Dr. S.K.Patil*
Prof. & Head, Deptt. of Biochemistry, CIMS. *
Hemoglobinopathies have traditionally been defined as a family of disorders caused either by production of a structurally abnormal hemoglobin molecule, synthesis of insufficient quantities of normal hemoglobin or rarely, both. Sickle-cell anemia (HbS), Hemoglobin C disease (HbC), and the thalassemia syndromes are representative hemoglobinopathies that can have severe clinical consequences. The first two conditions result from production of hemoglobin with an altered amino acid sequence, whereas the thalassemias are due to decreased production of normal hemoglobin.
  1. Organization of globin genes:
    In order to understand diseases resulting from genetic alterations in the structure or synthesis of the hemoglobin, it is necessary to grasp how the hemoglobin genes, directing the synthesis of the different globin chains, are structurally organized into gene families and how they are expressed.
    1. a-Gene family: The genes coding for the a - globin-like and ß - globin-like subunits of the hemoglobin chains occur in two 'separate gene clusters (or families) located on two different chromosomes. The a-gene cluster on chromosome 16 contains two genes for the a-globin chains, the gene that is expressed early in development as a component of embryonic hemoglobin, and a number of globin - like genes that are not expressed (that is their genetic information is not used to produce globin chains; they are called pseudogenes).
    2. ß-Gene family: A single gene for the ß-globin chain is located on chromosome 11, along with our other ß-globin-like genes the e-gene (which, like the gene, is expressed early in embryonic development), two genes (G and A that are expressed in fetal hemoglobin, HbF), and the d-gene that codes for the globin chain found in the minor adult hemoglobin Hb A2.
    3. Steps in globin chain synthesis: Expression of a globin gene begins in the nucleus, where the DNA sequence encoding the gene is transcribed. The RNA produced by transcription is actually a precursor of the messenger RNS (mRNA) that is used as a template for the synthesis of a globin chain. Before it can serve this function, two non-coding stretches of RNA (introns) must be removed from the mRNA precursor sequence, and the remaining three fragments (exons) reattached in a linear manner. The resulting mature MRNA enters the cytosol, where its genetic information is translated, producing globin.

  2. Sickle cell anemia (Hemoglobin S disease)
    Sickle-cell anemia, also called sickle-cell disease, is the most common disorder, resulting from the production of a variant hemoglobin. It occurs primarily in the black population, affecting 1 in 500 newborn black infants in the United States. Sickle-cell anemia is a homozygous recessive disorder occurring in individuals who have merited two mutant genes (one from each parent) that code for synthesis of the ß-chains of the globin molecules. The mutant ß-globin chain is designated ßs, and the resulting hemoglobin, a2ßs2, is referred to as HbS. The presence of the disease is not evident in an infant until sufficient HbF has been 16 replaced by HbS so that sickling can occur. Sickle-cell anemia is characterized by a lifelong hemolytic anemia, painful crises, and increased susceptibility to infections and other indications of poor circulation. Heterozygotes representing one of ten American Blacks, have one normal and one sickle-cell gene. The blood cells of such heterozygotes contain both HbS and HbA. These individuals have sickle-cell trait; they usually do not show clinical symptoms and can have a normal life span.
    1. Amino acid substitution in HbS ß-chains:
      A molecule of HbS contains two normal a-globin chains and two mutant ß-globin chain (ßs) in which glutamate at position six has been replaced with valine. Therefore during electrophoresis at alkaline pH, HbS migrates more slowly toward the anode (positive electrode) than does HbA. This altered mobility of HbS is due to the absence of the negatively charged glutamate residues in the two ß-chains, thus rendering HbS less negative than HbA. Electrophoresis of hemoglobin obtained from lysed red blood cells is routinely used in the diagnosis of sickle cell trait and sickle-cell disease.
    2. Sickling causes tissue anoxia:
      The substitution of the non-polar valine for a charged glutamate residue results in a pronounced decrease in the solubility of HbS in its deoxygenated form. The molecules aggregate to form fibers that deform the red cells into a crescent or sickle shape. Such sickled cells frequently block the flow of blood in the small diameter capillaries. This interruption in the supply of oxygen leads to localized anoxia (oxygen deprivation), which causes pain and eventually death (infarction) of cells in the vicinity. Several structural variants of hemoglobin result in the sickling phenomenon, but most of these are extremely uncommon.
    3. Variables that increase sickling:
      The extent of sickling and, hence the severity of disease is increased by any variable that increases the proportion of HbS in the deoxy state (that is, reduces the affinity of HbS for oxygen). These variables include decreased oxygen tension due to high altitude or flying in a nonpressurized plane, increased CO2 concentration, decreased pH and increased concentration of 2, 3-BPG in erythrocytes.
    4. Possible selective advantage of heterozygous state:
      The high frequency of the HbS gene among black Africans, despite its damaging effects in the homozygous state, suggests that a selective advantage exists for heterozygous individuals. For example, heterozygotes for the sickle-cell gene are less susceptible to malaria caused by the parasite plasmodium falciparum. This organism spends an obligatory part of its life cycle in the red blood cell. Because these cells in individuals heterozygous for HbS, as well as in homozygotes, have a shorter life span than the normal, the parasite cannot complete this stage of its development. This fact may provide a selective advantage to heterozygotes living in regions where malaria is a major cause of death. More recent study indicates that malarial parasite increases the acidity of erythrocytes (pH down by 0.4). The lower pH increases the sickling of erythrocyte to about 40% from the normally occurring 2%. Therefore the entry of malarial parasite promotes sickling leading to lysis of erythrocytes. Furthermore, the concentration of potassium ion is low in sickled cells, which is unfavorable for the parasite to survive. Sickle cell trait appears to be an adaptation for the survival of the individuals in malaria infested regions. Unfortunately, homozygous individuals, the patients of sickle cell anemia cannot live beyond 20 years.
  3. Diagnosis of Sickle cell anemia:
    1. Sickling Test: This is a simple microscopic examination of blood smear prepared by adding reducing agents such as sodium dithionite. Sickled erythrocyte can be detected under the microscope.
    2. Electrophoresis: When subject to electrophoresis in alkaline medium (pH 8.6), sickle cell hemoglobin (HbS) moves slowly towards anode (positive electrode) than does adult hemoglobin (HbA). The slow mobility of HbS is due to less negative charge, caused by the absence of glutamate residues that carry negative charge. In case of sickle-cell trait, the fast moving HbA and slow moving HbS are observed. The electrophoresis of hemoglobin obtained from lysed erythrocytes can be routinely used for the diagnosis of sickle cell anemia and sickle cell trait.
    3. By using Recombinant DNA Technology: Prenatal diagnosis for a genetic disease, one has to analyze DNA of family members of the afflicted individual. Families with history of severe genetic disease, for example, an affected previous child or near relative may wish to determine the presence of disorder in the developing fetus. Prenatal diagnosis allows for an informed reproductive choice it the fetus is the affected in many cases, diagnosis using DNA technology can achieve a high degree of reliability, but not complete certainly.
      • The genetic disorders of hemoglobin are the most common genetic disease in humans at present there is no satisfactory treatment for most of these disorders, and prenatal diagnosis is the only available method for limiting the number of afflicted individuals. Prenatal diagnosis of hemoglobinopathies has in the past involved the determination of the amount and kinds of hemoglobin synthesized in red cells obtained from fetal blood. For example, the presence of hemoglobin S in hemolysates indicated sickle cell anemia. However, the invasive procedures to obtain fetal blood have high mortality rate (approximately 5%), and diagnosis cannot be carried out until late in second trimester of pregnancy when hemoglobin S begins to be produced.
      • Current diagnostic techniques: Sickle cell anemia is an example of a genetic disease caused by a point mutation. The sequence altered by the mutation abolishes the recognition site of the restriction endonuclease Basu 36 that recognizes the sequence CCTNAGG (Where N is any nucleotide). Thus, the A to T mutation within a codon of the ßs-globin gene eliminates a cleavage site for the enzyme. DNA digested with Basu 36 yields a 1.1kb fragment, whereas 1.3 kb fragment is generated from the ßs as a result of the loss of one Basu 36 cleavage site. Diagnostic techniques for analyzing fetal DNA (from amniotic cells or chorionic villus sampling), rather than fetal blood, have proved valuable because they provide safe, early detection of sickle cell anemia as well as other genetic diseases. Thus the application of recombinant DNA techniques to prenatal diagnosis has given couples at risk information to make informed reproductive decisions.
  4. Management of sickle cell disease:
    Administration of sodium cyanate inhibits sickling of erythrocyte, cyanate increases the affinity of oxygen to HbS and lowers the formation of deoxy HbS. However it causes certain side effects like peripheral nerve damage. In patients with severe anemia, repeated blood transfusion is required. This may result in iron overload and cirrhosis of liver. Replacement of HbS with other forms of hemoglobin has been tried. Fetal hemoglobin (HbF) reduces sickling. Sickle cell disease awaits gene-replacement therapy!
  5. Hemoglobin C disease:
    Like HbS, HbC is a hemoglobin variant having a single substitution the sixth position of the ß-globin chain. In this case, however, a lysine is substituted for the glutamate (as compared to a valine substitution in HbS). This substitution causes HbC to move more slowly toward the anode than does HbA or HbS in electrophoresis technique. Patients homozygous for hemoglobin C generally have a relatively mild chronic hemolytic anemia. These patients do not suffer from infarctive crises, and no specific therapy is required.
  6. Hemoglobin SC disease:
    In this disease, some ß-globin chains have the sickle-cell mutation, whereas other ß-globin chains carry the mutation found in HbC disease. Patients with HbSC disease are doubly heterozygous, because both of their ß-globin genes are abnormal, although different from each other. Hemoglobin levels tend to be higher in HbSC disease than in sickle-cell anemia, and may even be at the low end of the normal range. The clinical course of adults with HbSC disease differs from that of sickle-cell anemia, in which patients generally have painful crises beginning in childhood. It is common for patients with HbSC disease to remain well (and undiagnosed) until they suffer an infarctive crisis. This crisis often follows childbirth or surgery and may be fatal.
  7. Thalassemia:
    The thalassemias are hereditary hemolytic diseases in which an imbalance in the synthesis of globin chains occurs. As a group they are the most common single gene disorders in humans. Normally, syntheses of the alpha and beta globin chains are coordinated so that each alpha globin chain has a beta globin chain partner. This leads to the formation of a2ß2 or hemoglobin A. In the thalassemias, the synthesis of either the a or ß-globin chain is defective. A thalassemia can be caused by a variety of mutations, including gene deletions, or substitutions or deletions of one to several nucleotides in the DNA. Each thalassemia can be classified as either a disorder in which no globin chains are produced (ao or ßo-thalassemia) or in which some chains are synthesized but at a reduced rate (a+ or ß+-thalassemia).
    1. a-Thalassemias are defects in which the synthesis of a-globin chains is decreased or absent. Because each individual's genome four copies of the ?-globin gene (two on each chromosome 16), there are several levels of ??-globin chain deficiencies. If one of the four genes is defective, the individual is termed a "silent carrier" of a-thalassemia, because no physical manifestations of the disease occur. If two a-globin genes are defective, the individual is designated as having a-thalassemia trait; if three are defective, the individual has hemoglobin H disease, a mildly to moderately severe hemolytic anemia; and if all four of the a-globin genes are defective, hydrops fetalis and fetal death result, because a-globin chains are required for the synthesis of HbF. The synthesis of unaffected-y and ß-globin chains continues resulting in the accumulation of-? tetramers in the newborn (?4 Hb Bart) or ß tetramers (ß4 HbH). Although these tetramers are fairly soluble, the subunits show no heme-heme interaction. Their oxygen dissociation curves are almost hyperbolic, indicating that these tetramers have high oxygen affinities. This makes them essentially useless as oxygen deliverers to the tissues.
    2. In ß-thalassemias, synthesis of ß-globin chains is decreased or absent, whereas a-globin chain synthesis is normal. a-globin chains cannot form stable tetramers and therefore precipitate, causing the premature death of cells initially destined to become mature red blood cells. Because there are only two copies of the ß-globin gene, individual with gene defects have either ß thalassemias trait (ß thalassemias minor) if they have only one defective ß-globin gene, for ß thalassemias major, if both genes are defective. Because the ß globin gene is not expressed until late in fetal gestation, the physical manifestations of ß thalassemias appear only after birth. Those individuals with ß thalassemias minor make some ß chains and usually do not require specific treatment. However, those infants born with ß thalassemias major have the sad consequence of being seemingly healthy at birth, but becoming severely anemic, usually during the first or second year of life. These patients require regular transfusion of blood. Although this treatment is life saving, the cumulative effect of the transfusion is iron overload (a syndrome known as hemosiderosis), which typically causes death between the ages of 15 and 25 years. Bone marrow replacement has been a boon to these patients in the last 10 years.
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