M. E. Gravely

Globin Chain Electrophoresis: a New Approach to the Determination of the Gγ/Aγ Ratio in Fetal Haemoglobin and to Studies of Globin Synthesis

Summary. Separation of globin chains by electrophoresis provides a simple and rapid method for the determination of the Gγ/Aγ ratio in human fetal haemoglobin, and of biosynthetic rates of the globin chains. Whole haemolysates were analysed by electrophoresis on polyacrylamide gels in urea, acetic acid and Triton X‐100. Electrophoresis of haemolysates from newborn infants led to four bands: Aγ, Gγ, β and α. The identity of these bands was indicated by examination of haemoglobins of known globin chain composition. In 15 samples, the %Gγ was similar by Triton gels and by amino acid analysis of the γCB‐3 peptide. Some mutant globin chains were also separable with the electrophoretic technique. Triton gel electrophoresis provides rapid analysis of very small amounts of haemoglobin, and permits examination of globin chain composition as well as globin synthetic ratios.

The Synthesis of Fetal Hemoglobin Types in red Blood Cells and in BFU-E Derived Colonies from Peripheral Blood of Patients with Sickle Cell Anemia, β+- and δβ-Thalassemia, Various forms of Hereditary Persistence of Fetal Hemoglobin, Normal Adults and Newborn

The biosynthesis of two types of human fetal hemoglobin (Hb F), namely Hb F with Gγ chains having glycine in position 136 and Hb F with Ay chains having alanine in position 136, was studied in blood samples and in cultures of erythroid precursors from blood of patients with different hemoglobinopathies. High pressure liquid chromatography (HPLC) was adapted to allow the separation of the methionyl-containing tryptic peptides GγT-15 and AγT-15 (which include the Gly → Ala polymorphism at position 136) from a digest of microquantities of 35S-methionyl labelled Hb F. This method was sensitive enough to quantitate the relative production of the Gγ and Aγ chains by erythroid colonies derived from cloned Burst Forming Units (BFU-E) which were cultured for 16 days on methylcellulose. The production of Hb F in these colonies was generally higher than the level of Hb F in blood except for subjects with the GγAγ-HPFH heterozygosity. The Gγ to Aγ ratio in the Hb F produced in cultures of cells from Gγδβ-thalassemia ...

alpha-thalassemia-2 and the variability of hematological values in children with sickle cell anemia.

Summary: Seventy children homozygous for Hb S (SS) and their 111 heterozygous (AS) parents were evaluated through their erythrocytic indices, hemoglobin composition, and occasionally through in vitro Hb chain synthesis values.Three groups of SS patients and of AS parents were identified based on differences in degree of microcytosis (MCV) and (degree of hypochromia (MCH) values. The level of Hb S in the Hb S heterozygotes showed a trimodal distribution. Five SS patients had an α-thalassemia homozygosity (α0α/α0α; βS/βS) which was characterized by a distinct microcytosis and hypochromia (MCV, ≤ 70 fl; MCH, ≤22 pg). Nine SS patients had an α-thalassemia heterozygosity (α0/α/αα; βS/βS) with an MCV value of 71 to 78 fl, and an MCH value of 21.3 to 26.5 pg. Four AS parents had an α-thalassemia-2 homozygosity with values of MCV ≤ 71 fl and MCH ≤ 23.5. The level of Hb S was < 31%. Thirty-nine AS parents had an α-thalassemia-2 heterozygosity characterized by an MCV value of 72 to 79 fl, an MCH value of 23.6 to 26.5, and a level of Hb S ranging between 31.0 and 36.8%.The Hb A2 level in SS patients was significantly correlated with the RBC counts and the MCV and MCH (r = 0.38, −0.52, and −0.47, respectively). Significant correlations in AS parents were also noted between the MCV, MCH, RBC, and Hb S percentages (r = 0.62, 0.68, and −0.49, respectively).Although the data are limited, the simultaneous occurrence of an α-thal-2 homozygosity seems to decrease the level of Hb F in sickle cell anemia. The presence of an α-thal-2 heterozygosity or homozygosity together with an SS or AS condition resulted in identifiable hematologic phenotypes.Speculation: The occurrence of microcytosis and hypochromia among SS patients in association with increased erythrocyte counts and Hb A2 percentages indicates the concomitant presence of α-thalassemia-provided β chain deficiencies have been excluded through biosynthetic experiments or through family studies. The α-thal-2 heterozygosity (α0α/αα; βS/βS), which is associated with a mild α chain deficiency, results in a slight decrease of the degree of microcytosis and hypochromia values. On the other hand, the α-thal-2 homozygosity (α0α/α0α; βS/βS) and perhaps also the α-thal-1 heterozygosity (α0α0/αα; βS/βS), which are associated with a moderate α chain deficiency, result in a distinct microcytosis and hypochromia. The high incidence of α-thal-2 among Black Americans requires careful consideration and may be more prevalent than an iron deficiency anemia in children with the SS and AS conditions.

Hb S, Hb G-PHILADELPHIA AND |[alpha]|-THALASSEMIA-2 IN A BLACK FAMILY

Summary: A Black family is described in which Hb S, Hb G-Philadelphia and α-thal-assemia-2 determinants occurred in different combinations. The propositus was a healthy fullterm neonate who had 46% Hb G-Philadelphia and about 5% Hb Barts in cord blood together with a relative microcytosis (MCV = 85 f1***) and hypochromia (MCH = 28 pg). This is consistent with a diagnosis 6f Hb G-Philadelphia trait in association with a homozyqous α-thalassemia-2 {a°α/a°αG βA/βA). The mother and another son also had Hb G-Ph1ladelphia in association with Hb S trait but with 373 Hb G-Philadelphia and with 391 Hb S. Hematological and biosynthetic studies confirm the assignment of the αα/αDα; βA/βAgenotype in both and that of the αα/αDα; βA/βA genotype in the father. Despite this evidence for a moderate α chain deficiency in the propositus, the biosynthetic α/non-α value in the neonatal period was a high 1.2. Similar values were observed in 8 control cord blood samples if the incubation was not delayed longer than 3 hours after collection (α /non-α = 1.28 ± 0.14). When the propositus was studied again, but at six months of age, the proportion of Hb G-Philadelphia in peripheral blood was unchanged, a marked microcytosis and hypochromia were observed, and a distinct deficiency of α chain synthesis (α /non-α = 0.56) was present.Speculation: The occurrence of a decreased number of active α chain genes due to the inheritance of α-thalassemia-2 determinants causes the proportion of the α chain variant Hb G-Philadelphia to Increase and the proportion of the β chain variant Hb S to decrease. Thus, the levels of these two abnormal hemoglobins help to define the number of active α chain genes and the occurrence of α-thalassemia. Despite the existence of a moderate α chain deficiency, the failure to detect any decreased in vitro synthesis of α chains in this neonate as well as the finding of excessive In vitro α chain synthesis in normal neonates suggest that during the transition from fetal to adult type of erythropoiesis, the synthesis of γ chains may decline more rapidly than that of α chains before the synthesis of β chains is established.

Hb-volga or α2β227(B9)Ala→Asp an unstable hemoglobin variant in three generations of a dutch family

Abstract An unstable hemoglobin was found in five members of three generation of a Dutch family. Chemical characterization indicated a single amino acid-substitution at position 27 of the β chain, α2β227(B9)Ala→Asp. The occurrence of this variant is associated with a hemolytic anemia with splenomegaly, methemoglobinemia, and the formation of Heinz bodies.

A new δ chain variant, haemoglobin-A2-melbourne or α2δ243Glu→Lys(CD2)

Abstract Haemoglobin-A 2 -Melbourne, α 2 δ 2 43Glu→Lys(CD2) , has been found in members of a family of Italian origin. This variant is a stable haemoglobin and its presence does not have clinical consequences.

Hb-Alberta or α2β2 (101(G3) GLU→GLY), a New High-Oxygen-Affinity Hemoglobin Variant Causing Erythrocytosis

Hb-Alberta has been found in a 51 year old Caucasian male with erythrocytosis. The substitution in this variant involves the glutamyl residue in position lOl(G3) of the β chain which is replaced by a glycyl residue. Hb-Alberta accounts for about 45% in the heterozygote, and readily forms hybrid tetramers with other hemoglobins. The oxygen affinity of Hb-Alberta is greatly increased, its Bohr effect reduced, and its subunit interaction greatly diminished.

Hemoglobinopathies observed in the population of the Southeastern United States (SE-USA).

A survey of nearly 250,000 citizens of Georgia and South Carolina conducted during the past twenty years has led to the detection of over 40 abnormal hemoglobins and several additional hemoglobinopathies. The presence of some of these hemoglobin abnormalities cause (severe) clinical symptoms but others remain undetected unless a specific search is initiated. The incidence of Hb S varies slightly among the populations of different areas, and appears to be the highest in the coastal counties of Georgia and South Carolina. A survey of over 17,000 persons of mainly high school and college age has shown that a significant number of cases with clinically significant hemoglobinopathies will remain undetected unless such surveys are actively promoted.

δβ-Thalassemia in a Mexican Family: Clinical Differences Among Homozygotes

Three delta beta-thalassemia homozygotes were found in a Mexican family. Both parents and two sibling had heterozygous delta beta-thalassemia with about 10% Hb F, mild microcytosis and mild hypochromia, while three siblings were normal. Hb F, which was the only Hb component in the homozygotes, had equal quantities of Ggamma and Agamma chains as in BgammaAgamma-delta beta-thalassemia. The homozygotes had comparable erythrocytic indices which were about the same as those of the heterozygotes. However, two were clinically and hematologically healthy but the third had a severe chronic hemolytic anemia and a more severe in vitro chain synthesis imbalance than her homozygous sisters. Comparison of these cases with other GgammaAgamma-delta beta-thalassemia homozygotes and with GgammaAgamma-HPFH homozygotes indicates the possibility that the proliferation of F-cell precursors may be defective in delta beta-thalassemia.

Hemoglobin Hofu or αβ [126 (H4) Val → Glu] Found in Combination with Hemoglobin S

Hb Hofu, αβ[126 (H4) Val → Glu], was found in 10 members of 2 apparently unrelated Valmiki families in central India. None showed evidence of hemolysis and hemoglobin levels were normal in most. In two individuals, Hb Hofu occurred in combination with Hb S, but neither had clinical manifestations of sickle cell disease. In samples containing Hb Hofu, the isopropanol precipitation test was positive. Quantitation of the hemoglobin fractions by DEAE-cellulose chromatography showed that Hb Hofu constituted a mean of 23-25% of the total whether in combination with Hb A or Hb S.