Severe hemolytic anemia associated with the homozygous state for an unstable hemoglobin variant (Hb Bushwick)

We have investigated a 13-year-old girl from first cousin parents who presented with severe hemolytic anemia. Hematologic studies showed unstable hemoglobin (Hb) disease (chronic Heinz body anemia), and DNA analysis showed that the patient was homozygous for the previously reported abnormal Hb called Hb Bushwick (beta 74E18 gly-->val). Hb Bushwick is unstable in vitro and in vivo. In addition, using globin chain biosynthetic studies, we show that the beta (Bushwick) chains are unstable. Six members of the patient's family were heterozygous for Hb Bushwick and had a compensated hemolytic disorder. By contrast, the homozygous patient had chronic anemia caused by a combination of hemolysis and ineffective erythropoiesis that was subject to severe exacerbation concomitant with infection. Thus, although unstable Hb disease is correctly regarded as dominant, we clearly see a dosage effect in its expression, whereby the homozygous state is still compatible with life although the red blood cells contain nearly 100% unstable Hb.     

Hb Bruxelles: alpha 2A beta (2)41 or 42(C7 or CD1)Phe deleted

Hb Bruxelles is a new beta-globin variant producing severe congenital Heinz body anemia. It results from the deletion of one of the two adjacent phenylalanines, beta 41 or beta 42, presumably by frameshift mutagenesis. Its whole blood oxygen affinity is significantly lowered.     

Hb Madrid [beta115(G17)Ala-->Pro] in a Korean family with chronic hemolytic anemia

Hb Madrid, in which the alanine residue at beta115 (G17) is replaced by proline, results in a moderately severe hemolytic anemia due to the disruption of an alpha helical region and the weakening of an alpha1 beta1 contact (1,2). It was first discovered in a single Spanish patient, by protein structural analysis, whose parents did not carry the abnormality (1). The second observation of Hb Madrid was in an American Black teenager by DNA analysis, who had the family history of chronic hemolytic anemia, but none of family members were available for evaluation (3).     

Hemoglobin Sendagi (beta 42 Phe----Val): a new unstable hemoglobin variant having an amino acid substitution at CD1 of the beta-chain

Hb Sendagi, a new unstable hemoglobin variant, was found in a Japanese male and his daughter, who have a moderate hemolytic anemia. The variant showed decreased stability upon heat and isopropanol precipitation tests. The variant did not separate from hemoglobin A by electrophoresis, but the abnormal beta-chain emerged ahead of the normal beta-chain on reverse-phase HPLC. Structural analyses revealed that the phenylalanine beta 42 (CD1), one of the critical amino acid residues in the heme pocket, had been replaced by valine. Oxygen equilibrium studies of the patient's hemolysates indicated that Hb Sendagi had a lowered oxygen affinity and a normal response to 2,3-diphosphoglycerate. Hb Hammersmith (beta 42 Phe----Ser) and Hb Louisville (Bucuresti) (beta 42 Phe----Leu) have previously been reported to have varying degrees of molecular instability and altered oxygen binding function. Hb Sendagi provides an additional model for elucidation of the role of the phenylalanine beta CD1 on the structure and function of hemoglobin.     

Studies on a family with Hb J Calabria (alpha 2 beta 2 64 (E8) Gly replaced by Asp).

Hb J Calabria is a fast moving hemoglobin variant which was found in an Italian family by Vecchio et al (1), and in a French family by Blouquit et al. who studied its functional properties (2). The original family described by Vecchio et al. in which both Hb J Calabria and beta-thalassemia were present has been reexamined and is the subject of the present study. Hematological and clinical features of the carriers are described. The heterozygous carriers of Hb J Calabria showed only mild variable subclinical anemia and levels of the abnormal hemoglobin ranging from about 33 to 42%. The Hb J Calabria/beta-thalassemia double heterozygote showed a moderate chronic hemolytic anemia with alterations of the RBC indices and morphology in addition to splenomegaly. The relationship between structural abnormality, functional properties and clinical expression of Hb J Calabria is discussed.     

Hemoglobin Saverne: a new variant with elongated beta chains: structural and functional properties

A 26-year-old French woman born in Saverne (France) was found to have Heinz body hemolytic anemia. Isoelectrofocusing showed the presence of an abnormal band amounting to 35% of the total hemoglobin concentration, suggesting a beta variant. Structural analysis of the abnormal beta chain showed an elongated C-terminal segment. Histidine 143 is replaced by a proline and the C-terminal sequence is identical to the corresponding segment of Hb Cranston. This new variant, named Hb Saverne, has beta chains composed of 156 amino acid residues. Studies of its functional properties showed that Hb Saverne is an unstable, high affinity variant with low cooperativity.     

Studies on a Family with HB J Calabria (α2 β2 64 (E8 GLY → ASP)

Hb J Calabria is a fast moving hemoglobin variant which was found in an Italian family by Vecchio et al. (1), and in a French family by Blouquit et al.who studied its functional properties (2). The original family described by Vecchio et al. in which both Hb J Calabria and β-thal-assemia were present has been reexamined and is the subject of the present study. Hematological and clinical features of the carriers are described. The heterozygous carriers of Hb J Calabria showed only mild variable sub-clinical anemia and levels of the abnormal hemoglobin ranging from about 33 to 42%. The Hb J Calabria/β-thal-assemia double heterozygote showed a moderate chronic hemolytic anemia with alterations of the RBC indices and morphology in addition to splenomegaly. The relationship between structural abnormality, functional properties and clinical expression of Hb J Calabria is discussed.     

The unstable Hb Hammersmith or alpha 2 beta 2(42)(CD1)Phe----Ser observed in an Indian child; identification by HPLC and by sequence analysis of amplified DNA.

We have identified the unstable hemoglobin variant present in a Chipewayan Indian patient with severe hemolytic anemia as Hb Hammersmith or alpha 2 beta 2(42)(CD1)Phe----Ser. Her parents were normal. Identification was greatly facilitated by the use of reversed phase high performance liquid chromatography for the isolation of the beta X chain and its tryptic fragments, and of sequence analysis of amplified DNA which readily identified a TTT(Phe)----TCT(Ser) mutation at codon 42.     

Hb Leslie, an unstable hemoglobin due to deletion of glutaminyl residue beta 131 (H9) occurring in association with beta0-thalassemia, HbC, and HbS.

A new unstable hemoglobin, Hb Leslie, has been observed in three generations of a Georgia family. The propositus, a 42-yr-old black veteran with hemolytic anemia and splenomegaly, has a hemoglobin variant with an electrophoretic mobility similar to that of hemoglobin F. The variant comprises about 85% of the total hemoglobin and was isolated by chromatography. Chemical analysis has identified the abnormality as a deletion of the glutaminyl residue in position 131 (H9) of the beta-chain. Deletion of this critical residue which participates in the alpha1beta1 contact causes decreased stability of the hemoglobin without significant changes in functional properties or morphologic abnormalities in the erythrocyte. Family studies revealed hemoglobin Leslie occurring in combination with beta0-thalassemia, HbS, and HbC. All persons with the various Hb Leslie combinations, including the propositus, have no clinical manifestations other than anemia. In some the anemia is fully compensated. There is no history of drug-associated hemolysis.     

Hb Leiden-beta (0) thalassemia in a Chinese with severe hemolytic anemia

The first case of Hb Leiden (alpha2beta2 6 or 7 Glu---O)-beta (0) thalassemia in a young patient with chronic severe hemolytic anemia, which improved after splenectomy, is described. His parents were Chinese. The patient's blood showed no Hb A or normal beta chains when no blood transfusion was given. His mother was heterozygous for beta(0) thalassemia, and his father and brother had a trait for the unstable Hb Leiden. The Hb Leiden level of the father was 22.6% and that of the brother was 19.3%. It is probable that the abnormal hemoglobin in this Chinese family resulted from an independent gene mutation, unrelated to the one found in 2 Caucasian families reported earlier.     

Hb Oslo [β42(CD1)Phe→Ile; HBB: c.127T>A]: A Novel Unstable Hemoglobin Variant Found in a Norwegian Patient

Unstable hemoglobin (Hb) variants are the result of sequence variants in the globin genes causing precipitation of Hb molecules in red blood cells (RBCs). Intracellular inclusions derived from the unstable Hb reduce the life-span of the red cells and may cause hemolytic anemia. Here we describe a patient with a history of hemolytic anemia and low oxygen saturation. She was found to be carrier of a novel unstable Hb variant, Hb Oslo [β42(CD1)Phe→Ile (TTT>ATT), HBB: c.127T>A] located in the heme pocket of the β-globin chain. Three-dimensional modeling suggested that isoleucine at position 42 creates weaker interactions with distal histidine and with the heme itself, which may lead to altered stability and decreased oxygen affinity. At steady state, the patient was in good clinical condition with a Hb concentration of 8.0–9.0?g/dL. During virus infections, the Hb concentration fell and on six occasions during 4 years, the patient needed a blood transfusion.     

Hb Bristol-Alesha Presenting Thalassemia-Type Hyperunstable Hemoglobinopathy

Hemoglobin (Hb) Bristol-Alesha is caused by a GTG --> ATG mutation at codon 67 in the Hb beta chain, resulting in abnormal beta globin chains with mutated molecules from normal beta67 valine (Val) to beta67 methionine (Met) or beta67 aspartate (Asp). We describe a Japanese child with this rare hemoglobinopathy and a very unstable Hb molecule phenotype. The diagnosis of hemolytic anemia was made when the patient was 6 months of age. Development of marked splenomegaly necessitated red blood cell transfusions twice a month. After splenectomy when the patient was 4 years of age, laboratory findings of hemolytic anemia became more prominent. Specific abnormal Hb molecules initially were not detected, and the alpha/beta globin synthesis ratio was abnormal at 2.22. After splenectomy, we identified the presence of abnormal beta-globin chains with a beta67Val:beta67Met:beta67Asp molecule ratio of 74:11:15. We speculate that the high fraction of the beta67Met molecule in this patient, compared with that in previously reported cases, caused extreme Hb instability, which resulted in thalassemic hyperunstable hemoglobinopathy and very severe clinical findings.     

HB Hakkari or α2β231(B13)LEU→ARG,A Severely Unstable Hemoglobin Variant Associated with Numerous Intra-Erythroblastic Inclusions and Erythroid Hyperplasia of the Bone Marrow

A severely unstable hemoglobin variant, Hb Hakkari or α₂β₂31 (B13)Leu→Arg, has been observed in a 5-year-old Turkish girl with a severe hemolytic anemia without Heinz body formation. A modest increase in liver and spleen size was present and the level of Hb F was a high 33%. The variant could not be observed in red cells and was only detected through sequencing of the amplified β-globin gene and also by hybridization with specific oligonucleotide probes. The parents were normal, and it is assumed that the variant occurred as a de novo mutation. Smears from bone marrow aspirates showed numerous inclusion bodies in the erythroblast and, as a result, a erythroid hyperplasia. It is suggested that the hemoglobin variant which is unstable and is readily losing its heme group because one of the heme binding sites has been lost, precipitates in the erythroblasts, thus interfering with the maturation process and causing the severe anemia.     

Hyperunstable hemoglobin Toyama [alpha 2 136(H19)Leu----Arg beta 2]: detection and identification by in vitro biosynthesis with radioactive amino acids

A previously reported case of congenital Heinz body anemia was reinvestigated. Heat denaturation, isopropanol testing, PCMB precipitation, isoelectricfocusing, and reversed phase high performance liquid chromatography on the red cell lysate from the patient gave either negative, or at most, questionable results. In vitro globin biosynthesis using peripheral blood with incorporation of 3H-leucine demonstrated the production of an abnormal alpha chain at the rate of about 1/3 that of the normal alpha chain. A substitution, alpha 136(H19)Leu----Arg, was elucidated by peptide mapping and radiosequencing of an abnormal tryptic peptide. The hemoglobin consisting of the abnormal alpha and normal beta chains eluted between Hb A2 and Hb A0 in anion exchange high performance liquid chromatography. It was barely detectable by this method, comprising less than 1/1000 of the amount of Hb A0, although it was produced at a level of 1/3 of that of HB A0 in terms of radioactivity. The daughter of the propositus was similarly afflicted and produced the same abnormal alpha chain. The son, who also produced the abnormal alpha chain, was essentially free from hemolytic manifestation. His red cells were microcytic and showed an alpha/beta synthetic ratio of over 2.     

Hemoglobin Sabine [beta 91 (F7) Leu-->Pro]: occurrence in a Sardinian individual with hemolytic anemia and inclusion bodies.

BACKGROUND. Hemoglobin (Hb) Sabine (beta 91 Leu-->Pro) is an unstable variant detected for the first time in a 16-year-old Scottish-English-German girl affected by moderately severe hemolytic anemia. A second case was described in a patient of Yugoslavian descent. We report another case of this Hb variant arising as a de novo mutation in a Sardinian patient. METHODS. Definition of the mutation was obtained by DNA direct sequencing on amplified beta-globin gene, as well as by structural analysis of the hemoglobin variant. RESULTS AND CONCLUSION. The patient presented a moderately severe hemolytic anemia with red blood cell inclusion bodies. Hemoglobin electrophoresis showed that quantitatively the abnormal fraction represented 9% of the total Hb amount. beta globin gene analysis revealed a single nucleotide substitution, T-->C, at codon 91, which gives rise to a leucine-->proline substitution. Structural analysis of the variant confirmed the amino acid substitution (Leu-->Pro) predicted by DNA sequencing.     

Hb Oegstgeest [alpha104(G11)Cys-->Ser (alpha1)]. A new hemoglobin variant associated with a mild alpha-thalassemia phenotype

A microcytic hypochromic anemic state was observed in an 8-year old Black female of Surinam origin during pre-operative Hb S [beta6(A3)Glu-->Val] screening. Her high zinc protoporphyrin (ZPP) level suggested a chronic iron depletion but, in contrast, the high red blood cell (RBC) count (5.85 x 10(12)/L) was indicative of a possible coexisting thalassemia. No abnormal hemoglobin (Hb) bands were present on high performance liquid chromatography (HPLC) or alkaline electrophoresis and the Hb A2 level was normal. Break point polymerase chain reaction (PCR) failed to reveal any of the common alpha-thalassemia (thal) mutations but selective DNA sequencing of both alpha-globin genes disclosed a TGC-->AGC transversion at codon 104 of the alpha1 gene. Cystine at codon 104 is involved in alpha/beta globin contact and has been described to be a critical amino acid of the alpha2 chain when substituted by a tyrosine (Hb Sallanches), inducing Hb H (beta4) disease in the homozygous state. Our heterozygous patient had a moderate anemia of 12.2 g/dL and a borderline haptoglobin suggesting some degree of hemolysis.     

Hb Southampton [beta106(G8)Leu-->Pro, CTG-->CCG] in an Argentinean boy

Hb Southampton (also known as Hb Casper) is characterized by the substitution of a leucine residue for a proline at codon beta106 (CTG-->CCG). This mutation breaks the G helix and severely distorts the tertiary structure of the molecule, producing an unstable hemoglobin (Hb) and severe hemolysis. We identified this hemoglobinopathy in a young patient with severe hemolytic anemia and hepatosplenomegaly.     

Clinical and hematologic features of beta0-thalassemia (frameshift 41/42 mutation) in Thai patients

BACKGROUND AND OBJECTIVES: Frameshift 41/42 mutation is the most common mutation of beta0-thalassemia found in Thailand. We studied clinical and hematologic features in 84 patients and relatives with frameshift 41/42 to determine whether it is possible to predict phenotypic severity from genetic factors. DESIGN AND METHODS: The clinical phenotypes and hematologic data of Thai patients with frameshift 41/42 were studied. Alpha-thalassemia, Hb Constant Spring (HbCS) genes and the presence of Xmnl-Ggamma polymorphism were studied in patients who had mild symptoms. RESULTS. Homozygotes for frameshift 41/42 and compound heterozygotes for frameshift 41/42 and beta0-thalassemia produced severe symptoms and have a thalassemia major phenotype. Combination of frameshift 41/42 and beta0-thalassemia or Hb E produced mild to moderate symptoms with thalassemia intermedia phenotype and severe symptoms with thalassemia major phenotype. The co-inheritance of beta-thalassemia or HbCS gene or the presence of Xmnl-Ggamma polymorphism was not associated with mild disease in patients with frameshift 41/42 and HbE. INTERPRETATION AND CONCLUSIONS: The clinical phenotype of homozygotes for frameshift 41/42 and compound heterozygotes for frameshift 41/42 and beta0-thalassemia could be used to predict a severe phenotype with thalassemia major. However, the clinical phenotype of compound heterozygotes of frameshift 41/42 and beta0-thalassemia or Hb E were variable and could not be accurately predicted. Associations between concomitant alpha-thalassemia or HbCS of the presence of Xmnl-Ggamma polymorphism and a mild clinical phenotype are not apparent, indicating the involvement of other ameliorating determinants or genetic modifications.     

Interaction of hemoglobin E and pyrimidine 5' nucleotidase deficiency

A Bangladeshi family is described in which the genes for both hemoglobin E (Hb E) and pyrimidine 5' nucleotidase deficiency are segregating. An individual homozygous for both these conditions has a severe hemolytic anemia, whereas family members who are homozygous for Hb E are asymptomatic and those homozygous for pyrimidine 5' nucleotidase deficiency have the mild hemolytic anemia that is characteristic of this disorder. Globin-chain synthesis experiments have shown that the mechanism underlying the interaction between these two genotypes is a marked decrease in the stability of Hb E in pyrimidine 5' nucleotidase-deficient red blood cells (RBCs). It has also been found that in the enzyme-deficient RBCs in which Hb E is highly unstable, free alpha-chains, though not beta E-chains, acoumulate on the membrane. In view of the increasing evidence that the hemolysis associated with pyrimidine 5' nucleotidase deficiency results not only from an increase in the level of erythrocyte pyrimidines, but also from inhibition of the hexose monophosphate shunt activity in young erythrocytes, it is likely that the marked instability of Hb E in the enzyme-deficient cells results from oxidant damage acting on a mildly unstable Hb variant. These observations may have important implications for the better understanding of the pathophysiology of Hb E/beta-thalassemia, globally the commonest important form of thalassemia.     

Early decline of hemoglobin can predict progression of hemolytic anemia during pegylated interferon and ribavirin combination therapy in patients with chronic hepatitis C.

Aim: Ribavirin, used to treat chronic hepatitis C, can induce hemolytic anemia, forcing the discontinuance of treatment. To establish a predictive measure to help circumvent this, we evaluated the relationship of hemoglobin (Hb) decline with the discontinuance of treatment during the progression of ribavirin‐induced anemia. Methods: One hundred and sixteen patients (71% male) with genotype 1 chronic hepatitis C were treated with pegylated interferon (PegIFN) α‐2b and ribavirin. The mean age was 50.6 years and 55% were IFN naïve. A decline of Hb concentration by 2 g/dL at two weeks from the start of the treatment (“2 by 2” standard) was adopted as the predictive factor for the progression of anemia. Results: By applying the “2 by 2” standard, with ΔHb ≥ 2 g/dL (34%, n = 39), treatment was discontinued in 12 cases (31%), three of which (8%) because of severe anemia. ForΔHb < 2 g/dL (64%, n = 76), treatment was discontinued in 11 (14%) cases; none due to severe anemia. Ten percent (4/39) of patients showed the minimum Hb ≤ 8.5 g/dL in the ΔHb ≥ 2 g/dL group, with none in the ΔHb < 2 g/dL group (P = 0.001). Furthermore, the patients with minimum Hb ≤ 8.5 g/dL were found only in the “2 by 2” standard‐positive and low CL/F (<15) group (4/29, 14%). Conclusion: Monitoring the Hb decline using the “2 by 2” standard can identify patients who are prone to developing severe anemia. Further prospective studies are needed using ribavirin reduction based on the “2 by 2” standard.