General background 3
Resistance mutants 4
Hemoglobin mutants 4
Sickle Cell Anemia and Genetics: Background Information 5
Connection of Sickle cell with Malaria 6
Introduction of Recombinant DNA technology in diagnosing disease: 8
Discussion (implementation of recombinant DNA technology to solve the issue) 9
This report targets to study a group of people that has developed resistance to malaria. It will focus on the use of recombinant DNA technology to study this cohort and gain insights into the nature of this observation. This study targets the investigation of the evolution of such resistant gene patterns and the implementation of recombinant DNA technology in solving the issue.
An extensive impact and high death rate have concluded malaria disease as the strongest evolutionary discriminating vigour in the recent human history. Genes responsible for malaria resistance in human proffer a strong affirmative selection in contemporary humans (Hedrick, 2011). This contagious disease may become a strong selective compel in the human populations. Variants found responsible for resisting malaria are categorised as loss-of-function mutants. The combined effects of developed changes in chromosomal alleles in globin locus and thalassaemia haplotypes are predicted for such genomic revolution. There are many developed and successful technologies exist that can investigate the cause of the problem from its root level (Hedrick, 2011).
Apart from geographic patterns other factors including gene ﬂow, genetic drift, mutation, duplication and the interaction among these factors also play a role in this type of evolutions. In the literature, two examples of population genetics have been provided, in which two malaria resistant variants in the same population are discussed. These are two alleles S and C at β-globin locus and then alleles at two different genes, the S allele at the β-globin locus and another on α-thalassemia variant at the α-globin locus (Hedrick, 2011).
Ferreira et al. stated in their work that Sickle human haemoglobin (Hb) confers a survival advantage to those living in endemic areas of malaria (Ferreira et al., 2011). Furthermore, he adds that this protective outcome is exerted irrespective of parasite load, exposing that sickle Hb confers host tolerance to infection caused by Plasmodium. This approach can facilitate the investigation of the case selection and prediction of the outcomes in the future generations for these variants (Hedrick, 2011).
Preservation of the sickle-cell Hb variant has been a model example of heterozygote advantage. On the other hand several other variants such as G6PD deﬁciency at an X-linked locus, β -globin (S, C and E) and a-thalassemia provides strong examples of gene selection. Several important general ﬁndings that may be a reason of genetic resistance to malaria in humans are as following (Hedrick, 2011):
1- A wide overlap of the past geographical allocation of malaria and human genetic variants is seen that presents resistance to malaria (Hedrick, 2011).
2-The existence of strong selective force for resistance to malaria resulted in high frequency in some unfavourable genetic diseases, for instance, sickle-cell anemia, G6PD deﬁciency, thalassemia and ovalocytosis (Hedrick, 2011).
3-Most of the variants that resist to malaria are loss-of-function type mutants. They result in altered and reduced expression or a distorted product. They own a large pleiotropic cost and result in disease.
4-At some loci involved variants nucleotide or codon speciﬁc while others are relatively common. Most of them are recent polymorphisms and might have cropped up in the last several thousands years, not from very ancient times.
5- A variety of unlinked genes is found to be responsible for resistance. Moreover, different populations exhibited different levels of resistance while living in the similar endemic area (Hedrick, 2011).
Numerous genes are responsible in genetically evolved resistance to malaria. These genetic factors also vary because of the variation in speciﬁc resistant variants, dissimilarity in malaria strain or species and varying effects of variants in a given host genotypes. Though, new technologies and methods have been commenced and successful in the identification of potential malaria resistance. According to data estimation from a malaria resistance analysis from Kenya, only 2.1% of the variation was accredited to sickle-cell variation, and 2% was attributed to α-thalassemia. While, 25% resistance was ascribed to functioning host genes, and 29% was due to unknown household effects (Hedrick, 2011).
Hemoglobinopathies are hereditary transmitted disorders of haemoglobin that result in abnormal structure and function containing structural hemoglobin variants and thalassemias. These inherited defects decrease the production while synthesis of α- and β-globins of the human adult haemoglobin. Although, hundreds of structural variants of adult hemoglobin have been recognized but only three of them, S, C and E at HBB locus, caught main attraction and reached considerable frequencies in any populations. According to Hedrick and other experts, the important structural variants are present only at two codons in the HBB gene. It suggests that mutants on the other sites of HBB locus with all sites existing at HBA loci are either detrimental or neutral in malarial environments. Another possibility is that it had not gone through mutation in malaria environments (Hedrick, 2011).
Several other described factors in the literature that are doubted for inducing resisting malaria in humans are:
- G6PD deﬁciency,
- Anomalies on red blood cell surface loci, (i.e., Duffy and ABO),
- Immune gene complex, such as HLA complex (HLA-Bw53 and DQw5) (Hedrick, 2011).
Sickle Cell Anemia and Genetics: Background Information
Sickle cell anemia originates due to a point mutation in its β globin gene. The hemoglobin protein is made up of four globin subunits; two alpha (α) and two beta (β). Two different genes, α and β globin, encodes these subunits, respectively. In this mechanism oxygen binds at the iron sites, so all the four globin chains must be synchronized to perform its function properly. Due to point mutation a non-polar amino acid valine is infused into the β globin chain in the place of glutamic acid. This mutation gives the Red blood cells a stiff and sometimes sickle-shaped appearance on releasing their oxygen. The sickle cell mutation creates a sticky area on β chains’ surface when oxygen is not adhered to it. Similar incidence happens with other molecules of sickle cell haemoglobin. So they stick on each other and polymerize in long fibers, giving the RBC a distorted sickle shape emergence (Chowning et al., n.d.).
The sickled cells, due to its shape, stuck in narrow blood vessels and blocks blood flow. Therefore, it creates a painful crisis in the bones and joints of disease sufferers. The chances of strokes, damage to lungs, heart, kidney and blindness also exist. This mutation cause mortality rate high, and most of the sufferers die before the onset of 20’s. Though, medical science has developed and found out the treatments to every disease and mutation, but it only could be successful to prolong the lives of these sufferers up to 40s and 50s (Chowning et al., n.d.). The two key genes, β globin alleles play a significant role in the inheritance of sickle cell anemia, namely A and S. Person having two normal A alleles (AA) exhibit normal haemoglobin and normal Red Blood Cells (Ferreira et al., 2011). Two alleles responsible for developing sickle cell anemia are mutant S alleles, SS. Heterozygous AS, for sickle cell allele (AS) turns out into both, normal and abnormal hemoglobin. Persons with heterozygous alleles are healthy, but they exhibit and may suffer several symptoms of sickle cell anaemia under specific circumstances, such as low blood oxygen or high elevation. Heterozygous (AS) individuals also act as a carrier of this trait. Because, both types of hemoglobin are manufactured in heterozygotes, and A and S both alleles are co-dominant (Chowning et al., n.d.; Genome Sciences Education Outreach, n.d.).
Connection of Sickle cell with Malaria
Various researches have revealed that in Africa, the frequency of this fatal disease 1/100 while in 1/500 (Chowning et al., n.d.). Now the question is that why is the rate of recurrence of such a potentially fatal disease at a large extent in Africa?
The answer is associated with another fatal disaster, malaria. The main symptoms of Malaria are severe headaches, chills and fever, vomiting and nausea that may result in anaemia and death. The pathogen of malaria is a protozoan parasite (Plasmodium) that is transmissible in humans through Anopheles mosquito. On the exposure of the malarial parasite in the bloodstream, the mutated red blood cells become sickled and die. These cells ensnare the parasites within and lessen the infection (Chowning et al., n.d.; Ferreira et al., 2011).
The people with normal hemoglobin who have AA genotype are more susceptible towards infection in comparison of AS heterozygotes people. Deaths of AA homozygotes contribute in the diminishing of alleles A from the gene pool. On contrary, people with the AS genotype are not affected by sickle cell anaemia and thus, show very low tendency of contracting malaria. They can stay alive and replicate in malaria-infected expanses. Therefore, both alleles A and S exist in the population, simultaneously. SS homozygotes exhibit sickle cell anaemia that results in early death. Consequently, S alleles are isolated from the gene pool (Chowning et al., n.d.). Thus, in malaria endemic regions, where malaria is prevalent, S allele provides a survival advantage to the people with only one copy of the allele (Genome Sciences Education Outreach, n.d.). This is the main reason detrimental S allele is maintained in the populace at a comparatively high frequency. This experience can be examined using recombinant DNA technology. Furthermore, apart from another mode of studies DNA technologies will help improving the condition and implementation of good allele to fight off the damaging fatal diseases (Chowning et al., n.d.).
In the diagnosed group of people, we attempted to review all the recent advancement in malarial population structure and gene flow. The focus of the study is based on three main areas. These areas include mosquito species identification, studies of the basis of resistance, and implementation of recombinant DNA technology for the analysis of reason at genetic level. Most of the data and predictions are relied on the previously cited literature.
Introduction of Recombinant DNA technology in diagnosing disease:
Recombinant DNA technology allows the separation of libraries of DNA sequences equivalent to either the entire genome of an entity or the articulated sequences of a given cell type. Gene-specific probes secluded from these libraries are helpful in identifying the DNA sequences and comparing their functionality and the consequences of rearrangements, mutations that result in genetic diseases. Genetic linkage mapping of the complete human genome constructed via DNA sequence polymorphisms facilitates the antenatal diagnoses of monogenic diseases, without an understanding of the biochemical defect. Recombinant DNA procedures are now applied in the identification of molecular defects in human that is responsible for somatic mutations linked with acquired infectious disease (Caskey, 1987; Davies, 1981). DNA differs in every organism, and that is due to the point mutations, insertions, deletions and repetitions. In polymorphism, more than one sequence variations exist at one DNA site (Chowning et al., n.d.). Alleles of a gene represent the best example of polymorphism, for instance, A and S alleles of β globin gene. Polymorphic sites are either within genes (as β globin alleles) or outside of protein-coding regions. Through taking advantage of polymorphic regions, new recognition sites can be shaped, or old sites eradicated. This disparity results in a unique pattern of restriction fragments when inserted in another person’s DNA. Following this recombinant DNA technique of restriction enzyme digestion, the DNA fragments are isolated through gel electrophoresis (Genome Sciences Education Outreach, n.d.). With the help of Southern Analysis method, special probes are attached to these target fragments. The generated DNA patterns are analyzed on a gel to investigate the foreign or patched up paternity cases (Caskey, 1987). If a polymorphic site is near to the suspected area accountable for the disease, it is said to be linked. While, polymorphic region with separable restriction enzyme lies within the responsible gene called ‘Within,' as in the case of sickle cell anemia. In 1978, Kan and Dozy revealed that the restriction enzyme Mst II, which incise normal β globin DNA at a specific site, cannot distinguish and cut DNA with sickle cell mutation. Mst II acknowledged the sequence CCTNAGG (where N = any nucleotide) (Kan & Dozy, 1978). Sickle cell disease is evolved because of a single point mutation on chromosome 11, β globin that transform CCTGAGG to CCTGTGG. Therefore, the A to T mutation triggers sickle cell anemia along with loss of the recognition site for restriction enzyme Mst II. Hence, the DNA from AA individuals, AS and SS generate different sizes of restriction (Kan & Dozy, 1978; Chowning et al., n.d.).
Discussion (implementation of recombinant DNA technology to solve the issue)
The research from Ferreira and Marguti opened the new ways to novel therapeutic involvements against malaria that continued to impose terrific social, medical and economic load to a large percentage of the human populace (Davies, 1981). The previous literature has cited that the sickle mutation is highly selected in the malaria prevalent areas of the world, often sometimes 10-40% of the population observed carrying this mutation. Such results endowed with significant insights for emerging treatment and cure for this shattering disease that has also been accused of a million premature deaths in sub-Saharan Africa (ScienceDaily, 2014). Despite several decades of research, the mechanism underlying this protective effect remained elusive. The sickle hemoglobin that is capable of resisting malaria is indirectly reducing the number of parasites, thus conferring some protection against the disease. The IGC team's results challenge this explanation (Chowning et al., n.d.).
Ferreira with his team, successfully established that genetically engineered one copy of sickle hemoglobin alike to sickle cell trait, do not surrender to cerebral malaria and generating resistance in humans. According to the Ferreira, the chief player in this protective mechanism HO-1 (heme oxygenase-1) enzyme releases the carbon monoxide gas. This gas confers protection against cerebral malaria via blocking accumulation of free heme after the plasmodium infection. During the process carbon monoxide when produced in response, protects the infected host from submitting to cerebral malaria without intervening parasitic life cycle into the red blood cells (Ferreira et al., 2011).
Scientists believe that this mechanism they for sickle cell trait may be a general mechanism acting in other red blood cell genetic diseases responsible in resistance to malaria in human populations (ScienceDaily, 2014).
Recombinant DNA technology is becoming a treatment option for diseases at every level, from exceptional metabolic disorders to prevalent cancers. Pairing stem cell expertise with recombinant DNA technology allows stem cells extracted from the patient to be customized in the laboratory and introduce a desired gene. For example, a normal β globin gene taken from a patient with sickle cell may be introduced into the DNA of normal cells to induce the resistance against various diseases.
The resistance to malaria in human given an immense source of data and provided an interesting example to study newly emerged mutational disease. The existence of the S allele in malaria-endemic areas of Africa is estimated around 16% (Hedrick, 2011). Though this frequency it little lower in US, due to controlled anti-malaria programmes. The observed elevation in the malaria cases in recent years has been the result of increased travelling, immigration and resistance to medication. The use of recombinant DNA technology has been opened up new paths to fight malaria, through Sickle cell allele from heterozygous haemoglobin. The wide purpose of recombinant DNA diagnostics will help in changing scenarios with the deep knowledge of sickle cell abnormalities and will help to diminish the malaria pathogen.
Thus, recombinant DNA technology has extended ability to identify the disease. Considerable advancement in the diagnostic procedures is accomplished, and significant insights are gained in diagnostic precision through such developments.
Caskey, C. T. 1987. Disease diagnosis by recombinant DNA methods. Science, 236(4806), 1223-1229.
Davies, K. E. 1981. The application of DNA recombinant technology to the analysis of the human genome and genetic disease. Human genetics, 58(4), 351-357.
Ferreira, A., Marguti, I., Bechmann, I., Jeney, V., Chora, Â., Palha, N., Rebelo, S., Henri, A., Beuzard, Y. and Soares, M. 2011. Sickle Hemoglobin Confers Tolerance to Plasmodium Infection. Cell, 145(3), pp.398-409.
Hedrick, P. W. 2011. Population genetics of malaria resistance in humans. Heredity, 107(4), 283-304.
Kan, Y. W., & Dozy, A. M. 1978. Polymorphism of DNA sequence adjacent to human beta-globin structural gene: relationship to sickle mutation. Proceedings of the National Academy of Sciences, 75(11), 5631-5635.
ScienceDaily, 2014. Mystery solved: How sickle hemoglobin protects against malaria. [online] Available at: http://www.sciencedaily.com/releases/2011/04/110428123931.htm [Accessed 7 Nov. 2014].
Genome Sciences Education Outreach. "Sickle Cell Anemia - University of Washington." http://gsoutreach.gs.washington.edu. N.p., n.d. Web. 7 Nov. 2014 <http://chroma.gs.washington.edu/outreach/genetics/download