Juvenile myelomonocytic leukemia (JMML) with the hematologic phenotype of severe beta thalassemia.

A 3-year-old Filipino-American child with recurrent fever, splenomegaly, anemia, and thrombocytopenia, was found to have a hemoglobin F level of 76.9%. His reticulocyte count was elevated (4.3%), and erythroblasts were present in his peripheral blood. The child's erythrocytes were microcytic (MCV 66.9 fl) but his serum ferritin level was normal. His bone marrow at initial presentation demonstrated normal cellularity without an increase in blast cells. The disease progressed with worsening anemia, leukocytosis, and thrombocytopenia, with increased blasts in his marrow and the appearance of a mediastinal mass. His liver, spleen, and lymph nodes were found to be infiltrated with myeloblasts, supporting a diagnosis of juvenile myelomonocytic leukemia (JMML). Analysis of the child's Hb F showed a Ggamma/Agamma ratio of 2.2, which was within the characteristic range for JMML. A globin synthesis study using blood reticulocytes showed an alpha/non-alpha globin synthesis ratio of 2.24, typical of severe homozygous beta thalassemia. Southern blot analysis of blood-leukocyte DNA from the patient and his parents demonstrated no apparent abnormality in the beta-globin gene promoter or coding regions. The elevated level of Hb F in this child with JMML appeared to be part of an acquired Cooley's anemia-like hematologic phenotype.

Hemoglobin-specific antibody in a multiply transfused patient with sickle cell disease.

Human hemoglobins (Hbs) are known to be immunogenic, and both normal and variant forms of Hb have been shown to stimulate antibody formation in a variety of animal species. In patients who are homozygous for the sickle Hb (HbS) mutation, transfusion of normal, HbA-containing erythrocytes provides a potential stimulus for HbA alloimmunization. We tested serum samples for the presence of anti-Hb antibody by a solid-phase enzyme-linked immunosorbent assay (ELISA) using Hb-coated polystyrene microtiter plates. Hb-bound antibody was identified using an antihuman IgG antibody. Serum samples from 89 patients with sickle cell disease were initially tested for evidence of Hb antibody. The serum from three individuals exhibited antibody activity against HbA with little or no activity against HbS. Only one of them, a multiply transfused adult with HbSS, was available for further study. When this patient's antibody was tested against a variety of normal and mutant Hbs using antibody either to human IgG or to kappa chains, the anti-Hb antibody demonstrated specificity for the region of the Hb beta chain corresponding to the site of the amino acid substitution of HbS. The level of activity of the patient's anti-HbA showed no significant change over 1.5 years of observation. The transfusion of erythrocytes containing Hb structurally different from that of the recipient appeared to be capable of stimulating the production of Hb-specific alloimmune antibody.

Fetal hemoglobin expression in transplant recipients of placental blood hematopoietic progenitor cells.

Patients who achieved bone marrow engraftment of cord blood-derived progenitor cells provided an opportunity to examine the expression of fetal Hb by neonatal hematopoietic progenitors in a postneonatal host. Cord blood cells from histocompatible siblings were successfully transplanted in two children with the Fanconi anemia syndrome. One of the transplant donors had heterocellular hereditary persistence of fetal Hb, apparently due to gamma-globin gene triplication; the other donor was hematologically normal. The G gamma/A gamma ratio of the patient who received his transplant from the donor with hereditary persistence of fetal Hb was markedly elevated, similar to that of the transplant donor's cord blood, and this ratio remained elevated in subsequent months. In the other child, the G gamma/A gamma ratio immediately after her transplant was typical of the normal newborn, and over the next several months it reverted to the adult pattern. Globin synthesis studies performed shortly after engraftment demonstrated ratios of fetal Hb/adult Hb synthesis in both patients that were typical of those of normal newborns. Over the next several months, both patients converted to the adult pattern. Fetal Hb to adult Hb switching in these patients seemed to follow a temporal sequence intrinsic to the transplanted neonatal progenitor cells, without discernible influence of postneonatal environmental factors. The program for Hb switching seems to be an inherent feature of neonatal hematopoietic progenitor cells.

Hb south Milwaukee [beta 105 (G7) Leu----Phe]: a newly-identified hemoglobin variant with high oxygen affinity.

Fifteen individuals among four generations of a family of English ancestry demonstrated elevated hemoglobin levels accompanied by leftward-shifted whole blood oxygen equilibrium curves. Five of the affected family members have required phlebotomies for relief of symptoms attributable to erythrocytosis. An abnormal hemoglobin or globin chain could not be isolated, but 43% of the beta chains of the affected individuals contained a Leu----Phe substitution at position 105 (G7). Oxygen equilibrium curves demonstrated a normal Bohr effect but decreased cooperativity.

Hb Mizuho [beta 68(E12)Leu----Pro]. Second occurrence identified in a Caucasian child with hemolytic anemia and dense erythrocyte inclusions.

Hb Mizuho [beta 68(E12)Leu----Pro] was identified in a child of Italian/Sicilian descent who exhibited severe, transfusion dependent hemolytic anemia which improved following splenectomy. The patient's peripheral blood smear, which prior to splenectomy demonstrated coarse erythrocytic basophilic stippling, showed large, dense erythrocytic hemoglobin inclusions following splenectomy. Whole blood oxygen equilibrium results were consistent with the presence of a hemoglobin component exhibiting increased oxygen affinity with decreased cooperativity. The abnormal beta chain was characterized by high performance liquid chromatography analysis of the isopropanol precipitable hemoglobin fraction.

Hemoglobin Warsaw (Phe beta 42(CD1)----Val), an unstable variant with decreased oxygen affinity. Characterization of its synthesis, functional properties, and structure.

In Hb Warsaw Val replaces the Phe normally present at the heme contact position beta 42 (CD1). This variant is unstable, and it readily undergoes methemoglobin formation. In DEAE-cellulose chromatography, the variant hemoglobin co-eluted with Hb A; a partially heme-depleted fraction of the variant, representing 5-6% of the total hemoglobin, eluted separately and in pure form. The heme replete form of Hb Warsaw exhibited decreased oxygen affinity with a normal Bohr effect and normal cooperativity and interaction with 2,3-diphosphoglycerate (DPG). The heme-depleted Hb Warsaw had a higher oxygen affinity than that of Hb A, decreased cooperativity and 2,3-DPG interaction, and a very low alkaline Bohr effect. Gel filtration of the heme-depleted form showed it to exist entirely as alpha beta dimers. Globin chain synthesis by Hb Warsaw-containing reticulocytes followed a balanced alpha/beta ratio. In short-term synthesis experiments, a major portion of incorporated radiolabeled L-leucine was recovered from the dimeric, heme-depleted Hb Warsaw fraction, suggesting that subunit association precedes the incorporation of heme into the beta subunits in the post-synthetic assembly of this hemoglobin. Structural analysis of deoxyhemoglobin containing roughly equal proportions of normal and variant beta chains showed that the replacement leaves a cavity next to the heme that is large enough to hold a water molecule, which may account for the instability of Hb Warsaw. The heme and the pyrrol nearest to ValCD1 tilt into the cavity. The resulting increase in the tilt of the proximal histidine relative to the heme plane, coupled with a possible stretching of the Fe-N epsilon bond may account for the low oxygen affinity.

Hb Warsaw (beta 42 Phe----Val): an unstable hemoglobin with decreased oxygen affinity. I. Hematologic and clinical expression.

Four members in two generations of a Polish-American family exhibited findings of congenital Heinz-body hemolytic anemia accompanied by cyanosis. Two of the affected family members have also developed severe pulmonary hypertension, with a fatal outcome in one of them. Blood from the affected individuals showed decreased oxygen affinity and contained elevated levels of methemoglobin. An unstable hemoglobin fraction underwent rapid precipitation following exposure of the red cell lysates to isopropyl alcohol or heat. This hemoglobin contained a newly identified abnormal beta chain with an amino acid substitution at the same position as that of Hb Hammersmith and Hb Bucuresti-Louisville.

The primary structure and functional properties of the hemoglobins of a ground squirrel (Spermophilus townsendii, Rodentia).

The hemoglobin of the ground squirrel Spermophilus townsendii consists of two components which are present in a ratio of ca. 2:1. The two hemoglobins have identical alpha-chains, but differ in their beta-chains. We present the primary structures of the alpha- and the two beta-globin chains. Following chain separation by chromatography on carboxymethyl-cellulose CM-52, the amino-acid sequences were established by automatic Edman degradation of the globin chains and the tryptic peptides, as well as of a peptide obtained by acid hydrolysis of the Asp-Pro bond of the beta-chains. The two beta-chains differ by only one amino-acid residue, Ala being present in the main and Asp in the minor component in position 58 (E2). The comparison with human hemoglobin showed only 14 exchanges in the alpha-chains but 33 in the beta-chains. Whereas no contact positions are affected in the alpha-chains, we found four such substitutions in the beta-chains, including one heme contact, two alpha 1/beta 1-contacts, and one alpha 1/beta 2-contact. It seems however, that the substitution found in the beta-chains has no effect on the oxygen affinity.

Modelling whole blood oxygen equilibrium: comparison of nine different models fitted to normal human data.

The ability of nine different models, prominent in the literature, to meaningfully characterize the oxygen-hemoglobin equilibrium curve (OHEC) of normal individuals was examined. Previously reported data (N = 33), obtained using the DCA-1 (Radiometer, Copenhagen), and new data (N = 8), obtained using the Hemox-Analyzer (TCS, Southampton, PA), from blood samples of normal, non-smoking volunteers were used and these devices were found to give statistically similar results. The OHECs were digitized and fitted to the models using least-squares techniques developed in this laboratory. The "goodness-of-fit" was determined by the root-mean-squared (RMS) error, the number of parameters, and the parameter redundancy, i.e., correlation between the parameters. The best RMS error did not necessarily indicate the best model. Most literature models consist of ratios of similar-order polynomials. These showed considerable parameter redundancy which made the curve fitting difficult. The best fits gave RMS errors as low as 0.2% saturation. The Hill model gave a good characterization over the saturation range 20%-98% with RMS errors of about 0.6% saturation. On the other hand, good characterizations over the entire range were given by several other models. The relative advantages and disadvantages of each model have been compared as well as the difficulties in fitting several of the models. No single model is best under all circumstances. The best model depends upon the particular circumstances for which it is to be utilized.

Hemoglobin Evanston (alpha 14 Trp----Arg). An unstable alpha-chain variant expressed as alpha-thalassemia.

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 indicate that the proposita in each of these families has the genotype--, alpha A/--, alpha Ev. These observations suggest that two separate mechanisms are contributing to the alpha-thalassemia-like expression of Hb Evanston : the newly synthesized alpha EV-chains are unstable and are subject to early proteolytic destruction; and the mutant alpha-allele is linked to an alpha-globin gene deletion.

Iron deficiency and sickle cell anemia.

In a patient with sickle cell anemia, iron deficiency was accompanied by hypochromic, microcytic RBCs, absence of bone marrow iron, and a low serum ferritin level. The mean corpuscular hemoglobin concentration (MCHC) was decreased (27.6 g/dL) and was associated with an extreme scarcity of sickled erythrocytes in blood smears. Iron therapy resulted in reticulocytosis and an increase in sickled erythrocytes. In vitro studies demonstrated a decrease in sickling of erythrocytes as a function of oxygen saturation of the blood when the patient was iron deficient. The whole blood oxygen dissociation curve showed a substantial decrease in oxygen pressure necessary to produce 50% saturation of hemoglobin at pH 7.4 and 37 degrees C (P50), indicating an increased oxygen affinity. These data suggest that a reduction of the MCHC induced by iron deficiency may ameliorate sickling.

Hemoglobin Milledgeville (alpha 44 (CD2) Pro leads to Leu): a new variant with increased oxygen affinity.

Hemoglobin Milledgeville, a new hemoglobin structural variant, was identified in three members of a black American family. The oxygen affinity of blood and hemoglobin samples from the affected individuals was markedly increased (p50 O2 of whole blood 11-15 mmHg at 37 degrees C, pH 7.4), and the abnormality was associated with mild erythrocytosis. The variant hemoglobin did not separate from Hb A by electrophoresis or by chromatography or isoelectric focusing, and efforts to isolate an abnormal globin chain were also unsuccessful. The Hb A2 fraction as well as Hb A from erythrocytes of affected individuals exhibited increased oxygen affinity, indicating that the altered oxygen equilibrium was the result of a hemoglobin alpha chain abnormality. Fractionation of trypsin and chymotrypsin digests of isolated alpha chains demonstrated a single abnormal peptide representing a Pro leads to Leu substitution at alpha 44 (CD2). Properties of Hb Milledgeville include low cooperativity (n = 1.1-1.4), a normal alkaline Bohr effect (delta logp50/delta pH = -0.62), and normal interaction with 2,3-diphosphoglycerate. The alpha CD2 proline residue normally participates in the formation of the alpha 1 beta 2 subunit interface in the deoxy quaternary conformation, but not in oxyhemoglobin; the leucine substitution may produce destabilization of the deoxy conformation with a resulting shift in equilibrium toward the oxy conformation.

Two new sickle cell syndromes: HbS, Hb Camden, and alpha-thalassemia; and HbS in combination with Hb Tacoma.

Hemoglobin variants having electrophoretic mobility more rapid than that of HbA were identified in combination with sickle hemoglobin in two patients at the Cook County Hospital. Neither individual had symptomatic hematologic disease. In one patient, the rapidly migrating hemoglobin had the amino acid substitution characteristic of Hb Tacoma (beta-40 arg leads to ser), a mildly unstable variant. In the other patient, Hb Camden (beta-131 gln leads to glu) was identified, and the hematologic findings also indicated that he has alpha-thalassemia trait. In the patient with HbS-Camden--alpha-thalassemia, globin synthesis was unbalanced (alpha/beta 0.66), and HbS represented only 19.5% of the total hemoglobin. The latter finding suggests that under conditions of limited alpha-chain availability beta Camden may combine with alpha subunits at least as efficiently as does betaA. HbS represented 56% of the hemoglobin of the patient with HbS Tacoma, although the rate of synthesis of beta Tacoma by her reticulocytes was consistently greater than that of betaS. A time-course synthesis study demonstrated a progressive increase in the specific activity of beta Tacoma in relation to that of betaS, suggesting that the unstable beta-chains of Hb Tacoma underwent selective intracellular degradation. This process appears to explain the disparity between the rates of synthesis of the two beta chains and the relative representation of HbS and Hb Tacoma in the patient's erythrocytes.

Hemoglobin Nigeria (alpha-81 Ser replaced by Cys):a new variant associated with alpha-thalassemia.

Hematologic evaluation of a Nigerian obstetrical patient disclosed the presence of sickle-cell trait as well as evidence of a hemoglobin alpha-chain abnormality. Hemoglobins containing the variant alpha-chain were isolated by DEAE-cellulose column chromatography, and analysis of the purified alpha-chain demonstrated a ser replaced by cys substitution at alpha-81. The abnormal alpha-chain represented approximately 45% of the total, and hemoglobins containing this alpha-chain appeared to have normal stability and functional properties. In addition to the abnormal hemoglobins that were identified in this patient, she also was found to have persistent microcytosis in the absence of iron deficiency, and the percentage of HbS in her erythrocytes was less than that usually present in individuals with sickle cell trait. These findings, together with a reduced alpha/beta globin synthesis ratio from her peripheral blood reticulocytes, indicated that the presence of alpha-thalassemia trait. Hematologic findings from members of the patients's family suggest that an alpha-thalassemia gene may be linked to that of the structurally abnormal alpha-chain.

Sickle cell syndromes. III. Silent-carrier alpha-thalassemia in combination with hemoglobin S and hemoglobin C.

Silent carrier alpha-thalassemia was identified in two individuals, one with sickle-cell trait and the other hemoglobin (Hb) C trait. Both are parents of a child with characteristic hematologic features of the Hb SC-alpha thalassemia syndrome, including microcytosis and an unbalanced pattern of globin synthesis. In contrast to the typical findings that accompany heterozygous Hb S or Hb C with concomitant alpha-thalassemia trait, neither of the parents had microcytosis nor a percent of the abnormal hemoglobin in their erythrocytes that was below the normal range. In both, however, globin synthesis of peripheral blood reticulocytes was unbalanced, consistent with mild alpha-thalassemia. These findings suggest that the alpha-thalassemia silent carrier may be hematologically indistinguishable from the nonthalassemic individual, even when hemoglobin S or C are present.

Three new variants of glucose-6-phosphate dehydrogenase associated with chronic nonspherocytic hemolytic anemia: G-6-PD Lincoln Park, G-6-PD Arlington Heights, and G-6-PD West Town.

Glucose-6-phosphate dehydrogenase (G-6-PD) deficiency was identified in three children who were evaluated because of chronic nonspherocytic hemolytic anemia. One child is of German extraction, another Puerto Rican, and the third Mexican. In each of the patients the hemolytic process was well compensated, but each had one or more episodes of anemia following exposure to an oxidant drug or with infections. The electrophoretic, functional, and kinetic properties of the mutant enzymes, derived both from the patients' erythrocytes and from cultured fibroblasts, allowed each to be distinguished from G-6-PD variants previously described.

Sickle cell syndromes. II. The sickle cell anemia-alpha-thalassemia syndrome.

Five American black patients, ages 1 to 16 years, with the sickle cell anemia-alpha-thalassemia syndrome are described. Each patient had persistent microcytosis not explained by iron deficiency, and in each family the presence of alpha-thalassemia in combination with sickle cell trait was demonstrated in one of the parents. In one patient, in whom the diagnosis of sickle cell anemia was established at birth, an elevated level of Barts (gamma4) hemoglobin was also found. In these patients levels of alkali-resistant hemoglobin and reticulocyte counts were similar to those of sickle cell anemia patients of comparable age; however, stained smears of their peripheral blood rarely showed the presence of irreversibly sickled cells. No major ameliorative effect of the alpha-thalassemia on the clinical expression of the sickle cell disease of these patients was evident.