Restriction endonuclease analyses of DNA from one Black G gamma A gamma-HPFH homozygote and four Black and one Indian G gamma A gamma-HPFH heterozygotes have identified three different HPFH types which are the result of large deletions including the delta and beta genes. Two of the types are comparable to those characterized previously, but the third, which is present in the Indian heterozygote, shows a distinct difference in the size of the deletion. The 5' end point of the deletion in this type III G gamma A gamma-HPFH extends 0.5-1.0 kb beyond the 5' end point of one of the Black types of HPFH (type I). Each of the three types is associated with a distinct ratio between the G gamma and the A gamma chains, an observation supported by family data. The highest ratio is found in the heterozygote with the Indian type III G gamma A gamma-HPFH, with 69.3% G gamma chains, while the averages for the other types were 50.7% G gamma (type I) and 32.3% G gamma (type II).

Additional data are presented that were obtained with a newly developed cation-exchange high-performance liquid chromatography (HPLC) method allowing the separation of numerous normal and abnormal human hemoglobin types. The method was found to be of considerable value for the diagnosis of certain hemoglobinopathies in the adult as well as in the newborn. Definitive differentiation between, AS, SS, S-beta+-thal, SD, SC, AC, C-beta+-thal, etc in cord bloods was readily accomplished even when the samples were collected on filter paper. Analyses of the hemoglobin in cells from Hb S and Hb C heterozygotes with different numbers of alpha chain genes (alpha alpha/alpha alpha; -alpha/alpha alpha; -alpha/-alpha) after separation by a Dextran density centrifugation technique failed to show significant changes in the relative quantitation of the variant hemoglobin types. Glycosylation of hemoglobin increased greatly with an increase in red cell age. Similar analyses of red cells from a young girl with Hb S trait and Hb H disease (the --/-alpha genic arrangement) gave comparable data. Some indication was obtained that her youngest cells contained some of the beta A4 and beta S4 tetrameric hemoglobins.

The presence of the alpha-globin frameshift mutant, Hb Wayne, in three generations of a second family is described. The data include a hematological evaluation of the four heterozygotes, structural characterization of the variant, the use of HPLC for the separation of tryptic and chymotryptic peptides, functional analyses of the isolated variant showing high affinity for oxygen and the (near) absence of a Bohr effect, and alpha chain gene organization analyses with restriction endonuclease technology suggesting that the Hb Wayne heterozygote has a full complement of four alpha globin genes.

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