- Jay Patel
Sickle cell anemia is one of the most common disorders which are inherited from a single gene disorder. Usually these disorders have been seen in African-Americans1. Sickle cell disease was first described in 1910 by dental student with pulmonary symptoms. Sickle cell shaped was first known as Herrick coined to describe the peculiar appearance of the red blood of the cell of this patient. Sickle cell anemia disorder involves in hemoglobin and normally red blood cells are round and smooth, so they can easily pass thru the vessels2. However, sickle cell disorder hemoglobin cells are sticky and stiff. So, it cannot move easily thru vessels. When sickle cells occur at least one of the beta-globulin subunits in hemoglobin replaced with hemoglobin S. Sickle cell anemia is inherited in an autosomal recessive pattern. So, both copies of a gene have each cell in mutation. Parents with individual carry autosomal recessive condition, so each parent carry one copy of a mutated gene. However, they are not showing the sign and symptoms of the condition. The hemoglobin has two parts: alpha and beta. Sickle cell patients have mutation in gene on chromosome 11 which is coded for beta subunits for hemoglobin protein2.
Figure 1: Showing where sickle cell mutation located on chromosome 111.
Figure 2 describe the hereditary nature of the sickle phenomenon. This pattern recognized in 1917 by some erythrocytes of certain person, when Emmel was the first occurrence of sickle in a father and son1. In 1923, the study of large Negro kindred result suggests that the huge phenomenon was due to dominant gene1. So, one person was responsible for sickle cell disorder. And the other person was just carrying the sickle cell anemia trait, but that person was not infected by sickle cell disorder. I am going to discuss about kindred 57 which is very unusual. In kindred 57, three siblings were infected with sickle cell disease. The oldest girl was diagnosed at the age of 5 and died at the age of 17 due to cardiac decomposition. And the other two brothers diagnosed at age 8 and 2. However, they were died at the age of 17 and 10. So when they examined the nine member kindred they found unusual interest. The mother had sickle trait, but she was not infected by sickle cell anemia. However, father had sickle cell train and he was infected by sickle cell disorder. The fathers’ brother and sister had similar but less pronounced1. As another example in kindred 62, the sickle cell anemia occur in 10 year boy who at the age 2.5 developed epigastria and back pain and was found to have hematologic findings consistence as far as they gone with the diagnosis of sickle cell anemia disease. They said that his hemoglobin was 3.5Gm. per cent at the time of his admission. Also, his erythrocyte count 1,260,000per cu.mm, and the leukocytes 50,400 per cu.mm1. However, he was treated with transfusions and discharged. These treatments allow him to away from hospitalizations seven and half years. He is treated as an usually mild case of the disease, but yet it is quite possible that in the absence of transfusion therapy he would have expired at age 2.5 year. The most reasonable alternative explanation of his anemia is that it is due to the interaction of the sickle cell trait with some unknown environmental or other genetic factor1.
Figure 2: The distribution of sickle cell disease and the sickle cell trait in the 75 kindreds included in this study
Figure 3 shows the simple example of how a sickle cell disease transfers. Sickle cell disease is an autosomal recessive disease. These means both parents have to pass the sickle cell trait to their children in order to infected children with sickle cell disorder3. If just one of the parents transfers the sickle cell trait, the children will have sickle cell trait, but he won’t be infected with a sickle cell disease. However, those children can pass the sickle cell disease to their children. So in other word, both parents have to have sickle trait. If one parent doesn’t have sickle cell trait, the children have less chance of getting sickle cell disorder3.
Figure 3: The simple view of how to pass the sickle cell disease from children thru parents.
Earlier researchers used the blot hybridization to confirm the δβ thalassemia and hereditary persistence of fetal hemoglobin which is the result of gene detection4. However, β-thalassemia and α-thalassemia are due to both gene deletions and point mutations. In the polymorphism for sickle cell disease for Hpa I restriction endonuclease site in American Blacks 3’ to the β-globin gene which was mostly associated with the sickle cell allele. Philips et al have combined the Hpa I analysis with a second polymorphisms found in the γ-globin genes, so they found out that extension of blot hybridization for prenatal diagnosis of sickle cell anemia to over 80% of couples at risk in 1980.4 Also, direct analysis of the sickle cell anemia should be possible by use of restriction enzyme whose recognition sequence is eliminated by sickle cell nutation.
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To identify the sickle disease they used the blot hybridization method. In this method, they isolated the high molecular DNA from peripheral blood lymphocytes. After that they determined the of fragment lengths which was 1.8kb for DNA fragment was resolved by digestion of recombinant DNA clone pBR322βPst with BamHI and electrophoresis through 0.8% agarose in TEA buffer. Then labeling the 38P-labling of the probe by Alu I digestion of pBR322βpst produces a 737-bp, 5’- specific β-globin fragment.4
As result, they found out that Hb S has been shown to be the result of a single nucleotide base mutation in the β-globin gene that converts the glutamic acid codon (GAG) at amino acid position 6 to one for valine (GTG).5 In the table 1 show that A to T trans version within the β-globin gene sequence affect a restriction endonuclease recognition site for both enzyme MnII and Dde I.4
When they analyzed the β-globin gene sequence for Dde I cleavage sites within the 5’end of the β-globin gene up to the BamHI site. However, this result suggest that Dde I cleavage site 33, 122, 210 and 411bp from reference site.6 The 411bp site was affected by the Sickle cell mutation which is shown in figure 4. Also, one fragment of 201bp represent the DNA restriction fragment 3’ from the sickle cell site and another fragment more than 170bp represent the region 5’ to the sickle cell mutation site.4
In the figure 5 shows that digestion of pBR322βPst with Alu I is represented in lane A. A fragment was approximately 855bp long and it’s specific to the 5’ region of the β-globin gene. It was expected from the restriction map presented. When subsequent redigestions with Hae II were performed and only fragment d gave the predicted. However, a slight discrepancy in the length of one Hae III fragment was observed which indicates the existence of an additional Alu I site in the 5’ intragenic region. This may be due to the creation of new Alu I site during the construction of this clone4. However, the actual length of the Alu I probe is 737bp rather than the anticipated 855bp. Blot hybridizations using 32P-labled Alu I fragment hybridized to Alu I, Hae III and Dde I digests of cloned β-globin gene have also given the expected banding patterns4.
By blot hybridization analyses were performed on Dde I-digested lymphocytes DNAs obtained from normal sickle cell and sickle cell train individuals. In the figure 6, autoradiograms of digested DNA from sickle cell shown. DNA from control individual AA shows both the 175bp and 201bp bands. However, sickle cell DNA (SS) shows the 376 bp bands. DNA from sickle cell trait individual (AS) shows expected combination of 175, 201, and 376 bp.4
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Sickle cell anemia is a disease that affects hemoglobin and the oxygen transport molecule in the blood. Some red blood cells become sickle-shaped and these elongated cells get stuck in small blood vessels so that parts of the body don’t get the oxygen they need. Sickle cell anemia is caused by a single code letter change in the DNA. This is turn alters one of the amino acids in the hemoglobin protein. Valine sits in the position where glutamic acid should be. The valine makes the hemoglobin molecules stick together and forming long fibers that distort the shape of the red blood cells, and this brings on an attack.1 The rigid HbS molecules bend red blood cells into a sickle (crescent) shape. The sickle-shaped cells die prematurely which can lead to a shortage of red blood cells (anemia). The sickle-shaped cells can also block small blood vessels causing pain and organ damage. Mutations in theHBBgene can also cause other abnormalities in beta-globin which leading to other types of sickle cell disease7. These abnormal forms of beta-globin are often designated by letters of the alphabet or sometimes by a name. In these other types of sickle cell disease, just one beta-globin subunit is replaced with hemoglobin S. The other beta-globin subunit is replaced with a different abnormal variant such as hemoglobin C or hemoglobin E.8 In hemoglobin SC (HbSC) disease, the beta-globin subunits are replaced by hemoglobin S and hemoglobin C. Hemoglobin C results when the amino acid lysine replaces the amino acid glutamic acid at position 6 in beta-globin (written Glu6Lys or E6K). The severity of hemoglobin SC disease is quite variable, but it can be as severe as sickle cell anemia.
Figure 7: Difference in amino acids between normal and sickle cell disease.
Hemoglobin E (HbE) is caused when the amino acid glutamic acid is replaced with the amino acid lysine at position 26 in beta-globin (written Glu26Lys or E26K). In some cases, the hemoglobin E mutation is present with hemoglobin S.3 In these cases, a person may have more severe signs and symptoms associated with sickle cell anemia such as episodes of pain, anemia, and abnormal spleen function. Other conditions known as hemoglobin sickle-beta thalassemia are caused when mutations that produce hemoglobin S and beta thalassemia occur together. The signs and symptoms of hemoglobin S-beta thalassemias are usually more severe than those of hemoglobin SC disease and may include severe pain and organ damage9. Chronic anemia is generally moderate and not a major source of morbidity for individuals with sickle cell disease. However the presence of a viral infection such as Parvovirus infection can lead to a temporary reduction in red blood cell production and cause more severe life threatening anemia. This is called an aplastic crisis. Primarily, morbidity in sickle cell disease arises from vaso-occlusive events or tissue damage resulting from obstructed flood flow. Some of the more common symptoms include pain crises, acute chest syndrome, cerebrovascular accidents, and splenic and renal dysfunction. Pain crises are episodes of excruciating musculoskeletal pain and acute chest syndrome is a life threatening pneumonia like illness. Cerebrovascular complications include transient ischemic attacks, ischemic strokes and hemorrhagic strokes sometimes associated with seizures. Splenic sequestration is a result of blood pooling and in life threatening instance it can be associated with severe anemia and hypovolemic shock. After early infancy individuals with sickle cell disease become susptible to bacterial infections due to functional asplenia and disordered humeral immunity10.
In the Table 2 shows that sickle cell disease is a major public health concern that has great impact on both individuals and society. Between 1990 and 1996 there were averages of 75000 hospitalizations per year in the united states among individuals with sickle cell disease. In the table there were showing that different mutation had equally chances of occur sickle cell anemia7.
Sickle cell anemia is genetic disorder characterized by the Hb S variant of the β-globin gene. More than 50000 Americans are affected with sickle cell disease making it one of the most prevalent genetic disorders in the United States of America. Individuals who are affected with sickle cell anemia have two copies of his variant (Hb, SS) and the primary hemoglobin present in their red blood cells is sickle hemoglobin. Individuals affected with other types of sickle cell disease are compound heterozygotes7. They possess one copy of the Hb S variant plus one copy of another β-globin gene variant such as Hb C or Hb β-thalassemia. These individuals produce a mixture of variant hemoglobin’s. Carrier individuals have one copy of sickle variant and one copy of the normal β-globin gene (Hb AS) producing a mixture of sickle hemoglobin and normal hemoglobin. The carrier state of sickle cell disease is often referred to as sickle cell trait. Although individuals with sickle cell trait do not express sickle cell disease. One study found that sickle cell trait may be a risk factor for sudden death during physical training. In addition, individuals with sickle cell trait are protected from malaria infection. The high frequency of the Hb S variant is believed to be a result of this protective effect9.
Kan and Dozy were the first to describe the diagnosis of sickle cell anemia in the DNA of affected individuals based on the linkage of the sickle cell allele to an Hpa I restriction fragment length polymorphism11. Later, it was shown that the mutation itself affected the cleavage site of both Dde I and Mst II and could be detected directly by restriction enzyme cleavage. A more general approach to the direct detection of single nucleotide variation by the use of allele-specific oligonucleotide hybridization. In this method, a short synthetic oligonucleotide probe specific for one allele only hybridizes to that allele and not to others under appropriate conditions. All of the above approaches are technically challenging and its require a reasonably large amount of DNA, and they are not very rapid. The polymerase chain reaction (PCR) developed provided a method to rapidly amplify small amounts of a particular target DNA11. The amplified DNA could then be readily analyzed for the presence of DNA sequence variation (e.g., the sickle cell mutation) by allele specific oligonucleotide hybridization, restriction enzyme cleavage, ligation of oligonucleotide pairs, or ligation amplification.
In conclusion, sickle cell disorder is well known autosomal recessive disorder which is happen on chromosome 11. The mutation in sickle cell anemia occurs when the sixth amino acid in the hemoglobin sequence changes from glutamic acid to valine, a point mutation where the adenine becomes thymine in the sixth codon. Mutations in theHBBgene can also cause other abnormalities in beta-globin, leading to other types of sickle cell disease. This method was highly recommended for identification of the sickle cell anemia. The occurrence of polymorphisms that create or eliminate Dde I recognition sites could change the expected blot hybridization pattern. However, they were using the 737bp assay fragments of 175 and 201 bp , they expect that occurrence of specific polymorphisms to be minimal. Also, those parents who both have sickle cell trait can pass thru the sickle cell disorder to their children. If anyone get the sickle cell disorder, that person blood shape will change to sickle shape blood. Sickle shape blood cannot move easily on blood vessel. The mutation in sickle cell anemia occurs when the sixth amino acid in the hemoglobin sequence changes from glutamic acid to valine, a point mutation where the adenine becomes thymine in the sixth codon. Mutations in theHBBgene can also cause other abnormalities in beta-globin, leading to other types of sickle cell disease.
- Neel, James V. “The inheritance of the sickling phenomenon, with particular reference to sickle cell disease.”Blood6.5 (1951): 389-412.
- Serjeant, Graham R., and Beryl E. Serjeant.Sickle cell disease. Vol. 2. New York: Oxford university press, 1992.
- Powars, D., L. S. Chan, and W. A. Schroeder. “The variable expression of sickle cell disease is genetically determined.”Seminars in hematology. Vol. 27. No. 4. Elsevier, 1990.
- Geever, Robert F., et al. “Direct identification of sickle cell anemia by blot hybridization.”Proceedings of the National Academy of Sciences78.8 (1981): 5081-5085.
- Solovey, Anna, et al. “Circulating activated endothelial cells in sickle cell anemia.”New England Journal of Medicine337.22 (1997): 1584-1590.
- Hicks, EdwardJ, et al. “IDENTIFICATION OF SICKLE CELL AND HÆMOGLOBIN C TRAITS IN CORD BLOOD.”The Lancet310.8042 (1977): 818.
- Ashley-Koch, Allison, Quanhe Yang, and Richard S. Olney. “Sickle hemoglobin (Hb S) allele and sickle cell disease: a HuGE review.”American Journal of Epidemiology151.9 (2000): 839-845.
- Powars, D. R., L. Chan, and W. A. Schroeder. “The Influence of Fetal Hemoglobin on the Clinical Expression of Sickle Cell Anemiaa.”Annals of the New York Academy of Sciences565.1 (1989): 262-278.
- Lan, N., Howrey, R. P., Lee, S.-W., Smith, C. A., Sullenger, B. A.Ribozyme-mediated repair of sicklebeta-globin mRNAs in erythrocyte precursors.Science 280: 1593-1596, 1998
- Cheung, M.-C., Goldberg, J. D., Kan, Y. W.Prenatal diagnosis of sickle cellsanaemia and thalassaemia by analysis of fetal cellsin maternal blood.Nature Genet. 14: 264-268, 1996
- Wu, Dan Y., et al. “Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia.”Proceedings of the National Academy of Sciences86.8 (1989): 2757-2760.
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