Hemoglobin Evanston (alpha 14 Trp----Arg). An unstable alpha- chain variant expressed as alpha-thalassemia. G R Honig, … , R A Spritz, M Guis J Clin Invest. 1984;73(6):1740-1749. https://doi.org/10.1172/JCI111382. Research Article A new hematologic syndrome with phenotypic features of mild Hb H disease was identified in three children from two unrelated black American families. Erythrocytes from each of these children contained Hb H (beta 4) and Hb Barts (gamma 4), as well as a slowly migrating hemoglobin fraction that made up 7-10% of the total hemoglobin. The parents of the affected children all showed mild thalassemia-like changes, with one of the parents in each family also expressing the variant hemoglobin; in the latter individuals the mutant alpha-chains made up less than 2% of the total, and were present mainly or exclusively in combination with delta-chains in the form of a slowly migrating Hb A2. Purified Hb Evanston showed an increased oxygen affinity, but its Bohr effect, cooperativity, and 2,3-diphosphoglycerate effect were normal. The mutant hemoglobin appeared to have normal stability to heat and to isopropanol, and the stability of its alpha-chain in an extended time course synthesis study also appeared to be similar to that of alpha A. However, the results from short- term globin synthesis studies, and from mRNA translation in vitro, suggest that the two types of alpha-chains were synthesized at relatively equal rates, with a major fraction of the newly synthesized variant alpha-chains undergoing rapid catabolism. The hematologic data taken in combination with DNA hybridization and globin synthesis findings […] Find the latest version: https://jci.me/111382/pdf Hemoglobin Evanston (a14 Trp -- Arg) An Unstable a-Chain Variant Expressed as a-Thalassemia George R. Honig, Mir Shamsuddin, Loyda N. Vida, Myrtho Mompoint, and Eric Valcourt Division of Hematology, the Children's Memorial Hospital, Chicago, Illinois 60614 Lemuel J. Bowie and Elizabeth C. Jones Evanston Hospital, Evanston, Illinois 60201 Patricia A. Powers and Richard A. Spritz University of Wisconsin, Department of Genetics, Madison, Wisconsin 53706 Margot Guis, Stephen H. Embury, John Conboy, Yuet W. Kan, and William C. Mentzer University of California, San Francisco, California 94110 Susan C. Weil, Roli K. Hirata, Jami Waloch, John F. O'Riordan, and Thomas K. Goldstick Northwestern University, Departments of Medicine, Pathology, and Chemical Engineering, Chicago, Illinois 60611 Atypctract. A new hematologic syndrome with to heat and to isopropanol, and the stability of its phenotypic features of mild Hb H disease was identified a-chain in an extended time course synthesis study also in three children from two unrelated black American appeared to be similar to that of aA. However, the results families. Erythrocytes from each of these children con- from short-term globin synthesis studies, and from mRNA tained Hb H (04) and Hb Barts (74), as well as a slowly translation in vitro, suggest that the two types of a-chains migrating hemoglobin fraction that made up 7-10% of were synthesized at relatively equal rates, with a major the total hemoglobin. The parents ofthe affected children fraction of the newly synthesized variant a-chains un- all showed mild thalassemia-like changes, with one of the dergoing rapid catabolism. The hematologic data taken parents in each family also expressing the variant he- in combination with DNA hybridization and globin syn- moglobin; in the latter individuals the mutant a-chains thesis findings indicate that the proposita in each of these made up <2% of the total, and were present mainly or families has the genotype -,a Al/,aEv. These observations exclusively in combination with 5-chains in the form of suggest that two separate mechanisms are contributing a slowly migrating Hb A2. Purified Hb Evanston showed to the a-thalassemia-like expression of Hb Evanston: (a) an increased oxygen affinity, but its Bohr effect, coop- the newly synthesized aEV-chains are unstable and are erativity, and 2,3-diphosphoglycerate effect were normal. subject to early proteolytic destruction; and (b) the mutant The mutant hemoglobin appeared to have normal stability a-allele is linked to an a-globin gene deletion. A preliminary report of this work was presented at the 1982 meeting Introduction of the American Society of Hematology. Address correspondence to Dr. Honig, University of Illinois, College The a-thalassemia syndromes result from deficient synthesis of of Medicine, Department of Pediatrics, 840 S. Wood St., Chicago, IL, the a-globin chains ofhemoglobin. The most frequent underlying 60612. cause for this group of disorders is the deletion of one or more Received for publication 7 November 1983 and in revised form 3 of the normal complement of four a-globin genes (1, 2). The February 1984. a-thalassemia phenotype has also been observed in association J. Clin. Invest. with certain a-chain structural mutants, and it has further been © The American Society for Clinical Investigation, Inc. shown that the thalassemia-like expression of these abnormal 0021-9738/84/06/1740/10 $1.00 hemoglobins may arise by several different mechanisms. In one Volume 73, June 1984, 1740-1749 group of these variants the mRNA products of the mutant 1740 Honig et al. a-globin genes appear to be unstable, causing the mRNA's to moglobins for oxygen equilibrium studies were isolated by DEAE-cel- be degraded and thereby prematurely terminating their function lulose chromatography in the oxy-form, and were concentrated by sed- as templates for globin chain synthesis (3, 4). Other a-globin imentation at 100,000 gfor 16 h. The hemoglobin solutions were dialyzed gene mutations that produce thalassemia-like hematologic against 0.05 M Bis-Tris buffer in 0.10 M NaCI, and adjusted to a final changes are expressed by the synthesis of highly unstable concentration of I mg/ml (equivalent to '60 MM in heme). An enzymatic reducing system (24, 25) was added to each of the samples to minimize a-chains, which undergo proteolytic destruction and are rapidly methemoglobin formation, and the oxygen equilibrium curves were removed from the erythroid cells (4-6). In a third group of determined at 300C. The data were digitized electronically (Tektronix a-chain variants, the mutant a-globin genes exist in linkage to Graphics Tablet; Tektronix, Inc., Beaverton, OR) and were characterized an a-globin gene deletion, so that the linked a-thalassemia de- by using the Hill equation, which was fitted by nonlinear regression terminant is expressed in conjunction with the hemoglobin over the range of 20 to 97% saturation. The smoothness of the sum-of- structural variant (7-12). squared-residuals function allowed the use of a Gauss-method nonlinear In this report we describe a newly identified a-chain mutant regression algorithm (26). that appears to have thalassemia-like expression as a result of Fluorometric studies of the purified hemoglobins were performed two separate mechanisms, i.e., as the result of an instability of with short path-length cuvettes and by a front surface technique (27). the newly synthesized variant a-chain, and because of the linkage For these determinations the hemoglobins were prepared in Tris-glycine buffer, pH 7.0, at a concentration of 90 uM. The measurements were of its gene with an a-globin gene deletion. made with a Perkin-Elmer MPF44B recording spectrofluorometer (Per- kin-Elmer Corp., Instrument Div., Norwalk, CT) with a temperature- Methods controlled cell holder. Globin synthesis determinations. In studies that used intact reticu- Hematologic measurements. Erythrocyte and hemoglobin measurements locytes, erythroid cell suspensions were prepared from freshly drawn were made with a model S Coulter counter (Coulter Electronics, Inc., blood samples and were supplemented with glucose, hemin, and a mixture Hialeah, FL), which was calibrated daily using a commercial standard. of amino acids excluding leucine, in a Tris-buffered medium at pH 7.4 Other hematologic determinations were by standard laboratory (28). L-[4,5-3H]leucine with a specific activity of 52 Ci/mmol (New methods (13). England Nuclear, Boston, MA) was added at a concentration of 10 MCi/ Hemoglobin studies. Stroma-free hemoglobin solutions were prepared ml. The cell suspensions were incubated in a shaking water-bath at 370C from washed erythrocytes as previously described (14). Electrophoresis for various times, as indicated for the individual experiments. of hemoglobins and globin proteins, and measurements of electrophoretic For studies of cell-free globin chain synthesis, polysomal RNA was mobilities were as described by Schneider and Barwick (15). Hemoglobin isolated from the reticulocytes that were contained in 5-6 ml of blood, (Hb)' A2 measurements were made by DEAE-cellulose microcolumn as previously described (6). The cell-free translation experiments using chromatography (16) and the alkali-resistant hemoglobin fraction was the purified RNA were carried out at 300C for 60 min, in an incubation estimated by the method of Singer et al. (17). Isolation and quantification mixture containing micrococcal nuclease-treated rabbit reticulocyte lysate of individual hemoglobins was by the DEAE-cellulose column chro- (29) (Bethesda Research Laboratories, Gaithersburg, MD), and supple- matography procedure of Abraham et al. (18) using glycine-containing mented with L-[35S]methionine (>600 Ci/mmole; Amersham Corp., buffers. Globin chain separations were by CM-cellulose column chro- Arlington Heights, IL). The newly synthesized globin chains were sep- matography with buffers prepared in 8 M urea (19) or by high perfor- arated by electrophoresis in 12% polyacrylamide gels containing 2% mance liquid chromatography (HPLC) according to Shelton et al. (20). Triton X-100 and 6 M urea (30). The radioactive protein bands were For hemoglobin stability testing stroma-free lysates were heated at 500C visualized by fluorography with ENHANCE (New England Nuclear), in phosphate buffer (21) or at 370C in buffer containing isopropanol according to the manufacturer's specifications.