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.

Two Mexican families from the State of Jalisco have been studied in which 11 members were carriers of Hb Tarrant. Ten subjects were Hb Tarrant heterozygotes producing about 25% of the abnormal hemoglobin. One 9-year-old boy was homozygous for Hb Tarrant. About 50% of his hemoglobin was of the variant type. The heterozygotes had mild erythrocytosis which was considerably more severe in the homozygote. The average P50 value for blood of the heterozygote was 15.1 mm Hg (controls: 22.5 mm Hg) while this value was decreased to 9 mm Hg in the homozygote. The clinical condition of the homozygote is compatible with a mild chronic tissue hypoxia.

A modification of a high pressure liquid chromatographic (HPLC) procedure is described that enables the complete separation and quantitation of the A gamma T, A gamma I, and G gamma chains in human fetal hemoglobin. The method, which is fast and accurate, requires 5 to 2000 micrograms Hb F. The purity of the Hb F is not essential and admixture of up to 70% adult Hb does not interfere with the determination. The method has been applied to the Hb F of 64 Black SS patients and 7 persons with the Hb S-HPFH (G gamma A gamma type) conditions. (A) Both “adult” G gamma to A gamma (2:3) and “newborn” G gamma to A gamma (3:2) ratios were observed in adult SS patients, 8 yr and older. Only 12% of the SS patients had the “newborn” ratio. This high G gamma to A gamma ratio may be due to a modification of the genetic switch mechanism that regulates the change of this ratio after birth. (B) Intermediate G gamma to A gamma ratios were only found in young SS patients, 5 yr of age or less. The results suggest a delayed switch of the newborn leads to adult ratio in sickle cell anemia. (C) The A gamma T chain was present in only 6% of all SS patients. One patient is homozygous for this variant chain. (D) Three of the 7 subjects with Hb S-HPFH were positive for the A gamma T chain. Its percentage was low, which suggests that the A gamma T chain gene is in trans of the HPFH determinant. (E) Quantitation of the three gamma chain types is also possible in the Hb F from Hb S heterozygotes with (nearly) normal Hb F levels. Such an analysis is useful for an evaluation of genetic conditions involving variations in the production of (different types of) Hb F.

High pressure liquid chromatography (HPLC) was applied to the HbF isolated from blood of numerous black patients with beta-thalassemia trait or homozygosity, G gamma-delta beta-thalassemia trait, G gamma A- gamma HPFH heterozygosity, or the G gamma-[delta+ beta+]-HPFH condition. The method allowed an accurate evaluation of the relative quantities of three types of gamma-chain (G gamma, A gamma I, A gamma T) in the fetal hemoglobins. The results have shown the following. (A) The incidence of the A gamma T-chain in beta-thal heterozygotes and G gamma A gamma-HPFH heterozygotes is about the same as has been observed in black newborn; about one of five blacks are heterozygous for this A gamma-chain variant. The A gamma T-chain was not detected in the nine G gamma-delta beta-thal heterozygotes nor in the eight G gamma-[delta+ beta+]-HPFH heterozygotes. (B) In most cases, the A gamma T-chain was produced by the A gamma gene in trans to the beta-thal or HPFH determinant. The contribution by the gamma-chain genes in trans to the beta-thal or HPFH determinant is about 15% of the total gamma-chain production in both conditions. (C) Three black beta-thal heterozygotes (and five additional relatives) had the A gamma T gene in cis to the beta-thal determinant. Four of these patients had a low levels of G gamma-chain (the “adult” level), and the contribution by the A gamma gene in cis to the beta-thal determinant was about three times that of the A gamma gene in trans. The four additional patients, all members of one family, had a high level of G gamma-chain (the “newborn” level), and the contribution of the A gamma gene in cis was half of that seen in the previously mentioned four patients while that of the A gamma gene in trans was essentially the same. These limited data suggest that the genetic anomaly causing high high G gamma levels in adult beta-thal heterozygotes is linked to the beta-thal determinant and that one of its primary effects is a decreased synthetic expression of the A gamma gene in cis to the beta-thal determinant.

A black family is described in which the β chain variant hemoglobin Leslie, the α chain variant hemoglobin Montgomery, and α-thalassemia-2 occurred in different combinations. All members studied had normal hematological values and no chronic ill health. The propositus had 11% hemoglobin Montgomery, 24% hemoglobin Leslie, and only about 0.5% of the Hb Montgomery–Leslie hybrid. The mother of the propositus was a Hb Leslie heterozygote with 31% Hb Leslie. Her father had 26% and her son had 17% Hb Montgomery, suggesting that the father is heterozygous for α-thalassemia-2; this designation was supported by data from in vitro biosynthetic studies. The β-Leslie chain generally shows reduced ability to pair with the normal α chain. However, in the propositus, who is heterozygous for both variants, the assembly of β-Leslie and α-Montgomery chains might be reduced even more, and the instability of the Hb Montgomery-Leslie hybrid appears much greater than was expected.

The synthesis of alpha and non-alpha chains (beta, delta, G gamma, and A gamma) was studied in cultures of peripheral blood mononuclear cells from eleven beta-thalassemia heterozygotes, two HPFH heterozygotes, and one HPFH homozygote. The synthesis of Hb F in the thalassemia colonies (average value: 12.6%) was comparable to that in normal adult colonies (average value: 12.6%) was comparable to that in normal adult colonies (average value: 12.2%). The percent G gamma chain in the Hb F varied greatly but a relationship between the G gamma chain percentage in the Hb F from colonies and that from peripheral blood was established. The relative synthesis of Hb A2 in colonies of beta-thalassemia heterozygotes (average value: 5.8%) was 1.6 times as much as that in colonies of normal adults (average value: 3.6%). Hb A2 and Hb A were absent in the colonies of the HPFH homozygote. The alpha/non-alpha (i.e., beta, gamma, and delta) ratio of the hemoglobins in the cultured cells of the beta-thalassemia heterozygotes and the alpha/beta and alpha/beta ratios of isolated Hb A and Hb A2 were about one (range 0.74 to 1.38). The alpha/gamma ratio of the Hb F synthesized in BFUe-derived colonies of the HPFH homozygote, however, was 1.5. These results suggest a deficiency in the in vitro culture system resulting in decreased levels of alpha-mRNA or in a partial inhibition of initiation of protein synthesis which is known to reduce the synthesis of alpha chains more than that of the beta chains.

Annals of the New York Academy of SciencesVolume 344, Issue 1 p. 253-261 THE RATIO OF Gγ AND Aγ CHAINS OF THE HEMOGLOBIN F SYNTHESIZED BY BFU-E-DERIVED COLONIES FROM BLOOD OF SUBJECTS WITH β+ THALASSEMIA AND RELATED HEMOGLOBINOPATHIES * T. H. J. Huisman, T. H. J. Huisman Department of Cell and Molecular Biology Medical College of Georgia Augusta, Georgia 30912Search for more papers by this authorG. D. Efremov, G. D. Efremov Department of Cell and Molecular Biology Medical College of Georgia Augusta, Georgia 30912Search for more papers by this authorA. L. Reese, A. L. Reese Department of Cell and Molecular Biology Medical College of Georgia Augusta, Georgia 30912Search for more papers by this authorJ. S. Howard, J. S. Howard Department of Cell and Molecular Biology Medical College of Georgia Augusta, Georgia 30912Search for more papers by this authorM. E. Gravely, M. E. Gravely Department of Cell and Molecular Biology Medical College of Georgia Augusta, Georgia 30912Search for more papers by this authorJ. B. Wilson, J. B. Wilson Department of Cell and Molecular Biology Medical College of Georgia Augusta, Georgia 30912Search for more papers by this author T. H. J. Huisman, T. H. J. Huisman Department of Cell and Molecular Biology Medical College of Georgia Augusta, Georgia 30912Search for more papers by this authorG. D. Efremov, G. D. Efremov Department of Cell and Molecular Biology Medical College of Georgia Augusta, Georgia 30912Search for more papers by this authorA. L. Reese, A. L. Reese Department of Cell and Molecular Biology Medical College of Georgia Augusta, Georgia 30912Search for more papers by this authorJ. S. Howard, J. S. Howard Department of Cell and Molecular Biology Medical College of Georgia Augusta, Georgia 30912Search for more papers by this authorM. E. Gravely, M. E. Gravely Department of Cell and Molecular Biology Medical College of Georgia Augusta, Georgia 30912Search for more papers by this authorJ. B. Wilson, J. B. Wilson Department of Cell and Molecular Biology Medical College of Georgia Augusta, Georgia 30912Search for more papers by this author First published: May 1980 https://doi.org/10.1111/j.1749-6632.1980.tb33666.xCitations: 7 * This work was supported by a research grant (HLB-05168) from the U.S. Public Health Service. Contribution 526 of the Medical College of Georgia Department of Cell and Molecular Biology. AboutPDF 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 onFacebookTwitterLinkedInRedditWechat Citing Literature Volume344, Issue1Fourth Cooley's Anemia SymposiumMay 1980Pages 253-261 RelatedInformation

A Black family is described in which Hb S, Hb G-Philadelphia and alpha-thalassemia-2 determinants occurred in different combinations. The propositus was a healthy fullterm neonate who had 46% Hb G-Philadelphia and about 5% Hb Bart's in cord blood together with a relative microcytosis (MCV = 85 fl) and hypochromia (MCH = 28 pg). This is consistent with a diagnosis of Hb G-Philadelphia trait in association with a homozygous alpha-thalassemia-2 (alpha 0 alpha/alpha 0 alpha G; beta A/beta A). The mother and another son also had Hb G-Philadelphia in association with Hb S trait but with 37% Hb G-Philadelphia and with 39% Hb S. Hemotological and biosynthetic studies confirm the assignment of the alpha alpha/alpha 0 alpha G; beta A/beta S genotype in both and that of the alpha alpha/alpha 0 alpha; beta A/beta A genotype in the father. Despite this evidence for a moderate alpha chain deficiency in the propositus, the biosynthetic alpha/non-alpha 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 (alpha/non-alpha = 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 alpha chain synthesis (alpha/non-alpha = 0.56) was present.

Peripheral blood mononuclear cells from normal adults, normal newborn infants, and from newborn and adult subjects with one of three γ chain variants ( G γF-Malta I, G γF-Port Royal, A γF-Hull) were cultured in vitro with erythropoietin. The 35 S-methionine-labelled hemoglobin from 13- to 15-day-old BFUe-derived colonies was studied by chromatography on columns of DEAE-cellulose and the quantities of Hbs A 2 , F x , and F 0 determined. The percentages of G γ and A γ chains in isolated Hb F x and Hb F 0 were determined using high-performance liquid chromatography (HPLC) of the tryptic peptides of these proteins. Calculation of these percentages was based on the total activities of the G γT-15 and A γT-15 peptides which contain one ( 35 S-labelled) methionyl residue each and can be separated by the HPLC procedure. The data show an increased synthesis of Hb F in the „adult”︁ colonies and a decreased synthesis in the „newborn”︁ colonies. The G γ to A γ ratio of the Hb F from adult colonies varied greatly. The percentages of G γ and A γ chains in the Hb F from adult colonies correlated with the percentages in the Hb F isolated from the Hb F of circulating red blood cells. The G γ to A γ ratio in the Hb F from newborn colonies was high as in the Hb F from cord blood samples. G γ and A γ chain abnormal Hb F variants were readily detectable in colonies from both adults and newborn. The G γ to A γ ratio in the Hb F of colonies of adult Hb F-Malta I and Hb F-Hull heterozygotes approached 1, but that of adult Hb F-Port Royal heterozygotes remained about as high as in colonies from newborn heterozygotes. The percent Hb F-Port Royal in the Hb F of adult colonies was twice that in the Hb F of newborn colonies. These results are discussed in the light of information from recent detailed studies of genomic DNA assuming controlling functions for the segments of DNA that are interspersed between the various structural genes.

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.

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.

Hb P-Nilotic which is produced by a hybrid of beta and delta genes was found in several members of a Sudanese family, three of whom had an associated beta-thalassemia. Chemical analyses confirmed the crossover between positions 22 and 50 of the beta delta P chain. The Hb p-Nilotic heterozygote had completely normal hematology, but the patients with the Hb P-Nilotic--beta-thalassemia condition had moderately severe clinical and hematological abnormalities which were considerably more pronounced than those in the father who had a beta-thalassemia heterozygosity. The absolute cellular contents of normal and abnormal non-alpha chains in these subjects and the results of in vitro chain synthesis analyses suggested that the thalassemia gene in this family is of the beta0 type and that the beta A gene which is present in cis to the beta delta P gene is incapable of being stimulated when the beta0-thalassemia determinant is present in trans. It is proposed that a number of recombination events produced a beta delta P hydrid gene with duplication of the beta A gene in cis as well as a change in an untranscribed strand of DNA which controls the expression of the beta A gene.

The biosynthesis of two types of human fetal hemoglobin (Hb F), namely Hb F with G gamma chains having glycine in position 136 and Hb F with A gamma 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 G gamma T-15 and A gamma T-15 (which include the Gly leads to Ala polymorphism at position 136) from a digest of microquantitites of 35S-methionyl labelled Hb F. This method was sensitive enough to quantitate the relative production of the G ygamma and A gamma 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 G gamma A gamma-HPFH heterozygosity. The G gamma to A gamma ratio in the Nb F produced in cultures of cells from G gamma delta beta-thalassemia or G gamma-HPFH heterozygotes was lower and that from A gamma-HPFH heterosygotes was higher than the ratios in the Hb F of the corresponding peripheral blood cells. Mixtures of G gamma and A gamma chains were present in cell cultures of SS patients, beta+-thalassemia homozygotes and G gamma A gamma-HPFH heterozygotes in a ratio similar to that in the Hb F of mature red cells. These data suggest that erythroblasts in BFU-E derived colonies reactivate all available gamma chain structural genes, both in cis and in trans to the abnormal determinant. Hb F biosynthesis by adult blood samples concerns primarily the G gamma chains. This was particularly striking for blood samples in which erythroblasts were absent and the biosynthesis took place in fetal reticulocytes. Thus, the F-retuculocytes in blood of A gamma-HPFH heterozygotes with about 5% Hb F of the A gamma type produced primarily Hb F with G gamma chains. Similar differences were observed for G gamma A gamma-HPFH heterozygotes and, less strinkingly, for SS patients. A satisfactory explanation for this observation has not yet been obtained.

The in vitro synthesis of hemoglobin chains was investigated in 34 scikle cell anemia (SS) patients and five patients with Hb S‐β o ‐thalassemia. Incubations were made for 30 minutes and for 120 minutes. Hematological and family data were also obtained. Although the 30‐minute α/non‐α total activity ratios were more widely distributed than the ratios at 120 minutes, a distinct classification of SS patients into groups without an α‐thalassemia, with a heterozygous α‐thal‐2, or with a homozygous α‐thal‐2 could not be made. Family studies indicated that four patients who had 30‐minute α/non‐α ratios below 0.82 and mean corpuscular volume (MCV) values below 70 fl had a homozygosity for both α‐thal‐2 and Hb S. They had mild hematological features of SS disease. Many SS patients with 30‐minute α/non‐α ratios between 0.8 and 1.0 and MCV values above 70 fl had an associated α‐thal‐2 heterozygosity. Their hematological features were similar to those of SS patients with four active α chain genes. It appears that an α‐thal‐2 heterozygosity (−α/αα; β S /β S ) does not alter the hematological expression of SS disease. An α‐thal‐2 homozygosity −α/−α;β S /β S results in a microcytosis similar to that seen in Hb S‐β o ‐thalassemia patients. The diagnosis of these α chain deficiencies in association with SS disease (by in vitro chain synthesis analyses) leaves several uncertainties.

Two unrelated families are reported in which beta-thalassemia trait occurred with a heterozygosity of Hb G-Philadelphia (alpha2 68(E17)Asn leads to Lys beta2) in one family and with Hb Rampa (alpha2 95(G2)Pro leads to Ser beta2) in the other. The percentage of Hb G-Philadelphia was not influenced by the simultaneous presence of a beta-thalassemai determinant, but that of Hb Rampa was descreased from 20% in the simple heterozygote to about 6% in persons with the Hb Rampa-beta-thalassemia combination. Data from in vitro recombination experiments with isolated alpha X, alpha A, and beta A chains, with heme attached, indicated a preferential formation of Hb A over Hb Rampa but not over Hb G-Philadelphia in conditions of relative beta-chain deficiency. This suggests that the rate of assembly of monomers to form dimers or tetramers can be an important mechanism of controlling the quantity of certain hemoglobin variants with critical substitutions in heterozygotes.

(1978). Short Communications: Hb Suresnes or α2 141(Hc3) Arg→His β2 in a Black family. Hemoglobin: Vol. 2, No. 2, pp. 187-189.

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.

A second instance of the unstable mutant Hb Nottingham (α;2β;2 (FG5) 98 Val→Gly) is reported in a 7-year-old boy. Because of splenomegaly, cholelithiasis, and frequent episodes of abdominal pain, he underwent a splenectomy and cholecystectomy at age 6. The surgery resulted in both an amelioration of his RBC destruction and an acceleration of his rate of growth.Biosynthetic studies were carried out using reticulocytes obtained from his peripheral blood. These analyses disclosed an exceedingly high specific activity ratio for Hb N/Hb A. In addition as the incubation proceeded, more radioactivity accumulated in the α; chain fraction than in the β; chains (β;A+β;N). This observation is presumed secondary to degradation of the unstable β;N chains.