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We have identified a 16 bp deletion in the 3' untranslated region of the alpha 2-globin gene, including the first nucleotide of the polyadenylation signal sequence. The propositus, her mother and one of her brothers, all heterozygotes for the above deletion, have mild microcytic anemia. This is the first description of a deletion in the alpha gene involving both the 3' untranslated region and the polyadenylation signal sequence, causing alpha-thalassemia.

We studied two members of an African American family with erythrocytosis. An abnormal hemoglobin variant with an electrophoretic pattern on cellulose acetate similar to Hb J was identified. The oxygen dissociation curve using whole blood was biphasic, dramatically left-shifted, and hyperbolic. Sequence analysis of DNA from the proband showed heterozygosity for a T-->A change at the first position of codon 145 in the beta-globin gene which results in the substitution of an asparagine residue for normal tyrosine. The second cycle of C-terminal amino acid sequence analysis of a mixture of alpha- and beta-globin chains showed tyrosine, aspartic acid, and small amounts of asparagine. Collectively, these results indicate the existence of a mutation at codon 145 of the beta-globin gene which encodes for asparagine instead of tyrosine, and that asparagine then undergoes a partial posttranslational deamidation to aspartic acid. This amino acid substitution corresponds to Hb Osler, which is a high oxygen affinity hemoglobin variant, initially described to be caused by a substitution of Tyr-->Asp at beta 145. Posttranslational amino acid modification may constitute an important component in the pathophysiology of hemoglobinopathies.

When first tested for abnormal hemoglobins, a 2-year-old boy, appeared to have Hb F, Hb S and Hb A2. Confirmatory testing revealed a beta chain variant inherited from his father and beta S from his mother. Analysis of tryptic peptides in conjunction with automated DNA sequence analysis showed that the variant hemoglobin was Hb Shelby [beta 131(H9)Gln-->Lys (CAG-->AAG)]. Heat and mechanical stabilities of various liganded Hb Shelby tetramers were compared to those of Hb A and Hb S. Oxy-Hb Shelby precipitated more readily than oxy-Hb A, but was much more stable than oxy-Hb S during mechanical agitation. In contrast, oxy-Hb Shelby was much less stable than oxy-Hb A and oxy-Hb S following heat treatment. Met-Hb Shelby was most unstable compared to other liganded forms of Hb Shelby, while deoxy- and carbonmonoxy-forms of Hb Shelby showed similar heat-induced precipitation rates. These data indicate that heat instability of Hb Shelby is accompanied by heme oxidation, and that denaturation by mechanical agitation occurs in the absence of heme oxidation. Hb Shelby, like Hb A, can form hybrids with Hb S which participate in polymer formation in vitro. However, Hb S/Hb Shelby hybrids copolymerized with Hb S less than A/S hybrids. Since the patient's MCHC value is normal, this finding coupled with the elevated Hb A2 and Hb F levels, both of which are known to inhibit polymerization of Hb S, may contribute to the patient's mild clinical presentation.

In an effort to clarify the role of amino acid hydrophobicity at the beta 6 position in sickling we have made recombinant hemoglobin tetramers containing beta 6 Val (Hb S) and beta 6 Leu (Hb Leu). Recombinant Hb S and Hb Leu had the same electrophoretic mobility, chromatographic behavior, and absorption spectrum. The deoxy form of both tetramers polymerized in high phosphate buffer (1.8 M) and exhibited distinct delay times prior to polymerization. The kinetics of polymerization for recombinant and native Hb S were similar, while recombinant Hb Leu polymerized more readily. The solubility of deoxy Hb Leu was less than deoxy Hb S, indicating that rapid polymerization and decreased solubility of deoxyhemoglobin is accelerated with increasing hydrophobicity at the beta 6 position.

We describe a new deletional form of gamma delta beta-thalassemia segregating in two generations of a family of Irish descent. Affected family members present with a beta-thalassemia minor phenotype, normal Hb A2 and Hb F levels. Genomic blotting analyses on DNA from affected family members show heterozygosity for a large deletion beginning at least 15 kb upstream of the 5' endpoint of the gamma delta beta-thalassemia-1 deletion, extending through the entire beta-like globin gene cluster, and continuing for at least 10 kb beyond the 3' endpoint of the deletion associated with the Spanish form of delta beta 0-thalassemia. This deletion is among the largest described so far, and removes at least 205 kb encompassing the entire beta-like globin gene cluster on chromosome 11.

There are three major African haplotypes associated with the sickle mutation: Benin (#19), Senegalese (#3), and Central African Republic (#20). Previous studies have suggested that the Xmn I site (-158 bp 5' to the G gamma gene) is associated with elevated levels of G gamma and with the Senegalese haplotype, while other investigators questioned this association. In order to clarify the issue, we have determined beta haplotypes, tested for the presence of the Xmn I site, and measured Hb F and G gamma expression levels in 143 American Black patients with sickle cell anemia. Haplotypes were determined using eight polymorphic sites in the beta-like globin gene cluster: Hinc II 5' to epsilon, Hind III in IVS-II G gamma and A gamma, Hinc II within and 3' to psi beta, Ava II in IVS-II of beta, and Hpa I and Bam HI 3' to beta. The G gamma /A gamma ratio was analyzed by high performance liquid chromatography using a C18 column. The Xmn I site was present in all 31 chromosomes with the Sengalese haplotype. Of the remaining 255 chromosomes with other haplotypes, only 2 (0.8%) had the Xmn I site present. There was significant correlation between the presence of the Xmn I site and increased G gamma /A gamma ratio in a dose-dependent manner. The Hb F level was not significantly increased in the presence of the Xmn I site. The data indicate that the Xmn I site maintains a G gamma /A gamma ratio typical of fetal life but does not necessarily cause elevation of Hb F. The latter seems to depend on factors other than the Xmn I site.

Previous work has suggested that the beta C mutation arose on a relatively rare beta A chromosome with a unique haplotype, and these data have been used to postulate a single origin of the beta C allele. Most characteristic of this haplotype has been the absence of a Hpa I recognition site 3' to the beta-globin gene. We studied 40 patients with Hb SC, 11 with Hb AC, and one with Hb CC. Complete haplotyping was possible on 44 beta C alleles and 3' haplotyping was possible on an additional four beta C alleles. Our results support the predominence of a unique beta C haplotype which is characterized by the absence of the Hpa I site. However, one patient who was homozygous for Hb C had one beta C allele with an atypical haplotype which contained the Hpa I site. It is unclear at this time whether or not this represents a second origin of the beta C mutation.

There are three main African haplotypes associated with the sickle mutation on chromosome 11. We have examined an Rsa I polymorphism 550 bp 5' to the beta-globin gene to study the degree of linkage disequilibrium between this Rsa I site and the three haplotypes. This Rsa I site is contained within the 10.3 kb or less area of randomization separating the 5'- and 3'-haplotype clusters. The beta S-containing chromosomes of the Benin and Senegal haplotypes are not cut, while those of the Central African Republic are cleaved by Rsa I at this site. Possible explanations of these findings are discussed.

There has been much progress in basic studies of the beta-thalassemia disorders in recent years, as well as in the practical aspects of prenatal diagnosis and clinical management. Alterations in a single or a few nucleotides account for most of the types of beta-thalassemia syndromes that have been characterized, and a variety of deletions have been found associated with beta O thalassemia, delta beta thalassemia, gamma delta beta thalassemia, and hereditary persistence of fetal hemoglobin (HPFH). Studies of nondeletional types of HPFH have revealed several single base changes 5' to the G gamma and A gamma genes, suggesting that these regions may be of major importance in the switchover from fetal to adult hemoglobin synthesis and in the relative production of the G gamma and A gamma globin chains during development. Evaluation of restriction enzyme polymorphisms and the assignment of haplotypes in the beta-like globin region of chromosome 11 have allowed delineation of the origin and distribution of thalassemia mutations, and have provided an important means for prenatal diagnosis. These studies have accumulated much new information about the function and expression of eukaryotic genes, and have served as a model for the investigation of human genetic disorders. The clinical management of patients with Cooley's anemia has benefited greatly from changes in transfusion practice, clear guidelines for splenectomy, and the availability of effective chelation therapy for iron overload. Current basic and clinical studies may lead to new approaches to management, treatment and eventual cures for this disease, including safe bone marrow transplantation, oral chelation treatment, and gene therapy.

We describe a rapid procedure for constructing cloned human genomic libraries from small amounts of peripheral blood. High molecular weight DNA is isolated from 5-20 ml peripheral blood, partially cleaved with Eco R1, and 8-22 kb fragments are cloned using bacteriophage Charon 4A and suitable E. coli host. Using the approach we have isolated and characterized several non-alpha globin clones from a Kurdish Jew with homozygous beta thalassemia. The ability to isolate suitable amounts of high molecular weight DNA from peripheral blood provides a relatively simple means of constructing human gene libraries representing a variety of hemoglobin disorders.