Sickle Beta-thalassemia Research Articles

1 The structure and function of normal and abnormal haemoglobins

This chapter reviews general aspects of the normal human haemoglobins which include those predominantly present in the embryo, the fetus and newborn baby, and in the normal adult. Special emphasis is given to factors which affect the levels of fetal haemoglobin in the adult because increased percentages of Hb F can be of great benefit to adults with certain haemoglobinopathies such as sickle cell anaemia and beta-thalassaemia. A review of the numerous Hb variants published since the discovery of Hb S in 1959 reveals a steady stream of newly detected abnormalities; most of these are the result of single-point mutations in the alpha-, beta-, gamma-, or delta-globin genes. Of the more than 600 variants listed in a repository, some 200 have clinical significance because of a decreased stability, a change in functional properties, among others. Methodology developed for the detection and quantitation of normal and abnormal Hb components has been greatly modified during the past 30 years; isoelectrofocusing and different fast developing micro chromatographic procedures are the methods of choice. Analyses of DNA isolated from the white cells has become most useful for the final characterization of the variants; this methodology consists of amplification of a desired segment of DNA and identification of a mutation with labelled oligonucleotide probes. Additional methods include sequence determination of this amplified DNA and identification of known mutations with an allele specific amplification procedure.

A Short-Term Trial of Butyrate to Stimulate Fetal-Globin-Gene Expression in the β-Globin Disorders

Fetal-globin (gamma-globin) chains inhibit the polymerization of hemoglobin S (sickle hemoglobin) and can functionally substitute for the beta-globin chains that are defective or absent in patients with the beta-thalassemias. Identifying safe mechanisms to stimulate fetal-hemoglobin production is therefore of great interest. Previous studies have shown that administering butyrate selectively stimulates the promoter of the human fetal-globin gene and leads to increases in gamma-globin--gene expression in the developing fetus, cultured cells, and animal models. To determine whether butyrate can stimulate fetal-globin production in humans, we treated three patients (3 to 13 years old) with sickle cell anemia and three patients (7 to 27 years old) with beta-thalassemia syndromes with a short course of intravenous infusions of arginine butyrate. The drug was infused continuously for either two or three weeks; the initial dose was 500 mg per kilogram of body weight per day. Globin-chain ratios, proportions of reticulocytes producing hemoglobin F (F reticulocytes), and levels of gamma-globin messenger RNA (mRNA) were determined before and during treatment. In all six patients, fetal-globin synthesis increased by 6 to 45 percent above pretreatment levels (P < 0.01). The proportion of F reticulocytes increased about twofold, and the level of gamma-globin mRNA increased twofold to sixfold. The increase in gamma-globin synthesis led to improvement in the globin-chain ratios in the patients with thalassemia. The treatment of one patient was extended for seven weeks, and her hemoglobin level increased from 4.7 to 10.2 g per deciliter (2.9 to 6.3 mmol per liter). Side effects were minimal; one patient had a transient increase in serum aminotransferase concentrations. In patients with beta-hemoglobinopathies butyrate, a natural fatty acid, can significantly and rapidly increase fetal-globin production to levels that can ameliorate beta-globin disorders. Further trials of this class of compounds are warranted to determine long-term tolerance and efficacy in patients with sickle cell anemia or beta-thalassemia.

Surgical Management of Children with Hemoglobinopathies

Sickle hemoglobinopathies include sickle cell disease, sickle-C disease, and sickle-beta thalassemia. Patients with these disorders commonly suffer a multitude of destructive events to vital organs, especially to the central nervous system, the spleen, the kidney, the lung, and the heart as a result of microvascular plugging by the sickled erythrocytes. Thoughtful preparation for anesthesia and operation, especially when directed by experienced individuals, can greatly reduce the hazard of inducing the sickle crises that formerly plagued individuals with sickle hemoglobinopathies who faced major operations under general anesthesia. The patient must be free of any acute illness, especially one involving the respiratory system. Adequate hydration preoperatively combined with avoiding perioperative hypoxia, hypothermia, and acidosis, the triggers for sickling, will go far toward avoiding sickle-induced complications. Modern transfusion therapy, consisting of multiple small transfusions of Hb A erythrocytes administered over several weeks prior to the operation, not only corrects the chronic anemia but suppresses erythropoiesis of cells containing Hb S in the patient's bone marrow and leaves him or her with a majority of cells containing Hb A. This provides a safety net in case a sickle-inducing insult occurs despite the best efforts to avoid one. Individuals with sickle hemoglobinopathies may require any of the operations common to all children, for example, herniorrhaphy, appendectomy, tonsillectomy, and circumcision, but a significant number will develop calcium bilirubinate cholelithiasis and possibly cholecystitis as a result of the continual increased load of bile salts resulting from the shortened lifespan of the cells containing Hb S. Also, although most individuals with Hb S will gradually suffer splenic infarction by late childhood, a significant number of infants will experience acute splenic sequestration crisis, a life-threatening entity, the recurrence of which is prevented by splenectomy. Several publications have demonstrated that such surgical procedures can be performed in large numbers of patients with sickle hemoglobinopathies without deaths and with minimal morbidity.

Facial bone infarcts in sickle cell syndromes.

Bone infarction in the sickle cell syndromes (sickle cell anemia, sickle beta thalassemia, and sickle C disease) is common in the long bones, but the facial bones, particularly the orbits, are infrequently involved. In a review of the literature, only 13 cases of facial bone infarcts in sickle cell syndromes were found. Seven episodes of facial bone infarcts--four orbital, two mandibular, and one in the zygomatic arch--in six patients encountered at the authors' institution are reported. Five patients had hemoglobin SS, and one had hemoglobin SC. Bone marrow scans were positive for infarction in five of the six episodes during which they were obtained, which highlights the usefulness of this technique in the diagnosis of facial bone infarction.

Diagnosis of Sickle Cell Anemia and β-Thalassemia with Enzymatically Amplified DNA and Nonradioactive Allele-Specific Oligonucleotide Probes

We have developed a simple and rapid nonradioactive method for detecting genetic variation and have applied it to the diagnosis of sickle cell anemia and beta-thalassemia. The procedure involves the selective amplification of a segment of the human beta-globin gene with oligonucleotide primers and a thermostable DNA polymerase, followed by hybridization of the amplified DNA with allele-specific oligonucleotide probes covalently labeled with horseradish peroxidase. The hybridized probes were detected with a simple colorimetric assay. We demonstrated the usefulness of this method in a retrospective analysis of two pregnancies at risk for beta-thalassemia and one at risk for sickle cell anemia, as well as in an analysis of nine DNA samples simulating three family sets.

Screening for sickle cell trait: the Veterans Administration National Sickle Cell Program.

Results of the Veterans Administration Sickle Cell Program for a period of 10 years are presented. We screened 370,250 patients; 404,341 attended educational sessions, and 38,347 had individual counseling sessions. Sickle cell trait was present in 6.4% of patients, and HbC trait was present in 1.8%. The clinically significant disorders HbSC disease, sickle cell anemia, and sickle beta thalassemia were present in 0.41% of individuals screened. A large number of uncommon variants were detected. The program enhanced the awareness of and the approach to evaluation of hemoglobinopathies.

Clinical and molecular correlations in the sickle/β+‐thalassemia syndrome

Sickle/beta thalassemia is a sickling disorder of varying severity which results from compound heterozygosity for sickle cell trait and beta-thalassemia trait. Clinical and genetic studies have shown an inverse correlation between the level of hemoglobin A and the severity of the disease. It has been suggested that the level of hemoglobin A may be a function of the severity of the beta-thalassemia defect. In this study, we use molecular biological techniques to test this hypothesis. We show that the interaction of the mildest of the beta+-thalassemia genes with the sickle gene results in a high level of hemoglobin A. However, the interaction in this case resulted in a severe sickling disorder in the absence of significant anemia. We hypothesize that a mild beta+-thalassemia gene may have two opposite effects on the clinical course of sickle/beta+ thalassemia: (1) A high level of hemoglobin A which probably confers a favorable antisickling effect and (2) decreased hemolysis leading to increased numbers of total circulating red cells, thereby increasing the blood viscosity and the propensity for sickling. The inheritance of heterozygous alpha thalassemia 2 in conjunction with the mild beta+-thalassemia gene and sickle gene in this patient may have further enhanced the latter effect and resulted in a severe sickling disorder.

9 Transfusion Support for Haemoglobinopathies

The indications and management of blood transfusion in the haemoglobinopathies have been reviewed. The sickle cell diseases that require transfusion support are sickle cell anaemia, sickle haemoglobin-C and -D diseases and sickle beta-thalassaemia. Homozygous beta-thalassaemia (Cooley's anaemia) is the major problem among the thalassaemias. The pathophysiology of the sickle cell disorders is largely based on the secondary effects of increased blood viscosity, whereas in the thalassaemias the defect is ineffective haematopoiesis. In the former the major problems occur as manifestations of vaso-occlusive crises with disseminated bone and abdominal pain, priapism, stroke and leg ulcers. Bone infarction and aseptic necrosis occur but the widespread bone changes, underdevelopment and haemochromatosis that complicate the thalassaemia are not prominent. Transfusion therapy in the sickle cell diseases is mainly episodic and is guided by the frequency of crises and the severity of vaso-occlusive complications. Partial exchange transfusion and the maintenance of haemoglobin A concentrations at 40 to 50 per cent is frequently indicated. In the thalassaemias, maintenance of haemoglobin levels is essential for normal growth and development. The problem of haemochromatosis is very serious. With hypertransfusion regimens the haemoglobin and haemotocrit are maintained above 12-13 g/dl and 35 per cent. The resulting benefit appears to be reduced blood volume, less iron turnover, and less intestinal iron absorption. The splenomegaly in these disorders is frequently associated with hypersplenism requiring well-timed splenectomy. Chronic and intensive chelation is necessary to prevent the ravages of iron overload. The availability of automated equipment for in vivo and ex vivo blood cell separation has brought new possibilities for improving the management of these haemoglobinopathies. It is feasible, but not as yet practical, to offer transfusions of neocytes (red cells with a mean age of 30 days) which have a 50 per cent longer survival than routine red cell preparations (mean age of 60 days). Neocytes can be prepared ex vivo from fresh routine blood donations using blood cell separator devices. The result is reduced transfusion requirements. A more recent suggestion for using the new technology is to remove the patient's oldest and most abnormal corpuscles on the basis of buoyant density and replacing them with neocytes . Thus the short-lived abnormal red cells would be removed before they could unload their iron. With automation it is possible to perform these procedures on an outpatient basis.

A case-control study in northern Liberia of Plasmodium falciparum malaria in haemoglobin S and beta-thalassaemia traits.

A case-control study was carried out on 558 patients with malaria attending a hospital in Yekepa, northern Liberia; 94 patients (16.8%) were aged at least ten years, probably because of a low level of protective immunity in town dwellers due to malaria control. The proportion of sickle cell traits (1.8%) among the patient group was lower than in the population (7.2%) served by the hospital (chi 2, 21.455, 1 df, P less than 0.001). A stratified analysis showed the relative risk for Plasmodium falciparum malaria in sickle cell trait over normal homozygotes, as 0.29 (upper 95%) confidence interval, 0.56). For beta-thalassaemia trait, the proportion among patients was 5.5% as against 9.0% in the general population (chi 2, 6.158, 1 df, 0.025 greater than P greater than 0.010). Stratified analysis gave a weighted relative risk for beta-thalassaemia heterozygotes of 0.49 (upper 95% confidence interval, 0.74). Although there were four beta-thalassaemia traits in the 10-14 year stratum with moderate to high parasitaemias, we consider that the overall results are consistent with relative resistance against P. falciparum malaria of both sickle cell and beta-thalassaemia heterozygotes in this population. No conclusions were possible from this investigation with regard to HbC and the malaria hypothesis. We found no evidence that P. falciparum malaria elevates HbA2 concentrations into the beta-thalassaemia range.

Laryngeal edema associated with sickle beta-thalassemia disease

Laryngeal edema associated with sickle beta-thalassemia disease

Newborn screening for hemoglobinopathies in New York State: Experience of physicians and parents of affected children

Responses of physicians and parents to New York State-mandated newborn screening for sickle cell disease were solicited and evaluated. The index group comprised 25 infants born in western upstate New York. Each was found to have either sickle cell disease, hemoglobin SC disease, sickle beta-thalassemia, or hemoglobin C disease. In nondirective interviews the following factors were assessed: clinical course, the physician's policies of disease treatment and family counseling, the parents' reactions to the diagnosis and their level of understanding and compliance with medical recommendations, and the physicians' and parents' views on newborn screening. Newborn screening for sickle hemoglobin makes early prophylaxis and prompt treatment possible. Some morbidity may have been averted, judging from parental understanding of medical needs. Parents and physicians agreed that newborn screening for hemoglobinopathies is a valuable public health program. Suggestions for improving the New York state program included a need to increase communication among the screening laboratory, the hospital, and the physician; encouraging physicians to educate parents more fully, provide genetic counseling, and test parents and siblings of the identified neonate; and, most important, provide a well-delineated mechanism for follow-up of every infant with a potentially symptomatic hemoglobinopathy.

Free alpha-globin pool in human bone marrow.

A pool of free alpha-globin chains was found in the bone marrow samples from three controls, two patients with beta-thalassemia trait, three with sickle beta-thalassemia, three with hemoglobin (Hb) Lepore trait, one with alphabeta-thalassemia, four with homozygous beta-thalassemia, and one doubly heterozygous for Hb Lepore and beta-thalassemia. The average percentage of newly synthesized alpha-chains found in the free alpha-globin pool was 6.2% in the controls and 33.0% in the patients heterozygous for thalassemia or Hb Lepore. These controls and patients had balanced beta- and alpha-globin synthesis in the bone marrow. In the homozygous patients and in the one patient doubly heterozygous for thalassemia and Hb Lepore, there was a marked deficit of beta-chain synthesis in the bone marrow and also a large pool of newly synthesized free alpha-chains. The function of this pool of free alpha-chains is not known, but it may be involved in the regulation of globin chain synthesis in normal patients and in the compensatory synthesis of beta-chains that occurs in the bone marrow of patients heterozygous for thalassemia or for Hb Lepore.

Synthesis of globin chains in sickle -thalassemia.

In five patients with sickle beta-thalassemia there was balanced alpha- and beta-globin synthesis in the bone marrow and decreased total beta-chain synthesis relative to that of alpha-chain in the peripheral blood. These findings are similar to those in patients with simple beta-thalassemia trait. Despite a range of hemoglobin concentrations from 6.8 to 12.5 g/100 ml in the patients with sickle thalassemia, there was no evidence of a significant excess of alpha-chains in the red cells of the bone marrow which could contribute to the hemolysis and anemia. In patients heterozygous for beta-thalassemia the capacity to synthesize beta-chain decreases more rapidly than that for alpha-chain. In nonthalassemic subjects the rates of beta- and alpha-chain synthesis decrease equally as the red cell matures. The beta(S)- and beta(A)-chains serve as convenient markers for globin synthesis due to the nonthalassemic and thalassemic alleles in patients with sickle beta-thalassemia. The unbalanced globin synthesis in the peripheral blood of these patients is explained by the decrease in relative synthesis of beta(S)-chain, in comparison with that of alpha-chain. This instability is not present in sickle cell trait. The beta(A)-chain synthesis was only unstable in the two patients who had the most marked anemia. The major mechanism for achieving balanced globin production in the bone marrow in the presence of one thalassemic gene appears to be increased synthesis of beta-chain due to the nonthalassemic allele. In addition, there may be a decrease of total alpha-chain synthesis in some patients.