As a science, the knowledge of genetics did not exist until 1865 – the same year that slavery was technically abolished in the United States. At that time, Gregor Mendel, an Austrian monk with a scientific mind, published his studies on inheritance in the edible pea. Mendel had discovered genes. He found that genes exist in pairs and that one of each pair is randomly transmitted by each parent to their offspring. He also found that genes are not changed by being inherited and that their effect is the same whether it is the male or female parent who transmits them. This pattern of inheritance, which is the pattern of inheritance for sickle cell disease, is called Mendelian inheritance.
Genes carry the blueprints for the bodies of all living things in a special code. Genes are made from DNA (deoxyribonucleic acid), a chemical that has a helical structure. How DNA duplicates itself and encodes information and how proteins are made from this code have been revealed in a series of stunning research achievements. One of the most important was the discovery of its structure in 1953 (technically by Watson and Crick at Cambridge University, but there is evidence that a female counterpart actually discovered it some time before.
Genes have two vital roles: they duplicate themselves for transmission from one generation to the next and they direct the manufacture of the body’s proteins. They determine how living things are made and how they will function. When protein is consumed, it is digested down to individual amino acids, and then rebuilt into protein chains required by the many areas of the body. This know-how resides in DNA. Which amino acid corresponds to which three-letter word of the DNA is called the genetic code. Therefore, genes are made up of DNA strands. Research has determined that all living things, from bacteria to humans, use the same genetic code, making it the oldest universal language on earth.
One of the most important proteins in the body is hemoglobin. Hemoglobin contains a total of 574 amino acids. In 1940, a young medical student at Johns Hopkins University, Irving Sherman, published his observation that when light was passed through the red blood cells of sickle cell patients, the sickled cells transmitted the light differently than the nonsickled cells. Dr. William Castle, the Harvard professor of medicine, who had figured out how pernicious anemia develops, understood the implications of Sherman’s observation, which suggested a special orientation of molecules inside the cells.
In a chance conversation during a train ride from Denver to Chicago, Castle mentioned Sherman’s observation to Linus Pauling, who had worked extensively with hemoglobin. Pauling, assuming that hemoglobin was at the heart of the sickling matter, followed up with experiments that compared hemoglobin samples from normal people, those with sickle cell trait, and people with sickle cell disease. By subjecting the hemoglobin samples to electrophoresis, a technique that separates proteins on the basis of their own electrical charge, Pauling found that he had two different hemoglobins in his samples. Normal individuals had one type, sickle cell disease patients had another, and those with sickle cell trait had both types. This was the first demonstration of a change in protein structure showing Mendelian inheritance and is of historic interest since this important discovery came from a study of sickle cell hemoglobin.
In 1956, from the same laboratory in Cambridge where Watson and Crick worked, Vernon Ingram set out to learn what the abnormality was in sickle cell hemoglobin and why it changed. He used an enzyme to break HbS and HbA into smaller pieces, which he then compared by electrophoresis. The two hemoglobins were identical in all but one of those pieces. Studying that piece further, Ingram discovered that HbS differed from HbA in a single amino acid: valine was substituted for glutamic acid in one position. Here, at last, was the thing that made sickle cell hemoglobin different from normal hemoglobin.
More than one gene serves as a blueprint for hemoglobin, however. Hemoglobin molecules consist of four subunits – two of each of the two different protein chains. These are designed as alpha and beta chains. Since one gene can code for only one protein, two different genes are needed to code for two different kinds of hemoglobin subunits. The chain responsible for sickle cell hemoglobin is the beta chain. In the beta chain, the 6th amino acid of HbA is glutamic acid, but the 6th amino acid of HbS is valine. The beta chain of HbA is 146 amino acids in length. Therefore, the tiniest change in, or the alteration of, just one of the three bases that code for the 6th amino acid is responsible for sickle cell disease.
Currently, those with sickle cell disease are treated for their symptoms only, since there is no cure for the disease. Research for a cure is progressing along several different avenues. These include the following:
- the development of anti-sickling agents;
- turning on the genes that increase the production of Hb F;
- bone marrow transplantations;
- gene therapy;
- Vitamin E (Although this is not a high priority on most lists, there are some efforts being made to study what effects vitamin E, and other nutrients, have on sickled cells)
Anti-sickling agents include those that would prevent HbS molecules from polymerizing on contact. Such drugs would have to concentrate in the red blood cell and interact with hemoglobin. Since there are so many hemoglobin molecules, large amounts of the drug would need to be taken. Therefore, it would be very imporant for such drugs to be nontoxic. Although several potential anti-sickling agents have been screened over the years, they have not met the requirements of a good drug – either they did not work as prescribed or they were more harmful than helpful. One drug that did look promising is vanillin, a chemical found in the vanilla orchid, as well as in other plants, and which can also be made synthetically. Vanillin concentrates in the red blood cell, interacts with hemoglobin, and inhibits polymer formation. Research continues with it.
Another way to prevent sickling would be to dilute HbS, which is less likely to polymerize when it is less concentrated. One way to dilute it would be to change the nature of the red cell membrane so that the cell would hold more water. One such cell-swelling drug is hydroxyurea. Red cells made during hydroxyurea treatments appear to contain an increased amount of fluid. In addition, the red blood cell population favourably changes and the cells most likely to cause clogging seem to disappear.
Researchers are developing other less toxic drugs for increasing fetal hemoglobin production. One of them is based on a chemical found in everyday foods – a food additive called butyrate. Butyrate is a simple fatty acid that is found in the body and used to enhance the flavor of foods.
Dr. Susan Perrine of the Children’s Hospital Oakland Research Institute, in California, began studying butyrate in the mid 1980s after reading a report that it turned on a gene in chickens that normally produces a blood protein in immature chicks. She also found that high levels of butyrate in mothers with diabetes delayed the changeover from fetal to adult hemoglobin in their children. However, simply eating butyrate in foods had no effect on hemoglobin, but an injectable preparation of arginine butyrate produced substantial increases in fetal hemoglobin in patients with sickle cell disease and beta-thalassemia without any harmful effects. A newer oral form (isobutyramide) is also being developed.
Turning on the genes that increase the production of HbF is another research program that currently holds the most promise for sickle cell sufferers. While it would not actually provide a cure, high levels of HbF could inhibit sickling and its painful consequences. This technique should be approached with caution because it is not specific for HbF genes, and other genes might also be turned on, including those that cause cancer.
As mentioned before, the drug hydroxyurea increases HbF by supressing bone marrow activity. When active marrow cells are suppressed, more of the less active marrow cells – the ones that make HbF-producing cells – go into red cell production. Although the drug appears to work, it does not do so uniformly nor does it work in all patients. The drug is also being tested clinically in combination with erythropoietin, a hormone that stimulates red blood cell production. When given in high doses, erythropoietin also stimulates the inactive stem cells to produce red blood cells.
Stem cells never stop their production of HbF genes. In January of 1995, a clinical trial of hydroxyurea was terminated after dramatic results. The drug, which was given as a pill to adults with severe sickle cell disease, reduced by half the number of painful episodes, hospitalizations, situations requiring blood transfusions, and occurrences of acute chest syndrome. Although the drug has been supported by the National Institutes of Health, it is still not advised for use in children or for women planning to have children.
However, hydroxyurea has some disadvantages. It is toxic to bone marrow and it can cause genetic mutations. Thus, it should not be used by people planning to have children. When it was used in combination with erythropoietin, a much smaller dose was needed and the combination increased the amount of fetal hemoglobin more rapidly than using the highest dose of hydroxyurea alone. Hydroxyurea continues to be tested, but it is actually being tested for a different effect – that involving HbF.
Bone marrow transplantation takes place under local anesthetic. Usually 500 to 700 ml of marrow is aspirated from the pelvic bones on an HLA-compatible donor (human lymphocyte antigen) or of the recipient himself during periods of complete remission. The aspirated marrow is filtered and then infused into the recipient in an attempt to repopulate the patient’s marrow with normal cells. This procedure has affected long-term, healthy survivals in about half the patients with severe aplastic anemia. Bone marrow transplantation may also be effective in treating patients with acute leukemia, certain immunodeficiency diseases, and solid tumor malignancies. Because this procedure carries serious risks, it requires strict adherence to infection protection and aseptic techniques. Success of the procedure depends on the stage of the disease and the bone marrow match. Chemotherapy, and possibly radiation treatments, will have to take place in order to remove cells that may cause the body to reject the transplant.
Gene therapy means the transplantation of “good” genes into people. It is considered to be the ultimate cure for all genetic diseases; and successful gene therapy would correct the original genetic injury for life. Gene therapy for sickle cell patients is a challenging process. First, copies of normal hemoglobin genes must be made and then inserted into the right kind of stem cells. Then, a huge number of them have to be made. Later, these stem cells must be put back into the marrow so they can implant and produce red blood cells that make enough HbA to prevent polymerization of the HbS gene. Even better, the gene therapy would have to inactivate the HbS gene.
The procedure does have some worrisome aspects to it. When the new gene inserts itself into the chromosome, there is yet no way to be certain that it would not activate a cancer gene or inactivate an essential or cancer-suppressing gene, for example. Therefore, it may be some time before gene therapy is used to treat sickle cell disease.
