Rare variants of Hb D Punjab, Hb O Arab and polymorphism of human hemoglobins

This report describes the occurrence, study and molecular diagnostics of 40 Hb O Arab beta 121 Glu Lys cases and 4 Hb D punjab beta 121 Glu Gln cases in Bulgaria. Hematological, morphological and clinical data for 12 patients with Hb O arab are listed. Among them we observed 7 simple heterozygotes for Hb O Arab/Hb A, two double heterozygotes-compounds for Hb O/beta+-thalassemia and three compounds for Hb O/beta 0-thalassemia (the latter assumed). Also, general hematological, morphological and clinical data are presented for 4 Hb D Punjab carriers, from which two are simple heterozygotes and two are assumed, as compounds for Hb D/beta 0-thalassemia. The consideration of heterozygosity, homozygosity for both abnormal hemoglobins and of the compound state of Hb O or Hb D/beta-thalassemia or HbS types let us suggest the relative neutrality of the variants and the limitation in their distribution, depending on genetic structure of populations, where they spread. It may be concluded that human hemoglobin is characterized by marked monomorphism. At the same time, the high frequency of HbS, HbE and HbC in some populations can be well explained by contemporary selectionism; the distribution of relatively neutral Hb D Punjab and Hb O Arab with some limitations can follow Kimura's neutralism concept.     

Twelve families with Hb 0 Arab in the Burgas district of Bulgaria observations on sixteen examples of Hb 0 β° thalassaemia

Summary 12 families with Hb 0 Arab from the Burgas district of Bulgaria were examined. Of the 262 members of the 12 families, 44 persons were simple Hb 0 Arab heterozygotes, 18 had -thalassaemia, and 16 were double heterozygotes for Hb 0 Arab and ^° thalassaemia.     

Double heterozygous Hb O Arab/beta-thalassemia in a Tunisian child

A study realized in a Tunisian family of 22 persons has revealed a double heterozygoty Hb O Arab/beta(0) thalassemia in a child of 16 years old. The father of this child presents a beta thalassemia and his mother is haemoglobine O Arab homozygote.     

First case of a single heterozygote of an abnormal hemoglobin,Hb Stanmore, [beta111(G13)Val-->Ala

We describe a hemoglobin beta-chain mutant detected incidentally in an unusual profile of glycated hemoglobin (HbA1c) measured by ion-exchange HPLC. Analysis of intact globin by electrospray ionization mass spectrometry (ESI-MS) and peptide analysis by on-line HPLC-ESI-MS-MS revealed the substitution, [beta111(G13)Val-->Ala], which was confirmed by DNA analysis. This was the second case of Hb Stanmore. As the first case combined beta(0)-thalassemia, and the family study in that case showed no case of Hb Stanmore without combined thalassemia, the case presented here is the first case of single heterozygote, and the first Japanese case. Hb Stanmore is isoelectrophoretically silent with only mild clinical symptoms, although stability by isopropanol test was positive.     

Hemoglobin E: Distribution and population dynamics

Hemoglobin E, an anomaly of the -chain of human hemoglobin, is widely distributed in Southeast Asia and adjacent areas. In some populations of Southeast Asia the frequency of the gene responsible for the production of HbE reaches values near 0.3. In view of the probable disadvantage of the HbE homozygote and the certain disadvantage of the double heterozygote for the HbE and -thalassemia genes an advantage of the heterozygote has to be postulated in order to explain the high gene frequencies. There is some evidence for and against malaria protection being the factor conveying heterozygote advantage. Data concerning genotype fitnesses in the triallelic system involving HbA, HbE and -thalassemia are scanty. Crude fitness data based on clinical observations and equilibrium calculations indicate that the system is unstable. Dynamic models of the three genes suggest that the HbE gene is replacing the -thalassemia gene in most Southeast Asian populations. The distribution of HbE and that of the austroasiatic group of languages are similar. This and several other aspects of the geographic and ethnic distribution of HbE are discussed in the last section of this review.     

Identification of abnormal hemoglobins by isoelectric focusing

Using IEF on slabs of acrylamide gel was adapted for screening of abnormal Hemoglobins which are at the same level by electrophoresis on cellulose acetate strips. This method is fast, inexpensive and allowed the simultaneous analysis of 70 samples of whole blood. The characterization technique of IEF allowed us to distinguish some rare variants like Hb O Arab, HbD and T gamma in B 0-thalassemia.     

Hemoglobin tacoma — A β-chain variant

A hitherto unknown inherited hemoglobin variant, Hb Tacoma, was discovered in three healthy members of a family of European extraction.Hybridization experiments with canine hemoglobin indicated a structural abnormality in the -chain. The variant was therefore designated as Hb _{2 2} ^Tacoma. Separation of Hb Tacoma from Hb A could only be clearly achieved by starch grain electrophoresis in TEB buffer at pH 8.6–9.0, where Hb Tacoma moved more rapidly towards the anode than Hb A; gradient column chromatography on DEAE cellulose and CM-Sephadex achieved partial separation. The proportion of abnormal hemoglobin in the heterozygote amounted to 43 per cent of the total hemoglobin. Hb Tacoma was less heat resistant and became more rapidly denatured in 8 M urea solution than Hb A, Hb C, or Hb S.On thin-layer starch gel electrophoresis in TCB buffer at pH 8.6 Hb Tacoma was associated with an electrophoretically slow, benzidine-positive component, Hb Tacoma-slow. The exact biochemical nature of this minor component could not be determined, although a polymeric product of Hb Tacoma is suspected.Heterozygosity for Hb Tacoma is associated with a raised Hb A₂ level without evidence of -thalassemia.A preliminary report of Hb Tacoma was presented at the Annual Meeting of the American Society of Human Genetics, Boulder, Colorado, August, 1964, and at the Seventh Annual Meeting of the American Society of Hematology, November, 1964, Seattle, Washington (Blood, 24, 848 (1964)).     

Universal newborn screening for Hb H disease in California

Newborn screening is an accepted public health measure to ensure that appropriate health care is provided in a timely manner to infants with hereditary/metabolic disorders. Alpha-thalassemia is a common hemoglobin (Hb) disorder, and causes Hb H (beta4) disease, and usually fatal homozygous alpha(0)-thalassemia, also known as Hb Bart's (gamma4) hydrops fetalis syndrome. In 1996, the State of California began to investigate the feasibility of universal newborn screening for Hb H disease. Initial screening was done on blood samples obtained by heel pricks from newborns, and stored as dried blood spots on filter paper. Hb Bart's levels were measured as fast-moving Hb by automated high-performance liquid chromatography (HPLC) identical to that currently used in newborn screening for sickle cell disease. Subsequent confirmation of Hb H disease was done by DNA-based diagnostics for alpha-globin genotyping. A criterion of 25% or more Hb Bart's as determined by HPLC detects most, if not all cases of Hb H disease, and few cases of alpha-thalassemia trait. From January, 1998, through June, 2000, 89 newborns were found to have Hb H disease. The overall prevalence for Hb H disease among all newborns in California is approximately 1 per 15,000. Implementation of this program to existing newborn hemoglobinopathy screening in populations with significant proportions of southeast Asians is recommended. The correct diagnosis would allow affected infants to be properly cared for, and would also raise awareness for the prevention of homozygous alpha(0)-thalassemia or Hb Bart's hydrops fetalis syndrome.     

Fetal haemoglobin level—effect of gender,age and haemoglobin disorders

Fetal haemoglobin (HbF) level shows significant variations in health and disease states. In this study we investigated Hb F level in 75 cord bloods, 1266 healthy individuals, 1582 Hb S heterozygotes, 464 sickle cell anaemia, 93 Hb S/2-thalassemia and 65 -thalassemia major patients. The age range of the study groups varied from newborn to over 60 years of age. Hb F level was measured by an alkali denaturation procedure and by radial immunodiffusion. The ratio of the level of G-globin chains to the level of A-globin chains (G/A) was determined in the patients group by high performance liquid chromatography. The Hb F level was significantly higher in the sickle cell anaemia and -thalassemia major patients compared to the Hb S heterozygotes and the normal individuals. Within each group Hb F level was higher in the female population compared to the age-matched male groups. This difference was statistically significant (P<0.05) in the sickle cell disease patients and -thalassemia major patients but not in the normal individuals. After the age of 30 years, the difference in the value of Hb F in the male and female population become more apparent (P<0.05) in the sickle cell disease and -thalassaemia major patients. No statistically significant sex differences were found in the G/Aratio in the patient groups, and the range of G/Aratio in the patients groups were similar to those in the control group.The results showed that age, sex and genetic disorders of haemoglobin are factors that affect Hb F level and indicate the possible involvement of an X-linked factor in control of Hb F production.     

The interaction of hemoglobin O Arab with Hb S and β+ thalassemia among Israeli Arabs

Summary We have studied 105 individuals in the village of Jasser El Zarka in the Northern Coast of Israel of whom 59% had at least one abnormal hemoglobin. Of the individuals studied 41% were AA, 13.3% AS, 28.6% AO_Arab, 10.5% SO_Arab, 0.9% SS, 38% Arab-⁺ Thal, and 1.9% Thal trait. The SO_Arab double heterozygotes were characterized by a normal mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC), and an increase of Hb F (11.7±4.3%) and 2,3-diphosphoglycerate levels (27.8 m/g Hb). The increase of Hb F is higher than the one seen among O_Arabs of other ethnic backgrounds. Their clinical course was moderately severe and osteoporosis was quite frequent. The interactions of Hb O_Arab and Hb S were studied in vitro and it was confirmed the Hb O_Arab lowers the minimal gelling concentration of mixtures with Hb S (as compared to mixtures of Hb S and Hb A), but that this effect is ionic-strength dependent. Our data are in conflict with previous claims that Hb O_Arab mixtures with Hb S polymerized almost as much as pure S. Oxygen association curves show a significant displacement of the p50 to the right, but the effect of oxygen dissociation is less apparent. The displacement was not nearly as significant as with SS cells, confirming our gelation data. Blood group determinations establish that these Arab populations had black African admixture.The Hb O_Arab/⁺ Thal double heterozygotes exhibit moderate anemia (10.3g% of Hb) and the percentage of Hb A was 17.2±1.8%. The fetal Hb was 5.4±2.1% and the 2,3-diphosphoglycerate level in two cases was 17.4 mol/g Hb. The only case of a homozygote SS had moderate anemia (10.3 g Hb%), 25.7% of Hb F, and a very benign course.     

Molecular characterization of β-thalassemia in Azerbaijan

We have analyzed the -thalassemia mutations in 99 chromosomes of 49 adults with -thalassemia major and of one with Hb S--thalassemia, who are regular patients at a large hematology clinic in Bakü, Azerbaijan. A total of 20 different mutants were identified; three [frameshift at codon 8 (-AA); IVS-II-I (GA); IVS-I-110 (GA)] were present in about two-thirds of all chromosomes. Most alleles are the same as found in Mediterranean populations; a few have an Asian origin or come from Kurdistan, Lebanon, Saudi Arabia, or a black population. One mutant [frameshift at codons 82/ 83 (-G)] might be specific for the Azerbaijanian population. Nearly all patients were transfused, which made quantitation of Hb F impossible; high^G values were present in the Hb F of those patients whose -thalassemia chromosome carried the C T mutation at position — 158 in the promoter of the ^G-globin gene.     

Intrinsic potential for high fetal hemoglobin production in a Druze family with β-thalassemia is due to an unlinked genetic determinant

Summary The mechanism for elevated production of fetal hemoglobin (Hb F) in a Druze patient with °-thalassemia intermedia was investigated. Heterozygous family members exhibited normal Hb F levels, suggesting that the increase in -gene expression in the propositus may be partly due to anemic stress. Erythroid progenitors of these family members cultured in vitro [burst forming units (erythroid); (BFUe)] showed elevated synthesis of Hb F, indicating the existence of a genetically determined intrinsic capacity for high Hb F production in this family. The propositus was found to be homozygous for a IVS2-position 1 mutation, on the background of Mediterranean haplotype I, which is not known to be linked to high Hb F production. Moreover, extensive molecular studies of the -globin gene cluster, including sequence analysis of the promoter regions of the -globin genes, did not reveal any cisacting mechanism that could account for the high Hb F production in the propositus. A young niece of the propositus with °-thalassemia major was recently discovered, who was homozygous for the same -globin allele and haplotype as the propositus. However, unlike her uncle, she does not have a high Hb F level and presents with a severe clinical course. Her inability to produce high Hb F suggests that the genetic determinant for increased -gene expression in the propositus is unlinked to the -globin gene cluster.     

Beta-thalassemia due to a T----A mutation within the ATA box

Sequence analyses of amplified DNA from a Yugoslavian patient with Hb Lepore-beta-thalassemia and from his father with a simple beta-thalassemia trait have revealed a T----A mutation within the ATA box at a position 30 base pairs upstream from the Cap site. The nucleotide substitution was confirmed through dot-blot analysis of amplified DNA with specific 32P-labeled synthetic oligonucleotide probes. The patient had a clinically severe condition; his Hb Lepore-beta-thalassemia was of the beta + type, as about 8-10% of the non-alpha chain was normal beta A. The same T----A mutation at nucleotide -30 was present on both chromosomes of a young Turkish patient who suffered from a thalassemia intermedia with a low level of Hb F (13.1%) and a relatively high beta A chain synthesis. These data are similar to those obtained for other types of beta +-thalassemia caused by comparable substitutions at positions 31, 29, and 28 base pairs upstream from the Cap site of the beta-globin gene.     

A β-thalassemia mutant caused by a 300-bp deletion in the human β-globin gene

Summary Larger deletions are a rare cause of -thalassemia. We report a further instance of a deletion comprising about 300 bp in a female heterozygote. Exon 1, part of IVS-1 and the 5 -globin gene promoter region are lost.     

Micropreparative thin-layer peptide maps in the molecular diagnosis of abnormal human hemoglobins

General factors determining the possibility of application of peptide maps in thin layer of microcrystalline cellulose as a micropreparative method in molecular diagnostics of abnormal hemoglobins were studied. The effects of absorbtional capacity of cellulose and amino acid impurities in it, choice of eluent and elution technique, peptide structure and extent of its modification in staining as well as completeness and specificity of globin chain enzymatic digestion on peptides extraction from thin layer were analysed. The results of structural identification of Hb D Punjab beta 121 Glu----Gln at a Cypriot; Hb O Arab; beta 121 Glu----Lys at a Bulgarian woman, living in Kalinin region (the second case in the USSR); Hb M Saskatoon beta 63 His----Tyr at a woman from Georgia (the second case in the USSR); Hb Buenos Aires beta 85 Phe----Ser at a Russian girl (the first case in the USSR and the third case in the world); Hb Dagestan alpha 60 Lys----Glu at two members of a Lesgin family from Dagestan; Hb Agenogi beta 90 Glu----Lys at a Hungarian woman; Hb Setif alpha 94 Asp----Tyr at three patients from Cyprus and Hb Detroit beta 95 Lys----Asn at an Azerbaijanian woman (the first case in the USSR and the second case in the world) are presented.     

On the chromatographic heterogeneity of human fetal hemoglobin.

Minor fetal hemoglobins in red cell hemolysates of newborn and adults with elevated levels of Hb F have been separated and quantitated by Biorex 70 column chromatography. In addition to Hb F1, other minor hemoglobin zones eluting before F1, pre-F1, and after F1, post-f1 have been observed. The relative amounts of the two pre-F1 zones and F1 are higher in the red cells of adults with 97--100% Hb F (homozygous hereditary persistence of fetal hemoglobin, homozygous deltabeta-thalassemia and homozygous beta0-thalassemia) than in the red cells of an adult with homozygous beta+-thalassemia with 66% Hb F, a child with a trisomy-D-13 having 38% Hb F, and in two newborn. Hb F was glycosylated in vitro with [14C]glucose or [14C] glucose 6-phosphate, and was acetylated using chicken reticulocyte lysate or a crude acetyltransferase preparation isolated from the same lysate with [14C]acetyl-CoA as substrate. Chromatographic analyses indicated that the Hb F1 zone can be formed both by glycosylation and acetylation of Hb F, and that pre-F1 zones can be products of the reaction of Hb F with phosphorylated glycolytic intermediates. Biosynthesis of minor hemoglobins in reticulocytes was studied with [14C]leucine in the presence and absence of cycloheximide and by pulse-chase. The resulting data indicate that Hb F1 synthesis is dependent upon Hb F synthesis and that the posttranslational modification may take place at an early stage in Hb F synthesis.     

Hemoglobinopathy: Molecular Epidemiological Characteristics and Health Effects on Hakka People in the Meizhou Region,Southern China

Background

Hemoglobinopathies are the most common inherited diseases in southern China. However, there have been only a few epidemiological studies of hemoglobinopathies in Guangdong province.

Materials and Methods

Peripheral blood samples were collected from 15299 “healthy” unrelated subjects of dominantly ethnic Hakka in the Meizhou region, on which hemoglobin electrophoresis and routine blood tests were performed. Suspected cases with hemoglobin variants and hereditary persistence of fetal hemoglobin (HPFH) were further characterized by PCR, DNA sequencing, reverse dot blot (RDB) or multiplex ligation-dependent probe amplification (MLPA). In addition, 1743 samples were randomly selected from the 15299 subjects for thalassemia screening, and suspected thalassemia carriers were identified by PCR and RDB.

Results

The gene frequency of hemoglobin variants was 0.477% (73/15299). The five main subgroups of the ten hemoglobin variants were Hb E, Hb G-Chinese, Hb Q-Tahiland, Hb New York and Hb J-Bangkok. 277 cases (15.89%, 277/1743) of suspected thalassemia carriers with microcytosis (MCV<82 fl) were found by thalassemia screening, and were tested by a RDB gene chip to reveal a total of 196 mutant chromosomes: including 124 α-thalassemia mutant chromosomes and 72 β-thalassemia mutant chromosomes. These results give a heterozygote frequency of 11.24% for common α and β thalassemia in the Hakka population in the Meizhou region. 3 cases of HPFH/δβ-thalassemia were found, including 2 cases of Vietnamese HPFH (FPFH-7) and a rare Belgian ^Gγ(^Aγδβ)⁰–thalassemia identified in Chinese.

Conclusions

Our results provide a detailed prevalence and molecular characterization of hemoglobinopathies in Hakka people of the Meizhou region. The estimated numbers of pregnancies each year in the Meizhou region, in which the fetus would be at risk for β thalassemia major or intermedia, Bart’s hydrops fetalis, and Hb H disease, are 25 (95% CI, 15 to 38), 40 (95% CI, 26 to 57), and 15 (95% CI, 8 to 23), respectively.     

Properties of a recombinant human hemoglobin double mutant: sickle hemoglobin with Leu-88(beta) at the primary aggregation site substituted by Ala.

A recombinant double mutant of hemoglobin (Hb), E6V/L88A(beta), was constructed to study the strength of the primary hydrophobic interaction in the gelation of sickle Hb, i.e., that between the mutant Val-6(beta) of one tetramer and the hydrophobic region between Phe-85(beta) and Leu-88(beta) on an adjacent tetramer. Thus, a construct encoding the donor Val-6(beta) of the expressed recombinant HbS and a second mutation encoding an Ala in place of Leu-88(beta) was assembled. The doubly mutated beta-globin gene was expressed in yeast together with the normal human alpha-chain, which is on the same plasmid, to produce a soluble Hb tetramer. Characterizations of the Hb double mutant by mass spectrometry, by HPLC, and by peptide mapping of tryptic digests of the mutant beta-chain were consistent with the desired mutations. The absorption spectra in the visible and the ultraviolet regions were practically superimposable for the recombinant Hb and the natural Hb purified from human red cells. Circular dichroism studies on the overall structure of the recombinant Hb double mutant and the recombinant single mutant, HbS, showed that both were correctly folded. Functional studies on the recombinant double mutant indicated that it was fully cooperative. However, its gelation concentration was significantly higher than that of either recombinant or natural sickle Hb, indicating that the strength of the interaction in this important donor-acceptor region in sickle Hb was considerably reduced even with such a conservative hydrophobic mutation.     

DNA polymorphisms in north Sardinian newborns and their linkage with abnormal γ globin gene arrangements and with βo-thalassemia

Fetal hemoglobin analysis and globin gene mapping have identified one type of beta(0)-thalassemia and four different gamma globin gene arrangements among newborn babies from the northern part of Sardinia. The beta(0)-thalassemia with a nonsense mutation at codon 39 was found on two chromosomes, each with a distinct pattern of polymorphic restriction sites; one had the A gamma T (A gamma 75 Ile----Thr) mutation, while the second did not. Four closely related haplotypes were identified for chromosomes with the A gamma T mutation. The gamma-thalassemia heterozygosity with the -GA gamma- hybrid gene fell into two categories. One apparently originated through crossing-over between mismatched chromosomes characterized by the most common haplotype, while the other had polymorphisms resembling those of a less frequently occurring chromosome. Chromosomes with the -G gamma-AG gamma-A gamma- triplication had polymorphic sites to be expected for this condition, being complimentary to the -GA gamma- thalassemias. Of the two additional gamma globin gene variations the -G gamma- G gamma- arrangement was associated with the chromosome with the most commonly occurring haplotype, while the chromosome with the -A gamma-A gamma- arrangement had a haplotype characteristic for that with the A gamma T mutation, which identified an -A gamma-A gamma T- arrangement. The incidental discovery of a silent beta-chain mutant, Hb Hamilton, with the Val----Ile substitution at position beta 11, in five newborns was also reported.     

Mitochondrial Changes in β0-Thalassemia/Hb E Disease

The compound β°-thalassemia/Hb E hemoglobinopathy is characterized by an unusually large range of presentation from essentially asymptomatic to a severe transfusion dependent state. While a number of factors are known that moderate presentation, these factors do not account for the full spectrum of presentation. Mitochondria are subcellular organelles that are pivotal in a number of cellular processes including oxidative phosphorylation and apoptosis. A mitochondrial protein enriched proteome was determined and validated from erythroblasts from normal controls and β°-thalassemia/Hb E patients of different severities. Mitochondria were evaluated through the use of mitotracker staining, analysis of relative mitochondrial genome number and evaluation of mitochondrial gene expression in addition to assay of overall cellular redox status through the use of alamarBlue assays. Fifty differentially regulated mitochondrial proteins were identified. Mitotracker staining revealed significant differences in staining between normal control erythroblasts and those from β°-thalassemia/Hb E patients. Differences in relative mitochondria number and gene expression were seen primarily in day 10 cells. Significant differences were seen in redox status as evaluated by alamarBlue staining in newly isolated CD34+ cells. Mitochondria mediate oxidative phosphorylation and apoptosis, both of which are known to be dysregulated in differentiating erythrocytes from β°-thalassemia/Hb E patients. The evidence presented here suggest that there are inherent differences in these cells as early as the erythroid progenitor cell stage, and that maximum deficit is seen coincident with high levels of globin gene expression.