A new cation-exchange HPLC method is described for the separation and quantitation of abnormal human Hbs. The method makes use of Synchropak CM 300, a silica support with carboxylic acid residues, and Bis-Tris-KCN-Na-acetate developers and allows a completion of the chromatogram in 50 to 90 min depending on the sample to be analyzed. Specific chromatographic profiles have been obtained for several beta, alpha, and delta chain variants. The method is useful not only for the quantitation of Hb F and Hb A2 (except in the presence of Hb E) but also provides easy differentiation between simple heterozygotes for a specific abnormality and persons with the same abnormality together with an additional beta-thalassemia heterozygosity. Probably the most important application of the new procedure is in the quantitation of Hb A and the beta chain variants S, C, O-Arab, and E in cord blood samples, thus facilitating the diagnosis of conditions such as AE, EE, AS, SS, S-beta +thal, AC, CC, C-beta +thal, SC, and SO in newborn babies.

Isopycnic separations of red cells from cord bloods, and from patients with sickle cell anemia, different forms of HPFH, S-β0-thalassemia, and a β+-thalassemia homozygosity were made in order to evaluate the distribution of Hb F and the relative levels of Gγ and Aγ chains over the cell fractions. As expected, the cord blood data showed decreased levels of both Hb-F and Gγ chains in the top cell fractions since the β → γ and high Gγ: Aγ low Gγ: Aγ switches are operative around the time of birth. Complete cell fractionations were made on the blood of three SS patients with low Gγ values (40%) and three SS patients with high Gγ values (60%). The proportion of Gγ chain was constant in all cell fractions. while the Hb-F level was higher in cells with higher densities. The difference in the quantities of the three types of γ chain in the fetal hemoglobins of two SS patients with an AγT heterozygosity, one having a low Gγ value and the other a high Gγ level, can be explained by assuming an alteration in a regulatory mechanism. Considerable variation both in the level of Hb F and in the percentage of Gγ chain was observed in two GγAγ-HPFH heterozygotes with a relatively low Gγ percentage of 30%; an inverse relationship was present between the two parameters. Such a phenomenon was not evident for the GγAγ-HPFH homozygote, and also did not exist in two additional GγAγ-HPFH heterozygotes with an associated α-thalassemia-2 heterozygosity who had a similar amount of Hb F but with higher Gγ values of about 50%. The difference in Gγ values between these two categories of GγAγ-HPFH could be due to a higher affinity of the Gγ chains over Aγ chains for a slightly decreased amount of α chains as in an α-thalassemia-2 heterozygosity. 2 heterozygosity.

The relative quantities of the three types of gamma chain (G gamma, A gamma I, A gamma T) were determined in 18 AS parents of selected SS patients, in 15 additional HbS heterozygotes, as well as in additional SS patients, and in 35 SS and 24 AS newborn babies. The low amount of HbF in all AS adults (less than 1%) made it necessary to further improve the isolation procedure of HbF, which was accomplished by introducing an HPL chromatographic method. The additional data for older SS patients confirmed the existence of two groups characterized by either low G gamma (40%) or high G gamma (60%) values in their HbF. A distinction into “high G gamma” and “low G gamma” producers could also be made for HbS heterozygotes. Family studies, however, make it unlikely that the “high G gamma” condition is inherited in a simple mendelian fashion assuming a change in a regulatory mechanism. The presence of the A gamma T mutation, occurring either in cis or in trans to the beta S mutation, has been used to evaluate the possible contribution by specific gamma-chain genes to the gamma-chain composition of the HbF of adult AS persons. No clear pattern because evident, suggesting that most of the gamma-chain of this small amount of HbF could originate from gamma-chain genes in cis or in trans to the beta S gene or from both sets of genes. It is speculated that heterogeneity among “F-cells” may be a primary cause of the observed differences in gamma-chain composition of HbF in HbS heterozygotes.

(1982). Hb Cheverly or α2β2 45(CD4)Phe a Ser in an Elderly Italian Male. Hemoglobin: Vol. 6, No. 4, pp. 419-421.

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 αG chain, and one to give the αMO 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.

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.

Two alpha-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 alpha-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 alpha-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 alpha-chain genes, of which one regulated the synthesis of the normal alpha chain, one was mutated to give the alpha G chain, and one to give the alpha MO chain. The amino acid substitutions in Hb G-Philadelphia and Hb Matsue-Oki are located in the tryptic peptide alpha T-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 alpha 68 Asn replaced by Lys substitution is readily demonstrable in Hb G-Philadelphia the elucidation of the alpha 75 Asp replaced by Asn replacement in Hb Matsue-Oki was greatly facilitated by the use of these microprocedures.

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.

An Indian family is described in which the father has a delta chain abnormal hemoglobin which is the result of a mutation of the delta gene in cis to a beta-thalassemia heterozygosity. The abnormality concerns a substitution of the Asp residue in position 99 (G1) by an Asn residue. A similar substitution has been found in the beta chain of Hb Kempsey (alpha 2 beta 2 99 Asp replaced by Asn). The observed abnormality results in a greatly increased oxygen affinity of this newly discovered Hb A2 variant.