In a survey of Italian newborn babies, one baby boy was found to have a new fetal hemoglobin variant which is characterized by a GluaGln substitution at position γ6(A3). This variant had an isoleucine at position γ75 and an alanine at position γ136.

The synthesis of Hb F was studied in the BFUe-derived colonies of 20 SS patients after incubation with 35S methionine for 24 hours, 13 days after the start of the experiments. Similar analyses were made for isolated reticulocytes from 28 SS patients; these cells were incubated for 2 hours and occasionally for 24 hours. The G gamma and A gamma percentages were determined in these two Hb F preparations and in the Hb F of circulating red cells using two different HPL chromatographic procedures. The data show an increased production of Hb F in the colonies mainly of patients with low blood Hb F values. A close correlation was present between the % G gamma chain in peripheral red cells and the G gamma percentages observed for the Hb F synthesized in the BFUe-derived colonies and that in reticulocytes. However, the values for the reticulocytes were significantly higher than those for the colonies. These data indicate a considerable variability among SS patients in their ability to produce Hb F in in vitro colonies which may in part be due to differences in the numbers of BFUe types in circulation. The variation in G gamma and A gamma levels in Hb F synthesized in reticulocytes and BFUe-derived colonies could be the result of slight differences in the stability of the corresponding mRNA's.

The synthesis of α and non-α chains of human hemoglobin (Hb) was studied in reticulocytes and in BFUe-derived cell colonies from patients with α chain or β chain deficiencies. The subjects included normal adults (αα/αα) with or without a β chain variant (Hb S, Hb Leslie) or an α chain variant (Hb G-Georgia); α-thalassemia-2 heterozygotes (α 0 α/αα) with an α chain variant (G-Georgia or G-Philadelphia); an α-thalassemia-1 heterozygote (α 0 α 0 /αα); α-thalassemia-2 homozygotes (α 0 α/α 0 α) with a β chain variant (Hb S), an α chain variant (G-Philadelphia), a Hb S homozygosity with Hb G-Philadelphia, or a Hb G-Philadelphia homozygosity; and three black β + -thalassemia homozygotes. Data from reticulocyte in vitro synthesis analyses showed the expected deficiencies. However, similar analyses of the Hb synthesized in cell colonies (even from the black β-thalassemia homozygotes) gave (nearly) balanced ∑α/∑non-α ratios. It is speculated that this balanced synthesis is due to a most effective proteolysis in the immature erythroblast which rapidly removes free α or β chains. The levels of Hb F and Hb A 2 were considerably increased in these proerythroblasts; a two- to threefold increase in the synthesis of Hb A 2 was observed over that seen in the reticulocytes.

Quantitative information about the three types of gamma chains (A gamma T, A gamma I, G gamma) has been obtained for the Hb F from 285 normal Black babies, 172 babies with a Hb S or Hb C heterogeneity, and from 150 babies and older patients with the SS, SC, or CC conditions by means of a high pressure liquid chromatographic microprocedure. The frequency of the A gamma T gene in the AA babies was 0.1035, while that in the SS patients was a low 0.0362. This low A gamma T frequency in the SS population adequately explains the lower percentage (13.2%) of A gamma T heterozygotes among AS newborn babies as compared to 17.9% among AA babies. The genotype with the A gamma T mutant in trans to the beta S mutation is presumed to occur about three times more frequently than that in which the A gamma T mutant is in cis to the beta S mutation. A study of family members of one SS patients who has an A gamma T homozygosity provided data supporting linkage of the A gamma T and beta S anomalies in some Hb S heterozygotes.

The synthesis of alpha and non-alpha chains of human hemoglobin (Hb) was studied in reticulocytes and in BFUe-derived cell colonies from patients with alpha chain or beta chain deficiencies. The subjects included normal adults (alpha alpha/alpha alpha) with or without a beta chain variant (Hb S, Hb Leslie) or an alpha chain variant (Hb G-Georgia); alpha-thalassemia-2 heterozygotes (alpha 0 alpha/alpha alpha) with an alpha chain variant (G-Georgia or G-Philadelphia); an alpha-thalassemia-1 heterozygote (alpha 0 alpha 0/alpha alpha); alpha-thalassemia-2 homozygotes (alpha 0 alpha 0/alpha 0 alpha) with a beta chain variant (Hb S), an alpha chain variant (G-Philadelphia), a Hb S homozygosity with Hb G-Philadelphia, or a Hb G-Philadelphia homozygosity; and three black beta +-thalassemia homozygotes. Data from reticulocyte in vitro synthesis analysis showed the expected deficiencies. However, similar analyses of the Hb synthesized in cell colonies (even from the black beta-thalassemia homozygotes) gave (nearly) balanced sigma alpha/sigma non-alpha ratios. It is speculated that this balanced synthesis is due to a most effective proteolysis in the immature erythroblast which rapidly removes free alpha or beta chains. The levels of Hb F and Hb A2 were considerably increased in these proerythroblasts; a two- to threefold increase in the synthesis of Hb A2 was observed over that seen in the reticulocytes.

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.

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.