Abnormal Beta Chain Research Articles

Erythrocyte microRNAs: a tiny magic bullet with great potential for sickle cell disease therapy.

Sickle cell disease (SCD) is a severe hereditary blood disorder caused by a mutation of the beta-globin gene, which results in a substantial reduction in life expectancy. Many studies are focused on various novel therapeutic strategies that include re-activation of the γ-globin gene. Among them, expression therapy caused by the fetal hemoglobin (HbF) at a later age is highly successful. The induction of HbF is one of the dominant genetic modulators of the hematological and clinical characteristics of SCD. In fact, HbF compensates for the abnormal beta chain and has an ameliorant effect on clinical complications. Erythropoiesis is a multi-step process that involves the proliferation and differentiation of a small population of hematopoietic stem cells and is affected by several factors, including signaling pathways, transcription factors, and small non-coding RNAs (miRNAs). miRNAs play a regulatory role through complex networks that control several epigenetic mechanisms as well as the post-transcriptional regulation of multiple genes. In this review, we briefly describe the current understanding of interactions between miRNAs, their molecular targets, and their regulatory effects in HbF induction in SCD.

DENATURING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY: AN EFFICIENT SCREENING APPROACH IN THE GENETIC DIAGNOSIS OF HEMOGLOBIN HAMMERSMITH

In this study we here present the first report of the detection of the rare beta-thalassemia mutation in the Taiwanese population: Hemoglobin Hammersmith (Hb Hammersmith). The mutation was detected by denaturing high-performance liquid chromatography (DHPLC) screening followed by automated direct sequencing. The mutation was found in an affected woman and her immature female fetus in the heterozygous state. Molecular abnormality of Hb Hammersmith results from an abnormal beta chain with an amino acid substitution (condon 42, TTT→TCT, Phe→Ser) in the beta-globin (HBB) gene with the clinical presentation of hemolytic anemia. Given known wide spectrum of beta-thalassemia alleles in the Taiwanese population, the present report further confirmed the high heterogeneity rate. This result indicated that the importance of the efficient screening approach by DHPLC for genetic diagnosis in beta-thalassemia.

EDMA 2000 as a matrix for high-performance liquid chromatography of human haemoglobin chains.

An ethylenedimethacrylate polymer-based matrix (EDMA 2000) is described that shortens the time for separation of haemoglobin (Hb) chains to 30 min, compared with the use of C4 large-pore Vydac columns, commonly used for reversed-phase HPLC of Hb variants. EDMA 2000 was successfully used for the isolation of an abnormal beta-chain of the unstable Hb recently characterized as Hb Nottingham.

Structure of the human adult hemoglobin minor fraction A1b by electrospray and secondary ion mass spectrometry. Pyruvic acid as amino-terminal blocking group

Hemoglobin A1b is a minor hemoglobin component from human hemolysate (less than 0.5% of total hemoglobin) whose structure has never been established. It was purified and studied by mass spectrometry. Electrospray ionization of its abnormal beta-chain indicated a 70-Da mass increase. Separation of the trytic digest by reversed-phase liquid chromatography revealed an abnormal beta T1 peptide. Cesium ion bombardment ionization produced a protonated molecular ion at m/z 1022.516, showing an additional C3H2O2 residue to normal beta T1. The amino acid sequences of both abnormal and normal beta T1 peptides were found identical by comparison of their collision activation spectra. Time course hydrolysis of abnormal beta T1 indicated a rapid loss of the modifying group, leading to normal beta T1. At least, mild treatment with acidic methanol showed an additional methylated site, comparatively with normal beta T1. All these results are consistent with a ketimine-linked pyruvic acid at the amino end of the beta-chain of hemoglobin.

Hb Isehara (or Hb Redondo) [β92(F8)HIS→SN]: An Unstable Variant with a Proximal Histidine Substitution at the Heme Contact

A 50-year-old Japanese female patient was found to have hemolytic anemia. Isoelectrofocusing of her hemolysate revealed two abnormal hemoglobin bands, one of which was very close to the Hb A2 band, and the other between the Hb A2 and Hb F bands. CM-cellulose column chromatography of the globin prepared from the abnormal hemoglobin showed that the abnormal chain eluted faster than the normal beta and delta chains; the beta X chain, however, did not separate from the normal beta chain in urea cellulose acetate electrophoresis. An instability test of the patient's hemolysate revealed the presence of an unstable component. Structural analysis of the abnormal beta chain indicated that the histidine residue at beta 92(F8) was replaced by an asparagine or aspartic acid residue. DNA amplified by polymerase chain reaction was sequenced by the dideoxy method. The nucleotide sequence of the beta 92 codon was AAC instead of CAC, suggesting that the amino acid substitution corresponded to His----Asn, which is the same as is found in Hb Redondo or beta 92(F8)His----Asn----Asp.

A third instance of the high oxygen affinity variant, HB heathrow [β103(G5)phe→leu]: Identification of the mutation by mass spectrometry and by DNA analysis

Hb. Heathrow [beta 103(G5)Phe- greater than Leu] was identified in an Englishman with a life-long history of polycythemia, his father had been similarly affected. A hemoglobin variant was suspected from the high oxygen affinity of the patient's blood. The Hb Heathrow abnormal beta chain was resolved from the normal beta chain by high performance liquid chromatography, and the abnormal peptide and the amino acid replacement were identified by mass spectrometry. The corresponding base change (C- greater than G at codon 103) was demonstrated by sequence analysis of the polymerase chain reaction amplified exon 2 of the genomic beta-globin gene. This is only the third known instance of Hb Heathrow.

HB Yokohama [β31(B13)LEU→PRO] Detected AS A de novo Mutation in a Yugoslavian Boy

Hb Yokohama [beta 31 (B13)Leu----Pro] was observed in a young Yugoslavian boy as a de novo mutation. The child exhibited severe transfusion-dependent hemolytic anemia. The variant was detected and quantitiated at 10.5% by reversed phase high performance liquid chromatography. In vitro globin chain synthesis showed a slight imbalance with an alpha/beta ratio of 1.38. Structural characterization of the abnormal beta chain was done by high performance liquid chromatographic analysis, on material obtained by high salt precipitation. The mutation was confirmed by sequencing of the amplified DNA.

HB Mizuho [β68(E12)LEU-PRO]. Second Occurrence Identified in A Caucasian Child with Hemolytic Anenia and Dense Erythrocyte Inclusions

Hb Mizuho [beta 68(E12)Leu----Pro] was identified in a child of Italian/Sicilian descent who exhibited severe, transfusion dependent hemolytic anemia which improved following splenectomy. The patient's peripheral blood smear, which prior to splenectomy demonstrated coarse erythrocytic basophilic stippling, showed large, dense erythrocytic hemoglobin inclusions following splenectomy. Whole blood oxygen equilibrium results were consistent with the presence of a hemoglobin component exhibiting increased oxygen affinity with decreased cooperativity. The abnormal beta chain was characterized by high performance liquid chromatography analysis of the isopropanol precipitable hemoglobin fraction.

Molecular characterization of beta-globin gene mutations in Malay patients with Hb E-beta-thalassaemia and thalassaemia major.

This study concerned the identification of the beta-thalassaemia mutations that were present in 27 Malay patients with Hb E-beta-thalassaemia and seven Malay patients with thalassaemia major who were from West Malaysia. Nearly 50% of all beta-thalassaemia chromosomes carried the G----C substitution at nucleotide 5 of IVS-I; the commonly occurring Chinese anomalies such as the frameshift at codons 41 and 42, the nonsense mutation A----T at codon 17, the A----G substitution at position -28 of the promoter region, and the C----T substitution at position 654 of the second intron, were rare or absent. Two new thalassaemia mutations were discovered. The first involves a frameshift at codon 35 (-C) that was found in two patients with Hb E-beta zero-thalassaemia and causes a beta zero-thalassaemia because a stop codon is present at codon 60. The second is an AAC----AGC mutation in codon 19 that was present on six chromosomes. This substitution results in the production of an abnormal beta chain (beta-Malay) that has an Asn----Ser substitution at position beta 19. Hb Malay is a 'Hb Knossos-like' beta +-thalassaemia abnormality; the A----G mutation at codon 19 likely creates an alternate splicing site between codons 17 and 18, reducing the efficiency of the normal donor splice site at IVS-I to about 60%.

Characteristics in tyrosine coordinations of four hemoglobins M probed by resonance Raman spectroscopy.

Resonance Raman spectra of four hemoglobins (Hbs) M with tyrosinate ligand, that is, Hb M Saskatoon (beta distal His----Tyr), Hb M Hyde Park (beta proximal His----Tyr), Hb M Boston (alpha distal His----Tyr), and Hb M Iwate (alpha proximal His----Tyr), were investigated in order to elucidate structural origins for distinctly facile reducibility of the abnormal subunit of Hb M Saskatoon in comparison with other Hbs M. All of the Hbs M exhibited the fingerprint bands for the Fe-tyrosinate proteins around 1600, 1500, and 1270 cm-1. However, Hb M Saskatoon had the lowest Fe-tyrosinate stretching frequency and was the only one to display the Raman spectral pattern of a six-coordinate heme for the abnormal beta subunit; the others displayed the patterns of a five-coordinate heme. The absorption intensity of Hb M Saskatoon at 600 nm indicated a transition with a midpoint pH at 5.2, whereas that of Hb M Boston was independent of pH from 7.2 to 4.8. The fingerprint bands for the tyrosinate coordination as well as the Fe-tyrosinate stretching band disappeared for Hb M Saskatoon at pH 5.0, and the resultant Raman spectrum resembled that of metHb A, while those bands were clearly observed for Hb M Boston at pH 5.0 and for two Hbs M at pH 10.0. These observations suggest that the unusual characteristics of the heme in the abnormal beta chain of Hb M Saskatoon result from the weak Fe-tyrosinate bond, which allows weak coordination of the proximal histidine, giving rise to the six-coordinate high-spin state at pH 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Inclusion body beta-thalassemia trait in a Swiss family is caused by an abnormal hemoglobin (Geneva) with an altered and extended beta chain carboxy-terminus due to a modification in codon beta 114

We have analyzed the sequence of the beta globin gene of a chromosome that is linked to the occurrence of an inclusion body beta-thalassemia characterized in the heterozygote by moderate anemia, severe red cell abnormalities, splenomegaly, inclusion body formation, elevated Hb A2 levels, and an increased in vitro alpha/beta chain synthetic ratio. The data indicate a change in codon 114 from CTG (Leu) to -GG that resulted in a frameshift and the presumed synthesis of an abnormal beta chain that is 156 residues long with a completely different C-terminal amino acid sequence. The change in codon 114 gives a -GGGCCC- sequence that creates a new ApaI site; the resulting 2.6-kilobase fragment has been observed in all subjects with this thalassemia condition. Protein structural analyses failed to demonstrate any trace of the abnormal beta chain, even in reticulocytes and nucleated red cells that were isolated by density gradient centrifugation. The inclusion bodies appear to contain mainly normal alpha chains. It is assumed that the structure of the beta-Geneva chain prevents it from combining with normal alpha chains; this results in a rapid breakdown of the abnormal protein during the early stages of red cell maturation and an accumulation of free alpha chains.

Inclusion Body β-Thalassemia Trait in a Swiss Family Is Caused by an Abnormal Hemoglobin (Geneva) With an Altered and Extended β Chain Carboxy-Terminus due to a Modification in Codon β114

Abstract We have analyzed the sequence of the beta globin gene of a chromosome that is linked to the occurrence of an inclusion body beta-thalassemia characterized in the heterozygote by moderate anemia, severe red cell abnormalities, splenomegaly, inclusion body formation, elevated Hb A2 levels, and an increased in vitro alpha/beta chain synthetic ratio. The data indicate a change in codon 114 from CTG (Leu) to -GG that resulted in a frameshift and the presumed synthesis of an abnormal beta chain that is 156 residues long with a completely different C-terminal amino acid sequence. The change in codon 114 gives a -GGGCCC- sequence that creates a new ApaI site; the resulting 2.6-kilobase fragment has been observed in all subjects with this thalassemia condition. Protein structural analyses failed to demonstrate any trace of the abnormal beta chain, even in reticulocytes and nucleated red cells that were isolated by density gradient centrifugation. The inclusion bodies appear to contain mainly normal alpha chains. It is assumed that the structure of the beta-Geneva chain prevents it from combining with normal alpha chains; this results in a rapid breakdown of the abnormal protein during the early stages of red cell maturation and an accumulation of free alpha chains.

A new Case of Hb Little Rock [β143(H21)His → Gln], a high affinity variant. Study during pregnancy

A new case of Hb Little Rock [beta 143(H21)His----Gln] is described. This high affinity variant (P50 = 15 mm Hg in whole cells) has a nearly normal cooperativity. The abnormal beta chain is readily detectable using urea-Triton X-100 electrophoresis. The altered beta T-14 peptide was separated by high performance liquid chromatography. The proposita was followed during a pregnancy. Oxygen unloading was reduced, but normal oxygen loading was maintained in the fetus. There were neither placental abnormalities, nor intrauterine growth retardation.

LFA‐1 β‐chain synthesis and degradation in patients with leukocyte‐adhesive proteins deficiency

The defective membrane expression of the adhesive protein family (LFA-1, Mo1 and p150,93) on leukocytes from certain patients with recurrent bacterial infections was shown to be secondary to the absence of synthesis of mature beta chain that is common to all three antigens (Springer et al., 1984, Lisowska-Grospierre et al., 1986). In all patients, studies of beta-chain biosynthesis that lead to this conclusion were performed using the monoclonal anti-beta chain antibody to isolate the beta subunit. Since this antibody detects the mature form of beta chain only, the potential presence of a precursor or of an abnormal beta chain in the patient's cells could not be tested. The availability of the polyclonal antibody to the purified beta subunit allowed us to re-examine the biosynthesis of the LFA-1 subunits in 3 affected children. In all 3 patients, the absence of membrane expression of the LFA-1, CR3 and p150,95 proteins was confirmed. The LFA-1 alpha-chain precursor of 170 kDa was detected in the lysates of PHA blasts of two children, but was not detected in the third. The beta-chain precursor of 85 kDa was isolated by the polyclonal anti-beta chain antiserum from the cytoplasm of phytohemagglutinin and Epstein-Barr virus-induced blasts of one patient. The same antibody precipitated some peptides of smaller mol. wt. from the cell lysates of 2 other patients. These results suggest that in this disorder the membrane nonexpression of the adhesive proteins is probably due to the structural abnormality of beta chain which, although synthesized, is rapidly degradated.

Hemoglobin Sendagi (β42 Phe↠Val): A New Unstable Hemoglobin Variant Having an Amino Acid Substitution at Cd1 of the β-Chain

Hb Sendagi, a new unstable hemoglobin variant, was found in a Japanese male and his daughter, who have a moderate hemolytic anemia. The variant showed decreased stability upon heat and isopropanol precipitation tests. The variant did not separate from hemoglobin A by electrophoresis, but the abnormal beta-chain emerged ahead of the normal beta-chain on reverse-phase HPLC. Structural analyses revealed that the phenylalanine beta 42 (CD1), one of the critical amino acid residues in the heme pocket, had been replaced by valine. Oxygen equilibrium studies of the patient's hemolysates indicated that Hb Sendagi had a lowered oxygen affinity and a normal response to 2,3-diphosphoglycerate. Hb Hammersmith (beta 42 Phe----Ser) and Hb Louisville (Bucuresti) (beta 42 Phe----Leu) have previously been reported to have varying degrees of molecular instability and altered oxygen binding function. Hb Sendagi provides an additional model for elucidation of the role of the phenylalanine beta CD1 on the structure and function of hemoglobin.

Structural and functional studies of hemoglobin Poissy alpha 2 beta 2(56) (D7) Gly----Arg and 86 (F2) Ala----Pro.

Hemoglobin Poissy alpha 2 beta 2(56) (D7) Gly----Arg and 86 (F2) Ala----Pro, is a new variant of the beta chain with two substitutions within the second exon of the corresponding gene. The electrophoretic mobilities are identical to those of Hb Hamadan alpha 2 beta 2(56) (D7) Gly----Arg as is the fingerprint of the tryptic hydrolysate of the two abnormal beta chains. The second substitution beta 86 Ala----Pro was detected by high-pressure liquid chromatography. Hb Poissy has a threefold increase in oxygen affinity with low Hill coefficient and diminished Bohr effect, which are restored to normal upon addition of 2,3-bisphosphoglycerate. Since the functional properties of Hb Hamadan (beta 56 Gly----Arg) have been described as normal, the abnormal function of Hb Poissy may be attributed to the beta 86 (F2) Ala----Pro substitution. Hb Poissy exhibits a mild instability and a greater reactivity of the thiol groups of the beta 93 (F9) Cys residues in the deoxy form than does Hb A. The oxidation rate of Hb Poissy is biphasic indicating a large inequivalence between the alpha and beta hemes. Thereafter NMR studies demonstrated that the beta 86 Ala----Pro substitution produces a displacement of the F helix closer to the heme plane and a large increase in the dynamic fluctuations of the tertiary structure on the proximal side of the beta hemes. These results lead to the conclusion that the beta 86 Ala----Pro substitution produces a destabilization of the F helix extending downwards to the FG corner and altering both the beta hemes and the alpha 1 beta 2 contacts.

Unique sensitivity of Hb Zürich to oxidative injury by phenazopyridine: Reversal of the effects by elevating carboxyhemoglobin levels in vivo and in vitro

Phenazopyridine (PAP) causes a hemolytic anemia in normal individuals who receive an overdose or in patients with decreased renal function given therapeutic doses. There are no reports of PAP-induced hemolysis in individuals with unstable hemoglobins. Therapeutic doses of PAP administered to a subject with Hb Zürich (His E7 (63) beta leads to Arg) caused a severe hemolytic anemia with many large Heinz bodies appearing in the red cells. Incubation of whole blood from three asymptomatic Hb Zürich subjects with PAP at a molar ratio of PAP/Hb of 1.3/1 produced a moderate to marked increase in methemoglobin (MHb) and Heinz body formation accompanied by a slight to moderate decrease in levels of reduced glutathione. The rates of MHb formation were proportional to the concentration of PAP. In two of the subjects the rates of formation of Heinz bodies and MHb were substantially higher than in the third subject. Red cells from five normal adults were not affected. In Hb Zürich red cells the affinity of the abnormal beta chain for carbon monoxide is much greater than that of the normal alpha and beta chains. The two subjects with higher rates of MHb and Heinz body formation were nonsmokers with in vivo carboxyhemoglobin (HbCO) levels of 4-6%. The third subject with low rates of MHb and Heinz body formation was a smoker with in vivo HbCO levels of 15-18%. Increasing levels of HbCO from 8.2% to 14.3% by the in vitro addition of CO caused a marked reduction in the rate of Heinz body formation and a moderate decrease in MHb formation. Rates of MHb formation similar to those observed in normal red cells occurred at HbCO percentages of 89.2 and 99.2. Red cells containing Hb Zürich are extremely sensitive to oxidative injury by PAP, either in vivo or in vitro. The degree of oxidative injury diminishes as the level of HbCO increases, a phenomenon that is enhanced by preferential binding of CO to the abnormal beta subunit of Hb Zürich.

Hemoglobin Great Lakes (β68 [E12] Leucine → Histidine): A New High-Affinity Hemoglobin

Abstract Hemoglobin Great Lakes, beta 68 (E12) Leu replaced by His is a new high oxygen affinity hemoglobin variant discovered in a 29-yr-old female having numerous hospitalizations for thrombophlebitis associated with mild erythrocytosis. The mutant hemoglobin has normal stability and normal electrophoretic mobility, but increased oxygen affinity (P-50 16.1 mm Hg at 37 degrees C, pH 7.4) and reduced cooperativity. The abnormal beta-chain could be separated on globin chain chromatography on carboxymethyl/cellulose in spite of the normal electrophoretic mobility of the intact hemoglobin. The leucyl residue at beta 68th position (E12) is in the middle of E-helix, which is part of the heme pocket and next to the valine (E11), which is the heme binding site. The substitution of proline for leucine in hemoglobin Mizuho resulted in the distortion of tertiary structure of the beta-chains and lead to a serious instability of hemoglobin molecule. However, the substitution of this residue by histidine in hemoglobin Great Lakes is not associated with hemoglobin instability.

Hemoglobin Great Lakes (beta 68 [E12] leucine replaced by histidine): a new high-affinity hemoglobin

Hemoglobin Great Lakes, beta 68 (E12) Leu replaced by His is a new high oxygen affinity hemoglobin variant discovered in a 29-yr-old female having numerous hospitalizations for thrombophlebitis associated with mild erythrocytosis. The mutant hemoglobin has normal stability and normal electrophoretic mobility, but increased oxygen affinity (P-50 16.1 mm Hg at 37 degrees C, pH 7.4) and reduced cooperativity. The abnormal beta-chain could be separated on globin chain chromatography on carboxymethyl/cellulose in spite of the normal electrophoretic mobility of the intact hemoglobin. The leucyl residue at beta 68th position (E12) is in the middle of E-helix, which is part of the heme pocket and next to the valine (E11), which is the heme binding site. The substitution of proline for leucine in hemoglobin Mizuho resulted in the distortion of tertiary structure of the beta-chains and lead to a serious instability of hemoglobin molecule. However, the substitution of this residue by histidine in hemoglobin Great Lakes is not associated with hemoglobin instability.

Hemoglobin Tampa:beta 79 (EF3) aspartic acid replaced by tyrosine.

Hemoglobin Tampa was detected in a 6-year-old male caucasian who is homozygous for this variant hemoglobin. The variant hemoglobin has an electrophoretic mobility between Hb F and Hb S on cellulose acetate (pH 8.5) and a mobility between Hb S and Hb C on citrate agar (pH 6.0). In acid buffer globin chain analysis revealed an abnormal beta chain with a mobility between the beta A and beta S chains, and in alkaline buffer the mobility of the chain was at the beta S position. Structural characterization of the variant beta chain indicates that aspartic acid is replaced with tyrosine at position 79, the site of a previously reported mutation, Asp replaced by Gly (Hb Hsi-Tsou). The clinical histories of the available family members including the homozygous propositus appear to be unremarkable.