Hb Bibba or alpha 2 136(H19)Leu-->Pro beta 2 in a Caucasian family from Alabama.

Several members of a large Caucasian family who presented with a congenital Heinz body hemolytic anemia were found to be carriers of the unstable Hb Bibba or alpha 2 136(H19)Leu-->Pro beta 2. Identification by protein analysis was hampered by the instability of the variant which complicated its isolation from shipped blood samples. Moreover, the detection of the CTG-->CCG mutation at codon 136 of the alpha 2 gene required the substitution of dGTP by dITP during the DNA sequencing process to prevent the occurrence of secondary structures and compressions in the sequencing gel. The first Hb Bibba heterozygote, characterized in 1968 (1), is believed to be a member of this family. The clinical expression of the disease is surprisingly variable.

Compound heterozygosity for two alpha-globin gene defects, Hb Taybe (alpha 1; 38 or 39 minus Thr) and a poly A mutation (alpha 2; AATAAA-->AATAAG), results in a severe hemolytic anemia.

We have identified two alpha-globin gene variations in an Arabian male with severe hemolytic disease through sequencing of amplified DNA of his alpha 2- and alpha 1-globin genes. One of the abnormalities involves a CAC (ACC or CCA) deletion between codons 36 and 41 of the alpha 1-globin gene. This leads to the synthesis of an abnormal alpha chain with one instead of two threonine residues at positions 38-39 and to the formation of the unstable Hb Taybe. The second variation is a mutation located in the poly A site of the alpha 2-globin gene (AATAAA-->AATAAG) which is common among Arabian people. Family studies have shown that the two variations are located on opposite chromosomes. The hemolytic disease in this man, resembling Hb H disease, is likely the result of a severe downregulation of both alpha-globin genes on the chromosome with the alpha 2 poly A mutation, and the instability of the alpha-Taybe chain being the product of an alpha 1-globin gene; this leaves only one alpha 2-globin gene normally active.

The differences in quantities of alpha 2- and alpha 1-globin gene variants in heterozygotes.

We have identified through sequencing of amplified DNA the mutations in the alpha 2- and alpha 1-globin genes in 63 individuals with a heterozygosity for an alpha chain abnormal haemoglobin (Hb). Moreover, we developed a reverse transcription/polymerase chain reaction (RT/PCR) based procedure for the determination of the alpha 2- and alpha 1-mRNA ratio in normal individuals. The numbers of alpha 2 and alpha 1 variants were nearly the same. The average percentage of the abnormal Hb in heterozygotes with alpha 2 mutations (23.5%) was slightly higher than that in heterozygotes with alpha 1 mutations (19.7%) (stable Hbs only). These percentages correspond to a ratio of alpha 2 to alpha 1 of 1.19 to 1 at the protein level. Variations in the number of active alpha-globin genes and in the stability of the variants (greatly) affected the percentages of the abnormal protein. The average ratio between the alpha 2- and alpha 1-mRNAs in 12 normal individuals was 2.6-2.75 to 1, about as expected from published data, and 2.0 to 1 for two persons with an alpha-thalassaemia-2 (alpha-thal-2) (-3.7 kb) heterozygosity. The high relative mRNA (alpha 2) level which is about twice the relative level of the alpha 2 protein suggests a less efficient translation of the alpha 2-mRNA.

Hb Fannin-Lubbock in five Spanish families is characterized by two mutations: beta 111 GTC-->CTC (Val-->Leu) and beta 119 GGC-->GAC (Gly-->Asp).

We have sequenced the amplified beta-globin genes of five, apparently unrelated, Spanish adults with a fast-moving hemoglobin variant, and observed a GGC-->GAC mutation at codon 119 which identified the abnormality as Hb Fannin-Lubbock or alpha 2 beta (2)119(GH2)Gly-->Asp. In addition, we found a GTC-->CTC change at codon 111 which leads to a Val-->Leu replacement at this location. Protein analysis of the beta A and beta X chains from one of these individuals confirmed that both mutations are located on the same chromosome. It is hypothesized that some other known variants may carry an additional mutation in one of their exons, resulting in a silent amino acid substitution which may have an effect on some physicochemical property. In the case of Hb Fannin-Lubbock, it appears likely that the Val-->Leu replacement at beta 111, rather than the Gly-->Asp replacement of beta 119, is the cause of the instability of the variant. The Hb Fannin-Lubbock variant in these Spanish families had a normal oxygen affinity.

Beta-thalassemia alleles and unstable hemoglobin types among Russian pediatric patients.

A recently initiated collaboration between Russian and American institutions has resulted in the characterization of several known or new beta-thalassemia alleles and unstable hemoglobin types. Nine known beta-thalassemia alleles were present which have also been found in Mediterranean, East Asian, and Black populations; the possibility of independent mutations for some of the rare alleles should be considered. Hb Durham-N.C./Brescia with a codon 114 (CTG-->CCG; Leu-->Pro) change was present in six members of two families. This condition and two new variants have the characteristics of a dominant type of beta-thalassemia heterozygosity with moderate anemia, Heinz body formation, splenomegaly, etc. One new beta-thalassemia allele is a frame-shift at codon 124 (-A), while another is characterized by the introduction of an extra proline residue (codon: CCA) between residues Thr (beta 123) and Val (beta 126) to give the sequence -Thr-Pro-Pro-Pro-Val-.

Two different mutations in codon 68 are observed in Hb G-Philadelphia heterozygotes.

Through sequencing of amplified DNA containing the appropriate alpha-globin genes we have identified the base substitution leading to the formation of Hb G-Philadelphia [alpha 68(E17)Asn Lys]. Three subjects (approximately 25% Hb G) had an ACC AAA change at codon 68 of the alpha 2-globin gene; the chromosome with this mutation carried two alpha genes (alpha alpha). Six subjects (approximately 33% Hb G) had an AAC AAG change at the same codon of the alpha 2 alpha 1 hybrid gene on a chromosome with the 3.7 kb deletion (-alpha 3.7). These results indicate two independent mutations which likely occurred in different populations; increase in the level of Hb G is primarily dependent upon the loss of one or more alpha-globin genes.

Quantities of alpha Q chain variants in heterozygotes with and without a concomitant beta-thalassemia trait.

We have analyzed the quantities of alpha x chain-containing hemoglobins (alpha 2 x beta 2 and alpha 2 x delta 2) in 14 heterozygotes for Hb Q-India [alpha 64(E13)Asp-->His] or Hb Q-Thailand [alpha 74(EF3)Asp-->His]; both amino acid replacements are the result of mutations in the alpha 1-globin gene. Five of these persons (three with Hb Q-India and two with Hb Q-Thailand) had an additional beta(0)-thalassemia heterozygosity. The average quantities for Hb Q + Hb Q2 in the four groups were 17.2% (alpha alpha Q/alpha alpha; beta A/beta A), 9.5% (alpha alpha Q/alpha alpha; beta A/beta(0) Th), 26.8% (-alpha Q/alpha alpha; beta A/beta A), and 16.95% (-alpha Q/alpha alpha; beta A/beta(0) Th). These variations can best be explained by a posttranslational control mechanism; an imbalance in the alpha A, alpha Q, and beta A chain ratio will favor the alpha 2 Q beta 2 formation when an alpha-thalassemia is present and will reduce its formation in the presence of a beta-thalassemia heterozygosity.

Hb Sinai-Baltimore or alpha 2 beta (2)18(A15)Val->Gly, a silent, mildly unstable beta chain variant detected by isoelectrofocusing and high performance liquid chromatography.

Isoelectrofocusing and high performance liquid chromatographic methods were used to study an abnormal hemoglobin present in a Black male infant and his mother. The variant, named Hb Sinai-Baltimore, focused slightly behind Hb A and separated incompletely from Hb A by cation exchange high performance liquid chromatography, while the separation of the beta A and beta X chains by reversed phase high performance liquid chromatography was complete. The variant was identified through an analysis of peptides in a tryptic digest of the isolated beta X chain and by sequencing of amplified DNA which included the beta-globin gene. The Val->Gly replacement at position beta 18 (codon 18; GTG->GGG) or at the last position of the A helix decreases the stability of the variant without affecting the hematological parameters of its carrier. The propositus was a compound heterozygote for Hb Sinai-Baltimore and Hb S; the relative quantities of the two variant chains were somewhat different from those of the beta X and beta A chains in the mother with the simple Hb Sinai-Baltimore heterozygosity. An uncertainty about the alpha-globin gene status in the child prevented a further evaluation of these differences.

Hb Alesha or alpha 2 beta (2)67(E11)Val-->Met: a new unstable hemoglobin variant identified through sequencing of amplified DNA.

We have identified a valine-->methionine mutation at position 67 of the beta chain in the hemoglobin of a young Russian patient with severe hemolytic disease, anemia, splenomegaly, Heinz body formation, and continued requirement for blood transfusions despite an early splenectomy. Sequencing of amplified DNA readily identified a GTG-->ATG mutation at codon 67. The introduction of the larger methionine residue into the heme pocket, and the loss of the bonds between valine at beta 67 and the heme group, adequately account for the severe instability of Hb Alesha and the serious clinical condition of its carrier.