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The Missing Gene in Sickle Cell Anemia


Sickle cell anemia (SCA) is a single gene condition that results from a homozygotic mutation at the beta-globulin locus of a single gene. Thus, it is a single genetic disease, but the phenotypic characteristics are complex due to a diverse range of clinical manifestations that might start early in childhood (especially early childhood mortality). Some phenotypic conditions might not be recognized. However, individuals who survive SCA in early childhood are likely to develop stroke, the severe effect of SCA common in more than 8% of the patients.

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Past studies

Studies have shown that the interaction of some unrecognized modifier genes determines an individual’s susceptibility to overt stroke. These “missing genes” need to be studied in order to examine how they act and hopefully determine their identifications. An important way of studying these genes is the use of SNPs. For instance, a study of 108 SNPs in 39 genes obtained from 1,398 persons with the sickle cell disease1.

It has been found that about 31 from 12 candidate genes can interact with the fetal Hb. This is thought to be one of the methods through which genes modulate the risk of overt stroke. In addition, the study found that three of the 12 genes are involved in the TGF-β pathway as well as the SELP. These systems are associated with stroke in the general population.

A relatively rare but interesting feature is the presence of a novel property mutation in SCD. A novel property mutation is a form of mutation that gives new properties to the encoded proteins. For instance, the hemoglobin chains of the sickle cells are proteins that obtain the ability to aggregate into long fibers, causing cell impairment and loss of function. In this case, the mutation leads to the gain of glycosylation. The point mutation creates a novel n-glycosylation site in the new protein, which confers the novel property on it.

More recently, induced pluripotent stem cells (iPSC) lines have become a popular method of studying genetic factors and interactions, especially due to the ability of these cell lines to differentiate into almost any type of cell2,3. In the recent past ZFNs have become important techniques for increasing the frequency of recombination of these cell lines. The in situ correction technique is important in correcting the missing genetic fatcors4.

Deficiencies noted

A major problem in using the above methods has been the diversity and large number of genes that are associated with the modification of the susceptibility to SCA. This makes it difficult to identify a particular gene or group of candidate genes. Secondly, the use of IPSCs and combination with ZNF is a relatively new idea, which is both technically and expensive. Nevertheless, it holds the future of identifying genes that interact to produce an observable effect.

Objectives of the study

The objective of the proposed study is to use in situ correction of SCA mutation in human iPSC lines with ZFNs in order to identify the missing genes in SCA homozygosity.

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Significance of the study

The information obtained from the proposed study is expected to contribute to the increasing body of knowledge about sickle cell anemia and the importance of using iPSC lines in gene targeting therapies.

Experimental design

The study is a quantitative research that aims to examine the interaction of candidate genes that modify the susceptibility to stroke in individuals with SCA. First, a culture of patient-derived IPSCs will be derived, modified and analyzed under the existing protocols of the university. Dermal fibroblasts from three patients of SCA will be obtained and infected with a polycystronic lentiviral vector. The fibroblasts will be seeded in MEF medium and infected with the vector after 24 hours5. They will be transferred to a human ESC medium and allowed to grow for two months.

Engineered ZFNs will be used to target the human β-globin gene, where arrays of Zn finger binding each of the target sites will be selected using OPEN method. The selected Zn fingers will be cloned on Xbal-BamHI fragment into the expression vector (ZFN). Elongation factor 1 α(EF1a) promoter6.

Then, a loop-out and a transient transfection of gene targeting method will be used in the iPSC cell lines. Isolation of the GFP-positive cells will be done through FAC sorting after seeding them with matrigel in mTeSR1 medium. Finally, the colonies will be picked manually and screened using the PCR method.

Data will be obtained through the southern blotting technique for separating genomic DNA after restriction digestion of the PCR products. 32P-labelled probes will be used for the hybridization. Karyotyping, teratoma formation analysis and expression analysis will be done to examine the cell behaviors.


  1. Sebastiani P, Ramoni MF, Nolan V, Baldwin CT, Steinberg MH. Genetic dissection and prognostic modeling of overt stroke in sickle cell anemia. Nature genetics 2005; 37: 435-440.
  2. Park IH, Zhao R, West JA et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 2008; 451: 141–146.
  3. Yamanaka S. Strategies and new developments in the generation of patient-specific pluripotent stem cells. Cell Stem Cell 2007; 1: 39–49.
  4. Sebastiano V, Maeder ML, Angstman JF, Haddad B, Khayter C, Yeo DT, et al. In situ genetic correction of the sickle cell anemia mutation in human induced pluripotent stem cells using engineered zinc finger nucleases. Stem Cells 2011; 29: 1717-1726.
  5. Saha K, Jaenisch R. Technical challenges in using human induced pluripotent stem cells to model disease. Cell Stem Cell 2009; 5: 584–595.
  6. Lowry WE, Richter L, Yachechko R et al. Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc Natl Acad Sci USA 2008; 105: 2883–2888.

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