Modification of Hemoglobin H Disease by Sickle Trait Katherine K. Matthay, … , Yuet Wai Kan, Dorothy F. Bainton J Clin Invest. 1979;64(4):1024-1032. https://doi.org/10.1172/JCI109539. Research Article The rarity of hemoglobin (Hb) H disease in combination with sickle trait may be due in part to the absence of actual Hb H in individuals who, nonetheless, have inherited the deletion of three α-globin genes. We describe here a boy with persistent microcytic, hypochromic anemia despite adequate iron stores, who exhibited splenomegaly with a normal reticulocyte count and only rare inclusions in circulating erythrocytes. Starch gel electrophoresis and isoelectric focusing at age 5 yr showed 21% Hb S, persistent Hb Bart's, but no Hb H. Recticulocyte α/non-α globin chain synthesis ratio was 0.58 at age 5. The mother (Asian) had laboratory evidence of α-thalassemia trait and the father (Black) had sickle trait. The nature of α-thalassemia in this patient was investigated both by liquid hybridization and by the Southern method of gene mapping, in which DNA is digested with restriction endonucleases and the DNA fragments that contained the α- globin structural gene identified by hybridization with complementary DNA. The patient had only one α-globin structural gene, located in a DNA fragment shorter than that found in normal or α-thalassemia trait individuals, but similar to that present in other patients with Hb H disease. Morphologic studies of bone marrow by light and electron microscopy revealed erythroid hyperplasia with inclusions in polychromatic and orthochromatic erythroblasts, suggesting early precipitation of an unstable hemoglobin. […] Find the latest version: https://jci.me/109539/pdf Modification of Hemoglobin H Disease by Sickle Trait KATHERINE K. MATTHAY, WILLIAM C. MENTZER, Jr., ANDREE M. Dozy, YUET WAI KAN, and DOROTHY F. BAINTON, Departments of Pediatrics, Medicine, and Pathology, University of California, San Francisco, San Francisco, California 94143 A B S T R A C T The rarity of hemoglobin (Hb) H dis- ence of a (3s gene may thus modify the usual clinical ease in combination with sickle trait may be due in part expression of Hb H disease. to the absence of actual Hb H in individuals who, none- theless, have inherited the deletion of three a-globin I NTRODUCTION genes. We describe here a boy with persistent micro- cytic, hypochromic anemia despite adequate iron Simultaneous inheritance of a-thalassemia and struc- stores, who exhibited splenomegaly with a normal re- tural abnormalities of the (3-chain frequently modifies ticulocyte count and only rare inclusions in circulating the usual clinical expression of the f8-globin mutation erythrocytes. Starch gel electrophoresis and isoelectric ( 1-13). For example, observation of the nature of sickle focusing at age 5 yr showed 21% Hb S, persistent Hb cell anemia in several individuals who have also in- Bart's, but no Hb H. Recticulocyte a/non-a globin herited a-thalassemia trait has suggested in some cases chain synthesis ratio was 0.58 at age 5. The mother (4-6), but not all (7, 8), a favorable effect of the a- (Asian) had laboratory evidence of a-thalassemia trait thalassemia gene on the clinical severity of sickle cell and the father (Black) had sickle trait. The nature of anemia. a-Thalassemia with sickle cell trait consis- a-thalassemia in this patient was investigated both by tently results in a lower than usual percentage of hemo- liquid hybridization and by the Southern method of globin (Hb)S (9-13). We have investigated a child with gene mapping, in which DNA is digested with restric- sickle trait and microcytic anemia whose Asian mother tion endonucleases and the DNA fragments that con- had a-thalassemia trait and whose Black father had tained the a-globin structural gene identified by hy- sickle trait. The very low percentage of Hb S suggested bridization with complementary DNA. The patient had that both a-thalassemia trait and sickle cell trait were only one a-globin structural gene, located in a DNA present, but the presence of splenomegaly, extreme fragment shorter than that found in normal or a-thalas- microcytosis, and bizarre erythrocyte morphology, fea- semia trait individuals, but similar to that present in tures not seen in a-thalassemia trait, stimulated further other patients with Hb H disease. Morphologic studies investigation. of bone marrow by light and electron microscopy re- Analysis of DNA from this child by molecular hy- vealed erythroid hyperplasia with inclusions in poly- bridization and gene-mapping techniques revealed chromatic and orthochromatic erythroblasts, suggest- that three of the four a-globin genes normally found ing early precipitation of an unstable hemoglobin. The were deleted, thus unequivocally demonstrating the lack of demonstrable Hb H may be the result of both presence of the Hb H genotype. Paradoxically, Hb H diminished amounts of (A available for Hb H formation was not identified in hemolysate from either peripheral (since one 8-globin gene is (3s) and the greater affinity blood or bone marrow, although inclusion bodies could of a-chains for (3A than 8s-globin chains leading to the be found in occasional erythrocytes in both places. The formation of relatively more Hb A than Hb S. The pres- biochemical nature of these inclusions was not deter- mined, but the appearance in early erythroid pro- This work was presented in part to the American Society of genitors suggested a greater degree of instability than Hematology, New Orleans, La., December 1978. that associated with Hb H (14-16). This patient, who Dr. Kan is an investigator of the Howard Hughes Medical Institute. Dr. Mentzer is the recipient of a U. S. Public Health represents the unusual coincidence of Hb H disease Service Research Career Development Award. with sickle trait, demonstrates how the inheritance of a Received for publication 9 April 1979 and in revised form j8-globin structural mutation can modify the phenotype 8 June 1979. of Hb H disease sufficiently to alter the usual clinical 1024 J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 0021-9738/79/10/1024/09 $1.00 Volume 64 October 1979 1024 -1032 picture. In such patients, accurate genetic analysis is Liquid hybridization. DNA from the patient, his mother, impossible unless the new techniques of molecular and controls with hydrops fetalis, Hb H disease, a-thalassemia trait, and normals was prepared from leukocytes, liver, or hybridization and gene mapping are used. spleen by sodium dodecyl sulfate-pronase digestion and phenol extraction. The RNA was digested with ribonuclease METHODS and the DNA reduced to 2 x 106 daltons by limited depurina- Hematologic values. Hemoglobin levels and erythrocyte tion (26, 27). a-globin complementary DNA (cDNA) was pre- indices were obtained with a model S Coulter counter (Coulter pared as described (26, 28). The a-cDNA (1,000 cpm) was Electronics Inc., Hialeah, Fla.). Bone marrow differential incubated for 76 h at 78°C in duplicate 20-1.l reaction mixtures counts were performed by counting at least 500 cells. Cellu- containing 100 ,ug cellular DNA, 8 pg a-cDNA, 500 cpm of lose acetate electrophoresis was performed in Tris-EDTA- [3H]dCTP-labeled unique sequence HeLa DNA as internal borate buffer at pH 8.4 (17). Hb A2 was determined by micro- control, 0.5 MI NaCl, 0.002 NI EDTA, and 0.04 I Tris-HCl chromatography (18). Fetal hemoglobin was determined by (pH 7.4). The percentage of a-cDNA annealed was assayed alkali denaturation (19). Hb H preparations were made by by batchwise elutioi wvith hydroxylapatite (29). mixing one part of fresh whole blood with two parts of 1% Restriction endonuclease analysis of DNA. DNA was pre- brilliant cresyl blue (BCB)l in 0.9% NaCl and incubating at pared from the leukocytes of the patient, his mother, a-thalas- 37°C for 1-3 h. semia trait, silent carrier, Hb H disease, and normal controls, Hemoglobin and globin analyses. Globin chain svnthesis as described (30). 10 yg of human DNA and 1 ug of X-DNA wvas measured in vitro with fresh heparinized peripheral blood (as internal size marker) were digested for 4 h at 370C with or bone marrow, as described by Kan et al. (20). L-[3H]leucine- 1.25 U of Eco RI or Hpa I per microgram of DNA. The buffer labeled globin chains precipitated from cell-free hemolvsate for Eco RI digestion contained 100 mX1 Tris-HCI (pH 7.5), were separated on carboxymethyl cellulose columns in 8 MI 50 mM NaCl, 6 mM MIgCl2, and 6 mnM 2-mercaptoethanol, urea at pH 7.2 using a linear Na2HPO4 gradient. The rela- and for HpaI digestion, 6 mnM Tris-HCI (pH 7.5), 26 mM NaCl, tive synthesis of a-chain to ,- and y-chain was expressed as the 6 mM \1gCI2, and 6 mnM 2-mercaptoethaniol. The samples ratio of the total radioactivity of the a- to non-a-globin peaks. wvere precipitated in alcohol, dried and resuspended in 30 ILI Starch gel electrophoresis was carried out at 4°C for 3 h at of 5 mMI Tris (pH 7.5), 0.1 mM EDTA. The digested DNA's 25 mA and 200 V according to the method of Smithies (21) with were electrophoresed in 0.8% agarose and then transferred to 0.006 \I Na2HPO4 buffer for the gel and 0.04 NI Na2HPO4 nitrocellulose filters (31). The filters were hybridized with buffer for the electrodes, both at pH 7.0. The gel was then 32P-labeled a- and 8-globin cDNA (2 x 108 epm/pLg) for 2 d sliced horizontally and stained with benzidine. and extensively washed and autoradiographed, as de- Thin-layer isoelectric focusing in polyacrylamide gel was scribed (30).