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Sickle Cell Anemia, Thalassemia, and Other Genetic Diseases
Summary: Yuet Wai Kan's laboratory is investigating safer DNA tests for genetic disorders, a mouse model of sickle cell anemia, and the control of gene expression.
Our laboratory has been studying sickle cell anemia, thalassemia, and other genetic diseases that are important worldwide. These diseases are significant health problems in the Mediterranean region, Africa, and Asia. In the United States, they are found in people of African, Italian, Greek, and Asian descent. Approximately one out of eight African Americans carries the sickle cell gene. The β-thalassemia gene is common in people of Mediterranean, Middle Eastern, and Asian origin. The severe form of α-thalassemia occurs almost invariably in people from Southeast Asia. Although African Americans and other ethnic groups also carry the α-thalassemia gene, they have the mild form and usually do not suffer any deleterious consequences.
Homozygous states of these autosomal-recessive diseases cause severe anemia. Patients with sickle cell anemia suffer from frequent painful crises, thrombotic episodes, and infections. Management consists primarily of treatment of complications. Prophylactic penicillin therapy prevents death from infections during childhood, and recent studies showed that hydroxyurea therapy decreases the frequency of painful crises and the number of hospital admissions. The severe form of α-thalassemia is associated with third-trimester fetal death or stillbirth from hydrops fetalis. Homozygous β-thalassemia patients usually require regular blood transfusions and iron chelation therapy. Although the carriers of these diseases are asymptomatic, they can readily be identified by blood tests. Until there is a cure, prevention can be accomplished by genetic screening and counseling together with prenatal diagnosis.
Most of the mutations that give rise to these diseases have been defined, allowing for improvement in understanding of the diseases, development of diagnostic tests, and design of new therapeutic approaches. In the 1970s, our laboratory introduced several DNA tests for genetic diseases, which were then applied to prenatal diagnosis. Currently, DNA is obtained by amniocentesis or chorionic villus sampling. Although these procedures are quite safe, they are still invasive and carry a small, but not completely negligible, risk for the fetus. Hence there are efforts to develop effective tests that are completely noninvasive for the fetus.
Safer DNA Tests
It has been known for some time that fetal cells can leak into the maternal circulation early in pregnancy. Early attempts at isolating fetal lymphocytes from the maternal blood were successful, but it was found that they can remain in the maternal circulation for many years, making misdiagnosis possible. Trophoblasts can also be found in the maternal blood, but they are hard to find because they are large and multinucleated and are liable to be trapped in the maternal circulation. Most investigators are directing their efforts to identifying fetal nucleated red cells, as these cells have a short life span and will not be carried forth to the next pregnancy.
We are interested in prenatal diagnosis of sickle cell anemia, thalassemia, and other single-gene disorders. To detect these genotypes in the fetus, pure fetal cells uncontaminated by maternal cells are needed. From ~20 ml of maternal blood, up to 20 fetal nucleated cells can be identified. They are removed from microscope slides, and polymerase chain reaction assays are performed on the DNA prepared from them. To date, fetuses at risk for sickle cell anemia, β-thalassemia, and cystic fibrosis have been correctly genotyped in this manner. However, the procedure for isolating the few fetal nucleated red cells from millions of maternal blood cells is tedious and time consuming. We are investigating two possible methods of making the test more robust, practical, and clinically useful worldwide. First, specific antibodies against nucleated fetal cells are being generated by the phage display technique. This will improve the enrichment of the fetal cells. Second, we are developing an image analysis system that will identify these fetal cells and retrieve them by laser capture microdissection. These methods, which will greatly facilitate noninvasive prenatal diagnosis, can be applied to many genetic diseases.
Mouse Model of Sickle Cell Anemia
A mouse model in which the mouse hemoglobin is replaced completely with the human sickle hemoglobin will be useful for the study of the disease and to test drugs that inhibit the sickling process. These mice can also be used to search for drugs that stimulate fetal hemoglobin synthesis, which is effective in alleviating the sickling process. To achieve this, we have interbred four strains of mice: (1) mice whose endogenous α-globin genes have been knocked out, (2) mice whose endogenous β-globin genes have been knocked out, (3) human α-globin transgenic mice, and (4) human βs-globin transgenic mice. The βs-globin transgene was derived from a 240-kilobase yeast artificial chromosome that contains the whole β-globin gene cluster, including the locus control region (LCR), all the β-globin-like genes and the intergenic regions. Since the control of globin expression involves the interaction of trans factors and cis-acting elements, including those in the intergenic regions, these animals may more truly reflect the human condition. Studies of various drugs to stimulate fetal hemoglobin synthesis in these mice are in progress.
The Transcription Factor NFE2 and Related Genes
The transcription factor NFE2, which binds to the core elements in the LCR, is important for the control of globin gene expression. We have isolated several proteins that bind to this core element: NFE2, Nrf1 (NFE2-related factor 1), and Nrf2. NFE2 is specific to hematopoietic tissue, but Nrf1 and Nrf2 are expressed in hematopoietic and many other tissues. Work in cultured cells and in transgenic mice has shown that the NFE2 core element is a major enhancer of globin gene expression; however, mice lacking the gene for the transcription factor NFE2 have minimal anemia. They die instead from bleeding, because their megakaryocytes fail to mature.
Nrf1-null mice are anemic and die in utero at about day 15 of gestation. Preliminary studies indicate that the microenvironment in the fetal liver of these mice does not support hematopoiesis. Nrf2-null mice have no phenotype, and they appear to grow and reproduce normally. Recent studies show that Nrf1 and Nrf2 are also important transcription factors for a group of antioxidant genes as well as genes responsible for generating glutathione. Mouse embryo fibroblasts prepared from mice lacking these genes are killed when exposed to oxidants. These transcription factors control the expression of a number of antioxidant genes and genes that are important for the generation of glutathione. When Nrf2-knockout mice are exposed to a number of drugs, they die of a disease that resembles acute respiratory distress syndrome. They are also more susceptible to drugs such as acetaminophen. The roles of Nrf1 and Nrf2 in protection against antioxidants, DNA damage, and cancer predisposition are being investigated.
This work is supported in part by grants from the National Institutes of Health.
Last updated June 27, 2001
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