Human biochemical genetics and the term of the inborn errors of metabolic genetic based disease were established in 1902 [1]. Albinism is one prototypic defect studied by Garrod, where the deficiency of the enzyme tyrosinase in the hair, skin and eye prevent the synthesis of the pigment melanin. However, the most studied genetic disease is the Sickle cell anemia [2]. The World Health Organization (WHO) (1982) estimated that about five percent of the world populations are carriers of genes for clinically important disorders of hemoglobin. Third of a million severely affected homozygotes or compound heterozygotes are born each year. For more details refer to Weatherall et al. [3] and the references within. Hemoglobin is the oxygen carrier tetrameric molecule and can be found in vertebrate red blood cells, in some invertebrates and in the root nodules of legumes [1,4]. Each subunit is composed of a polypeptide chain, globin, and a prosthetic group, heme, which is an iron-containing pigment that combine with oxygen and gives the molecules its oxygen-transporting ability. Sickle cell anemia is a global disease and for the Mediterranean and the Africans communities is a local disease [5]. Livingstone, has described in detailed the roles which affect the percentage and the distribution of the Sickle cell anemia in West Africa. From the time of specifying the role of the heredity (the most critical one) to producing artificial blood and artificial oxygen carrier, the science and the scientist did not stop [6]. Additionally, scientists, particularly, those from the Sickle cell anemia endemic regions and countries, have summarized their experiences as well as the experience gained from their communities in controlling the disease side effect. There is a need for better diagnosis and simplifying the existing information for the public. Such information could be introduced as simple advice, to the communities in the west of sub- Sahara in Africa and Mediterranean region [7,8]. However, Sickle cell anemia patients will be nearly impossible to be recovered because it is a genetically based degenerative disease. That is mean each somatic cell have the responsible gene. At least its full treatment will not be in the coming years. This study tries to show that bioinformatics tools must give more concern to the DNA sequences, where DNA can show that prototype mutants are existed. The early detection of such mutants will enable us to avoid such built in serious genetic disease.

The nucleotides sequences collection

Normal β globin DNA sequence was the start point of this study. The sequence has been obtained from the www.ncbi.nlm.nih.gov nucleotides database and has transferred to the nucleotides blast search. These nucleotide database search nucleotides using a nucleotide query (Blast.ncbi.nlm.nih.gov/Blast.cgi) [9].

The nucleotides sequences adjustment

After the search is complete twenty-six sequences have been selected based on the sickle mutant variation (Table 1). The sequences then have been saved in one file using Fasta format.

Table 1: DNA sequences used in this study.

The used nucleotides sequences

Alignment has been done using ClustalX 2.1 [10]. The nonidentical regions have been removed as well as the so diverse sequences. Only one diverse sequence has left for demonstration. After removing the odd sequences, the total partial sequences become as seventeen.

Nucleotides translation

The sequences constituent of nucleotides (for each one) has been translated to amino acids using Blastx (search protein database using a translated nucleotide query) and have been rechecked using the translation option in Bioedit software version 7.2.5 (Frame 3) [11]. The obtained translated sequences then collected and putted in one file as FASTA format and saved. The final used DNA and Protein partial sequences used in this study are represented : (1) gi-47124545, (2) gi- 46854767, (3) gi-13549111, (4) gi-6003533, (5) gi-40886940, (6) gi- 49456780, (7) gi-29445, (8) gi-183944, (9) gi-29436, (10) gi-23268448, (11) gi-28302128, (12) gi-164697558, (13) gi-49168543, (14) gi- 187940240, (15) gi-13937928, (16) gi-6003531 and (17) gi-193244942.

Identity calculation

Antheprot have been used to determine the % of similarity upon alignment using both of the Clustal W option in the software [12].

Identifying the prototype mutants

Manual counting for the number of different nucleotides and amino acids either in each block as in Figures 1 and 2 or for each sequence for both of the DNA and the protein alignments.

Figure 1: Multiple alignment of the primary sequences of seventeen partial β globin DNA (nucleotides) sequences.

Figure 2: Multiple alignment of the primary sequences of seventeen partial β globin protein (amino acids) sequences.

Phylogenetic trees

Mega 6 has been used to generate phylogenetic trees for each of the alignment of the DNA and protein sequences using Maximum Parsimony method [13,14]. The Mega 6 trees for both of the DNA and protein sequences have been merged in one file using the software SplitTree 4 (version 4.13.1.) [15]. The comparison between the two trees has been done using the software Dendroscope (Version 3.2.10.) [16].

The biological system is sensitive for the chemical structure. Enzymes could be so specifics. Other protein forms could be also very sensitive. The red blood cell structures changes from ring to sickle shape without the oxygen. This cause blood to clots and deprives vital organs from their supply of blood, resulting in pain, intermittent illness, and often, a shortened life span. The only difference between normal and sickle cell hemoglobin is that in each β- chain, one glutamic acid replaced one valine. Valine, unlike glutamic acid, contains a nonpolar group. The result is a hydrophobic “sticky” region that can interact with hydrophobic region on neighboring molecules, producing clumping. A slight change in the β-globin 3D structure will induce a change in the configuration of it when it interacts with its neighboring subunits of the hemoglobin [17]. The DNA and protein alignments were represented as in Figure 1 and 2. Both of the DNA alignment and the protein alignment similarity have been calculated using Antheport 6.3.14 software. The variation in the DNA sequences is clearer that it is more than that in the protein. However, the % of similarity between the protein sequences is higher from that between the DNA sequences. Where the similarity in the DNA sequences was 75.585% while in the protein sequences was 69.388%. That is because on calculating the % of the similarity the number of the nucleotides is a critical factor where it exceeds the number of the amino acids and the percentage between them is 3:1. That causes the error in representing the differences between the similarities of both. For that, the number of the different nucleotides as well as the number of the different amino acids has been calculated manually. The data have been summarized in Table 2. The data represented the differences between blocks and the complete sequence(s) of the β-globin as in Table 2. For better comparison another strategy by building a phylogenic tree for both of the DNA sequences and the protein sequences have been followed. The numbers of nucleotides which are different for each sequence in each block have been written in the right part of the sequence. The differences by each one block and the total differences for both of the protein and the DNA sequences have been summarized in Table 2. Such manual analysis for the data enables detecting the prototype mutant. Amino acids which have been changed due to the change happened in the nucleotide have been neglected to represent only the prototype mutants. Then a comparison is done between both. At this point, the variations become clearer as in Figures 1 and 2 and Table 2. Maga 6 has been used to build the phylogenic tree of each of the DNA and the protein while SplitsTree 4 (Version 4.13.1) software was used to build two phylogenic trees in one file and Software. Dendroscope (Version 3.2.10) was used to compare both of the trees. The different trees are shown in Figures 3-5.

Table 2: Manual counting for nucleotides and amino acids, which show differences from the alignments.

Figure 3: Maximum Parsimony analysis of the DNA.

Figure 4: Maximum Parsimony analysis of the Protein.

Figure 5: 1Phylogenic trees comparison for both of tree 1 and 2.

For the DNA sequences the differences was inferred using the Maximum Parsimony method. Tree 1 and 2 (Figure 3) out of 10 most parsimonious trees (length=72) is shown. The consistency index is 1.000000 (1.000000), the retention index is 1.000000 (1.000000), and the composite index is 1.000000 (1.000000) for all sites and parsimonyinformative sites (in parentheses). The MP tree was obtained using the Subtree-Pruning-Regrafting (SPR) algorithm [13] with search level 0 in which the initial trees were obtained by the random addition of sequences (10 replicates). The tree is drawn to scale, with branch lengths calculated using the average pathway method [13] and are in the units of the number of changes over the whole sequence. The analysis involved 17 nucleotide sequences. Codon positions included were 1^st+2^nd+3^rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 295 positions in the final dataset. The differences analyses were conducted in MEGA6 [14].

The differences were inferred using the Maximum Parsimony (MP) method. Tree 2 (Figure 4) out of 10 most parsimonious trees (length = 12) is shown. The consistency index is 1.000000 (1.000000), the retention index is 1.000000 (1.000000), and the composite index is 1.000000 (1.000000) for all sites and parsimony-informative sites (in parentheses). The MP tree was obtained using the Subtree-Pruning- Regrafting (SPR) algorithm [13] with search level 0 in which the initial trees were obtained by the random addition of sequences (10 replicates). The tree is drawn to scale, with branch lengths calculated using the average pathway method [13] and are in the units of the number of changes over the whole sequence. The analysis involved 17 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 80 positions in the final dataset. The differences analyses were conducted in MEGA6 [14].

Recently, Amara 2014 has represented the hypothesis that the DNA is the subject of mutant change due to the various stresses and factors mutation, reproduction, adaptation, epigenetic and it should be given more concern. Yes, the protein made function, but it might exist a prototype mutant (incomplete mutant) which is ready to be a true mutant not detected yet. And will not be detected under the protein level or even by translating the nucleotides to protein. Each nucleotide might be critical in any investigation concerning genetic disease so it is recommended to use nucleotides sequence for searching the distribution of any genetic disease in any population. This study highlight that such change is not supporting Darwin theory about the evolution, even it against it. Existence of two alleles could dilute many genetic disorders if one is correct. For, that the marriage outside the family, safe a lot and protect from the degenerative diseases as well as many other genetic related illness. In contrast, marriage from the same genetic pool increases the genetic disorder probability. Verses to Darwin hypothesis, Alleles differences enable variations (color, length. etc.) but disable species alteration. It becomes clear that Darwin has clearly succeeded to discover the role of the genetic material in the variation of the offspring phenotypes. However, he has failed in relating that to the species alteration, which might not existed at all. The hemoglobin molecule has a Quaternary structure that consists of four polypeptide chains, known as globin. While most genes are existed in pair each in one chromosome, it enables variation, as well as enable saving the host if one gene is defect. Even so if the two essential genes are defecting, that cause fatal disease such as the degenerative disease and put a big question mark about our understanding to the evolution. Genes constituents have narrow range of change if compared by their mobility within the same species, and partially with the sexually reproduced (have x and y chromosome) organisms include humans. Narrow range of change enables variation but major change is restricted, that safe the whole species feature. It has been expected that the regions where Sickle cell anemia become as an endemic disease that the health individuals must be subjected to DNA analysis before marriage. For better investigation for any pro-mutant, individuals which expected to be in risk, such those in endemic area must subjected to examination using complete sequencing for the β-globin gene. While the scientific research is aiming to solve problems, here this study introduces some steps should be followed by the government where Sickle disease is existed:

1. Marriage from relatives should be avoided.

2. Marriage from Foreigner should be encouraging.

3. Balanced foods contain antioxidants must be used.

4. Natural Edible plant proved traditionally effective with the Sickle cell anemia patient should be investigated and should be available in the market.

5. Control Sickle cell disease selective agent, particularly the malaria, to increase the Genetic pool where the correct gene should be higher than the defect one. In such case the defect one will be disappeared by correct marriage as a result of the time factor (more than successful generation).

6. More sophisticated determination roles are in need to determine both of the Sickle anemia patient or those are more able to acquire the genetic illness or in better word to transfer it to their generations. Complete sequence for β1, β2, α1 and α2 globin are in need; particularly in the endemic regions.

This study concerning with Sickle cell anemia disease. Different bioinformatics software have been used to investigate the various types of β globin under both of the DNA and the protein level. The sequences, which have been used in this study have been reduced aiming to investigate the differences in sequences have nearly no gaps. The DNA and the protein have been compared using phylogenic tree comparison.