Abstract
Sickle cell disease (SCD) is a group of innate red blood cell disorders that affect their function. These disorders arise from alterations in the beta globin gene that encode the production of globin, which is the protein component of hemoglobin. Consequently, there is impaired transport of oxygen alongside other complications. Various forms of treatment exist in the treatment of SCD including hydroxyurea, bone marrow transplants, and blood transfusions among others. However, the invention of personalized medicine provides a lasting solution to developing permanent cure of SCD. This paper describes the process of screening and genetic counseling provided to a 17-year-old African American boy wishing to have personalized treatment for SCD. Blood and skin samples were tested. The patient was found to be homozygous for sickle cell hemoglobin. The reprogramming process showed normal cell behavior thus confirming that the treatment would be beneficial to the patient. The patient received genetic counseling explaining the logic of the tests and the subsequent treatment steps.
Introduction
Sickle cell disease (SCD) denotes a cluster of hereditary red blood cell illnesses. It is among the most common autosomal recessive disorders globally (Bölke & Scherer, 2012). The disorder arises when there is a transmutation in the order of nucleotides in the gene that ciphers the beta chain of the human hemoglobin. A single nucleotide change from adenine to thiamine results in a change in the sixth amino acid from glutamate to valine. This substitution leads to the production of aberrant hemoglobin denoted as Hb S. In sickle cell anemia, an individual inherits two defective copies of the hemoglobin gene from both parents leading to homozygous Hb SS.
Such hemoglobin has tendencies of clumping, which causes erythrocytes to stiffen and assume a sickle shape. In the absence of adequate oxygen, the sickle red blood cells are highly predisposed to clustering, which blocks blood vessels particularly capillaries and leads to some of the symptoms observed in the ailment. Sickle cell anemia (homozygous Hb SS) represents about 60%-70% of SCD in the United States (Sun, Liang, Abil, & Zhao, 2012). Other varieties of SCD occur when the defective Hb S gene is passed down to the offspring alongside other uncharacteristic β-globin chain modifications. They include sickle-hemoglobin C disease (Hb SC) and thalassemias (alpha and beta thalassemia). Other atypical types such as D-Punjab, O-Arab, and E have also been reported (Homer & Oyeku, 2016).
The formation of red blood cells occurs in the bone marrow through a process known as erythropoiesis, which occurs under the influence of the hormone erythropoietin. In the initial stages of fetal development, erythropoiesis occurs in the mesodermal cells of the yolk sac (Homer & Oyeku, 2016). The process shifts to the liver after the first trimester and afterward to the bone marrow after seven months. Erythropoiesis continues to take place in the bone marrow of all the bones until the age of 5 years. Thereafter, the tibia and femur remain the active sites of hematopoiesis until the age of 25 after which the cranial bones, pelvis, vertebrae, sternum, and ribs take over the process for the remaining part of life. Nevertheless, some illnesses can force erythropoiesis to happen outside the bone marrow in the spleen or liver, which is a condition known as extramedullary erythropoiesis (Homer & Oyeku, 2016).
The symptoms of SCD include sporadic vaso-occlusive crisis and longstanding hemolytic anemia. Tissue ischemia causes extensive pain in addition to the destruction of organs such as bones, liver, brain, eyes, lungs, kidneys, and joints. The earliest symptom of sickle cell disease in young children is the swelling of limbs. The abnormal red blood cells may accumulate in the spleen thereby impairing the functions of the spleen. Consequently, such children may be susceptible to some bacterial contagions. Prolonged destruction of erythrocytes causes anemia, accumulation of bilirubin (jaundice), deferred growth and sexual development.
Conventional treatment of SCD includes antibiotics to prevent bacterial infections, analgesics for pain management, hydroxyurea to boost erythrocyte production and cut down on the need for frequent blood transfusions, supplemental oxygen, and blood transfusions. Bone marrow transplant is also beneficial for children who find suitable donors (Romero et al., 2013). Nevertheless, finding a donor that matches the human leucocyte antigen (HLA) of the patient is an uphill task that complicates the procedure (Bolaños-Meade et al., 2012). Therefore, expanded bone marrow stromal cells derived from the patient’s mesenchymal stem cells are being targeted in the improvement of osteonecrosis treatment in SCD patients (Lebouvier et al., 2015).
Personalized medicine is a field of medicine that entails the provision of tailored medical treatment based on the patients’ risks and projected responses. Patients with a given disorder are stratified into different groups based on established characteristics. The aim of this paper is to describe the process that a genetic counselor would use to determine whether a patient with SCD is a suitable candidate for personalized medicine. The patient, in this case, is a 17-year-old African-American boy who has recently heard of personalized medicine in SCD and would wish to receive the treatment.
Materials and Methods
Approximately 4 ml of blood was removed from the patient’s vein into a collection tube containing EDTA. Samples of the skin tissues were obtained through a skin biopsy. To determine whether the patient was an ideal candidate for personalized SCD treatment, it was necessary to screen the patient for SCD and determine the precise form of the disease because SCD encompasses a group of related hemoglobin disorders.
A rapid test as described by Kumar et al. (2014) was done to determine the density of erythrocytes. This process was done by first developing an aqueous multiphase system (AMPS) to be used for erythrocyte separation. Seven percent of polyethylene glycol (PEG) with an approximate molecular weight of 20 kDa weight by volume was mixed with 10.3% Ficoll having a molecular weight of about 400 kDa. A separate three-phase gradient medium was prepared by mixing 3% PEG (MW of 20 kDa), 10% dextran (MW of 500 kDa) and 5% polymer of incompletely hydrolyzed polyvinyl acetate (MW of 3 kDa) (Kumar et al., 2014). The polymer consisted of 75% hydroxyl and 25% −OCOCH3 groups.
The media were made isotonic by the addition of sodium chloride after which the pH was adjusted to about 7.4, which is the physiological pH, through the addition of concentrated sodium hydroxide or hydrochloric acid and measuring the ensuing pH using a pH meter. Nycodenz, a compound with a high density and low-osmolality was added to the media to amplify the density of each system to the required range (Kumar et al., 2014). The media were mixed on a vortex mixer to ensure uniform blending of the constituents. The correct densities were confirmed by an oscillatory U-tube densitometer. The blood sample was added to the density media and centrifuged at 13,700 × g for 10 minutes. The separation of the blood was used to diagnose the precise type of SCD.
To determine the suitability of the patient for personalized treatment of SCD, part of the patient’s blood was reprogrammed into human induced pluripotent stem cells (iPSCs). Gene editing using was done using the zinc finger nuclease editing technique as described by Kumar et al. (2014). TAL effector nucleases (TALENs), which is a group of synthetic nucleases that identify long, precise DNA sequences, can also be used in the editing of DNA sequences (Sun & Zhao, 2014). The purpose of editing was to repair the cells to correct the defective gene and allow for the differentiation of the cells. The progress of the cells in the different stages was observed to ascertain that healthy cells were formed.
Results
Centrifugation of blood through the gradient media yielded a visual separation of sickled erythrocytes. It was observed that the patient’s blood moved from the loading position through the media and formed distinct layers. The proportion of dense red blood cells was present in significant quantities accounting for about 10% of the blood cells. The density of the erythrocytes was 1.120 g/cm3. This observation corresponded to a diagnosis of SCD, in particular, Hb SS. Therefore, the patient was homozygous for sickle cell anemia. Normal hemoglobin is known to have a very small amount of dense cells comprising about 2% or less as indicated in figure 1 (Kumar et al., 2014).
The genetic testing showed that the patient’s pluripotent stem cells responded favorably to the zinc finger nucleases. The expression of the cells regulated by small quantities of the drug. Therefore, the patient was a suitable candidate for the personalized treatment of sickle cell anemia using zinc finger nucleases.
The patient received genetic counseling explaining why it was necessary to carry out the described tests before deciding on the correct treatment approach. First, the patient was informed that sickle cell disease was a group of different disorders and that it was necessary to determine the correct form of the disease that affected the patient before the right treatment could be developed for him. His results showed that he was homozygous for Hb SS, meaning that he had received faulty beta hemoglobin genes from both parents. It was further explained that though most patients with sickle cell anemia (Hb SS) possessed two copies of defective Hb SS, each person’s risk and responses to treatment differed.
The differences were determined by susceptibilities encoded into the patient’s genome at birth in addition to the lifestyle and environmental conditions of the patients. There was no guarantee that the drugs capable of curing cure another patient would be effective for the patient. In fact, there was a likelihood that the drugs would result in more harm than good. Therefore, it was necessary to determine how the risks varied and the dynamics of response to treatment to prevent adverse drug reactions in the course of treatment. The risks and treatment response, which corresponded to the biology of each patient’s disease, would then be used as the basis of personalized medicine to come up with a customized treatment approach (Schork, 2015). The patient was also informed that personalized medicine helped to cut down the cost of treatment by increasing the probability of the effectiveness of a chosen therapeutic approach.
Discussion
The meticulous reprogramming of wild-type and sickly cells to become prototypes for the tissues affected sickle cell disease provides researchers with a new platform for relating the biology and drug sensitivity of affected and healthy cells. Nevertheless, apart from the technical setbacks of precise and reproducible reprogramming, the molecular origin of a disorder may be different between patients suffering from a disease that is known to affect a specific gene. Different behavior in two iPSC lines from a patient with a given disorder may cause confusion as to whether the difference is attributable to disease-pertinent genetic variations or a consequence of the reprogramming process (Chun, Byun, & Lee, 2011). Therefore, this problem is addressed by generating isogenic pairs of wild-type and diseases iPSCs that vary only in the disease-linked gene. Developing the right treatment requires a gene correction scheme for patient-obtained iPSCs that causes only the disease-associated mutation without additional genetic modification.
Various studies have shown that hiPSCs preserve the genetic expression of the parental cells. However, it is unknown whether the origin of the cells has an impact on the safety and operation of the hiPSCs. For these reasons, it is important to obtain human induced pluripotent stem cells from different sources and investigate the impact of the source on the safety, precision and differentiation aptitudes (Ferreira & Mostajo-Radji, 2013). The idyllic source of the cell should be easily accessible with minimal risk procedures. In addition, the cells should be present in large quantities and amenable to reprogramming with high efficiencies. Studies have shown that tissues from the mesoderm origin such as blood and fibroblasts as well as ectodermal tissues such as keratinocytes, and melanocytes are useful, hence the use of blood and skin cells in the study.
Personalized medicine is entrenched in the supposition that diseases vary in their etiologies, rates of advancement and reaction to drugs. Each person’s disease is inimitable. Therefore, each person requires special treatment.
References
Bolaños-Meade, J., Fuchs, E. J., Luznik, L., Lanzkron, S. M., Gamper, C. J., Jones, R. J., & Brodsky, R. A. (2012). HLA-haploidentical bone marrow transplantation with posttransplant cyclophosphamide expands the donor pool for patients with sickle cell disease. Blood, 120(22), 4285-4291.
Bölke, E., & Scherer, A. (2012). Sickle cell disease. Canadian Medical Association Journal, 184(3), 201-201.
Chun, Y. S., Byun, K., & Lee, B. (2011). Induced pluripotent stem cells and personalized medicine: Current progress and future perspectives. Anatomy & Cell Biology, 44(4), 245-255.
Ferreira, L. M., & Mostajo-Radji, M. A. (2013). How induced pluripotent stem cells are redefining personalized medicine. Gene, 520(1), 1-6.
Homer, C. J., & Oyeku, S. O. (2016). Sickle cell disease. American Journal of Preventive Medicine, 51(1), 3-4.
Kumar, A. A., Patton, M. R., Hennek, J. W., Lee, S. Y. R., D’Alesio-Spina, G., Yang, X.,… & Whitesides, G. M. (2014). Density-based separation in multiphase systems provides a simple method to identify sickle cell disease. Proceedings of the National Academy of Sciences, 111(41), 14864-14869.
Lebouvier, A., Poignard, A., Coquelin-Salsac, L., Léotot, J., Homma, Y., Jullien, N.,… & Rouard, H. (2015). Autologous bone marrow stromal cells are promising candidates for cell therapy approaches to treat bone degeneration in sickle cell disease. Stem Cell Research, 15(3), 584-594.
Romero, Z., Urbinati, F., Geiger, S., Cooper, A. R., Wherley, J., Kaufman, M. L.,… & Jin, X. (2013). β-globin gene transfer to human bone marrow for sickle cell disease. The Journal of Clinical Investigation, 123(8), 3317-3330.
Schork, N. J. (2015). Personalized medicine: Time for one-person trials. Nature, 520(7549), 609-611.
Sun, N., & Zhao, H. (2014). Seamless correction of the sickle cell disease mutation of the HBB gene in human induced pluripotent stem cells using TALENs. Biotechnology and Bioengineering, 111(5), 1048-1053.
Sun, N., Liang, J., Abil, Z., & Zhao, H. (2012). Optimized TAL effector nucleases (TALENs) for use in treatment of sickle cell disease. Molecular BioSystems, 8(4), 1255-1263.