Α-chain Variant Research Articles

Haptoglobin subtypes in Norway and a review of HP subtypes in various populations.

Isofocusing and immunoblotting of reduced serum samples identify the common haptoglobin alpha-chain variants 1S, 1F, 2FS, 2SS, 2FF, 3, as well as several rare alpha- and beta-chain variants. The gene frequencies found in 6,668 unrelated persons involved in Norwegian paternity cases were: HP*1S: 0.22, HP*1F: 0.16, HP*2FS: 0.58, HP*2SS: 0.04, HP*2FF: 0.004, HP*3: 0.0004, other HP* alpha variants: 0.0004, HP* beta variants: 0.0008. The corresponding gene frequencies in 153 unrelated Norwegian Saamis (Lapps) were: HP*1S: 0.19, HP*1F: 0.07, HP*2FS: 0.70, HP*2SS: 0.04. Norwegians and Norwegian Saamis differed both in phenotype and allele distribution. An earlier Norwegian population study has shown a lower HP*1 frequency in the north than in the south. This regional difference in haptoglobin gene distribution was reflected in the present material as a lower 1F frequency, indicating a Saamish influence in northern Norway. Furthermore, the relatively low 2FF frequency in the north coincides with the lack of observed 2FF genes in the Saamish population. Non-Scandinavians involved in Norwegian paternity cases did not differ from the rest of the material. A review of published haptoglobin gene frequencies shows the 1F frequency to be a good indicator of ethnic origin, and that 2FF and 2SS frequency determinations may also be valuable in genetic population studies.

Plasma desorption mass spectrometry of haemoglobin tryptic peptides for the characterization of a Hungarian α-chain variant

S-Aminoethylated-alpha A and -beta A globin tryptic peptides separated by reversed-phase high-performance liquid chromatography have been analysed by plasma desorption mass spectrometry. Almost all the expected alpha A and beta A tryptic fragments were tentatively assigned relative to the known globin chain sequences based on the molecular weight obtained by plasma desorption mass spectrometric analysis of the purified peptides. The application of plasma desorption mass spectrometry for structure elucidation of a haemoglobin alpha-chain variant revealed the first case of Hb Hasharon in Hungary.

Forensic aspects of haptoglobin: Electrophoretic patterns of haptoglobin allotype products and an evaluation of typing procedure

A procedure used for haptoglobin (Hp) typing in paternity cases has been evaluated. All serum samples have been subtyped with a one-dimensional isoelectric focusing/immunoblotting method, and samples with rare or questionable patterns have been further examined by two-dimensional electrophoresis with isoelectric focusing in the first dimension followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. The electrophoretic Hp-patterns of common and rare alpha- and beta-chain variants are shown, including allotype patterns of two new beta-chain variants and three new alpha-chain variants. Retyping of nearly 2000 individuals at intervals between 1 to 12 months revealed a typing error frequency of about 0.3%, which is considered acceptable, provided new blood samples are required in every case of paternity exclusion. Comparison of typing results obtained with the present procedure and with routine starch gel electrophoresis in more than 5000 serum samples gave conflicting results in 6 samples. The sensitivity of the described one-dimensional subtyping method was slightly better than that of starch gel electrophoresis. In 4110 unrelated individuals, involved in cases of disputed paternity the Hp 2SS 0.038, Hp 2FF 0.004, and Hp 3 (Johnson) 0.0005. These allele frequencies give a theoretical paternity exclusion rate of 32.5%, which is in accordance with the observed rate in 2200 paternity cases with more than 600 non-fathers. It is concluded that the present procedure represents a definite improvement for Hp subtyping in practical paternity diagnostics. Preliminary results with retyping of weak Hp patterns using a staining technique involving the biotin/avidin complex indicate that the sensitivity of the one-dimensional subtyping method may be substantially increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Separation of normal and abnormal hemoglobin chains by reversed-phase high-performance liquid chromatography

A review is presented of the elution patterns on reversed-phase columns of the normal and abnormal globin chains of different hemoglobin types, including 16 β-chain variants, 7 α-chain variants, 9 γ-chain variants, and 4-variants with fusion or hybrid chains. Separations appear to be based primarily on differences in hydrophobicity. The method is ideally suited for the detection of abnormal globin chains, their quantitation and their isolation. Semi-quantitative data based on the calculation of the δ non-α ratios allow the detection of β-thalassemic conditions in situations where the quantitation of hemoglobin A 2 by other procedures is impossible or complicated.

Structural and functional studies of Hemoglobin Wayne: An elongated α-chain variant

Hemoglobin Wayne (Hb Wayne) is a frame-shift, elongated α-chain variant that exists in two forms, with either asparagine or aspartic acid as residue 139. Oxygen equilibrium studies showed that stripped Hb Wayne Asn and Hb Wayne Asp possessed high oxygen affinity ( P 1 2 = 0.60 and 0.23 mmHg at pH 7, respectively), were non-co-operative and have a markedly reduced Bohr effect ( −Δ log P 1 2 / pH (7 to 8) = 0.34 and 0.10, respectively). Adding organic phosphate results in a decreased oxygen affinity and increased Bohr effect for both Hbs Wayne. The overall rate of carbon monoxide binding at pH 7 ( l′ = 5.6 × 10 6 m −1 s −1) was similar for both stripped Hbs Wayne and was 25-fold more rapid than that of stripped Hb A. When organic phosphate was added, Hb Wayne Asn exhibited a homogeneous slower rate of carbon monoxide binding ( l′ = 2.6 × 10 6 m −1 s −1), whereas Hb Wayne Asp showed heterogeneous binding ( l′ = 6.1 × 10 6 and 2.6 × 10 6 m −1 s −1 for fast and slow phases, respectively). The rates of overall oxygen dissociation and oxygen dissociation with carbon monoxide replacement for both Hbs Wayne were found to be slow compared to Hb A and uniquely different from each other. Similarly, sedimentation velocity experiments indicated that, although Hb Wayne Asn and Hb Wayne Asp were both less tetrameric than Hb A, each hemoglobin exhibited a distinct degree of oxygen-linked subunit dissociation. These observed differences in the allosteric properties of Hb Wayne Asn and Hb Wayne Asp appeared to be directly attributable to residue 139. The equilibrium and kinetic data are consistent with the X-ray diffraction analysis of Hb Wayne Asp, which shows that the C terminus of the deoxytetramers are severely disordered, a condition that results in major destabilization of the T conformation and disruption of normal hemoglobin function.

Duplication of the hemoglobin alpha-chain gene in sheep: characterization of a new alpha-chain variant present in animals possessing the alpha Leu and the II alpha His chains.

The structural characterization of a third type of alpha chain, detected in the hemoglobin of sheep also possessing the alpha Leu and the II alpha His chains, is reported. The new alpha-chain variant is an allele of the common alpha Leu chain controlled by the 1 alpha locus and differs from it in the replacement of the serine residue at position 8 with an alanine (I alpha 8 Ser leads to Ala). The alanine variant was observed in 38 of 206 sheep, whose hemolysates were analyzed by isoelectric focusing, and was found exclusively among the 57 animals also possessing the II alpha His chain.

Hb Etobicoke, alpha 85(F5) Ser leads to Arg found in a newborn of French-Indian-English descent.

During a recent testing program for hemoglobin (Hb) abnormalities in cord bloods of newborns in the State of Georgian an α-chain variant was discovered in a male Caucasian infant of French-Cherokee Indian-English descent. The hematological values of the infant at five weeks were: Hb 8.9 g/dl, RBC 2.99 × 1012/1; PCV 0.28 1/1, MCV 93 fl, MCH 30.4 pg, and MCHC 31.2%. Corresponding results on both parents were normal. Although the abnormal Hb was not found in either parent, paternity studies were not done. The variant had a mobility of Hb G on cellulose acetate electrophoresis at PH 8.6 and could not be detected by citrate agar electrophoresis at pH 6.1 (1). DEAE-cellulose chromatography with glycine-KCN-NaCl developers (2) redily separated the abnormal Hbs from Hb A, Hb F, and Hb F1 partially separated Hb Fx from Hb X. The combined concentration of the Hb Fx was 16% in the cord blood red cell lysate. A repeat chromatogram at five weeks of age showed improved separation and gave the concentration of Hb Fx as ...

A Case of Hemoglobin Iwata [α87(F8)HIS → ARG] in China

Hemoglobin Iwata was discovered in Japan in 1980 (1). Recently, we detected a slow-moving α-chain variant by cellulose acetate electrophoresis (2) in a family of the Han ethnic group during a survey carried out among students in Huhehot, Inner Mongolia. Structural analyses of the abnormal hemoglobin (Hb) demonstrated that it was Hb Iwata [α87 (F8) His→Arg]. This is the first example of this variant to be found in China.

Identification of hemoglobin G-Philadelphia (α68 Asn→Lys) and homoglonin matsue-oki (α75 Asp→Asn) in a black infant

Two α-chain variants, Hb G-Philadelphia and Hb Matsue-Oki, were present in members of a relatively large black family from South Carolina. The four Hb G-Philadelphia heterozygotes averaged 35.6% Hb G, suggesting the presence of an α-thalassemia-2 condition in cis to the Hb G mutation, which was confirmed by DNA structural analysis. The seven Hb Matsue-Oki heterozygotes averaged 22.2% Hb MO and likely have four active α-chain genes. One infant was a compound heterozygote for the two Hb variants which could not be separated from each other. The quantity of Hb G plus Hb MO was 58% by DEAE-cellulose chromatography and 69% by chain analyses. These results and the family data indicate that this child had three active α-chain genes, of which one regulated the synthesis of the normal a chain, one was mutated to give the α G chain, and one to give the α MO chain. The amino acid substitutions in Hb G-Philadelphia and Hb Matsue-Oki are located in the tryptic peptide αT-9, which is 29 amino acid residues long. Structural analyses of these abnormalities made use of high-pressure liquid chromatography for the separation of both tryptic and thermolytic peptides and of a highly sensitive ultra-micro sequencing procedure. Although the α68 Asn→Lys substitution is readily demonstrable in Hb G-Philadelphia the elucidation of the α75 Asp→Asn replacement in Hb Matsue-Oki was greatly facilitated by the use of these microprocedures.

Organization of alpha-chain genes among Hb G-Philadelphia heterozygotes in association with Hb S, beta-thalassemia, and alpha-thalassemia-2.

The percentages of the α-chain variant Hb G-Philadelphia (Hb G) or α2 68 Asn→Lysβ2 were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another with lower values (33.9±3.4%, N=63). Among the newborn heterozygotes, two babies fell in the category with the higher proportion of Hb G while 16 babies gave values between 25 and 34%. Studies of α-chain gene organization on the parents of one neonate with a Hb G level of 27% at birth and 37% at 8 months excluded the presence of chromosomes with triplicated α-chain genes which could lead to the α0αG/ααα genotype. Rather, these studies on five Hb G heterozygotes from three families confirmed the linkage between Hb G and a specific type of α-thalassemia-2 associated with the presence of a 16-kbp Bgl II fragment which most probably carries the αG locus since it has been found in 19 Hb G heterozygotes studied to date. The presence of an α-thal-2 heterozygosity and three α-chain genes (α0αG/αα) was confirmed among Hb G heterozygotes with lower proportions of this variant. It is likely that the even lower values found in some newborn could arise through defective assembly of αG-γ dimers. The presence of an α-thal-2 homozygosity and two active α-chain genes, one on each chromosome (α0αG/α0α), was confirmed among heterozygotes with the higher proportion of Hb G. One of each of these categories was present in each of the three families investigated. This type of variability in the number of active α-chain genes due to a heterozygosity or a homozygosity for α-thalassemia-2 explains the trimodality of Hb S percentages among heterozygotes and the atypical hematological or biosynthetic features among patients with β-thalassemia and sickle-cell syndromes.

Toward a system for detecting somatic-cell mutations. V. Preparation of fluorescent antibodies to hemoglobin Hasharon, a human α-chain variant

Antibodies against the abnormal human hemoglobin, Hb Hasharon ( α 47Asp→His), were raised in horse and purified by absorption against Sepharose 4B to which normal hemoglobins or Hb Hasharon were bound. The purified, non-precipitating antibodies were tested for specificity against normal hemoglobins and Hb Hasharon by immunodiffusion in the presence of anti-horse IgG, and by exposing mixtures of normal and Hb Hasharon-containing red cells to the antibodies after conjugation of the latter with fluorescein isothiocyanate. The ease with which antibodies specific for different variant hemoglobins have been prepared, and their potential for identifying individual erythrocytes that contain these hemoglobins by virtue of somatic mutation, underscore their value as aids to detection and analysis of mutational events in human subjects.

Post-translational control of human hemoglobin synthesis The role of the differential affinity between globin chains in the control of mutated globin gene expression

The interactions between β-thalassemia and the humen hemoglobin (Hb) α-chain variants, Hb Hasharon, Hb O Indonesia and Hb J Paris, and between α-thalassemia and the β-chain variants, Hb S, Hb C and Hb G San José, which are characterized by preferential decrease of the abnormal Hb level in peripheral blood, have been studied. Both biosynthesis studies in reticulocytes and determination of the relative affinity of abnormal chains for normal complementary chains by in vivo recombination experiments, involving globin chains previously isolated in their native form, have been carried out in order to provide insights on the molecular events following the synthesis of the mutant chains under conditions of complementary chain deficiency. Furthermore, we have measured the relative affinity for complementary chain of β D Los Angeles - and α J Rovigo - chains , the level of which does not decay in thalassemic carriers, and of α Legnano - and β Osu Christiansborg - chains , which have not yet been observed in association with thalassemias. Our experiments indicated that the differential affinity for β-chains is not always the major post-translational control mechanism which regulates the level of certain α-chain variants in β-thalassemic heterozygotes, and that preferential removal of abnormal chains by proteolytic enzymes is likely to play an important role in most cases. On the other hand, the low affinity of certain variant β-chains for α-chains may offer an explanation for the low level of certain β-chain variants in peripheral blood of non-thalassemic carriers, as well as to their decrease under conditions of relative α-chain deficiency (α-thalassemias).

A new human hemoglobin variant: Hb Bari ( α2 45 (CD3) His → Gln β2)

An α-chain variant hemoglobin was found in the hemolysate of a 21-year-old healthy male living in Bari (Puglia, Italy). Structural studies demonstrated a previously unreported amino acid substitution, α 2 45 (CD3) His → Gln β 2, involving a distal heme contact. The new variant has been named Hb Bari. Its electrophoretic behavior was the same as for Hb A; it was stable to both isopropanol and heat denaturation and exhibited normal functional properties, with respect to whole blood and stripped hemolysate studies. The level of Hb Bari was about 20% in the observed carrier. No relative was available for further investigations.

Erythropoiesis in normal and mutant chick embryos

Hemoglobin D Davis (Hb D D), an autosomal codominant in chickens, the α D-globin chain of Hb M of primitive cells and Hb D of definitive erythrocytes. Erythropoiesis and Hb synthesis was investigated in normal, heterozygous, and homozygous Hb D D mutant embryos (stages 15–44) and adults. The time of appearance, morphology, relationships to developmental changes, and number of primitive and definitive cells were determined. Primitive hemoglobins between stages 17 and 44 showed four components, P1, P2, E, and M (or M D), on high-resolution isoelectric focusing gels. Comparison of P1 P2 ratios in the four phenotypes indicated that homozygous Hb D D embryos had an increased proportion of Hb P2 relative to Hb P1 between stages 17 and 35. This difference coincided with an increase in the number of large primitive cells. In all phenotypes the proportions of primitive hemoglobins decreased after stage 25 and they were not detected after stage 40. Basophilic definitive erythroblasts were present in cell suspensions from all phenotypes between stages 24 and 25. Hb A, the major Hb and Hb D, the minor Hb, of definitive cells of embryos and adults were detected by isoelectric focusing of lysates by stage 29. Definitive cells from late embryos of all phenotypes had higher proportions of Hb D (or Hb D D) than did red cells from corresponding adult birds. Heterozygous Hb D D embroys and adults had both Hb D and Hb D D. Hb D D comprises about 30% of the total minor Hb rather than 50% expected for heterozygosity at a single locus. In this respect heterozygous Hb D D chick embryos and adult birds are similar to certain heterozygous α-chain variants in humans. A minor Hb, H, found in lysates of later embryos disappears in lysates of normal chicks 65 days after hatching, but was present in the circulation of homozygous Hb D D chicks until at least 195 days after hatching. Additionally, several minor Hb components which may be asymmetrical hybrids or derived precursors of Hb A and Hb D (or Hb D D) were observed. This study provides the precise developmental stages when the switchover of erythroid cell populations and hemoglobins in the chick embryo occurs. This is the first investigation of an α-globin chain mutant which is synthesized during all stages of red cell development and may be a useful animal model for the study of hemoglobinopathies in vertebrates.

A new haemoglobin variant: Haemoglobin anantharaj alpha 11 (A9) lysine → glutamic acid

Four heterozygotes for a fast α-chain variant in a Thai family were detected on starch gel electrophoresis during a survey study on iron deficiency anaemia in a rural area not far from Bangkok. They were healthy and had normal haematological profiles except for the presence of around 44% abnormal pigment, quantitated by cellulose acetate electrophoresis. The structural characterization of the variant by globin chain separation, peptide mapping, and amino acid analyses of the abnormal peptides indicated that lysine residue 11 (A9) of α-chain was replaced by glutamic acid. This mutation has not been previously described and it is proposed that it be called Haemoglobin Anantharaj.

The occurrence of haemoglobin-J (Tongariki) and of thalassaemia on Karkar Island and the Papua New Guinea mainland.

The known α-chain variant Hb-J (Tongariki) was found to occur at relatively high frequency in several villages on the island of Karkar, about 15 km from the New Guinea mainland. The same variant was also found in an adjacent coastal region of the mainland, but not in a large number of blood samples collected in the Central Highlands. This is the third Pacific location in which Hb-J (Tongariki) has been found, and this variant may be a useful genetic marker for the study of Melanesian populations. Hb-J (Tongariki) is a ‘high-level ’ α-chain variant; its mean proportion in heterozygotes is 41 %. Three homozygotes were found in which Hb-J (Tongariki) and Hb-J 2 (α J 2 δ 2 ) were the sole major and minor constituents respectively. These findings are of interest in relation to current studies on the possible duplication of the human α-chain gene locus.

Hemoglobin Constant Spring, and unusual alpha-chain variant involved in the etiology of hemoglobin H disease.

Annals of the New York Academy of SciencesVolume 232, Issue 1 p. 168-178 HEMOGLOBIN CONSTANT SPRING, AN UNUSUAL α-CHAIN VARIANT INVOLVED IN THE ETIOLOGY OF HEMOGLOBIN H DISEASE J. B. Clegg, J. B. Clegg Department of Haematology Nuffield Unit of Medical Genetics University of Liverpool Liverpool, EnglandSearch for more papers by this authorD. J. Weatherall, D. J. Weatherall Department of Haematology Nuffield Unit of Medical Genetics University of Liverpool Liverpool, EnglandSearch for more papers by this author J. B. Clegg, J. B. Clegg Department of Haematology Nuffield Unit of Medical Genetics University of Liverpool Liverpool, EnglandSearch for more papers by this authorD. J. Weatherall, D. J. Weatherall Department of Haematology Nuffield Unit of Medical Genetics University of Liverpool Liverpool, EnglandSearch for more papers by this author First published: May 1974 https://doi.org/10.1111/j.1749-6632.1974.tb20582.xCitations: 27AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume232, Issue1Third Conference on Cooley's AnemiaMay 1974Pages 168-178 RelatedInformation

Homozygous State for Hb Constant Spring (Slow-moving Hb X Components)

Abstract A 12-yr-old Malay boy who was studied because his youngest brother and both his parents had the slow-moving Hb X components (earlier reported to lead to Hb H disease when combined with α-thalassemia) was found to be homozygous for the same slow-moving components. He had splenomegaly and a just palpable liver, mild anemia with microcytosis, hypochromia, slight morphologic changes of the red blood cells, and slight reticulocytosis. Of eight children in the family, six had the trait for the abnormality, one was normal, and one, the propositus, was homozygous. Structural studies of the isolated abnormal hemoglobin showed it to be identical to Hb Constant Spring (Hb CoSp), an α-chain variant with 172 residues instead of the usual 141, the additional 31 being attached to the C-terminal end. In addition to the abnormal α variant for which the propositus was homozygous, he also had normal Hb A and normal Hb A2 with normal α-chains. If the theory that Hb CoSp is due to a structural mutation affecting the terminator codon is correct, this case provides evidence for a duplication of the gene for α-chain production. Results of study of several erythrocyte enzymes are also reported.

Hemoglobin Siam ?2 15arg?2: A new ?-chain variant

A 28-year-old, healthy male of chinese ancestry with normal hematologic findings was found to have an abnormal hemoglobin moving more slowly than hemoglobin F in starch gel electrophoresis. The relative amounts of the abnormal component, hemoglobin A2 and slow hemoglobin A2, as determined by cellulose acetate electrophoresis, were 15.2, 1.64, and 0.56% respectively. The hemoglobin chain dissociation by p-chloromercuribenzoate treatment confirmed that it was an α-chain variant. The abnormal globin chain was fractionated by carboxymethyl cellulose chromatography. Peptide mapping revealed that the amino acid alteration occurs in αTp III (tryptic peptide No. 3, residues 12 to 16 of the α-chain). Special stains and amino acid analyses of the abnormal peptide indicated that glycine at residue 15 was replaced by arginine, and the hemoglobin is thus α215argβ2. Since the mutation has not been previously described, it is proposed that it be called “Hemoglobin Siam”.

Interaction entre les hémoglobines Stanleyville II et S dans une famille du Zaïre. Etude de l'hybride Stanleyville II/S (α 278 lys β 26 val)

Summary Two generations of an African family with haemoglobin Stanleyville II, an α-chain abnormality, and haemoglobin S, a β-chain abnormality, are described. The propositus, aged of 7 years, presents a sickle-cell syndrome of moderate severity and a glucose-6-phosphate dehydrogenase deficiency; he has two mutants of adult haemoglobin, both abnormal, which were demonstrated to be haemoglobin S and an α-chain variant of the latter, haemoglobin Stanleyville II/S or α Sta-II 2 β 8 2 ; in this variant lysine replaces the normal asparagine at the 78th (EF 7) residue of the α-chains. This patient is therefore heterozygous for the α Sta-II chains and homozygous for the β S chains. His father is a sickle-cell trait carrier; the mother and a brother have four adult haemoglobins: Hb-A, Hb-Stanleyville II, Hb-S and Hb-Stanleyville II/S and are therefore doubly heterozygous for the α Sta-II and β S abnormalities; another brother is heterozygous for haemoglobin Stanleyville II, whereas a sister is homozygous for haemoglobin S and has a clinically severe sickle-cell anaemia. This family belongs to the Ngbandi tribe, situated along the Ubangi river in the North of the Republic of Zaire and is of Sudanese ancestry, as seems to be the case for the four other families with haemoglobin Stanleyville II observed up to now. The pattern of inheritance of the α and β variants of haemoglobin in this family provides a further indication that the chromosomic loci ruling the structure of the α and β chains segregate independently and are not situated on the same chromosome. The levels of the α Sta-II chains of the propositus and of some members of this family are in agreement with the hypothesis of a linkage of some α variants of haemoglobin with α-thalassaemia; furthermore the linkage of the α Sta-II chains with α-thalassaemia provides an explanation of the moderate severity of the sickle cell anemia syndrome, as well as of the hypochromia of the propositus, since α-thalassemia depresses the synthesis of the normal α-chains and consequently the concentration of haemoglobin S in the red cells.