes.