Elias Schwartz

Construction of Human Gene Libraries from Small Amounts cf Peripheral Blood: Analysis of β-Like Globin Genes

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.

Denatured hemoglobin in sickle erythrocytes.

To study the nature of numerous inclusion bodies seen in red cells from patients with sickle cell disease (Hb SS), we have prepared red cell ghosts free of oxyhemoglobin and analyzed them by spectrophotometric and heme extraction methods. The absorption spectrum in the visible region of the ghost suspensions was typical of hemichromes. The spectrum was similar to that of denatured hemoglobin repared by treatment of oxyhemoglobin S with mechanical shaking or heat. Similar treatment of cells containing only normal hemoglobin (Hb AA) showed a very small amount of denatured hemoglobin, approximately one-fifth of the amount in Hb SS cells. The amount of denatured hemoglobin determined after solution of membrane with 2.5% sodium dodecyl sulfate was 0.158+/-0.070% (1 SD) of the total cellular heme in Hb SS patients. In controls, the amount was 0.030+/-0.016%. Persons with Hb AA and reticulocytosis did not have an elevated amount of membrane-associated heme. In patients with hereditary spherocytosis and autoimmune hemolytic anemia, denatured stromal hemoglobin was normal or slightly elevated before and after splenectomy. The increased amount of denatured hemoglobin in Hb SS red cells may be related to the instability of sickle oxyhemoglobin.

Differential expression of Fcγ RIIA, Fcγ RIIB and Fcγ RIIC in hematopoietic cells: Analysis of transcripts

Abstract Fcγ receptors (Fcγ R) are glycoproteins that function in the immune response through their ability to bind the Fc portion of immunoglobulin G. Of the three human Fcγ R classes, Fcγ RII is most widely distributed among hematopoietic cells and is the only Fcγ R class present on platelets and megakaryocytes. There are three different genes coding for Fcγ RII: Fcγ RIIA, Fcγ RIIB and Fcγ RIIC. Alternative splicing of at least two of these genes results in the production of multiple transcripts. Combining Northern blot analysis with reverse transcription-PCR, we analyzed steady state levels of Fcγ RII mRNA in the megakaryocytic, myeloid and lymphoid lineages. We determined that megakaryocytic cells predominantly contain Fcγ RIIA mRNA; Fcγ RIIA transcripts with and without the transmembrane exon (Fcγ RIIa1 and Fcγ RIIa2, respectively) are present in comparable amounts. In contrast, B lymphocytes do not express Fcγ RIIA mRNAs, but do contain both Fcγ RIIB transcripts, Fcγ RIIb1 and Fcγ RIIb2, as well as the Fcγ RIIC transcript, Fcγ RIIc. Myelomonocytic cells contain mRNAs from all three Fcγ RII genes, predominantly the Fcγ RIIa1 transcript, both Fcγ RIIb1 and Fcγ RIIb2 transcripts and Fcγ RIIc. Lineage-specific expression of the Fcγ RII genes implies both differential regulation of expression and differential function in diverse cells.

Structure of platelet glycoprotein IIIa. A common subunit for two different membrane receptors.

The platelet membrane glycoprotein IIb/IIIa complex is a member of a family of alpha/beta heterodimers that function as receptors for adhesive proteins. In this report we describe the structure of the human beta subunit GPIIIa deduced from an analysis of 4.0 kb of overlapping cDNA sequences isolated from a human erythroleukemia (HEL) cell cDNA expression library. A continuous open reading frame encoding all 788 amino acids for GPIIIa was present. The deduced amino acid sequence included a 26-residue amino-terminal signal peptide, a 29-residue transmembrane domain near the carboxy terminus, and four tandemly repeated cysteine-rich domains of 33-38 residues. An exact correspondence of 128 amino acids from seven human platelet GPIIIa fragments with HEL GPIIIa indicates that HEL and platelet GPIIIa are the same gene product. The HEL GPIIIa sequence was compared with the sequences of the beta subunit for the human LFA-1/Mac-1/p150.95 complex and human endothelial cell GPIIIa, revealing a 38% similarity with the former and virtual identity with the latter. Northern blot analysis using RNA from both HEL and endothelial cells revealed two GPIIIa transcripts of 5.9 and 4.1 kb. However, HEL RNA, but not endothelial cell RNA, contained a transcript for GPIIb. This indicates that the GPIIIa-containing heterodimers in platelets and endothelial cells are not identical structures, but are members of a subfamily within the human family of adhesion protein receptors sharing an identical beta subunit.

Depletion of excessive liver iron stores with desferrioxamine

TO determine the therapeutic effect of long‐term, intensive iron chelation therapy, we studied liver iron content and histology in four children with thalassaemia major during 52‐83 months of intensive therapy with desferrioxamine. The initial biopsies obtained prior to or within 21 months after beginning chelation therapy had Grade IV iron staining, with heavy iron deposition present in parenchymal and reticuloendothelial cells. Subsequent biopsies, obtained when serum ferritin levels had fallen to 71‐246 μg/1, contained Grade 0 or Grade I stainable iron. Little or no iron was present in parenchymal or reticuloendothelial cells. The liver iron concentration, measured by magnetic susceptibility, returned to normal or nearly normal levels. Hepatic fibrosis did not progress during treatment with desferrioxamine. These findings demonstrate that intensive and sustained chelation therapy with desferrioxamine will remove excessive liver iron and preserve hepatocellular structure.

Rapid removal of excessive iron with daily, high-dose intravenous chelation therapy

We investigated the value of high-dose intravenous iron chelation therapy with deferoxamine as an alternative to conventional subcutaneous therapy in eight patients receiving regular transfusions who had massive iron stores, including two with clinical heart disease. Six to twelve grams of deferoxamine was infused daily for 12 hours over 12 to 25 months through externalized central venous catheters or implanted reservoirs. Serum ferritin levels decreased by 56% to 99%. Liver iron concentrations, measured by magnetic susceptibility in two patients, were 1234 and 2438 micrograms/gm wet weight (22.1 and 43.6 mumol/gm wet weight) after treatment for 17 and 25 months, respectively. A patient with congestive heart failure and a patient with severe ventricular dysrhythmias no longer required cardiac medication after 12 to 24 months of chelation therapy. Three episodes of bacteremia and three episodes of cellulitis accounted for a catheter-related infection rate of 0.14 per 100 patient-days. The catheter removal rate was 0.20 per 100 patient-days. No patient experienced serious visual, auditory, or other toxicities. We conclude that in some patients receiving regular erythrocyte transfusions, high-dose intravenous chelation therapy with deferoxamine is superior to conventional subcutaneous treatment.

Trial of low doses of aspirin as prophylaxis insickle cell disease

The effects of low doses of aspirin on the frequency and severity of painful vaso-occlusive crises were evaluated in children with sickle hemoglobinopathies. Aspirin was compared with placebo in 49 patients in a double-blind crossover study. Careful monitoring of patients revealed an average of 1.1 painful crises per patient year. During the 21 months of study, 70% of patients had a maximum of two painful crises, and 25% experienced four or more. The frequency and severity of crises were not affected by aspirin therapy. In view of aspirins demonstrated effect on platelet function, we suggest that platelets do not contribute to the initiation or progression of the vaso-occlusive process.

Chromosomal localization of the genes for the vitronectin and fibronectin receptors alpha subunits and for platelet glycoproteins IIb and IIIa.

The integrins, a family of related membrane receptors involved in cell-cell and cell-matrix interactions, are heterodimeric complexes of alpha and beta subunits. To begin to understand the evolution of these complexes, we studied the genomic organization of several alpha and beta integrin subunits. Using both somatic cell hybrids and an in situ hybridization technique, we have determined the chromosomal location of the genes for the alpha subunits of the vitronectin receptor (VNR alpha), the fibronectin receptor (FNR alpha), and for the alpha subunit of the platelet glycoprotein IIb/IIIa complex, GPIIb. In addition, we have determined the chromosomal location of the gene for the beta subunit of the GPIIb/IIIa heterodimer, GPIIIa. Our studies indicate that the alpha subunits do not localize to a single locus, but that each is found on a different chromosome. The gene for VNR alpha is located on chromosome 2, the gene for FNR alpha is on chromosome 12q11----13, and the gene for GPIIb is on chromosome 17q21----23. In contrast to the chromosomal dispersion of the alpha subunits, the genes for GPIIb and GPIIIa are physically close, with the gene for GPIIIa also located on chromosome 17q21----23. These studies indicate that the genes for the alpha subunits of the integrin family have been dispersed during evolution while GPIIb and GPIIIa are in close physical proximity. This physical proximity of GPIIb and GPIIIa may be involved in the concurrent expression of these proteins by megakaryocytes, and may result in linkage disequilibrium between these two genes, which would limit the use of restriction length polymorphisms in linkage studies of GPIIb/IIIa abnormalities in small kindreds.

α-Thalassemia in the American Negro

In Italian and Chinese patients with the alpha-thalassemia syndromes the production of alpha-chain of normal hemoglobin is decreased relative to that of beta-chain in reticulocytes. In this study the relative rates of alpha- and beta-chain synthesis were determined in members of three Negro families with alpha-thalassemia. Two of the families had members with hemoglobin H disease and alpha-thalassemia trait, while the mother of several children with alpha-thalassemia trait in the third family was doubly heterozygous for alpha-thalassemia and an alpha-chain mutant. The alpha/beta ratios of globin synthesis in the patients with hemoglobin H disease and alpha-thalassemia trait indicated less severe biochemical defects in the peripheral blood than those previously determined in Italian and Chinese patients. In the third family, there was a heterogeneity of expression of the gene for alpha-thalassemia, including patients with normal red cell indices and synthesis ratios. These findings differ from those previously described in patients with alpha-thalassemia from other racial groups. Hydrops fetalis due to homozygous alpha-thalassemia may not occur in the Negro because of the relatively mild thalassemic defect.

The silent carrier allele: β thalassemia without a mutation in the β-globin gene or its immediate flanking regions

Abstract A molecular genetic analysis has been performed using as subjects an Albanian family in which the father is a silent carrier, the mother has high Hb A 2 -β thalassemia trait, and both children have β thalassemia. Nucleotide sequence analysis of the daughters paternal β-globin gene and its flanking regions failed to reveal any base changes of known functional significance. When introduced into HeLa cells the gene was expressed at normal levels with proper processing of RNA. Haplotype analysis revealed that the affected son and daughter inherited different ϵγδβ-globin gene clusters from the father. The silent carrier allele is not due to a mutation within the β-globin structural gene or its flanking regions and as such represents a novel form of β + thalassemia.