Hb A2 Variant Research Articles

Known and new hemoglobin A2 variants in Thailand and implication for β-thalassemia screening

BackgroundWe reported molecular and hematological characteristics of δ-globin chain variants and addressed diagnostic consideration of complex hemoglobinopathies caused by their interactions with α- and β-thalassemias. MethodsStudy was done on four unrelated Thai subjects with second Hb A2 fractions. Hb analysis was carried out using automated HPLC and capillary electrophoresis. Mutations were identified by DNA analysis. Novel diagnostic methods based on PCR-RFLP and allele specific PCR were developed. ResultsHb analysis revealed Hb A2 variant in all cases. DNA analysis of δ-globin gene identified the Hb A2-Melbourne [δ43(CD2)Glu→Lys] in combination with α+-thalassemia, α0-thalassemia and β0-thalassemia in the first three cases, respectively. Analysis of the remaining case identified a novel δ-Hb variant namely the Hb A2-Lampang [δ47(CD6)GAT→AAT; Asp→Asn] found in association with Hb E and α+-thalassemia. These mutations could be identified using PCR-RFLP and allele specific PCR assays developed. ConclusionsIt is necessary to recognize the Hb A2 variant and to combine the amounts of Hb A2 and Hb A2-variant for a total Hb A2 value to make better diagnostic of these complex syndromes. Co-inheritance of these multiple globin gene defects could lead to complex hemoglobinopathies requiring comprehensive Hb and molecular assessments.

Analysis of sickle hemoglobin

Background: Sickle Hemoglobin is the most common pathological hemoglobin mutation worldwide which forms sickle shape or elongated forms on deoxygenation. According to malaria hypothesis, there is resistance against malaria by the heterozygous carrier state. There is spread of Hb S allele from the highly malarial regions to most other regions worldwide. The study was conducted as Sickle Hemoglobin has been frequently seen in our institute. Materials and Methods: This study was conducted in the department of pathology, Maharajgunj medical campus, Tribhuwan University Teaching hospital, from January 2011 to January 2013. All the relevant data were obtained from the archives of the department. Hemoglobin electrophoresis was performed by Cellulose Acetate Electrophoresis at alkaline pH Method. Sickling test was done using sodium dithionite. Results: A total of 35 cases were diagnosed as sickle cell disorder. The male: female ratio was 2.5:1with the commonest age group 11-20 years (42.8%). Sixty percent showed hemoglobin between 6-8 gm/ dl. Tharu, Chaudhary and Tharu, Rana (91.3%) were the commonest ethnic group with both sickle cell anemia and trait. Lama, Neupane and Baral suffered only from trait. Mean corpuscular volume ranged from 82 to 94 fL. On peripheral blood smear reversible and irreversible sickle cells were present in 88.56 % of cases and 11.42% were devoid of sickle cells. Sickling test was positive in all cases. On cellulose acetate electrophoresis 22 (62.85%) cases had Hb S, Hb F and Hb A2 or HbS and Hb A2 where as 13(37.1%) showed Hb S, Hb A and Hb A2 variants. Conclusion: The commonest ethnic group of Tharus(91.3%), Chaudhary and Rana are from Terai region of Nepal which is a malarial zone. However 8.4% of cases were seen in other ethnic group who are from non-endemic region. DOI: http://dx.doi.org/10.3126/jpn.v3i6.8989 Journal of Pathology of Nepal (2013) Vol. 3, 437-440

Normal Hb A2 β-Thalassemia Trait: Frameshift Mutation (HBB: c.187_251dup) in Cis with the Hb A2’ δ-Globin Gene Missense Mutation (HBD: c.49G>C)

We report the case of a father and daughter who are heterozygous for a duplication of 65 bp within exon 2 of the β-globin gene, resulting in an altered and truncated β-globin chain that is predicted to be non functional. The β-globin gene mutation is in cis with the common Hb A2 ′ missense mutation of the δ-globin gene (HBD: c.49G>C), resulting in β-thalassemia (β-thal) trait with normal levels of Hb A2. This is the second report of this β0-thal mutation, and both families were associated with the Hb A2 ′ variant and normal levels of Hb A2. Laboratories should be aware of the rare occurrence of β-thal trait with normal levels of Hb A2.

Iron deficiency does not compromise the diagnosis of high HbA2 thalassemia trait

Hemoglobinopathies are the only genetic disease in which it is possible to detect carriers using hematologic findings rather than DNA analysis. Complete screening is based on the detection of red cell indices, HbA2, HbF and hemoglobin variant values. The classical phenotype of heterozygous β-

First Description of a Hb A2 Variant in Thailand. Identification of Hb A2-Melbourne [δ43(CD2)Glu→Lys] in Thai Individuals

We report the molecular and hematological identifications of a Hb A2 variant found for the first time in Thailand. The subjects were two unrelated Thai women who had normal hematological features. In the first case, hemoglobin (Hb) high performance liquid chromatography (HPLC) analysis identified, in addition to Hb A, Hb A2 (1.6%) and a minor Hb variant (0.9%) separating after Hb A2. Analysis using capillary electrophoresis (CE) demonstrated 1.3% Hb A2 and 1.0% minor variant in zone 1. DNA analysis showed a single nucleotide mutation at codon 43 of the δ-globin gene (HBD:c.130G>A) causing a glutamic acid to lysine substitution corresponding to Hb A2-Melbourne, originally documented in an Italian subject, but not previously described in Thailand. The mutation could be confirmed by allele specific polymerase chain reaction (PCR) assay. Further screening using this technique in our series identified another case of a Thai woman with a double heterozygosity for Hb A2-Melbourne and α+-thalassemia (α+-thal). A β-globin gene haplotype associated with this Thai Hb A2-Melbourne gene was also established.

An insight into the suspected HbA2' cases detected by high performance liquid chromatography in Pakistan

BackgroundHemoglobin A2' (delta 16 Gly → Arg) is globally the commonest delta chain variant of HbA2. It is clinically and hematologically silent but its sole importance lies in the underestimation of HbA2 quantity during the workup of β-thalassaemia trait. High performance liquid chromatography (HPLC) identifies it as a small S-window peak with a mean retention time of 4.59 ± 0.03 minutes. This study aims at describing the frequency of detection of HbA2' by HPLC in Pakistan and its confirmation at a molecular level. Potential HbA2' cases were identified by a retrospective review of 10186 HPLC chromatograms in year 2006. Prospective samples were collected for polymerase chain reaction (PCR) amplification, restriction digestion and nucleotide sequencing.FindingsOne hundred and ninety two potential cases (1.89%) of HbA2' were detected on HPLC, having mean retention time of 4.59 ± 0.05 minutes. Sixty four (0.6%) new cases were suspected of having co-existing β-thalassaemia trait when the quantity of S-window peaks was taken into account. Thirteen samples with presumed HbA2' on HPLC were subjected to molecular analysis and the said mutation (δ 16 GGC → CGC) was not detected in any sample.ConclusionsIt is concluded that diagnosis of HbA2' on HPLC alone is not justified, as evidence of the presence of this delta chain variant in Pakistani population is yet to be proven. Such small S-window peaks should be either disregarded or confirmed at molecular level, and only then should influence the diagnosis of β-thalassaemia trait. Further studies are required to determine the true nature of these peaks.

HbA2-Partinico or δ(A2)Pro→Thr, a new genetic variation in the δ-globin gene in cis to the β+ thal IVS-I-110 G>A, and the heterogeneity of δ-globin alleles in double heterozygotes for β- and δ-globin gene defects

The study of the alleles of the delta-globin gene is relevant to the prevention of beta-thalassemia homozygosis; in fact, the increase of the HbA2 is an invaluable hematological marker of the beta-thalassemia heterozygosis and the double heterozygosis for alleles of delta- and beta-globin genes can cause the decrease of the HbA2 up to normal or borderline values. We carried out the characterization of alleles of the delta- and beta-globin genes, restriction fragment length polymorphism (RFLP) haplotype background, and hematologic phenotype in 23 double heterozygotes belonging to 18 unrelated families. A wide heterogeneity of the delta-globin alleles was detected; seven known alleles in trans to the beta-globin gene defects were revealed in 17 out of 18 families, while a new allele in cis to a beta-thalassemia allele was detected in one family. Moreover, the relative frequency of the delta-mutants was quite different from that found among heterozygotes. The new allele delta-cod 5 CCT>ACT, in cis to the allele beta(+) thal IVS-I-110 G>A, was found in five carriers of a Sicilian family. The new variant delta5(A2)Pro-->Thr, named HbA2-Partinico upon the origin of the family, was detected with high-performance liquid chromatography; it overlapped the HbA2 peak which was partially split. The double in cis heterozygotes had increased percentage of normal and variant HbA2 of comparable size. The variant originated most likely from a new mutational event because it was associated with RFLP haplotype I, commonly found with the beta(+) thal IVS-I-110 G>A, even if crossing over or gene conversion cannot be excluded.

Molecular evidences of single mutational events followed by recurrent crossing-overs in the common δ-globin alleles in the Mediterranean area

The human δ-globin gene (HBD) is one of the β-like globin genes expressed in adults. In the Mediterranean countries the carriers of δ-thalassemia defects or Hb A2-variants are >1% and about 40/70 known alleles have been found in families with this ethnic origin. The scope of this study was to investigate the variability of the gene and of the chromosomal background in order to highlight the origin and spreading of the δ-globin gene alleles in the Mediterranean area. We carried out the characterization of the δ-globin gene alleles and of RFLP-haplotypes, SNPs and one microsatellite associated with them in 231 carriers originating principally from East Sicily. Seventeen alleles were identified, of which five were new. The chromosomes associated with mutated alleles from unrelated carriers were 158; the allele Hb A2-Yialousa accounted for about 75% of relative frequency, Hb A2-Mitsero for about 8%. The alleles were associated with RFLP 5'-haplotypes “− − − −” or “+ − + +”, prevalent in the Mediterranean area, except Hb A2-Mitsero associated with the 5'-haplotype “Benin” “− − − +” and the Hb A2' associated with “+ − − +”, both of African origin. Each allele showed linkage with one haplotype with these exceptions. The Hb A2-Yialousa showed heterogeneity of the 5'-haplotype in 2/58 chromosomes; the Hb A2-Mitsero showed SNPs and Aγ-microsatellite typical of a “Benin” haplotype found associated with the Hb C and Hb S chromosomes; the Hb A2-Yialousa (14/58 chromosomes), Hb A2-Mitsero, Hb A2-Pylos, Hb A2-Fitzroy showed heterogeneity in the 3'-haplotypes and β-globin gene SNPs. The Hb A2-Coburg was found associated with the haplotype “+ − + +/+ +” different from that already reported “− − − −/+ −”. With the exception of this last allele, the linkage of each mutation with a core of RFLPs or SNPs around or inside the δ-globin locus suggested the unicentric origin of the mutations followed by recurrent recombination events causing the chromosomal background heterogeneity.

Mutational spectrum of delta‐globin gene in the Portuguese population

The phenotype of increased Hb A2 typical of beta-thalassaemia (beta-thal) carriers can be reduced to normal or borderline values because of the co-inheritance of a delta-globin gene (HBD, MIM #142000) mutation, which may lead to misinterpretation of diagnostic results. To know the spectrum of delta-globin mutations in the Portuguese population we performed a mutational analysis of the delta-globin gene in a group of 51 Portuguese beta-thal carriers presenting microcytosis, hypochromia and a normal/borderline Hb A2 level and in another group of 15 individuals suspected to have delta-globin structural abnormalities. The heterozygosity for the beta(+)IVS-I-6T-->C (HBB:c. 92+6T>C) mutation was the main cause for the mentioned atypical beta-thal carrier phenotype. Furthermore, eight individuals were double heterozygous for one common beta-thal mutation and the delta(+)Cd27G-->T mutation (Hb A2-Yialousa; HBD:c.82G>T). One of them also presented a novel delta-globin gene promoter mutation,-80G-->A (HBD:c.-130G>A), responsible for about 25% decrease of the promoter activity in transient expression assays. One the other hand, in the other group of 15 individuals suspected to have delta-globin structural abnormalities observed by biochemical methods, some known Hb A2 variants were identified - Hb A2' (HBD:c.49G>C), Hb A2-Babinga (HBD:c.410G>A), and Hb A2-Wrens (HBD:c.295G>A), and the novel Hb A2-Fogo [delta64(E8)(Gly-->Ser); (HBD:c.193G>A)]. This novel Hb A2 variant was observed segregating in linkage with Hb E (HBB:c.79G>A) in a three generation family. In conclusion, six different delta-globin mutations were found, being two of them new molecular defects. All delta-alleles identified were found linked to the expected beta-globin cluster haplotype. All mutations caused a low Hb A2 level and through this could lead to misdiagnosis when inherited together with a beta-thal allele.

A 65 bp duplication/insertion in exon II of the -globin gene causing 0-thalassemia

We describe a patient originating from Ghana who had combined heterozygous alpha (4.2)thalassemia, alpha alpha alpha anti3.7 triplication, the common delta globin variant HbA2' and a new 65 bp duplication/insertion in exon II of the b globin gene causing beta (0)-thalassemia.

The Detection and Diagnosis of Hemoglobin A2' by High-Performance Liquid Chromatography

Hemoglobin (Hb) A2' is a hematologically silent variant of HbA2 that is detected easily by high-performance liquid chromatography (HPLC), where it elutes in the S window. Our purposes were to define diagnostic criteria for the HbA2' trait using the Variant II (Bio-Rad, Hercules, CA) and to determine the prevalence of HbA2' in a metropolitan patient population. All Hb screens (N = 5,862) performed during a 26-month period were reviewed for new hemoglobinopathies. We identified 57 cases of HbA2' trait, making it the fourth most prevalent Hb variant detected in this population after HbS, HbC, and beta-thalassemia minor For HbA2' trait cases, the mean HbA2 level was 1.7% (SD, 0.17%), and the mean HbA2' level was 1.3% (SD, 0.18%). Six possible HbA2'/beta-thalassemia double heterozygotes were identified, for whom the sum of the HbA2 and HbA2' exceeded 4% of total Hb. Hb variants that might interfere with detection of HbA2' include HbS, glycosylated HbC, and HbG2. Diagnostic criteria proposed for the HbA2' trait by HPLC are HbA2 of 2% or less, S window peak of 1% to 2%, no previous diagnosis of HbS, and absence of HbG and HbC.

Two New δ-Globin Mutations: Hb A2-Ninive [δ133(H11)Val → Ala] and A δ+-Thalassemia Mutation [− 31 (A → G)] in the Tata Box of the δ-Globin Gene

Two new δ-globin gene mutations have been detected: one leads to a fairly stable Hb A2 variant differing electrophoretically only minimally from normal Hb A2, and the second causes a δ+-thalassemia (thal) phenotype.

Interaction of erythroid spectrin with hemoglobin variants: implications in β-thalassemia

Among the few studies, producing contradictory results, done on the interaction of erythroid membrane skeletal spectrin with hemoglobin (Hb), none has been able to provide a quantitative estimate of the association of spectrin with Hb. In this work, studies on the interactions of erythroid spectrin with Hb have been elaborated upon using a novel fluorescence technique. The concentration-dependent change in the fluorescence intensity of fluorescein-conjugated spectrin (F-spectrin) in presence of oxy-Hb indicated binding with a dissociation constant of ∼20 μM that has been directly evaluated from the increase in the extent of quenching of the fluorescein fluorescence of F-spectrin by reverse titration with the increasing concentrations of different Hb samples isolated from both normal and β-thalassemic patients. The Hb compositions, with major components of the normal HbA, the fetal HbF, and the variant HbA2, of each individual were estimated using the Variant HPLC device of Bio-Rad. Results of the present study indicated that the dissociation constant, K d, of spectrin binding to Hb decreased from 19.5 ± 2 μM in normal individuals to of 6.5 ± 0.5 μM in the presence of 73% HbA2 along with coeluted variants in the blood samples of patients suffering from β-thalassemia, indicating differential interactions of the Hb variants with spectrin.

Origin of Hb A2′ (Hb B2) [δ16(A13)Gly→Arg (GGC→CGC)

On a field trip to the Dogon country (le Pays Dogon) in central Mali, we detected a high frequency of the Hb A2 abnormality, reaching higher numbers among blacksmiths (up to 12.4%) living in the same villages. In this report, by direct nucleotide sequencing and employing a polymerase chain reaction‐restriction fragment length polymorphism approach, we show that the Hb A2 variant observed in the Dogon population is indeed Hb A2′, also called Hb B2, and that in all of the cases the abnormal δ‐globin gene is linked to a unique haplotype. The same haplotype was found linked to Hb A2′ in the Herero population belonging to the South African Bantu‐speaking Blacks from Namibia. Although the unique origin of this mutation in Africa is a possibility, a recurrent mutational event cannot be excluded because the linked β cluster haplotype is one of the two major haplotypes found in all African populations. A study of populations from other regions of Africa is required to clarify this issue.

Hb A2-MONREALE [δ146(HC3)His → Arg], A NOVEL δ CHAIN VARIANT DETECTED IN WEST SICILY

We report a novel mutation, Hb A2-Monreale [δ146(HC3)His → Arg], detected by cation exchange high performance liquid chromatography in a family from West Sicily. The mutation is due to a CA T → CGT substitution at codon 146 of the δ-globin gene. The two carriers had reduced levels of normal Hb A2 (1.1%), but comparable levels (0.9%) of the Hb A2 variant. Most likely the new variant has the same characteristics as Hb Cochin-Port Royal [β146(HC3)His → Arg], that is stable but has a 75% reduction of the Bohr effect. The finding of the new variant increases the genotype heterogeneity of the δ-globin gene in the Mediterranean area, and is relevant to the study and prevention of Cooley's Anemia.

Expression Studies of δ-Globin Gene Alleles Associated with Reduced Hemoglobin A2 Levels in Greek Cypriots

We previously identified five delta-globin gene alleles associated with reduced hemoglobin (Hb) A2 (Trifillis, P., Ioannou, P., Schwartz, E., and Surrey, S. (1991) Blood 78, 3298-3305). We have now evaluated functional consequences of the changes after expression in COS-1 cells to monitor effects on RNA splicing. In addition, variant Hb A2 tetramers were expressed in yeast to assess effects of amino acid changes on oxygen binding and stability to heat and mechanical agitation. The G --> T change at codon 27 and the A --> G change in IVS-2 both affect RNA splicing, whereas the C --> T change at codon 97 and the AT deletion in IVS-2 have no effect. Oxygen equilibrium curves of the Hb A2 variants expressed in yeast were similar to that of wild type Hb A2. None of the three variant Hb A2 tetramers (Thr --> Ile at codon 4 (Hb deltaT4I), Ala --> Ser at codon 27 (Hb deltaA27S), and Arg --> Cys at codon 116 (Hb deltaR116C)) showed decreased heat stability compared with Hb A2, whereas the Hb deltaT4I variant showed highest instability to mechanical agitation. Co-expression in yeast of alpha-globin chain and the delta-chain variant containing a Leu --> Pro change at codon 141 yielded no identifiable tetramers, suggesting lack of assembly or severe tetramer instability. These studies show the probable cause for decreased Hb A2 for two alleles is due to defective splicing, whereas decreased protein stability, increased tetramer association with red cell membranes, increased interdisulfide bond formation of delta-chains, which inhibits assembly with alpha-chains, and/or reduced assembly is suggested for the other three alleles.

Polymerization of Three Hemoglobin A2 Variants Containing Valδ6 and Inhibition of Hemoglobin S Polymerization by Hemoglobin A2

To understand determinants for hemoglobin (Hb) stability and Hb A2 inhibition of Hb S polymerization, three Valdelta6 Hb A2 variants (Hb A2 deltaE6V, Hb A2 deltaE6V,deltaQ87T, and Hb A2 deltaE6V, deltaA22E,deltaQ87T) were expressed in yeast, and stability to mechanical agitation and polymerization properties were assessed. Oxy forms of Hb A2 deltaE6V and Hb A2 deltaE6V,deltaQ87T were 2- and 1.6-fold, respectively, less stable than oxy-Hb S, while the stability of Hb A2 deltaE6V,deltaA22E,deltaQ87T was similar to that of Hb S, suggesting that Aladelta22 and Glndelta87 contribute to the surface hydrophobicity of Hb A2. Deoxy Hb A2 deltaE6V polymerized without a delay time, like deoxy Hb F gammaE6V, while deoxy Hb A2 deltaE6V,deltaQ87T and deoxy Hb A2 deltaE6V,deltaA22E,deltaQ87T polymerized after a delay time, like deoxy Hb S, suggesting that beta87 Thr is required for the formation of nuclei. Deoxy Hb F gammaE6V,gammaQ87T showed no delay time and required a 3.5-fold higher concentration than deoxy Hb S for polymerization, suggesting that Thr effects on Valdelta6 Hb A2 and Valgamma6 Hb F variants are different. Mixtures of deoxy Hb S/Hb A2 deltaE6V,deltaQ87T polymerized, like deoxy Hb S, while polymerization of Hb S/Hb A2 deltaE6V mixtures was inhibited, like Hb S/Hb F gammaE6V mixtures. These results suggest alpha2betaSdelta6 Val, 87 Thr hybrids and Hb A2 deltaE6V,deltaQ87T participate in Hb S nucleation, while only 50% of alpha2betaSdelta6 Val hybrids and none of the Hb A2 deltaE6V participate. These findings are in contrast to those of mixtures of Hb S with Hb F gammaE6V or Hb F gammaE6V,Q87T, which both inhibit Hb S polymerization. Our results also suggest participation in nucleation of some alpha2betaSdelta hybrids in A2S mixtures but not alpha2betaSgamma hybrids in FS mixtures.

Hb A2-Manzanares or alpha 2 delta 2 121 (GH4) Glu replaced by Val, an unstable delta chain variant observed in a Spanish family.

A slowly moving variant of Hb A2 was present in a 25-year-old Spanish woman and her mother, and was found to have a Glu replaced by Val substitution at position 121 of the delta chain. The variant was unstable and precipitated gradually during anion exchange chromatography. The effect of this substitution on the stability of the protein is apparently more severe for Hb A2 than for Hb A because Hb Beograd (beta 121 Glu replaced by Val) has normal physicochemical properties.

Hemoglobin NYU, a delta chain variant, alpha 2 delta 2 lys.

A minor hemoglobin (Hb) component with the electrophoretic properties of the delta-chain variant Hb A(2') was encountered in two unrelated families of Russian-Jewish ancestry. This minor component, designated Hb NYU, was shown to result from the substitution of lysine for asparagine at delta(12). We have confirmed studies of others that hemoglobin A(2') isolated from the hemoglobin of some African subjects, results from the replacement of the normal glycine at delta(16) by arginine. Thus for interpretations of the incidence of delta-chain variants in different populations, electrophoretic data are not sufficient. In members of one of the families in the present study, the visual estimations of normal Hb A(2) and of Hb NYU on starch-gel electrophoretic patterns suggested the presence of delta-thalassemia. In hemolysates of one of the heterozygotes for Hb NYU, hemoglobin A(2) was not demonstrable with starch-gel electrophoretic methods but was readily recovered by column chromatography in approximately the amounts expected for delta-chain heterozygotes.

OXYGEN EQUILIBRIA OF RED CELL HAEMOLYSATES OF ADULT INDIVIDUALS, HETEROZYGOUS FOR DIFFERENT RARE ABNORMAL HAEMOGLOBINS.

IN the past few years, oxygen equilibria of various normal and abnormal adult human haemoglobin types have been studied. No differences were observed between normal Hb-A (&α2β2) and the β-chain abnormalities Hb-S (ref. 1), Hb-C (ref. 2) and Hb-E (ref. 3). Investigations of the isolated Hb-A2′ (α2δ2) and its abnormal variant Hb-A2 revealed a considerable increase in oxygen affinity when compared with normal Hb-A (ref. 4). Hb-Lepore, which is composed of normal α-chains and ‘β’ chains being related to the β-chain of Hb-A as well as to the δ-chain of Hb-A2 (ref. 5), showed a high affinity for molecular oxygen similar to that of Hb-A2 (ref. 1). The affinities of the α-chain lacking abnormal haemoglobin types Hb-H (β4) and Hb-Bart's (γ4) for molecular oxygen were found to be extremely high, while no Bohr effect or haem–haem interactions were detectable6,7.