Sickle Cell Disease is an autosomal recessive disease (Ashley-Koch et al, 2000) that presents itself in children anywhere from 6 months to 2 years of age (Costa and Coran, 2016). Two copies of Hb S or one Hb S copy with another Hb variant is needed to have the disease expressed (Ashley-Koch et al, 2000). At the “-globin gene locus, the S allele is different from the wild type because of a substitution of an A (wild type) to a T (mutant) at the second position of the 6th codon of the ” chain gene (Saiki et al, 2005). This results in valine being produced instead of glutamic acid (Saiki et al, 2005).
In people of African and Mediterranean descent, there is a high frequency of sickle cell anemia (Ashley-Koch et al, 2000). A hallmark of the disease is that the red blood cells are sickle shaped red blood cells because they become dehydrated, rigid, and deformed (Blouin et al., 2000). Some of the common symptoms of sickle cell anemia are chronic anemia (where there are not enough healthy red blood cells), acute chest syndrome (a vaso-occlusive crisis of the pulmonary vasculature), stroke, splenic and renal dysfunction, pain crises, and susceptibility to bacterial infections (Ashley-Koch et al, 2000).
In 1978, Deisseroth mapped the disease using a DNA/cDNA molecular hybridization assay that detects the presence of a globin gene if it is present in 10% of the cells (Deisseroth et al, 1978). The clones, 157-BMPT-4 and 157-BNPT-1, that they used in this study were derived by the fusion of human fibroblast GM 126[26, XY, t(1, 15)(p36; ql)] with the mouse fibroblast tsClAOH (Deisseroth et al, 1978). Following that, they isolated the hybrid cells in Dulbecco’s modified Eagle’s medium (Deisseroth et al, 1978). They used Giemsa 11 strains to identify specifically the mouse and human chromosomes (Deisseroth et al, 1978).
When they used the purified DNA from a hybrid clone containing ” globin genes and the ” globin cDNA, they found no significant hybridization (Deisseroth et al, 1978). This means that the ” globin cDNA is free enough from the ” cDNA, which means that if they detect something it is just ” globin (Deisseroth et al, 1978). They also did the reverse with the ” globin genes and ” globin cDNA and found no significant hybridization (Deisseroth et al, 1978). This shows that the ” and ” probes were specific to their sites respectively (Deisseroth et al, 1978). In their results, they discarded the chromosomes that did not have the presence of human ” globin genes that were in greater than 10% of the metaphase spread in the hybrid cell line (Deisseroth et al, 1978). This left them with the chromosomes 6, 8, 9, 11, and 13 (Deisseroth et al, 1978). Only chromosome 11 was present in 100% of the hybrid clones that were positive for the ” globin gene (Deisseroth et al, 1978). The other four chromosomes were present in less than 10% of the hybrid cells.
To clone the gene, the restriction endonucleases for Eco RII, Hae III, Hinf, Bam HI, Alu I, T4 endonuclease IV, bacterial alkaline phosphatase, and purified T4 polynucleotide kinase were made or obtained (Marotta et al, 1977). Then using detergent and phenol extraction, sickle cell or “-thalassemic immature RNA was prepared form membrane-free red cell hemolysates (Marotta et al, 1977). A sucrose gradient centrifugation or an oligo(dT)-cellulose column chromatography were used to isolate globin mRNA from immature RNA (Marotta et al, 1977). ” and ” mRNA comes from immature sickle cell red blood cells when ” mRNA came from “-thalassemic immature sickle cell red blood cells (Marotta et al, 1977). The RNA’s were used as templates to create their respective double-stranded cDNAs using avian myeloblastosis virus RNA-dependent DNA polymerase (Marotta et al, 1977). The globin cDNAs were digested with restriction endonucleases and the resulting fragments were separated by electrophoresis (Marotta et al, 1977). wet gel was covered with a cellophane wrap and autoradiographed to locate [32P]cDNA fragments (Marotta et al, 1977). The isolated fragments were treated and made ready for a second round of digestion with second endonuclease (Marotta et al, 1977). The fragments of [32P]cDNA that came from the second endonuclease was analyzed by partial digestion (Marotta et al, 1977). The samples electrophoresed in a pH buffer at 4000 V for 1 hour (Marotta et al, 1977). Using displacement chromatography on thin layer chromatography plates, the materials were resolved in the second dimension (Marotta et al, 1977). The plates dried then were autoradiographed (Marotta et al, 1977). The nucleotide sequences were determined by direct inspection of autoradiographs (Marotta et al, 1977). They then prepared endonuclease IV digests of globin [32P]cDNA by synthesizing single stranded [32P]cDNA by using [“-32P]dCTP as the triphosphate (Marotta et la, 1977). The DNA was incubated for 16-18 hours at 37*C with the endonuclease IV in a mixture (Marotta et al, 1977). Using displacement chromatography on thin layer chromatography plates, the products were fractionated in the second dimension (Marotta et al, 1977). After the chemical degradation and autoradiography of the cDNA, a prominent band was extracted and preferential cleavage treatment was used (Marotta et al, 1977).
The double stranded “- and “- cDNA taken from sickle cell mRNA and the “- cDNA taken from “-thalassemic mRNA was put under the conditions used for transcription and the products were representative of the translated and 3′-terminal untranslated regions of the template mRNA (Marotta et al, 1977). [32P]cDNA fragments were cut by Hae III endonuclease, electrophoresed and viewed by autoradiography (Marotta et al, 1977). The fragments gotten from “- cDNA cut by Hae III shows four dark bands (H-1, H-2, H-3, and H-4) and one lighter band (H-5) (Marotta et al, 1977). They estimated the lengths of the five bands based on the polyacrylamide gels compared to SV40 DNA markers of known molecular weight (Marotta et al, 1977). To order the Hae III fragments, they compared the single cuts of the double stranded cDNA cut by Hae III with the double cut made by Hae III and Eco RI or Hae III and Bam HI (Marotta et al, 1977). The comparison showed that of the 5 Hae III fragments one was cut by Eco RI, Fragment H-3, and one was cut by Bam HI (fragment H-2) (Marotta et al, 1977). They located the Eco RI position at the mRNA triplet codon positions 121 and 122 (Marotta et al, 1977). The located Bam HI at the codon positions 99 and 100 (Marotta et al, 1977). They thought that the Hae III fragment that was cut by Bam HI overlapped the codons 99 and 100 (Marotta et al, 1977). They also knew that there had to be at least one Hae III site between Eco RI and Bam HI in the “-cDNA (Marotta et al, 1977). They studied the “-globin amino acid sequence and used the genetic code to narrow it down to only one possible place for the Hae III site in the correct region where it overlaps the codons for amino acids 114 and 115 (Marotta et al, 1977). Based on the estimated lengths of the H-2 (122 nucleotides) and H-3 (81 nucleotides) there was only one place that of the H-2 proximal end that the Hae III site could be was at the codons 74 and 75 of the “-globin mRNA (Marotta et al, 1977). This allowed them to place the H-2 and H-3 Hae III fragments in order relative to themselves and the “-globin mRNA chain (Marotta et al, 1977). They placed H-4 based on the facts that the sequence K3-2 (derived from the Hae III site of H-3) was located at the mRNA codon positions of 141 and 142, that the K3-2 sequence overlaps the unique cRNA products T17 and T12 which are located from codons 141 to 148, and that the Alu I cut of H-4 had yielded cDNA sequence K4-1 which is located at codons 150 and 151. These places the H-4 fragment is located adjacent next to H-3 and that it extended out into the 3′ terminal noncoding region of the “-globin mRNA (Marotta et al, 1977). They placed the H-5 fragment to the 3′ terminal of the “-globin mRNA was established by isolating it with a snake venom diesterase that contains the cDNA sequence K5-1 which corresponds to cRNA products T64, T55b, and T43 (Marotta et al, 1977). These experiments allowed them to order the Hae III fragments of the “-globin gene (Marotta et al, 1977).
Blouin et al used transgenic mice that have a unique model of SCD (sickle cell disease), SAD-1 which expresses HbSAD, sickled hemoglobin, and also has a disease that is biochemically, cellularly, and pathologically close to human sickle cell disease (Blouin et al, 2000). Patients with SCD that have increased levels of HbF have lower levels of sickling (Blouin et al, 2000). To find out the level of HbF expression needed to combat sickling in vivo, they mated the SAD mice to mice that have transgenic lines that carry 2 types of A”-globin gene recombinants (Blouin et al, 2000). The matings created double-transgenic mice, A”SAD, that expressed both “- and “SAD-globin genes and were predicted to produce adults that had red blood cells that were sickled hemoglobin and HbF (Blouin et al, 2000). They found that as the HbF levels increased the affected hematological parameters of the SAD mice improved (Blouin et al, 2000).
Cole-Strauss et al corrected the mutation by using an RNA-DNA oligonucleotide (Cole-Strauss et al, 1996). They designed a chimeric oligonucleotide that was a single molecule that was capable of folding back onto itself allowing it to form a duplex structure (Cole-Strauss et al, 1996). It was composed of DNA residues with 2 RNA residue blocks surrounding 5 short DNA residues (Cole-Strauss et al, 1996). When it was in the duplex form one strand held all the DNA residues and the other strand held the RNA-DNA blocks (Cole-Strauss et al, 1996). The internal sequence was complementary to the “-globin sequence that extends over the “-globin mutation site with one exception of a T base (Cole-Strauss et al, 1996). The RNA-DNA blocks were centered over the T base in the mutant sequence (Cole-Strauss et al, 1996). They introduced the chimeric molecule into lymphoblastiod cells that were homozygous for the “S mutant allele by using a commercial liposome formulation (Cole-Strauss et al, 1996). After 6 hours, they cells were assayed to see if they were corrected (Cole-Strauss et al, 1996). They were labeled corrected if they didn’t see the loss of a Bsu 361 restriction site which is lost in the A-to-T switch in the “S mutant (Cole-Strauss et al, 1996). In the mutant allele, the 1.2 kbp fragment is replaced by a 1.4 kbp (Cole-Strauss et al, 1996). When they assayed the cell, they saw both the 1.2 kbp and 1.4 kbp fragments which suggests that the rescue was partially successful (Cole-Strauss et al, 1996).
Direct analysis of fetal DNA can be used to diagnose Sickle cell anemia (Saiki et al, 2005). Using amniocentesis or chorionic villus sampling, DNA is obtained then treated with restriction endonuclease that distinguishes between the “-S mutation and the “-A wild type thus producing “-a and “-s specific restriction fragments that can be resolved using a southern transfer and hybridization with a “-globin probe (Saiki et al, 2005).