Translation of human globin mRNA: globin synthesis in cells containing Hb Leiden.

Most structurally abnormal hemoglobins are present in smaller amounts than HbA in the erythrocytes of heterozygous subjects. In the presence of a hemoglobinopathy, alpha and beta globin synthesis remains balanced with equal production of the two types of chains. In reticulocytes of subjects with Hb Leiden (beta 6 or 7 glu leads to 0) there is greater production of alpha than beta globin in vitro (beta/alpha = 0.67), and slightly more beta A is synthesized than beta Leiden (beta A/beta Leiden = 1.28). Differences in specific mRNA content, rates of initiation of chain synthesis, or rates of chain elongation could be responsible for such differential polypeptide synthesis. In the present study, the ribosomal assembly of beta A, beta Leiden, and alpha globin chains was examined in peripheral blood. The translation times of the three chains did not differ significantly (average times: beta A = 65.4 sec, beta Leiden = 70.8 sec, alpha = 53.5 sec). These results indicated that an altered rate of translation was not the source of the anomalous globin synthesis observed in vitro in cells containing Hb Leiden. The experiments suggested that the observed imbalance in alpha/beta production was due to either differential rates of initiation of globin chain synthesis or quantitative differences in the amounts of the specific mRNAs present in the cells.     

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.     

Hydrops fetalis due to an unusual form of Hb H disease

The occurrence of Hb H hydrops fetalis is reported for the first time. The mother has zeta-alpha thalassemia 1 (zeta zeta alpha alpha/----) and the father has non-deletion alpha thalassemia [zeta zeta alpha alpha/zeta zeta (alpha alpha)T]. The complete deletion of the zeta alpha cluster on one chromosome was confirmed by quantitation of alpha and zeta gene numbers, the normal alpha and zeta gene patterns arising from the remaining normal chromosome, and the decreased alpha/beta globin chain ratio of 0.57. The non-deletion alpha thalassemia defect could only be identified by the imbalanced alpha/beta globin chain ratio of 0.65 in the presence of normal gene numbers and patterns. The newborn was markedly anemic, unlike those with classical Hb H disease, because the non-deletion alpha thalassemia defect is more severe than alpha thalassemia 2. The decreased zeta genes during fetal life might have additional deleterious effects. In this family, the distinct BamHI restriction fragment length polymorphism in the hypervariable region of the zeta genes may be used for future prenatal diagnosis.     

Molecular basis for alpha-thalassemia associated with the structural mutant hemoglobin Suan-Dok (alpha 2 109leu----arg)

Hemoglobin (Hb) Suan-Dok (alpha 109Arg) is a rare alpha-globin structural mutation that is linked to an alpha-thalassemia (alpha-thal) determinant. When inherited in trans to an alpha-thal-1 mutation (-), it results in Hb H disease associated with low levels (9%) of the Suan-Dok Hb. The nature of the thalassemic defect associated with the alpha SD mutation has been investigated by structural and functional studies. Sequence analysis of the cloned Suan-Dok allele showed a missense mutation (T----G) at codon 109 in an otherwise normal alpha 2-globin gene. When the alpha 2SD-globin gene was introduced into mouse erythroleukemia cells, the steady state alpha-globin messenger RNA (mRNA) level was equivalent to the alpha A-globin gene control. Although in vitro translation of a synthetic alpha 2SD-globin mRNA generated levels of alpha globin equivalent to alpha 2A-globin mRNA at early time points, the ratio of alpha SD to alpha A globin decreased markedly at later time points. These data suggest that the thalassemic defect associated with the Suan-Dok mutation results from a significant instability of the alpha SD globin.     

High expression of human beta S- and alpha-globins in transgenic mice: hemoglobin composition and hematological consequences.

A line of transgenic mice (alpha H beta S-11; where alpha H is human alpha-globin) was created in which the human beta S and human alpha 2 globin genes, each linked to the beta-globin locus control region, were cointegrated into the mouse genome. On a normal genetic background, the transgenic mice produced 36% human beta S-globin chains with an alpha H/beta S ratio of 1.3. Higher levels of beta S were achieved by breeding the transgenic mice with mutant mice carrying a mouse beta major-globin gene deletion. Mice heterozygous for the beta major deletion (alpha H beta S[beta MD]; MD, mouse deletion) had 54% beta S with an alpha H/beta S ratio of 1.0; mice homozygous for the beta major deletion (alpha H beta S[beta MDD]) had 72.5% beta S and an alpha H/beta S ratio of 0.73. Because mouse alpha chains inhibit hemoglobin (Hb) S polymerization, we bred the mice to heterozygosity for a mouse alpha-globin deletion. These mice (alpha H beta S[alpha MD beta MDD]) had an increased alpha H/beta S ratio of 0.89 but expressed 65% beta S. Expression of the human genes cured the thalassemic phenotype associated with the murine beta major deletion. Transgenic alpha H beta S[beta MDD] mice had normal hematocrit and Hb and somewhat elevated reticulocytes (6% vs. 3% for control), whereas the mice carrying the alpha-globin deletion (alpha H beta S[alpha MD beta MDD]) had a normal hematocrit and Hb and more elevated reticulocytes (10.3 +/- 7.6% vs. 3.4 +/- 1.0%). Expression of the transgene restored a normal distribution of erythrocyte densities when compared to thalassemic mice; however, the average mean corpuscular Hb concentration of alpha H beta S[beta MDD] mice increased to 35.7 g/dl (vs. control 33.7 g/dl) whereas that of alpha H beta S[alpha MD beta MDD] mice was further elevated to 36.3 g/dl. The intrinsic oxygen affinity was increased in transgenic mouse erythrocytes at 280 milliosmolal, and the PO2 at midsaturation of alpha H beta S[alpha MD beta MDD] erythrocytes was higher than that of alpha H beta S[beta MDD] cells (37.4 +/- 2 vs. 33.5 +/- 1 mmHg). The higher values of the mean corpuscular Hb concentration and intrinsic PO2 at midsaturation, which favor in vivo sickling, may explain the slightly more severe hematological picture in alpha H beta S[alpha MD beta MDD] mice. We conclude that the transgenic mouse with high Hb S expression does not exhibit adult anemia but does have abnormal hematological features: increased erythrocyte density, high oxygen affinity, and reticulocytosis with increased stress reticulocytes.     

Variations in Globin Chain Synthesis in Hereditary Persistence of Fetal Haemoglobin

Globin synthesis was studied in four Negro families including 10 members with Hb A-HPFH and four with Hb S-HPFH. The beta/alpha specific activity ratios in 10 of these HPFH heterozygotes were similar to those of the control group. In two patients with Hb A-HPFH, the beta/alpha ratio was slightly decreased in one (0.84) and clearly decreased in another (0.78). In two of the patients with Hb S-HPFH the ratios were clearly decreased (0.71 and 0.75). The extended range of beta/alpha ratios in these 14 patients is similar to that of Negro patients with beta-thalassaemia trait. These studies indicate that a decreased beta/alpha ratio may be found in HPFH, as well as in beta-thalassaemia. Bone marrow globin synthesis was measured in two patients with Hb S-HPFH and decreased peripheral blood beta/alpha ratios, and in one with Hb A-HPFH and a normal peripheral blood beta/alpha ratio. In each patient the (beta+gamma)/alpha ratio of radioactivities as well as the beta/alpha specific activity ratio was close to 1 and therefore balanced, indicating more rapid decay of beta-chain synthesis relative to alpha-chain during red cell maturation or extremely rapid destruction of newly synthesized excess alpha-chains in the bone marrow.     

Hb Yokohama [beta 31 (B13)Leu----Pro] detected as a de novo mutation in a Yugoslavian boy.

Hb Yokohama [beta 31 (B13)Leu----Pro] was observed in a young Yugoslavian boy as a de novo mutation. The child exhibited severe transfusion-dependent hemolytic anemia. The variant was detected and quantitiated at 10.5% by reversed phase high performance liquid chromatography. In vitro globin chain synthesis showed a slight imbalance with an alpha/beta ratio of 1.38. Structural characterization of the abnormal beta chain was done by high performance liquid chromatographic analysis, on material obtained by high salt precipitation. The mutation was confirmed by sequencing of the amplified DNA.     

Variations in Globin Synthesis in Delta-Beta-Thalassaemia

Peripheral blood globin synthesis studies were done in 11 patients with delta beta-thalassaemia trait, Hb S-delta beta-thalassaemia or delta beta/betao-thalassaemia from two black and two Caucasian families. All patients had elevated Hb F and normal or decreased Hb A2 levels and 10 had family studies confirming the diagnosis. In addition, four unrelated non-thalassaemic patients with elevated Hb F levels also had peripheral blood globin synthesis studies. The beta/alpha specific activity globin synthesis ratios in the three blacks with delta beta-thalassaemia trait were 0.60--1.04. In the four Caucasians with delta beta-thalassaemia traint, the beta/alpha ratios were 0.58--1.02. These results demonstrate a wide range of ratios overlapping those of normal controls (0.99 +/- 0.06). The betas/alpha ratios in three blacks with Hb S-delta beta-thalassaemia ranged from 0.66 to 1.00, similar to those of patients with delta beta-thalassaemia trait. In the black patient with delta beta/betao-thalassaemia, the gamma/alpha ratio was 0.67. The beta/alpha peripheral blood ratios in the four non-thalassaemia patients with elevated Hb F ranged from 1.00 to 1.11, similar to those of normal controls. These studies indicate that a decreased beta/alpha ratio is not an invariable finding in delta beta-thalassaemia in blacks or Caucasian patients and that globin synthesis data alone is insufficient to diagnose definitively heterozygotes for delta beta-thalassaemia or to distinguish this trait from non-thalassaemic haematological disorders associated with a normal percentage of Hb A2 and an elevated level of Hb F.     

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.     

The Importance of the Genetic Picture and Globin Synthesis in Determining the Clinical and Haematological Features of Thalassaemia Intermedia

Twelve carriers of thalassaemia intermedia were studied. Their clinical and haematological picture was distinctly different from that in both heterozygotes and homozygotes for beta thalassaemia. Several genetic patterns were found responsible for thalassaemia intermedia: beta/delta beta thalassaemia, alpha 2 beta/beta thalassaemia-heterocellular HPFH. In a few subjects the genetic picture indicated that the patients were homozygous for beta thalassaemia, in spite of the mildness of the clinical situation. The lack of genetic uniformity was refelcted in very wide Hb A2 (2.5--8.7%) and Hb F (7.5--96.9%) ranges, as opposed to the noticeable degree of biochemical uniformity indicated by the very similar imbalance of globin chain synthesis: 0.33-0.54 for the non-alpha/alpha chain ratio in the peripheral blood. The mean for this parameter (0.43 +/- 0.05) was significantly different (P less than 0.001) from that observed in heterozygous carriers (0.60 +/- 0.10) and homozygous carriers (0.11 +/- 0.05) for beta thalassaemia. The marrow blood displayed a comparable pattern. It is therefore suggested that the severity of thalassaemia is attributable to the degree of chain synthesis imbalance.     

Further evidence of a quantitative deficiency of chain-specific globin mRNA in the thalassemia syndromes.

Formamide gel electrophoresis separates the mRNA fraction from reticulocyte polyribosomes of adult humans into two major RNA species with migratory rates identical to those of the alpha- and beta-globin mRNAs of the rabbit. That these two RNAs of human origin are the globin mRNAs is further supported by the deficiency of the presumed beta mRNA in reticulocyte polyribosomes of fetuses and premature infants, whose cells make gamma chains in preference to beta chains. The globin mRNAs of reticulocyte polyribosomes from patients with hematological disorders were estimated by scanning the stained formamide gels. In contrast to individuals with either hemolytic anemia without hemoglobinopathy or sickle cell anemia who had beta mRNA to alpha mRNA ratios of approximately one, a patient with Hb S-beta-thalassemia had a ratio of beta mRNA to alpha mRNA of 0.75 while two subjects with homozygous beta-thalassemia had severe deficiencies of beta mRNA. Conversely, a patient with alpha-thalassemia (Hb H disease) had a ratio of beta mRNA to alpha mRNA on reticulocyte polyribosomes of 6. These data provide further evidence of a quantitative deficiency of chain-specific globin mRNA in patients with the thalassemia syndromes.     

Association of Hb Q-Thailand with homozygous Hb E and heterozygous Hb Constant Spring in pregnancy

Hemoglobin (Hb) Q-Thailand [alpha74(EF3): Asp-->His] is an abnormal Hb found mainly in China and South-east Asian countries. Association of the alpha(Q-Thailand) allele with alpha-thalassemia has important implications in diagnosis. We report the hitherto undescribed conditions of this variant in two unrelated pregnant Thai women. Routine Hb analyses using high-performance liquid chromatography identified abnormal Hb migrating after Hb A(2) in addition to a homozygous Hb E in the proband 1 and to a heterozygous Hb Constant Spring (Hb CS) in the proband 2. Further alpha-globin gene analysis identified that the variant was caused by the GAC to CAC mutation at codon 74 of the alpha1-globin gene corresponding to the Hb Q-Thailand, detected in cis to the 4.2 kb deletional alpha-thalassemia 2 in both cases. Interaction of the alpha(Q-Thailand) with the beta(E) globin chains in the proband 1 leads to a Hb variant, namely the Hb QE. Family study of the proband 1 showed that her non-pregnant sister had the same genotype but her father was a double heterozygote for Hb E and Hb Q-Thailand in whom both Hb Q-Thailand and Hb QE were detected. Genotype-phenotype relationships observed in these families with complex hemoglobinopathies are presented and compared with those of simple homozygote for Hb E, heterozygote for Hb CS and heterozygote for Hb Q-Thailand found in other unrelated subjects. A simple DNA assay based on allele-specific polymerase chain reaction for simultaneous detection of the Hb Q-Thailand mutation and the 4.2 kb deletional alpha-thalassemia 2 determinant was developed and validated.     

Recent advances in the molecular understanding of non-transfusion-dependent thalassemia

Thalassemias are a group of inherited autosomal recessive hematologic disorders that occur because of defects in the alpha (α)- and beta (β)-globin genes of adult hemoglobin (Hb). An imbalance in the synthesis of one or more of the globin chains can result in a wide spectrum of phenotypes depending on the type and amount of globin synthesized and additional genetic modifiers. In patients with thalassemia intermedia, a condition known as non-transfusion-dependent thalassemia (NTDT), transfusion requirements are absent or episodic. Non-transfusion-dependent thalassemia includes β-thalassemia intermedia, HbE β-thalassemia, and α-thalassemia intermedia, also known as Hb H disease. This article focuses on the molecular features and genetic mutations specific to NTDT.     

Alpha Thalassaemia in Sicily: Haematological and Biosynthetic Studies

Eight Sicilian patients with Hb H disease and their families have been studied. The standard haematological tests and the alpha/beta chain synthesis ratios showed significantly different results in the patients with Hb H disease as compared with alpha thalassaemia carriers, except for Hb A2 values. There was no significant difference in the mean RBC, MCV, Hb A2, Hb A1 and Hb F of alpha thalassaemia carriers compared with normal controls. On the contrary significant difference was found between the mean alpha/beta chain synthesis ratio of alpha thalassaemia carriers and that of the normal controls; however, the extensive overlapping of alpha/beta values between these two conditions make this parameter insufficiently discriminant. No correlation was found between MCV, MCH, RBC and alpha/beta chain synthesis ratio in patients with alpha thalassaemia trait, suggesting that the ratio cannot be used to distinguish between carriers of a mild gene ('silent' carrier) and carriers of the more severe alpha thalassaemia gene. A possible genetic model for alpha thalassaemia in Sicily is presented.     

Further cases of Hb Hirosaki in two Japanese families

Hb Hirosaki (alpha 43(CE1)Phe----Leu) was recently found anew in two Japanese families. The probands had chronic hemolytic disorders, while two other carriers of the same Hb in one family did not exhibit increased hemolysis. Hb Hirosaki comprised 5 and 2% of the total Hb in the splenectomized and non-splenectomized proband, respectively, while it comprised only 1% in the two clinically silent carriers. The expected nucleotide substitution was found in the alpha 2 (rather than alpha 1) globin gene in 1 proband. The abnormal globin was synthesized (with incorporation of [3H]-leucine) at about 30 and 20% of the total alpha-chain in reticulocytes of the probands and in those of the silent carriers, respectively. The specific radioactivity of the abnormal chain was about 20-times higher than that of the normal counterpart in the non-splenectomized patient and silent carriers (7-times in the splenectomized patient). The total alpha/beta radioactivity ratio was much higher than unity. The newly synthesized Hb Hirosaki had a slightly high isoelectric point and appeared just in front of normal Hb A in anion-exchange chromatography, but the electric charge of aged Hb Hirosaki was indistinguishable from that of Hb A. These results suggest that Hb Hirosaki is quite unstable, and is rapidly denatured and degraded after biosynthesis. The physical number of the alpha globin genes was normal in all subjects.     

Effects of alpha-thalassemia-2 on the developmental changes of hematological values in children with sickle cell disease from Georgia

The hematology and pathophysiology of sickle cell disease during the postnatal development of younger hemoglobin (Hb) S homozygotes (SS) could be considerably affected by a variability of alpha globin gene numbers. We have documented longitudinal developmental changes of hematological values and hemoglobin composition on 147 patients with SS (alpha alpha/alpha alpha), 64 with SS (-alpha/alpha alpha), and 9 with SS (-alpha/-alpha) between the ages of 1 and 15 years. Non-steady-state data were excluded from these analyses. The number and organization of alpha globin genes was established by gene mapping. As anticipated, mean corpuscular volume and erythrocyte counts correlated with alpha globin gene numbers throughout the 15-year age interval. On the other hand, SS children with alpha alpha/alpha alpha, -alpha/alpha alpha, -alpha/-alpha had similar hemoglobin concentrations up to the ages of 5-10 years. Around the age of 7, the SS patients with -alpha/-alpha developed a higher Hb concentration than that of the SS (-alpha/alpha alpha), which in turn was higher than that of the SS (alpha alpha/alpha alpha). The emergence of this difference coincided with a developmental increase of the mean corpuscular hemoglobin concentration (MCHC) in patients with SS (alpha alpha/alpha alpha) and the decline of Hb F % under 15%. This newly observed developmental change of the MCHC could lead to increased hemolysis and anemia after the age of 5-10 years. It occurs to a smaller extent among SS (-alpha/alpha alpha) or not at all among SS (-alpha/-alpha) such that these two categories of patients have less severe hemolysis and higher hemoglobin levels at older ages. Although the proportion of Hb F was independent of alpha globin gene numbers, the absence of Hb Bart's suggested that alpha-thalassemia promotes the intracellular assembly of Hb F over Hb S tetramers. Thus, the interaction of alpha-thalassemia and Hb F in young SS patients may be more complex than revealed by Hb F levels in cell lysates. Among older SS children (greater than 7 years) alpha-thalassemia and Hb F levels exceeding 15% appear to have additive effects in diminishing the rate of hemolysis.     

Decreased alpha globine mRNA in nucleated red cell precursors in alpha thalassemia.

The alpha thalassemias are associated with a decrease in alpha chain synthesis. Hemoglobin H (HbH) disease is a moderately severe form of alpha thalassemia characterized by the production of 5%--20% of HbH, while alpha thalassemia trait is a milder form of alpha thalassemia. In two patients with HbH disease, the ratio of alpha chain synthesis to beta chain synthesis (alpha/beta ratio) was decreased in both bone marrow cells and reticulocytes. When isolated mRNA from bone marrow cells and reticulocytes was translated in a heterologous cell-free system, the alpha/beta ratios were lower than the intact cell ratios. These findings were confirmed by hybridization of the mRNA of both marrow cells and reticulocytes using purified alpha and beta cDNA probes. In the intact cells of two patients with alpha thalassemia trait, the alpha/beta ratios were also decreased and were similar in marrow cells and reticulocytes. Cell-free studies of translatable mRNA also demonstrated decreased alpha/beta ratios, but, unlike the HbH studies, the cell-free alpha/beta ratios were similar to the intact cell ratios. One hybridization study utilizing peripheral blood mRNA had an alpha/beta ratio consistent with the cell-free ratios. These results indicated that, in both HbH disease and alpha thalassemia trait, there was decreased alpha globin mRNA present in both nucleated red cell precursors and reticulocytes. In addition, the data suggested that there may be translational mechanisms that operate in intact HbH cells which attempt to balance globin chain production. In alpha thalassemia trait cells, no such controls appeared to be active and globin chain synthesis was directly proportional to the amount of alpha and beta globin mRNA in the cells.     

Hemoglobin North Shore: a variant hemoglobin associated with the phenotype of beta-thalassemia

Hemoglobin (Hb) North Shore (beta 134 val leads to glu) is a mutant hemoglobin that is associated with the phenotype of mild heterozygous beta-thalassemia. Heterozygotes are characterized low normal hemoglobin levels or mild anemia, microcytosis, increased HbA2, and 34%-38% Hb North Shore. The mechanism of the anemia and microcytosis associated with Hb North Shore was explored by studies of hemolysate thermal instability, peripheral blood globin biosynthesis, and whole blood oxygen affinity. Hb North Shore was mildly heat unstable in comparison to normal adult hemolysate. Pulse labeling of reticulocytes with 3H- leucine showed an alpha/beta ratio of 1.35 (normal 1.0). The beta North Shore/alpha ratio was 0.22-0.27, which was less than expected on the basis of gene dosage and less than that seen for most beta-chain variants. The beta A/alpha ratio was 0.50, as would be expected. The beta North Shore/alpha ratio was 0.26 after a 15-min pulse and did not decrease during 120 min of chase. These findings suggest that suboptimal synthesis rather than posttranslational degradation is responsible for the thalassemic phenotype associated with this variant hemoglobin. These observations parallel the findings in heterozygous HbE. It is not presently known whether the thalassemia phenotype is conferred by the structural mutation itself or by another mutation cis to the beta North Shore gene.     

Globin chain synthesis ratios in sideroblastic anaemia

Globin synthesis ratios were measured on reticulocytes from nine patients with primary acquired sideroblastic anaemia (SA), four patients with hereditary or congenital SA, two patients with secondary acquired SA and three patients with iron deficiency (ID). Ten of the samples from patients with SA and all the samples from patients with ID had normal ratios. Samples from three patients had significantly abnormal ratios, one from a patient with SA and acquired Hb H disease (alpha/beta 0 X 26), one from a patient with secondary acquired SA (alpha/beta 0 X 88), and one from a patient who went on to develop acute myeloblastic leukaemia (alpha/beta 1 X 36). Globin synthesis was stimulated by 100 microM haem similarly in normal, SA and ID reticulocytes. Any limitation of globin synthesis in SA and ID is therefore not easily reversible by adding haem. Inhibition of haem synthesis in nonsideroblastic reticulocytes using 4 mM isonicotinic acid hydrazide for 1 h incubation affected neither total globin synthesis nor the alpha/beta ratio. These results contradict the view that decreased haem synthesis decreases globin chain synthesis and decreases the alpha/beta globin chain synthesis ratios in human reticulocytes. Previously reported findings that haem could reverse globin chain synthesis inhibition in SA were good evidence for a primary deficiency of haem synthesis in the erythroblasts of these patients. Our inability to substantiate these findings emphasizes the need for a re-evaluation of the aetiology of sideroblastic anaemia.