# α Globin Hemoglobinopathy as A Case Study: Mutants and Prototype Mutants: Are They Absent α0, Reduced α+ or Recombinant?

Amro Abd Al Fattah Amara

► To cite this version:

Amro Abd Al Fattah Amara. α Globin Hemoglobinopathy as A Case Study: Mutants and Prototype Mutants: Are They Absent α0, Reduced α+ or Recombinant?. Cellular & Molecular Medicine: Open access, 2017, α Globin Hemoglobinopathy as A Case Study: Mutants and Prototype Mutants: Are They Absent α0 Reduced α+ or Recombinant?, 04 (01), 10.21767/2573-5365.100033. [https://hal.science/hal-04035593](https://hal.science/hal-04035593)

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# α Globin Hemoglobinopathy as A Case Study: Mutants and Prototype Mutants: Are They Absent α⁰, Reduced α⁺ or Recombinant?

## Abstract

Recently the existence of prototype mutants in *B* globin genes was proved. In this study a further and similar investigation was performed. The investigation concerns the *Homo sapiens* *α* globin. The prototype mutants were screened in the *α* globin DNA sequences. The results proved their existence. A selected *α* globin fragment contains the prototype mutant was used for searching both of the nucleotide and protein databases. The used fragment consists of nine nucleotides GGC AAG GTT and contain two silent mutants with the following amino acids sequences GKV. Searching the Blast protein database for the existence of correct mutants using the V00493.7 *Homo sapiens* 27 amino acids fragment was led to find 50 different sequences. Interestingly the protein sequences show similarity with marsupials, even-toed ungulates, odd-toad ungulates, placental, bats, rodents, carnivores, whales and dolphins; primates, insectivores, rabbits and hares. Species responsible for human bit or blood transfer are located and suggest gene recombination between different species occure through blood transfer. The restriction map for the 81 nucleotides for four nucleotide sequences (one prototype mutant, two correct mutants, and one normal) proved change in the restriction enzymes profile of the *α* globin sequences. That might be a proof for the existence of reduced *α⁺* globin (also in case of *β⁺* globin) macromolecules. The protein modelling in the selected different fragments show differences in the 3D structure and in their hydrophobicity. The unexpected exo-sources for the *α* globin put another load on the possibility of the haemoglobin illness solving. However, avoiding marriage from relatives, and from the same ethnic groups might be the best choice. Additionally avoiding blood contact with animals have compatible mutants is recommended. Further investigations are in need to prove/disprove the role of the gene recombination between species through blood in the transfer of the *α* globin mutants or even other ones.

**Keywords:** *α* Globin prototype; Gene recombination; Nucleotides database; Protein database

Received: May 05, 2017; Accepted: June 29, 2017; Published: June 30, 2017

## Introduction

The quaternary structure of the hemoglobin macromolecules consists of four globin polypeptide chains. Like any of the human genes some variant of the globin molecules are existed which controlled by basic genetic rolls. Hemoglobin diseases are a global disease. WHO estimated that 5% of the world populations have hemoglobin disorders [1].

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]. Livingstone in 1989 described that the disease is concentrated in some parts of the world particularly where the malaria is existed [5] and/or where excessive marriage from relatives over generations is existed as suggested by Amara in 2013 and 2015 [6,7]. Both of the described reasons are responsible for the image of the disease in the Mediterranean region and West Africans [5,7,8].

The hemoglobin molecules are highly sensitive to the O₂/CO₂ exchange. The sensitivity level could be in range can

## Amro Abd Al Fattah Amara*

Department of Protein Research, Genetic Engineering and Biotechnology Research Institute, Scientific Publishing, City for Scientific Research and Technological Applications, Universities and Research Center District, New Borg El-Arab, Alexandria, Egypt

**\*Corresponding author:**

Al-Fattah Amara AA

Head of the Protein Research Department, Genetic Engineering and Biotechnology Research Institute, Head of the office of the Scientific Publishing, City for Scientific Research and Technological Applications, Universities and Research Center district, New Borg El-Arab, Alexandria, Egypt.

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amroamara@web.de

amroamara@protonmail.fc

Tel: +203-4593422

Fax: +203-4593497

**Citation:** Amara AAAF (2017) *α* Globin Hemoglobinopathy as A Case Study: Mutants and Prototype Mutants: Are They Absent *α⁰*, Reduced *α⁺* or Recombinant? Cell Mol Med. Vol. 3 No. 2: 8
find differences between O₂ and CO₂ molecules. The folded hemoglobin macromolecules are able to differentiate between O₂ and CO₂. Its four-subunit composition of the normal adult hemoglobin (HbA) are α₂β₂. The other variants collectively can be found in the fetal hemoglobin (HbF:α₂γ₂), sickle cell hemoglobin (HbS:α₂S₂), and a minor adult hemoglobin (HbA2:α₂δ₂). Amara in 2015 suggested that prototype mutant in β globin could be a hidden source or sleeping mutants that could be converted to complete mutants (in the future). Accumulating, different types of prototype mutants might be avoided if detected early by avoiding marriage between individuals harboring the responsible genes (from each other) [7]. Even that the primary structures of the β, γ, and δ chains of human hemoglobin are highly conserved, human have stages of modifications concerning the functional hemoglobin. In general; fetus initially synthesizes a ζ₂ε₂ tetramer. After the first trimester, ζ and γ subunits will be replaced by α and ε subunits. While synthesis of β subunits begins in the third trimester, β subunits do not completely replace γ subunits to yield adult HbA (α₂β₂) until some weeks postpartum. For more details refer to Harper's Illustrated Biochemistry in 2003 [9].

Over 800 known mutant of the human hemoglobins are both extremely rare and benign, presenting no clinical abnormalities. Hemoglobinopathy is a term given for the mutants that compromise biologic function [9,10]. It is still not fully proved if the subunit is completely absent (α⁰ or β⁰) or whether its synthesis is reduced (reduced α⁺ or β⁺) [9,11-13].

Like any genes hemoglobin genes governed by the same genetic and molecular biology roles. Gillbert wrote about the gene recombination in the hemoglobin during the differentiation "If recombination occurs between two regions of homology that are in different genes (unaligned recombination or unequal crossing over), individual genes can be duplicated or lost in the resulting daughter DNA. A good example is the globin gene family. If recombination occurs between two similar (but not identical) genes, the resulting DNA will be rearranged so that one progeny is a gene or two short while the other offspring has a few too many. Again, no DNA has actually been lost; it's just been redistributed between offspring" [14]. In the adage of the molecular biology and the genetic engineering, one should not kick out the basic genetic roles which might introduce simpler solutions [6].

This study was started with a single aim to prove only the presence of prototype mutants in the α globin such as that proved by Amara in 2015 [7] in case of β globin genes, however the findings extend that aim and strong evidences about gene recombination between species was found.

# Materials and methods

## 4.1 The *Homo sapiens* α globin nucleotides sequences collection

Human α globin DNA sequence (AF349571.1 *Homo sapiens* haemoglobin alpha-1 globin chain (HBA1) mRNA, complete cds) was the start point of this study. For more details about the source gene refer to the information provided in the AF349571.1 document. The sequence was obtained from the www.ncbi.

nlm.nih.gov nucleotides database and has transferred to the nucleotide blast search. These nucleotide database search nucleotides using a nucleotide query (Blast.ncbi.nlm.nih.gov/Blast.cgi) [15].

>AF349571.1

cccacagactcagagaacccaccatggtgctgtctcctgacgacaagaccaacgt caaggccgcctggggtaaggtcggcgcgcacgctggcgagtatggtcggagggccctg gagaggatgttcctgtccttccccaccaccaagacctacttcccgcacttcgacctgagc cacggctctgcccaggttaagggccacggcaagaaggtggccgacgcgctgaccaacg cccgtggcgcacgtgacgacatgccaacgcgctgtccgcctgagcgacctgcac ggcacaagcttcgggtgaccgggtcaacttcaagctcctaagccactgcctgctgg tgaccctggccgccccacctccccgccgagttcacccctgcgggtcacgcctccctgga caagttcctggcttctgtgagcacgtgctgacctccaaataccgttaagctggagcctc ggtggccatgcttcttggccctttg

# The nucleotides sequences adjustment

After the search was completed 100 nucleotide sequences were obtained. The non-human nucleotide sequences were removed and only 21 human α globin nucleotide sequences were selected. The sequences were saved in one file as FASTA format. Aiming to exclude similar and repeated names sequences the "Data Analysis in Molecular Biology and Evolution" DAMBE ver 6.4. 58. software was used. Further four similar sequences were removed, and a sum of 18 sequences were kept for further investigation.

# Nucleotides sequences alignment

The 18 obtained sequences were aligned using Clustal Xver. 2.1 [16] BioEdit ver 7.2.3. and the sequences were investigated on the GeneDoc 2.7. [17]. Four nucleotide sequences were selected, three contain mutants and one is normal.

# Identifying the prototype mutants and select the targeted fragment

Manual selection for the silent mutant (prototype mutant) was performed as described by Amara in 2015 [7]. Four nucleotide sequences were selected. Two sequences, each contain a single correct mutant (single amino acid change) and one sequence contain silent two mutants, and one is normal. The nucleotide fragments which contain the specified mutants (81 nucleotides) were specified and selected for further investigations.

# Restriction map

The four nucleotide sequence for the used fragments (81 nucleotides) which represent the AF349571.1; AK223392.1; LT548101.1 and V00493.1 were used against the restriction enzyme database in each of Clone Manager version 4.01 software [18], pDRAW32.10 software [19] and Serial Cloner software 2.6.1. [20]. In each program each sequence was put in single txt file. In clone 4 and Serial Cloner software 2.6.1. the nucleotide sequence was adjusted to FASTA format. In pDRAW32.10 software the nucleotide sequence was adjusted without name as a text file. The restriction map for each sequence using each software was performed. The results were obtained and saved as image in case of the pDRAW32.10 software and Serial Cloner software 2.6.1 or in table as in Clone Manager version 4.01 software.
## Tracing the different restriction maps

The restriction maps were screened manually for the existence of different restriction sites.

## Screening the blast nucleotide database for similar mutants

The nucleotide database was screened for the existence of similar mutants using nucleotide query to prove their possible existence. The sequence which specified to contain prototype mutant was used (V00493.1). The obtained data were adjusted to remove any duplicated sequences or similar names (as above) using DAMBE ver 6.4.58 software. Then the rest were aligned using BioEdit ver 7.2.3. software and the alignment was represented using the GeneDoc 2.7. software. The similar mutants were given different colour (green) by manual highlighting.

## Nucleotides translation

The selected nucleotide fragment sequences (81 nucleotides) were translated to 27 amino acids using Blastx (search protein database using a translated nucleotide query) and were rechecked using the translation option in BioEdit software ver 7.2.3 software [21].

## Screening for correct mutants using 27 amino acids sequence

The following nucleotide sequences (under line) which has prototype mutant in the original nucleotide sequence was translated.

"(V00493.1 Homosapiens messenger mRNA for hemoglobin alpha chain: CCCACAGACTCAGAGAGAACCCACCATGGTGCTGTCTCCTGCCGACAAGACCAACGTCAAGGCCGCCTGGGGCAAGGTTGGCGCGCACGCTGGCGAGTATGGTGCGGAGGCCCTGGAGAGGATGTTCCTGCCTTCCCCACCACCAAGACCTACTTCCCGCACTTCGACCTGAGCCACGGCTCTGCCCAGGTTAAGGGCCACGGCAAGAAGGTGGCCGACGCGCTGACCAACGCCGTGGCGCACGTGGACGACATGCCCCAACGCGCTGTCCGCCCTGAGCGACCTGCACGCGCACAAGCTTCGGGTGGACCCGGTCAACTTCAAGCTCCTAAGCCACTGCCTGCTGGTGACCCTGGCCGCCACCTCCCCGCCGA GTTCACCCCTGCGGTGCACGCCTCCCTGGACAAGTTCCTGGCTTC TGTGAGCACCGTGCTGACCTCCAAATACCGTTAAGCTGGAGCCTC GGTAGCAGTTCCTCC)" The translated amino acids are MVLSPADKTNVKAAWGKVGAHAGEYGA were used for screening Swissport Database to find any change in the two targeted amino acids (G and V).

1 ATG GTG CTG TCT CCT GCC GAC AAG ACC AAC GTC AAG GC C GCC TGG GGC AAG GTT GGC GCG CAC GCT GGC GAG TAT G GT GCG 81

1 Met Val Leu Ser Pro Ala Asp Lys Thr Asn Val Lys Ala Ala Trp Gly Lys Val Gly Ala His Ala Gly Glu Tyr Gly Ala 27

ATGGTGCTGTCTCCTGCCGACAAGACCAACGTCAAGGCCGCCTG GGGCAAGGTTGGCGCGCACGCTGGCGAGTATGGTGCGMVLSP ADKTNVKAAWGKVGAHAGEYGA

The number of searched sequences output was adjusted to give 100 results. The obtained sequences were alignment using Swissport Database search tools and the phylogenetic tree was performed (Figure 1). Additionally the tree was visualized offline as circular tree using Tree Figure Drawing Tool Version 1.4.2 software after adjusting the species names. The tree obtained from the Swissport Database summarised the species based on their types. The alignment result was saved for offline manipulation.

## Refining the protein sequences

The obtained 100 protein sequences were refined using DAMBE ver 6.4.58 software to remove the repeated and similar names and sequences. Then manual remove for the sequences which have no change in the targeted GKV region was performed. At the end out of 100 protein sequences only 50 were obtained. Manually additional two sequences were loaded (AF349571.1 *Homo sapiens* and AK223392.1 *Homo sapiens*). Full alignment for the 52 sequences was performed and the phylogenetic tree was obtained using Mega 6 software (Figures 2 and 3).

## Phylogenetic tree generation and visualization

Phylogenetic tree and the alignment for selected proteins and nucleotides of the α globin were generated using different software (few only were represented). One phylogenetic tree was obtained from the online BLAST pairwise alignments option in Swissport Database. The offline used software for trees generation were, BioEdit ver 7.2.3., Mega 6 software, DAMBE ver 6.4.58 software and Clustalx ver 2.1. Tree Figure Drawing Tool Version 1.4.2 was used for visualizing each of the obtained trees [22]. Mega 6 software was used to give statistical report

## Protein models generation

Model for each of AF349571.1; AK223392.1; V00493.1 and Q862A7.3 α globin 27 amino acids fragments were generated. The Hemoglobin pdb file (P69905) was used to obtain single α globin macromolecule by the aid of the software Discovery Studio 4.1. Client 4.1.0.14169 (Accelrys software Inc.) software. The α globin used protein sequence was as the follow: The SEQUENCE 142 AA; 15258 MW; 15E13666573BBBAE CRC64; MVLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFP HFDLSHGSAQVKGHGKKVADALTNAVAHVDDMPNALSALSDLHA HKLRVDPVNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTV LTSKYR.

The software Modeller v 9.8 was used for generating model for each protein fragment (27 amino acids) [23]. For better 3D structure, the background of the images have been converted to white and the 3D image have been adjusted and saved [24] (Figures 4 and 5). The protein fragment was represented as ribbon and the surface structure of the hydrophobicity of the fragment was performed using the Discovery Studio 4.1. Client 4.1.0.14169 (Accelrys software Inc.) software. Only the 3D structure and the hydrophobicity were used to visualize the protein 3D differences due to the different amino acid constituents in the selected fragment.
<figure><img src="image_3.png" /></figure>

(a)

<figure><img src="image_4.png" /></figure>

(b)

<figure><img src="image_5.png" /></figure>

(c)

<figure><img src="image_6.png" /><figcaption>**Figure 2** 37 nucleotide sequences where prototype sequence was searched. The highlighted green letters show the region of the silent mutant coded nucleotides.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Figure 3** Phylogenetic tree was performed using BLAST pairwise alignment Database Swissport.</figcaption></figure>

## Results and Discussion

Hemoglobin diseases are impotent due to their health and economic side effect. Hemoglobin diseases are genetically based. They are hard to be repaired, at least in the few coming years or till significant progress happened in the gene therapy [6]. Each somatic cell has the defect gene. β globin macromolecules proved to have prototypes mutants in their nucleotide sequences [7]. This study represents an investigation concerning α globin. The

study aims to prove or disprove that α globin macromolecules have prototypes mutants. The investigation strategy is based on specifying some mutants on the α globin DNA then translating them. Finding silent mutation is the start point to find others converted to complete mutants. That was performed by manual tracing the prototype mutants obtained from the *Homo sapiens* nucleotide sequence alignment as in **Figure 6**.

The study was started by searching the blast protein database against a complete α globin sequence for 100 nucleotide
<figure><img src="image_8.png" /><figcaption>**Figure 4** 100 aligned amino acids sequence to the searched 27 amino acids.</figcaption></figure>

sequences. The result contains *Homo sapiens* and other species
genes. 21 *Homo sapiens* genes only were located and were
selected **(Figure 6)**. The *Homo sapiens* genes were adjusted to
FASTA format and were investigated using the DAMBE software.
The software enables removing of similar sequences and
detecting sequences with similar names. Three similar sequences

were removed. The 18 different sequences were subjected
to alignment (Figure 7). The result proves the existence of
different mutants in the different sequences. Four sequences
were selected. Two sequences each contain a single nucleotide
mutant; one contains two nucleotide mutants and one did not
contain mutants (in the selected part). The *Homo sapiens* selected
<table><caption>**Figure 5** Alignment for the 52 protein fragment sequences.</caption><thead><tr><th></th><th></th><th></th></tr></thead><tbody><tr><td>V00493.1</td><td>Homo sapiens: _27_AA</td><td>MVLSPADKTNVKAAWGKVGAHAGEYGA</td><td>27</td></tr><tr><td>AF349571.1</td><td>Homo sapiens: _27_AA</td><td>MVLSPDDKTNVKAAWGKVGAHAGEYG</td><td>26</td></tr><tr><td>AK223392.1</td><td>Homo sapiens: _27_AA</td><td>MVLSPADKTHVKAAWGKVGAHAGEYGA</td><td>27</td></tr><tr><td>Q862A7.3</td><td>Pipistrellus abramus: _27_AA</td><td>MVLSPADKTNVKAAWDKVGGHAGDYGA</td><td>27</td></tr><tr><td>P01958.2</td><td>Equus caballus: _27_AA</td><td>MVLSAADKTNVKAAWSKVGGHAGEYGA</td><td>27</td></tr><tr><td>P07402.1</td><td>Macaca speciosa: _27_AA</td><td>-VLSPADKTNVKAAWDKVGGHAGEYGA</td><td>26</td></tr><tr><td>P01939.3</td><td>Otolemur crassicaudatus: _27_AA</td><td>MVLSPPTDKSNVKAAWEKVGAHAGDYGA</td><td>27</td></tr><tr><td>P11757.1</td><td>Myotis velifer: _27_AA</td><td>-VLSPADKTNIKAAWDKVGAHAGDYGA</td><td>26</td></tr><tr><td>P01962.1</td><td>Tapirus terrestris: _27_AA</td><td>-VLSPPTDKTNVKAAWSKVGSHAGEYGA</td><td>26</td></tr><tr><td>P01937.1</td><td>Nycticebus coucang: _27_AA</td><td>-VLSPADKTNVKAAWEKVGSHAGDYGA</td><td>26</td></tr><tr><td>Q9XSE9.3</td><td>Equus przewalskii: _27_AA</td><td>MVLSAADKTNVKAAWSKVGGHAGEFGA</td><td>27</td></tr><tr><td>P01940.1</td><td>Tarsius bancanus: _27_AA</td><td>-VLSPADKTNVKAAWDKVGGHAGDYGA</td><td>26</td></tr><tr><td>P18970.2</td><td>Ailuropoda melanoleuca: _27_AA</td><td>MVLSPADKTNVKATWDKIGGHAGEYG</td><td>26</td></tr><tr><td>P28780.1</td><td>Taphozous georgianus: _27_AA</td><td>-VLSPADKTNVKAAWEKVGGHAGDYGA</td><td>26</td></tr><tr><td>P01938.2</td><td>Loris tardigradus: _27_AA</td><td>-VLSPADKTNVKTAWEKVGGHAGEYGA</td><td>26</td></tr><tr><td>P11753.1</td><td>Cynopterus sphinx: _27_AA</td><td>-VLSPADKTNVKAAWDKVGGNAGEYGA</td><td>26</td></tr><tr><td>P08258.1</td><td>Mandrillus sphinx: _27_AA</td><td>-VLSPADKKNVKAAWDKVGGHAGEYGA</td><td>26</td></tr><tr><td>P68235.2</td><td>Ursus maritimus: _27_AA</td><td>MVLSPADKSNVKATWDKIGSHAGEYG</td><td>26</td></tr><tr><td>P01948.2</td><td>Oryctolagus cuniculus: _27_AA</td><td>MVLSPADKTNIKTAWEKIGSHAGEYGA</td><td>27</td></tr><tr><td>P18975.2</td><td>Panthera leo: _27_AA</td><td>MVLSADKNNVKACWGKIGSHAGEYGA</td><td>27</td></tr><tr><td>P14259.2</td><td>Otolemur crassicaudatus: _27_AA</td><td>MVLSPPTDKSIVKAAWEKVGAHAGDYGA</td><td>27</td></tr><tr><td>P01960.2</td><td>Equus zebra: _27_AA</td><td>MVLSAADKTNVKAAWSKVGGNAGEFGA</td><td>27</td></tr><tr><td>P18969.2</td><td>Ailurus fulgens: _27_AA</td><td>MVLSPADKTNVKSTWDKLGGHAGEYG</td><td>26</td></tr><tr><td>P01933.1</td><td>Cercacebus atys: _27_AA</td><td>-VLSPDDKKHVKAAWGKVGEHAGEYGA</td><td>26</td></tr><tr><td>P09908.1</td><td>Phoca vitulina: _27_AA</td><td>-VLSPADKTNVKATWDKIGGHAGEYG</td><td>25</td></tr><tr><td>P09906.1</td><td>Rhinoceros unicornis: _27_AA</td><td>-VLSPPTDKTNVKTAWSHVGAHAGEYGA</td><td>26</td></tr><tr><td>P41330.2</td><td>Arctocephalus galapagoensis: _27_AA</td><td>MVLSPADKTNVKTTWDKLGGHAGEYG</td><td>26</td></tr><tr><td>P01961.3</td><td>Equus hemionus kulan: _27_AA</td><td>MVLSAADKTNVKAAWSKVGGNACDFGA</td><td>27</td></tr><tr><td>P24659.1</td><td>Macroderma gigas: _27_AA</td><td>-VLSPADKANVKAAWDKGGQACDYGA</td><td>26</td></tr><tr><td>P20854.1</td><td>Ctenodactylus gundi: _27_AA</td><td>-VLSAADKTNVKAAWDKIGGHAGEYGA</td><td>26</td></tr><tr><td>P07405.1</td><td>Felis catus: _27_AA</td><td>-VLSAADKSNVKACWGKIGSHAGEYGA</td><td>26</td></tr><tr><td>P09904.1</td><td>Physeter_catodon: _27_AA</td><td>-VLSPADKTNVKAAWAKVGNHADFGA</td><td>26</td></tr><tr><td>P41329.2</td><td>Arctocephalus galapagoensis: _27_AA</td><td>MVLSPADKTNIKTTWDKLGGHAGEYG</td><td>26</td></tr><tr><td>P10892.1</td><td>Lutra_lutra: _27_AA</td><td>-VLSPADKTNVKSTWDKIGGHAGEYG</td><td>25</td></tr><tr><td>P01956.1</td><td>Rousettus aegyptiacus: _27_AA</td><td>-VLSSADKTNIKAAWDKVGGNAGEYGA</td><td>26</td></tr><tr><td>P60530.2</td><td>Canis_latrans: _27_AA</td><td>MVLSPADKTNIKSTWDKIGGHAGDYG</td><td>26</td></tr><tr><td>P15163.1</td><td>Leptonychotes_weddellii: _27_AA</td><td>-VLSPADKTNVKTTWDKIGGHAGEYG</td><td>25</td></tr><tr><td>P01951.1</td><td>Talpa_europaea: _27_AA</td><td>-VLSGTDKSNIKAAWDKVGAHAGEYGA</td><td>26</td></tr><tr><td>P18976.1</td><td>Panthera_pardus_saxicolor: _27_AA</td><td>-VLSSADKNNVKACWGKIGSHAGEYGA</td><td>26</td></tr><tr><td>B3ECWC7.1</td><td>Tamias_merriami: _27_AA</td><td>-VLSPADKTNVKAAWEKVGGHGAAYGA</td><td>26</td></tr><tr><td>P01957.1</td><td>Procavia_capensis_habessinica: _27_AA</td><td>-VLSAADKNNVKCAWEKVGTHAGEYGA</td><td>26</td></tr><tr><td>Q7M2Y4.1</td><td>Chalinolobus_morio: _27_AA</td><td>-VLSPADKSNVKAAWDKVGGSACDYGA</td><td>26</td></tr><tr><td>P10778.1</td><td>Odobenus_rosmarus_divergens: _27_AA</td><td>-VLSPADKTNVKTTWDKLGGHAGEYG</td><td>25</td></tr><tr><td>P11755.1</td><td>Tadarida_brasiliensis: _27_AA</td><td>-VLSPEDKNNVKAAWSKVGGOACDYGA</td><td>26</td></tr><tr><td>P09839.1</td><td>Macrotus_californicus: _27_AA</td><td>-VLSAADKCNVKAAWDKVGGOAGEYGA</td><td>26</td></tr><tr><td>P01953.1</td><td>Meles_meles: _27_AA</td><td>-VLSPADKANIKATWDKIGGHAGEYG</td><td>25</td></tr><tr><td>P08852.1</td><td>Marmota_marmota_marmota: _27_AA</td><td>-VLSPADKTNVKAAWEKIGGHGAAYGA</td><td>26</td></tr><tr><td>P15448.1</td><td>Mellivora_capensis: _27_AA</td><td>-VLSPSDKANVKATWDKIGGHAGEYG</td><td>25</td></tr><tr><td>P14389.1</td><td>Pteropus_alecto: _27_AA</td><td>-VLSSTDKSNVKAAWDKVGGHVCEYGA</td><td>26</td></tr><tr><td>B3EWD9.1</td><td>Tamias_striatus: _27_AA</td><td>-VLSPADKTNLKAAWHKLGHGCEYGA</td><td>26</td></tr><tr><td>P18971.2</td><td>Balaenoptera_acutorostrata: _27_AA</td><td>MVLSPPTDKSNVKATWAKIGNHAEYGA</td><td>27</td></tr><tr><td>P19645.1</td><td>Paguma_larvata: _27_AA</td><td>-VLSSADKNNIKATWDKIGSHAGEYGA</td><td>26</td></tr></tbody><tfoot><tr><td colspan="4">VLS DK n6K W k6G hag 5G</td></tr></tfoot></table>
<figure><img src="image_9.png" /><figcaption>**Figure 6** Maximum parsimony analysis of taxa for the 52 protein fragment sequences.</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>**Figure 7** Different restriction maps as performed by pDRAW software.</figcaption></figure>
nucleotides sequences were subjected to logical analysis for the
mutant and the prototype mutants as in **(Figure 6)**.

Four sequences were selected represent each of

1. AF349571.1 *Homo sapiens* haemoglobin alpha-1 globin chain (HBA1) mRNA, complete cds

2. AK223392.1 *Homo sapiens* mRNA for alpha 2 globin variants, clone: FCC107C11

3. V00493.1 *Homo sapiens* messenger mRNA for haemoglobin alpha chain

4. LT548101.1 TPA_inf: *Homo sapiens* GLNC1 gene for globin C1

Searching for the existence of similar prototype mutants in the nucleotides blast database that prove their existence as in **Figure 7**. The nucleotide sequences were translated to their amino acids each of AF349571.1 and AK223392.1 show correct single amino acid change due to a single nucleotide change. Sequence V00493.1 show two nucleotides changes but no amino acid change (silent mutants) or as suggested by Amara in 2015 prototype mutants (**Table 1**) and one is normal.

By taking the protein fragment where the prototype mutants were specified and searching in the protein database for the probable existence of complete mutants, the results proved the existence of complete change in the specified amino acids as in (Figures 1-3,8). That proves the possibility of changing the prototype mutant (silent mutants) to complete correct mutants (amino acids changes). Unexpected result was emerged that the mutants were founds in organisms either able to bit the human or able to transfer blood from other species. That might be open the window for new explanation for the role of the natural gene transfer by insects and animals as in **Figure 8**. The symptoms of the rabies (the disease transferred by doges) including changing the infected patient behavior. Rabies is an acute viral disease of the nervous system of warm-blooded animals (usually transmitted by the bite of a rabid animal); rabies is fatal if the virus reaches the brain).

The V00493.1 nucleotide sequence contains two nucleotide
changes and given no amino acids change which means that
these two mutants are silent mutants. This sequence attracts
the attention for investigating if those two silent mutants were
mutated in any other sequence of the sequences located in the
nucleotide database. The nucleotide database was searched
using the selected 81 V00493.1 nucleotide sequence and the
results proved the existence of similar mutants as in **Figure 7**. The
similar mutants were highlighted by green color (**Figure 7**). The
selected fragment sequences constituent of 81 nucleotides were
translated to 27 amino acids using Blastx (search protein database
using a translated nucleotide query) and were rechecked using
the translation option in BioEdit software version 7.2.3 software.
The translated 81 amino acids (V00493.1) protein fragment was
used to search similar protein using Swissport database against
100 results. The obtained results prove the existence of correct
mutants representing those two suspected amino acids (GKV). As
above the obtained 100 sequences were preliminarily evaluated

using DAMBE ver 6.4.58 software. The total sequences finally
become as 50 sequences and additional two sequences were
added manually (AF349571.1 and AK223392.1) as in **Figures 2**.
The first obtained 100 sequences were subjected to phylogenetic
tree analysis and proved that species other than the *Homo
sapiens* are represented **(Figure 1)**. Both of the 100 and the 52
sequences which were used either for the tree **(Figures 1 and
3)** generation or for the alignments prove some facts **(Figure 2)**:

1. Prototype mutants could become full correct mutants

2. There is possibility for gene transfer within species.

3. The α globin genes prototype mutants are more likely to change to full mutants by the activity of different types of genetic modifications including epigenetic, cross-over, recombination, splicing, methylation, blood transfer etc.

In **Figure 3** which represents the phylogenetic tree of the 52-selected sequence, the evolutionary history was inferred using the Maximum Parsimony method. The most parsimonious tree with length=94 is shown. The consistency index is 0.457447 (0.451613), the retention index is 0.653061 (0.653061), and the composite index is 0.298741 (0.294931) for all sites and parsimony-informative sites (in parentheses). The MP tree was obtained using the Subtree-Pruning-Regrafting (SPR) algorithm (pg. 126 in ref. [25]) with search level 0 in which the initial trees were obtained by the random addition of sequences (10 replicates). The analysis involved 52 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 25 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 [26].

Even this study proves that more resources for the globin
mutation are existed but solutions also could be suggested based
on the type and the source of those mutants. For example, the
prevention of the existence of heterozygote alleles is so simple,
just by totally avoiding the marriage between the offspring of the
homozygote patients for several generations [6,7]. As in the folk
experiences; for seven-generation [6]. The marriage out of the
hemoglobin patients' carrier will lead to diluting the probability
of the existence of the gene generation after generation.
Even it might finally lead to the disappear of the disease. The
species which showed alignment with the 27 searched amino
acids are: V00493.1 *Homo sapiens*; AF349571.1 *Homo sapiens*;
AK223392.1 *Homo sapiens*; Q862A7.3 *Pipistrellus abramus*;
P01958.2 *Equus caballus*; P07402.1 *Macaca speciosa*; P01939.3
*Otolemur crassicaudatus*; P11757.1 *Myotis velifer*; P01962.1
*Tapirus terrestris*; P01937.1 *Nycticebus coucang*; Q9XSE9.3 *Equus
przewalskii*; P01940.1 *Tarsius bancanus*; P18970.2 *Ailuropoda
melanoleuca*; P28780.1 *Taphozous georgianus*; P01938.2 *Loris
tardigradus*; P11753.1 *Cynopterus sphinx*; P08258.1 *Mandrillus
sphinx*; P68235.2 *Ursus maritimus*; P01948.2 *Oryctolagus
cuniculus*; P18975.2 *Panthera leo*; P14259.2 *Otolemur
crassicaudatus*; P01960.2 *Equus zebra*; P18969.2 *Ailurus fulgens*;
P01933.1 *Cercacebus atys*; P09908.1 *Phoca vitulina*; P09906.1
*Rhinoceros unicornis*; P41330.2 *Arctocephalus galapagoensis*;
P01961.3 *Equus hemionus kulan*; P24659.1 *Macroderma giga*;
P20854.1 *Ctenodactylus gundi*; P07405.1 *Felis catus*; P09904.1
<table><caption>**Table 1** Logical analysis for the existence of prototype mutants.</caption><thead><tr><th>Index</th><th>Description</th></tr></thead><tbody><tr><td>>1-AF349571.1 (complete)</td><td>CCCACAGACTCAGAGAGAACCCACCATGGTGCTGTCTCCTGACGACAAGACCAACGTCAAGGCCGCCTGGGGTAAGGTCGCGCGCACGCTGGCGAGTATGGTGCGGAGGCCCTGGAGAGGATGTCCTGTCCITCCCCACCACCAAGACCTACTTCCCGCAC1TCGACCTGAGCCACGGCTCTGCCCAGG1TAAGGGCCACGGCAAGAAGGTGGCCGACGCGCTGACCAACGCCGTGGCGCACGTGGACGACATGCCCAACGCGCTGTCCGCCCTGAGCGACCTGCACGCGCACAAGCTTCGGGTGGACCCGGTCAACTTCAAGCTCCTAAGCCACTGCCTGCTGGTGACCCTGGCCGCCACCTCCCGCCGAG1TCACCCCTGCGGTGCACGCCTCCCTGGACAAGITCCTGGCITCTGTGAGCACCGTGCTGACCTCCAAATACCGTTAAGCTGGAGCCTCGGTGGCCATGC1TCT TGCCCCT1TGG</td></tr><tr><td>>1-AF349571 (81 nucleotides and 27 amino acids)</td><td>1 ATC CIO CSC 1C2 CC2 CAC CAC AAC ACC AAC CIC AkOCCC GEC !GC GCI MC CSC GCE CCC CAC Ca GCE CAC 2AI 0= CCC SI 1 Mat Val Le. See Pee Asp Asp Lys She Ask Val Lys Ala Ala Sep Cly Lys Val Cly Ala Nis Ala Cly Cl. Iye Cly Ala 27 ATGGTGCTGTCTCCTGACGACAAGACCAACGTCAAGGCCGCCTGGGGTAAGGTCGGCGCGCACGCTGGCGAGTATGGTGCGMVLSPDDKTNVKAAWGKVGAHAGEYG</td></tr><tr><td>>7-AK223392.1 (complete)</td><td>GCGCGCACGCTGGCGAGTATGGTGCGGAGGCCCTGGAGAGGATGTCCTGTCC1TCCCCACCACCAAGACCTACTTCCGCAC1TCGACCTGAGCCACGGCTCTGCCCAGG1TAAGGGCCACGGCAAGAAGGTGGCCGACGCGCTGACCAACGCCGTGGCGCACGTGGACGACATGCCCAACGCGCTGTCCGCCCTGAGCGACCTGCACGCGCACAAGCTTCGGGTGGACCCGGTCAACTTCAAGCTCCTAAGCCACTGCCTGCTGGTGACCCTGGCCGCCACCTCCCGCCGAG1TCACCCCTGCGGTGCACGCCTCCCTGGACAAGITCCTGGCITCTGTGAGCACCGTGCTGACCTCCAAATACCGTTAAGCTGGAGCCTCGGTGGCCATGC1TCT TGCCCCTT</td></tr><tr><td>>7-AK223392.1</td><td>1 AIC CSC CSC 2C2 CC2 GCC CAC AAC ACC CAC CSC AAC CCC CCC DSC 0 AAC CSC CrX GEC VC CC2 CrX CAC nT CO[ CCC 11 1 Mat Val La. Sae PCO Ala A.) Lya She N" Val Lya Ala Ala 20 Cly Lyn Val Cly Ala Nla Ala Cly Cl. Iyc Cly Ala 27 ATGGTGCTGTCTCCTGCCGACAAGACCCACGTCAAGGCCGCCTGGGGTAAGGTCGGCGCGCACGCTGGCGAGTATGGTGCGMVLSPADKTHVKAAWGKVGAHAGEYG</td></tr><tr><td>>13-V00493.1 (complete)</td><td>CCCACAGACTCAGAGAGAACCCACCATGGTGCTGTCTCCTGCCGACAAGACCAACGTCAAGGCCGCCTGGGGCAAGGTTGGCGCGCACGCTGGCGAGTATGGTGCGGAGGCCCTGGAGAGGATGTTCCTGTCCCTTCCCCACCACCAAGACCTACTTCCGCACCTTCGACCTGAGCCACGGCTCTGCCCAGGTTAAGGGCCACGGCAAGAAGGTGGCCGACGCGCTGACCAACGCCGTGGCGCACGTGGACGACATGCCCAACGCGCTGTCCGCCCTGAGCGACCTGCACGCGCACAAGCTTCGGGTGGACCCGGTCAACTTCAAGCTCCTAAGCCACTGCCTGCTGGTGACCCTGGCCGCCACCTCCCGCCGAGTTCACCCCTGCGGTGCACGCCTCCCTGGACAAGITCCTGGCTTCTGTGAGCACCGTGCTGACCTCCAAATACCGTTAAGCTGGAGCCTCGGT</td></tr><tr><td>>13-V00493.1</td><td>1 ATG GTG CTG TCT CCT GCC GAC AAG ACC AAC GTC AAG GCC GCC TGG GGC AAG GTT GGC GCG CAC GCT GGC GAG TAT GGT GCG 81 1 Met Val Leu Ser Pro Ala Asp Lys Thr Asn Val Lys Ala Ala Trp Gly Lys Val Gly Ala His Ala Gly Glu Tyr Gly Ala 27 ATGGTGCTGTCTCCTGCCGACAAGACCAACGTCAAGGCCGCCTGGGGCAAGGTTGGCGCGCACGCTGGCGAGTATGGTGCGMVLSPADKTNVKAAWGKVGAHAGEYG [Prototype mutant; the amino acids still normal]</td></tr><tr><td>>14-LT54810.1 (complete)</td><td>CCCACAGACTCAGAGAGAACCCACCATGGTGCTGTCTCCTGCCGACAAGACCAACGTCAAGGCCGCCTGGGGTAAGGTCGGCGCACGCTGGCGAGTATGGTGCGGAGGCCCTGGAGAGGATGTTCCTGTCCCTTCCCCACCACCAAGACCTACTTCCGCACCTTCGACCTGAGCCACGGCTCTGCCCAGGTTAAGGGCCACGGCAAGAAGGTGGCCGACGCGCTGACCAACGCCGTGGCGCACGTGGACGACATGCCCAACGCGCTGTCCGCCCTGAGCGACCTGCACGCGCACAAGCTTCGGGTGGACCCGGTCAACTTCAAGCTCCTAAGCCACTGCCTGCTGGTGACCCTGGCCGCCACCTCCCGCCGAGTTCACCCCTGCGGTGCACGCCTCCCTGGACAAGITCCTGGCTTCTGTGAGCACCGTGCTGACCTCCAAATACCGTTAA</td></tr><tr><td>>14-LT54810.1</td><td>1 ATG GTG CTG TCT CCT GCC GAC AAG ACC AAC GTC AAG GCC GCC TGG GGT AAG GTC GGC GCG CAC GCT GGC GAG TAT GGT GCG 81 1 Met Val Leu Ser Pro Ala Asp Lys Thr Asn Val Lys Ala Ala Trp Gly Lys Val Gly Ala His Ala Gly Glu Tyr Gly Ala 27 ATGGTGCTGTCTCCTGCCGACAAGACCAACGTCAAGGCCGCCTGGGGTAAGGTCGGCGCGCACGCTGGCGAGTATGGTGCGMVLSPADKTNVKAAWGKVGAHAGEYG</td></tr></tbody></table>

Physeter catodon; P41329.2 Arctocephalus galapagoensis; P10892.1 Lutra lutra; P01956.1 Rousettus aegyptiacus; P60530.2 Canis latrans; P15163.1 Leptonychotes weddellii; P01951.1 Talpa europaea; P18976.1 Panthera pardus saxicolor; B3EWC7.1 Tamias merriami; P01957.1 Procavia capensis habessinica; Q7M2Y4.1 Chalinolobus morio; P10778.1 Odobenus rosmarus divergens; P11755.1 Tadarida brasiliensis; P09839.1 Macrotus californicus; P01953.1 Meles meles; P08852.1 Marmota marmota marmota; P15448.1 Mellivora capensis; P14389.1 Pteropus alecto; B3EWD9.1 Tamias striatus; P18971.2 Balaenoptera acutorostrata; P19645.1 Paguma larvata; V00493.1 Homo sapiens; AF349571.1 Homo sapiens; AK223392.1 Homo sapiens; Q862A7.3 Pipistrellus-abramus; P01958.2 Equus-caballus; P07402.1 Macaca-speciosa; P01939.3 Otolemur-crassicaudatus;

P11757.1 Myotis-velifer; P01962.1 Tapirus-terrestris; P01937.1 Nycticebus-coucang; Q9XSE9.3 Equus-przewalskii; P01940.1 Tarsius-bancanus; P18970.2 Ailuropoda-melanoleuca; P28780.1 Taphozous-georgianus; P01938.2 Loris-tardigradus; P11753.1 Cynopterus-sphinx; P08258.1 Mandrillus-sphinx; P68235.2 Ursus-maritimus; P01948.2 Oryctolagus-cuniculus; P18975.2 Panthera-leo; P14259.2 Otolemur-crassicaudatus; P01960.2 Equus-zebra; P18969.2 Ailurus-fulgens; P01933.1 Cercacebus-atys; P09908.1 Phoca-vitulina; P09906.1 Rhinoceros-unicornis; P41330.2 Arctocephalus-galapagoensis; P01961.3 Equus-hemionus-kulan; P24659.1 Macroderma-giga; P20854.1 Ctenodactylus-gundi; P07405.1 Felis-catus; P09904.1 Physeter-catodon; P41329.2 Arctocephalus-galapagoensis; P10892.1 Lutra-lutra; P01956.1 Rousettus-aegyptiacus; P60530.2 Canis-latrans;
<figure><img src="image_11.png" /><figcaption>**Figure 8** Restriction map performed by Serial Cloner software 2.6.1.</figcaption></figure>
P15163.1 Leptonychotes-weddellii; P01951.1 Talpa-europaea; P18976.1 Panthera-pardus-saxicolor; B3EWC7.1 Tamias-merriami; P01957.1 Procavia-capensis-habessinica; Q7M2Y4.1 Chalinolobus-morio; P10778.1 Odobenus-rosmarus-divergens; P11755.1 Tadarida-brasiliensis; P09839.1 Macrotus-californicus; P01953.1 Meles-meles; P08852.1 Marmota-marmota-marmota; P15448.1 Mellivora-capensis; P14389.1 Pteropus-alecto; B3EWD9.1 Tamias-striatus; P18971.2 Balaenoptera-acutorostrata and P19645.1 Paguma-larvata.

The restriction maps of the four nucleotide sequences for the used fragments (81 nucleotides) which represent the AF349571.1; AK223392.1; LT548101.1 and V00493.1 were used against the restriction enzyme database in each of Clone Manager version 4.01 software [18], pDRAW32.10 software [19] and Serial Cloner software 2.6.1.[20]. The results were summarized in **Figures 4 and 5** and in **Table 2**. The results show different enzyme profiles. The most interesting result is the appearance of new enzymes that

<table><caption>**Table 2** Restriction map performed by Clone 4 software.</caption><thead><tr><th>V00493.1</th><th>LT548101.1</th><th>AF349571.1</th><th>AK223392.1</th></tr></thead><tbody><tr><td>Accll 1 57</td><td>Accll 1 57</td><td>Accll 1 57</td><td>Accll 1 57</td></tr><tr><td>Acil 1 38</td><td>Acil 1 38</td><td>Acil 1 38</td><td>Acil 1 38</td></tr><tr><td>Aeul 1 41</td><td>Aeul 1 41</td><td>Aeul 1 41</td><td>Aeul 1 41</td></tr><tr><td>Aorl 1 41</td><td>Aorl 1 41</td><td>Aorl 1 41</td><td>Aorl 1 41</td></tr><tr><td>Apyl 1 41</td><td>Apyl 1 41</td><td>Apyl 1 41</td><td>Apyl 1 41</td></tr><tr><td>AtuBl 1 41</td><td>AtuBl 1 41</td><td>AtuBl 1 41</td><td>AtuBl 1 41</td></tr><tr><td>BceRI 1 57</td><td>BceRI 1 57</td><td>BceRI 1 57</td><td>BceRI 1 57</td></tr><tr><td>Bepl 1 57</td><td>Bepl 1 57</td><td>Bepl 1 57</td><td>Bepl 1 57</td></tr><tr><td>BsaJI 1 41</td><td>BsaJI 1 41</td><td>BsaJI 1 41</td><td>BsaJI 1 41</td></tr><tr><td>BsmAl 1 9</td><td>BsmAl 1 9</td><td>BsmAl 1 9</td><td>BsmAl 1 9</td></tr><tr><td>Bsp211l 1 36</td><td>Bsp211l 1 36</td><td>Bsp211l 1 36</td><td>Bsp211l 1 36</td></tr><tr><td>BspNI 1 41</td><td>BspNI 1 41</td><td>BspNI 1 41</td><td>BspNI 1 41</td></tr><tr><td>BspRI 1 36</td><td>BspRI 1 36</td><td>BspRI 1 36</td><td>BspRI 1 36</td></tr><tr><td>BspWI 1 47</td><td>BspWI 1 47</td><td>BspWI 1 47</td><td>BspWI 1 47</td></tr><tr><td>BssHII 1 56</td><td>BssHII 1 56</td><td>BssHII 1 56</td><td>BssHII 1 56</td></tr><tr><td>BstGII 1 41</td><td>BstGII 1 41</td><td>BstGII 1 41</td><td>BstGII 1 41</td></tr><tr><td>BstNI 1 41</td><td>BstNI 1 41</td><td>BstNI 1 41</td><td>BstNI 1 41</td></tr><tr><td>BstUI 1 57</td><td>BstUI 1 57</td><td>BstUI 1 57</td><td>BstUI 1 57</td></tr><tr><td>BsuRI 1 36</td><td>BsuRI 1 36</td><td>BsuRI 1 36</td><td>BsuRI 1 36</td></tr><tr><td>Cfol 2 56, 58</td><td>Cfol 2 56, 58</td><td>Cfol 2 56, 58</td><td>Cfol 2 56, 58</td></tr><tr><td>Ctl 1 36</td><td>Ctl 1 36</td><td>Ctl 1 36</td><td>Ctl 1 36</td></tr><tr><td>Cthll 1 41</td><td>Cthll 1 41</td><td>Cthll 1 41</td><td>Cthll 1 41</td></tr><tr><td>CviJl 1 36</td><td>CviJl 1 36</td><td>CviJl 1 36</td><td>CviJl 1 36</td></tr><tr><td>Dsall 1 36</td><td>Dsall 1 36</td><td>Dsall 1 36</td><td>Dsall 1 36</td></tr><tr><td>DsaV 1 41</td><td>DsaV 1 41</td><td>DsaV 1 41</td><td>DsaV 1 41</td></tr><tr><td>EcoRll 1 41</td><td>EcoRll 1 41</td><td>EcoRll 1 41</td><td>EcoRll 1 41</td></tr><tr><td>Fbrl 1 37</td><td>Fbrl 1 37</td><td>Fbrl 1 37</td><td>Fbrl 1 37</td></tr><tr><td>Fnu4H 1 37</td><td>Fnu4H 1 37</td><td>Fnu4H 1 37</td><td>Fnu4H 1 37</td></tr><tr><td>FnuDI 1 36</td><td>FnuDI 1 36</td><td>FnuDI 1 36</td><td>FnuDI 1 36</td></tr><tr><td>FnuDII 1 57</td><td>FnuDII 1 57</td><td>FnuDII 1 57</td><td>FnuDII 1 57</td></tr><tr><td>FnuDIII 2 56, 58</td><td>FnuDIII 2 56, 58</td><td>FnuDIII 2 56, 58</td><td>FnuDIII 2 56, 58</td></tr><tr><td>HaeIII 1 36</td><td>HaeIII 1 36</td><td>HaeIII 1 36</td><td>HaeIII 1 36</td></tr><tr><td>Hhal 2 56, 58</td><td>Hhal 2 56, 58</td><td>Hhal 2 56, 58</td><td>Hhal 2 56, 58</td></tr><tr><td>HinPl 2 56, 58</td><td>HinPl 2 56, 58</td><td>HinPl 2 56, 58</td><td>HinPl 2 56, 58</td></tr><tr><td>Maell 1 29</td><td>Maell 1 29</td><td>Maell 1 29</td><td>Maell 1 29</td></tr><tr><td>Msp67l 1 41</td><td>Msp67l 1 41</td><td>Msp67l 1 41</td><td>Msp67l 1 41</td></tr><tr><td>Mval 1 41</td><td>Mval 1 41</td><td>Mval 1 41</td><td>Mval 1 41</td></tr><tr><td>Mvnl 1 57</td><td>Mvnl 1 57</td><td>Mvnl 1 57</td><td>Mvnl 1 57</td></tr><tr><td>NgoPl 1 36</td><td>NgoPl 1 36</td><td>NgoPl 1 36</td><td>NgoPl 1 36</td></tr><tr><td>Pall 1 36</td><td>Pall 1 36</td><td>Pall 1 36</td><td>Pall 1 36</td></tr><tr><td>SciNI 2 56, 58</td><td>SciNI 2 56, 58</td><td>SciNI 2 56, 58</td><td>SciNI 2 56, 58</td></tr><tr><td>ScrFl 1 41</td><td>ScrFl 1 41</td><td>ScrFl 1 41</td><td>ScrFl 1 41</td></tr><tr><td>Secl 1 41</td><td>Secl 1 41</td><td>Secl 1 41</td><td>Secl 1 41</td></tr><tr><td>Sfal 1 36</td><td>Sfal 1 36</td><td>Sfal 1 36</td><td>Sfal 1 36</td></tr><tr><td>Ssoll 1 41</td><td>Ssoll 1 41</td><td>Ssoll 1 41</td><td>Ssoll 1 41</td></tr><tr><td>Sual 1 36</td><td>Sual 1 36</td><td>Sual 1 36</td><td>Sual 1 36</td></tr><tr><td>TaqXI 1 41</td><td>TaqXI 1 41</td><td>TaqXI 1 41</td><td>TaqXI 1 41</td></tr><tr><td>Thal 1 57</td><td>Thal 1 57</td><td>Thal 1 57</td><td>Thal 1 57</td></tr><tr><td>Zanl 1 41</td><td>Zanl 1 41</td><td>Zanl 1 41</td><td>Zanl 1 41</td></tr></tbody></table>
could cut in the nucleotide sequence of V00493.1 sequence as in **Figures 4 and 5** and in **Table 2**. Those new restriction enzymes might suggest the existence of α⁰ globin or reduced α⁺ structure. That might happened at the gene level and represented after the protein expression. The *Homo sapiens* 27 amino acids fragment sequence of AF349571.1, AK223392.1, V00493.1 and Q862A7.3 3D model was built using Modeller v 9.8 software and Discovery Studio 4.1. Client 4.1.0.14169 (Accelrys software Inc.) software. Their 3D structures and their hydrophobicity surface show differences due to their amino acids constituents as in **Figures 9 and 10**.

## Conclusion

In conclusion, this study proves the existence of the prototype mutants in the *Homo sapiens* and other species of the α globin. The prototype mutants present in full mutants.

The protein structure is modified due to such mutants as well as the restriction map. The existence of the homology between *Homo sapiens* and the other species particularly the blood transferring ones suggest gene transfer and recombination between species. The facts and the suggestions and the results obtained from this

<figure><img src="image_12.png" /><figcaption>**Figure 9** α globin protein 3D model.</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>**Figure 10** Protein fragments models (AF349571.1, AK223392.1, V00493.1, Q862A7.3) and surface analysis for the hydrophobicity.</figcaption></figure>

preliminary study need more prove at the cell level to prove or disprove their correction however, they highlight the power of the bioinformatics as science to touch some scientific facts by linking finding and the probability to things one could find in our life but not proved scientifically yet.

## Conflict of Interest

The authors have no conflict of interests to declare.

## Financial Disclosure

Institutional support

## Acknowledgement

The author acknowledges the City of Scientific Research and Technological Applications for supporting this work.

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