Hb F-Forest Park, a new A gamma variant with two amino acid substitutions, 75(E19)Ile----Thr and 73(E17)Asp----Asn, which can be identified in adults by gene-mapping analysis.

The discovery is reported of a new fetal hemoglobin (Hb) variant which has an abnormal A gamma globin chain with two substitutions, namely 73(E17) Asp----Asn and 75(E19) Ile----Thr (the latter is also seen in the common A gamma T chain). This A gamma T variant was present in a female Caucasian newborn; its quantity at birth was 12.2% of the total Hb F (including F, F1 and F-Forest Park). Extensive gene-mapping analyses with a battery of restriction enzymes and probes identified normal globin gene arrangements in the baby and several relatives, but a -G gamma-G gamma-globin gene arrangement was present in the father, paternal grandmother and half-sister. The Hb F-Forest Park anomaly could be detected in the father, paternal grandfather, half-brother and the baby through digestion of their DNA's with SfaNI and hybridization with the gamma IVS-II probe, because the G----A base substitution at codon 73 leads to loss of a restriction site and to the occurrence of an abnormal fragment.

Hb natal or alpha 2(minus Tyr-Arg) beta 2: a high oxygen affinity alpha chain variant with a deleted carboxy-terminus resulting from a TAC----TAA (Tyr----terminating codon) mutation in codon alpha 140.

The discovery is reported of a fast-moving alpha chain variant (Hb Natal) which is characterized by a shortened alpha polypeptide chain because of the deletion of the Tyr-Arg carboxy-terminal residues. Through amplification of appropriate segments of DNA and hybridization with synthetic oligonucleotide probes, it was possible to detect a C----A mutation in codon 140 of the alpha 2 globin gene, which causes a change in the codon for tyrosine to a terminating codon. Hb Natal or alpha 2 (minus Tyr-Arg) beta 2 has a high affinity for oxygen without a Bohr effect and heme-heme interaction. These results provide direct evidence for the importance of the tyrosine residue at alpha 140 in the oxygenation-deoxygenation process.

Hb J-Antakya or alpha 2 beta (2)65(E9)Lys----Met in a Turkish family and Hb complutense or alpha 2 beta (2)127(H5)Gln----Glu in a Spanish family; correction of a previously published identification.

Almost 10 years ago we reported in this journal the characterization of Hb Hacettepe or alpha 2 beta (2)127(H5)Gln----Glu. Unfortunately, we have to conclude that the original characterization of this Turkish variant was in error. The corrected data are presented in this short communication. The variant (alpha 2 beta (2)65(E9)Lys----Met) was (re)named Hb J-Antakya, after the city where the family resides. An abnormal Hb, observed in a Spanish family and named Hb Complutense, had the beta 127 Gln----Glu substitution, erroneously assigned to the Turkish variant.

Hb Doha or alpha 2 beta 2[X-N-Met-1(NA1)Val----Glu]; a new beta-chain abnormal hemoglobin observed in a Qatari female.

Structural analysis of a fast-moving hemoglobin variant, present in three members of a Qatari family, identified a Val----Glu substitution at position 1 (NA1) of the beta-chain. The introduction of this glutamic acid residue prevents the removal of the initiator methionine, thus extending the N-terminus by one residue to Met-Glu-His-Leu-Thr-. The methionine residue is blocked by an as yet not completely identified molecule. The presence of the variant in a heterozygote does not have clinical consequences.

Quantitative and qualitative comparisons of spontaneous and radiation-induced specific-locus mutation in the ad-3 region of heterokaryon 12 of Neurospora crassa.

The data from forward-mutation experiments to obtain specific-locus mutations at two closely linked loci in the adenine-3 (ad-3) region of heterokaryon 12 (H-12) of Neurospora crassa have been used to determine the relative frequencies and mutational spectra of ad-3 mutants occurring spontaneously and those induced by 7 different radiation treatments. Previous studies have demonstrated that specific-locus mutants at these two loci result from 5 major genotypic classes, namely two classes of gene/point mutations (ad-3AR and ad-3BR), and 3 classes of multilocus deletion mutations ([ad-3A]IR, [ad-3B]IR and [ad-3A ad-3B]IR). Two different approaches were used to compare spontaneous mutation in the ad-3 region with that induced by 7 different radiation treatments (UV, 32P, 447 MeV protons, 85Sr, 250 kVp X-rays, 39 MeV helium ions, and 101 MeV carbon ions). These comparisons included X2-tests on the numbers of ad-3 mutants resulting in the following two sets of ratios: (1) gene/point mutations and multilocus deletion mutations; and (2) complementing and non-complementing ad-3BR mutants. Combination of the data from these two methods of comparison has demonstrated that each of the 7 radiation treatments induced a spectrum of ad-3 mutants that is statistically different from the spontaneous spectrum. In addition, these same two methods of comparison have been used to compare the mutagenic effects of each of the 7 radiation treatments with each other. Combination of the data from these two methods of comparison demonstrated that the majority of radiation-induced specific-locus mutations: (90.5% (19/21 of the pairwise combinations)) are qualitatively different from each other. We conclude that the mechanisms by which various radiations modify DNA tend to exhibit fundamental differences from each other and from the processes involved in spontaneous mutation.

Quantitative and qualitative aspects of spontaneous specific-locus mutation in the ad-3 region of heterokaryon 12 of Neurospora crassa.

The data from forward-mutation experiments to obtain specific-locus mutations at 2 closely linked loci in the adenine-3 (ad-3) region of heterokaryon 12 (H-12) of Neurospora crassa have been tabulated to determine the frequency of spontaneous ad-3 mutations and to determine the percentages resulting from each of the 2 major genotypic classes: gene/point mutations and multilocus deletion mutations. Gene/point mutations at the ad-3B locus (ad-3BR) have been characterized to determine the percentage showing allelic complementation to obtain a presumptive identification of the genetic alteration in each mutation at the molecular level. Data from experiments performed at 2 different laboratories have been compared to assess the interlaboratory reproducibility of quantitative data on H-12. No difference was found between the frequencies of spontaneous specific-locus mutations in the ad-3 region. Genetic analysis of 172 ad-3 mutants demonstrated that specific-locus mutations in the ad-3 region result from both gene/point mutations (82.0% [141/172]), and multilocus deletion mutations (14.5% [25/172]). Heterokaryon tests for allelic complementation demonstrated that 52.5% (53/101) spontaneous ad-3BR mutants show allelic complementation, and result from single base-pair alterations. In addition, 100% (25/25) of the spontaneous multilocus deletion mutations result from the 3 smallest sized genotypic subclasses. The implications of the present experimental data for the validation of the ad-3 specific-locus assay system in Neurospora are discussed.

Alpha-thalassemia and the production of different alpha chain variants in heterozygotes.

The production of five alpha chain variants (Hb G-Georgia, Hb St. Luke's, Hb Lloyd, Hb Montgomery, and Hb G-Philadelphia) in heterozygotes was evaluated through hematological observations, hemoglobin quantification, and biosynthetic studies. All heterozygotes for Hb St. Luke's and Hb Lloyd and most heterozygotes with Hb G-Georgia and Hb Montgomery had normal hematology and average sigma alpha/beta values of about 1.1. They were assigned a normal genotype (alpha alpha G/alpha alpha), although the proportions of Hb St. Luke's and Hb G-Georgia were low (10 to 13%) and those of Hb Lloyd and Hb Montgomery twice as high (20%). Data from short-term incubations confirmed this genotype for some of these heterozygotes. Isolated Hb St. Luke's and Hb G-Georgia gave low alpha G/beta values (0.2 and 0.3) indicating that these Hb variants were defective at the level of Hb assembly. Isolated Hb Montgomery and Hb G-Philadelphia, however, gave higher alpha G/beta values of 0.6 and 0.8, respectively. A second type of variability existed among Hb G-Georgia (20 vs. 13%), Hb Montgomery (28 vs. 20%), and Hb G-Philadelphia (47 vs. 34%) heterozygotes, in whom the levels of Hb G differed. The occurrence of higher levels of these three alpha chain heterozygosities was associated with hematological or biosynthetic evidence of a mild or moderate alpha chain deficiency due to an alpha-thalassemia-2 heterozygosity (alpha alpha G/alpha O alpha or alpha O alpha G/alpha alpha) or a homozygosity (alpha O alpha G/alpha O alpha), respectively.

Hb F-Columbus-Ga or alpha 2 G gamma 2 94(FGl) Asp replaced by Asn.

A new gamma chain variant with an electrophoretic mobility at pH 8.1 between those of Hb S and Hb C was isolated and quantitated by DEAE-cellulose chromatography. It was readily identified with the use of various micro-chromatographic and sequencing procedures as alpha 2 G gamma 2 94(FGl)Asp replaced by Asn. The hemoglobin was named Hb F-Columbus-Ga. The quantity of this G gamma chain variant (as % total gamma chain) was about 39% and the percentages of the normal G gamma and A gamma I chains were 37% and 24%, respectively.

Hb Albany-GA or alpha 2(11)(A9)Lys leads to Asn beta 2.

A fast-moving alpha chain abnormal hemoglobin (Hb J) was observed in a black teenager from southern Georgia. Its quantity, i.e. the sum of Hb J and Hb J2, was 23.8%. Its presence did not cause any hematological or clinical abnormalities. Tryptic peptides from a digest of the alpha-J chain were separated by HPLC. Amino acid analysis of these peptides and sequence analysis of the abnormal T-2,3 nonapeptide identified a Lys leads to Asn substitution at position alpha 11(A9).

Hemoglobin Hamilton or alpha 2 beta 2 11(A8)Val leads to Ile: a silent beta-chain variant detected by Triton X-100 acid-urea polyacrylamide gel electrophoresis.

A silent beta-chain hemoglobin variant, not detectable by starch gel or agar gel electrophoresis was found using Triton X-100 acid-urea polyacrylamide gel electrophoresis. The abnormal beta-X chain had a more anodic electrophoretic mobility; 38% of the total beta chain was of the abnormal type. Structural analysis using high-performance liquid chromatography and microsequencing procedures indicated a valine to isoleucine substitution at position beta 11(A8). This anomaly did not change the functional properties of the hemoglobin molecule. A mild reticulocytosis was observed in the propositus.

Hb F-Tokyo or alpha 2G gamma 2 34(B16)Val----Ile, a silent gamma chain variant detected by reverse phase high performance liquid chromatography.

Hb F-Tokyo with a Valine----Isoleucine replacement at position gamma 34(B16) is a G gamma chain variant which was discovered by reverse phase chromatography as this method permitted the nearly complete separation of the three types of gamma chain. The chemical characterization was greatly facilitated by the use of a larger, preparative, HPLC column which allowed the isolation of sufficient quantities of the different gamma chains.

Hb A2-Zagreb or alpha 2 delta 2(125)(H3)Gln replaced by Glu, a new delta chain variant in association with delta beta-thalassemia.

The structural identification of a new delta chain variant is described. The abnormal Hb A2 was found in two members of a family from Zagreb, Yugoslavia. The propositus also had a delta beta-thalassemia heterozygosity.

Hb Wayne, the frameshift variant with extended alpha chains observed in a Caucasian family from Alabama.

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.

Heterogeneity of the hemoglobin of the Ohrid trout (Salmo L. typicus).

We have analyzed the hemoglobins of five individual trout from the Ohrid Lake (Salmo L. typicus) by electrophoretic methods, by reversed-phase high-performance liquid chromatography, and by limited structural analyses. The two major classes of hemoglobin are type I (35% of total) and type IV (65%). Type IV is the major oxygen-transporting hemoglobin; it consists of three types of beta chain (in about equal quantities) and three types of alpha chain (one major and two minor types). Several structural differences have been observed between these three beta (IV) chains and between the three alpha (IV) chains, suggesting a complex genetic system governing the synthesis of these proteins. Moreover, a few amino acid substitutions occur at positions involved in contacts between chains, which suggests that differences in oxygen affinity may exist between these various type IV hemoglobins. Type I hemoglobin is less complex because it contains one type of beta chain and two alpha chains; the latter two differ in numerous positions, suggesting duplications of the alpha (I)-globin gene. The alpha and beta chains of type I hemoglobin differ considerably from the alpha and beta chains of type IV hemoglobin, indicating the existence of alpha (I)- and beta (I)-globin genes separate from the alpha (IV)- and beta (IV)-globin genes.

Hb S, Hb G-Philadelphia and alpha-thalassemia-2 in a Black family.

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.

Hemoglobin Montreal: a new variant with an extended beta chain due to a deletion of Asp, Gly, Leu at positions 73, 74, and 75, and an insertion of Ala, Arg, Cys, Gln at the same location.

The unstable hemoglobin Montreal with a deletion of three amino acid residues (Asp, Gly, Leu) at positions 73, 74, and 75 of the beta chain and an insertion of four residues (Ala, Arg, Cys, Gln) at the same location was observed in a 7-year-old Canadian boy suffering from a moderate hemolytic anemia. The introduction of an extra amino acid residue and of other changes in the crevice where the heme group is located is the likely cause of the instability of this hemoglobin variant. The above listed changes were detected through analyses of tryptic peptides of the beta-Montreal chain, sequencing of amplified DNA, and hybridization of amplified DNA with appropriate, 32P-labeled, oligonucleotide probes. It is suggested that a mispairing involving the AGTG sequences at codons 66 and 67 and at codons 72 and 73 of the normal beta gene caused a repetition of a 16-bp segment, while a deletion of 10 nucleotides due to recombination or slippage followed by a second short deletion during DNA repair resulted in the modified sequence of the beta-Montreal gene.

The identification of five rare beta-chain abnormal hemoglobins by high performance liquid chromatographic procedures.

The detection, quantitation, and characterization of five relatively rare beta chain abnormal hemoglobins mainly by high performance liquid chromatographic procedures are described. The variants involved are Hb City of Hope (beta 69 Gly----Ser), Hb Austin (beta 40 Arg----Ser), Hb Leiden (beta 6 or 7 Glu----0), Hb Louisville (beta 42 Phe----Leu), and Hb Presbyterian (beta 108 Asn----Lys). Some clinical and hematological data are also included.

Hb Davenport or alpha 2(78)(EF7)Asn----His beta 2.

Hb Davenport is a new, stable alpha chain variant, that was detected in two members of a Caucasian family living in Iowa. Hematological data were within normal limits. It is characterized by an Asn----His replacement at position alpha 78. Mild acidic hydrolysis of the large alpha T-9 peptide, separation of the resulting fragments by reversed phase high performance liquid chromatography, and sequence analysis of a few of these peptides greatly facilitated the identification of this substitution.