Introduction
----------------------------------------------
----------------------------------------------
Overview
Oxygen is an essential molecule to most living things on Earth. Hemoglobin molecules function to assist in utilization of this molecule in different ways (Hardison, 1998). In plants, hemoglobins are used for intracellular transport of oxygen within the electron transport chain (Hardison, 1998). Bacteria Fungi and Protists also use these molecules intracellularly to transfer oxygen and other molecules in respiratory chain reactions (Hardison, 1998). In multicellular organisms like birds and mammals, hemoglobin is used for the transfer of oxygen from tissue to tissue via the blood stream. When blood is transported to the lungs, the hemoglobin molecules located in red blood cells bind oxygen molecules. From the lungs, these oxygenated red blood cells are transported to all the tissues of the body.
The human hemoglobin molecule is constructed of 2 types of subunits, the α and β-globin subnits. The human α globin and β-globin subunit genes are located on two different chromosomes, 16 and 11 respectively (Protein-NCBI, 2011a; Protein-NCBI, 2011b). These two genes are said to have descended from a common ancestor approximately 450 million years ago from the jawed vertebrates (Hardison, 1998). For the purpose of this annotation, the human β-globin gene (HBB) and protein product will be explored. Topics such as gene location, gene and protein structure, mutations and disease will be explored. A phylogenetic analysis of the HBB gene will also be conducted to study homology between different groups of living organisms. NCBI Blast searches (Altschul et al., 1990), a
multiple alignment tool called ClustalW (Larkin et al., 2007) and the phylogenetic analysis tool, Phylip (U of W, Phylip, 2011) will be used to conduct this analysis. We hypothesize that Human Beta globulin will be well conserved throughout mammals, birds and amphibians; with mammals and amphibian being more closely related to birds, and most distant from each other. The furthest from Human Beta globulin will be the nematode, yeast, and bacterial genes.
The human hemoglobin molecule is constructed of 2 types of subunits, the α and β-globin subnits. The human α globin and β-globin subunit genes are located on two different chromosomes, 16 and 11 respectively (Protein-NCBI, 2011a; Protein-NCBI, 2011b). These two genes are said to have descended from a common ancestor approximately 450 million years ago from the jawed vertebrates (Hardison, 1998). For the purpose of this annotation, the human β-globin gene (HBB) and protein product will be explored. Topics such as gene location, gene and protein structure, mutations and disease will be explored. A phylogenetic analysis of the HBB gene will also be conducted to study homology between different groups of living organisms. NCBI Blast searches (Altschul et al., 1990), a
multiple alignment tool called ClustalW (Larkin et al., 2007) and the phylogenetic analysis tool, Phylip (U of W, Phylip, 2011) will be used to conduct this analysis. We hypothesize that Human Beta globulin will be well conserved throughout mammals, birds and amphibians; with mammals and amphibian being more closely related to birds, and most distant from each other. The furthest from Human Beta globulin will be the nematode, yeast, and bacterial genes.
HBB Gene Location
The genomic sequence of the HBB gene was retrieved using the Nucleotide data base offered on the NCBI website (Nucleotide, NCBI- Human Beta Globin Genomic Sequence, 2011). The map view of the data base was used to acknowledge the location of the gene on chromosome 11 at locus 11p 15.5 (Fig. 1) (NCBI Map Viewer- Human HBB gene locus, 2011). The entire genomic code of the beta globin gene family was retrieved from Nucleotide
(NG_000007.3).
(NG_000007.3).
Figure 1. Ideogram of Human Chromosome 11. The red mark in the diagram indicates the location of the HBB gene. (NCBI Map Viewer- Human HBB gene locus, 2011)
HBB Gene Structure
The HBB gene family is located at locus 11p 15.5 and is comprised of 5 functional genes and 1 pseudogene, 5’-epsilon, gamma G, gamma A, beta 1 pseudogene, delta, beta-3’ (Fig. 1) (Nucleotide, NCBI- Human Beta Globin Genomic Sequence, 2011)(Gene, NCBI- Human Beta globin ID 3043, 2011). The genes of this family are expressed at different stages of development.
Figure 1: Human HBB gene cluster. (Hardison, 1998)
HBB Gene Expression
The normal adult hemoglobin tetramer consists of two alpha chains and two beta chains. These large family of proteins related to hemoglobin’s are encoded at five different chromosomal locations in humans. For example the α-like globin gene is expressed at chromosomal location 16p13.3. The β-like globin gene is expressed at chromosomal location 11p15.5 (UCSC Genomic Bioinformatics, 2011). In humans the expression of α-like and β-like globin genes between multigene complexes is coordinated so that equal amounts of
the two types of globins are produced in all erythroid cells.
Not all genes of the HBB gene family are expressed or functional in humans at one point in development. Because of this, mutations can occur in
almost every amino acid position in the HBB and HBA genes (Hardison, 2008). Both gamma globin and the single epsilon genes are expressed in early embryonic and fetal erythroid tissues. In adult life the beta and delta globin genes are expressed (Entrez, 2011). All these functional or active genes have at least three exons and two introns which are found in the erythroid globin
genes.
the two types of globins are produced in all erythroid cells.
Not all genes of the HBB gene family are expressed or functional in humans at one point in development. Because of this, mutations can occur in
almost every amino acid position in the HBB and HBA genes (Hardison, 2008). Both gamma globin and the single epsilon genes are expressed in early embryonic and fetal erythroid tissues. In adult life the beta and delta globin genes are expressed (Entrez, 2011). All these functional or active genes have at least three exons and two introns which are found in the erythroid globin
genes.
Beta Globin Protein Structure
The messenger RNA (mRNA) code was also located in the Nucleotide database and is 626 nucleotides in length (NM_000518.4) (Nucleotide, NCBI- Human HBB mRNA, 2011). After the introns are spliced out of the pre-mRNA, the mature mRNA is translated into the human β-globin polypeptide which is 147 amino acids in length (NP_000509.1)(Protein-NCBI, 2011b). The human β-globin’s secondary structure is in the form of multiple alpha helices attached by non-helical segments (Fig. 1). The helical segments are formed by hydrogen bonds (Hardison, 1996). Normal folding of β-globin into this alpha helix allows for the formation of the tertiary structure of each of the globin molecules.
Figure 1: Beta Globin Molecule
The quaternary structure produces the mature hemoglobin molecule containing two α-globin chains and two β-globin chains. An iron containing heme molecule is tightly bound to each polypeptide. One oxygen molecule binds to each heme molecule (Hardison, 1998; US Library of Medicine, 2011) (Fig.
2).
2).
Figure 2: Quaternary Protein Structure of Human Hemoglobin. (MNIMBS, 2010)
Red Molecules represent α-globin
Blue molecules represent β-globin
Green molecules symbolize heme
Blue molecules represent β-globin
Green molecules symbolize heme
HBB Mutations
Beta-thalassemia
Beta-thalassemia is a result of deletions in the HBB gene. This deletion can encompass only the β-globin gene or it can include the whole β-globin gene cluster (OMIM - Beta-Thalassemia, MIM# 613985, 2011). Beta-zero-thalassemia results when there are no detectable β- globin chains present. When there are low detectable amounts of the β-globin chains available, the condition is called Beta-plus-thalassemia. The absence or low concentration of β-globin causes the subsequent decrease in the normal complete human Hemoglobin molecule (HbA) and hence anemia in the patient (OMIM - Beta-Thalassemia, MIM# 613985,
2011). The most common populations affected are people of the Middle East, Mediterranean, India, Far East, Central Asia and Transcaucasus (OMIM - Beta-Thalassemia, MIM# 613985, 2011).
Sickle Cell Anemia
Sickle cell anemia is a growing health concern because statistics have shown that approximately 7% of the population has the mutation and between 300,000 and 400,000 affected children are born each year (Rusanova et al., 2011). It is the most common disorder caused by a single nucleotide change (GAG → GTG) resulting in the sixth amino acid change from glutamic acid to valine (Rusanova et al., 2011; Kitayama Cabral et al., 2011; Ashley-Koch et al., 2000). This mutation results in a gene product called HbS which is a malformed protein (Ashley-Koch et al., 2000). The HbS protein subunits do not fold properly and form a long, rigid molecule which forces the red blood cells into a sickle shape (Fig 1). Primarily, these sickle shaped blood cells prematurely die and cause anemia. Affected blood cells also block small blood vessels and cause organ damage and pain (Ashley-Koch et al., 2000; US Library of Medicine, 2011; Nucleotide, NCBI- Human HBB mRNA, 2011; OMIM - Sickle Cell Anemia, MIM# 603903, 2011).
Beta-thalassemia is a result of deletions in the HBB gene. This deletion can encompass only the β-globin gene or it can include the whole β-globin gene cluster (OMIM - Beta-Thalassemia, MIM# 613985, 2011). Beta-zero-thalassemia results when there are no detectable β- globin chains present. When there are low detectable amounts of the β-globin chains available, the condition is called Beta-plus-thalassemia. The absence or low concentration of β-globin causes the subsequent decrease in the normal complete human Hemoglobin molecule (HbA) and hence anemia in the patient (OMIM - Beta-Thalassemia, MIM# 613985,
2011). The most common populations affected are people of the Middle East, Mediterranean, India, Far East, Central Asia and Transcaucasus (OMIM - Beta-Thalassemia, MIM# 613985, 2011).
Sickle Cell Anemia
Sickle cell anemia is a growing health concern because statistics have shown that approximately 7% of the population has the mutation and between 300,000 and 400,000 affected children are born each year (Rusanova et al., 2011). It is the most common disorder caused by a single nucleotide change (GAG → GTG) resulting in the sixth amino acid change from glutamic acid to valine (Rusanova et al., 2011; Kitayama Cabral et al., 2011; Ashley-Koch et al., 2000). This mutation results in a gene product called HbS which is a malformed protein (Ashley-Koch et al., 2000). The HbS protein subunits do not fold properly and form a long, rigid molecule which forces the red blood cells into a sickle shape (Fig 1). Primarily, these sickle shaped blood cells prematurely die and cause anemia. Affected blood cells also block small blood vessels and cause organ damage and pain (Ashley-Koch et al., 2000; US Library of Medicine, 2011; Nucleotide, NCBI- Human HBB mRNA, 2011; OMIM - Sickle Cell Anemia, MIM# 603903, 2011).
Figure 1: Sickle cell and normal phenotype of the human red blood cell. (Gabriel, Abram M.D. & Jennifer Przybylski, 2010)
Individuals whom carry only one HbS allele have what is called sickle cell trait. In this state, individuals do not express sickle cell but are carriers of the disease. Individuals with two copies of the HbS gene are affected by the disease (Ashley-Koch et al., 2000; OMIM - Sickle Cell Anemia, MIM# 603903, 2011). Specifically, there are different haplotypes, or specific mutations recognized by restriction endonucleases in the beta gene cluster (Kitayama Cabral et al., 2011; Ashley-Koch et al., 2000; Cardoso and Guerreiro, 2010). The most commonly occurring haplotypes are named after the location of which they were discovered: Bantu (or Central African Republic (CAR)), Senegal, Saudi Arabia-India, Benin and Camaroon (Fig. 2) (Kitayama Cabral et al., 2011; Ashley-Koch et al., 2000; Gabriel, Abram M.D. & Jennifer Przybylski, 2010; Cardoso and Guerreiro, 2010).
Figure 2: Distribution of SCA haplotypes in Africa and surrounding areas. Individual pie charts for each area studied represent relative frequencies of the haplotypes. CAR = Central African Republic. (Gabriel, Abram M.D. & Jennifer Przybylski, 2010)
Haplotypes linked to the HbS allele assist in predicting sickle cell anemia complications in patients (Cardoso and Guerreiro, 2010). Patients with the Bantu (CAR) haplotype are present with the most severe clinical symptoms, whereas the Senegal and Arab-India haplotypes have the mildest symptoms. The Benin and Cameroon haplotypes are intermediate in severity (Kitayama Cabral et al., 2011; Cardoso and Guerreiro, 2010). The gradation regarding symptom severity is closely tied to the fetal hemoglobin levels in the blood, where the Senegal and Arab-India haplotypes produce the highest levels and the Bantu mutation presents with the lowest concentration in the blood (Kitayama Cabral et al., 2011). Fetal hemoglobin levels are thought to neutralize the negative effects of the mutated beta globin gene product (Gabriel, Abram M.D. & Jennifer Przybylski, 2010).
Medicines and Genetic
Treatments
To combat sickle cell disease, medicines are being tested to increase the fetal hemoglobin levels of gamma globin molecules found in the beta globin gene cluster. Chemicals, such as hydroxyurea (aka hyroxycarbamide) and Azacytamide, have been shown to boost the levels of gamma globin and in turn also prevent polymerization of the HbS protein subunits and hence sickling of the blood cells (OMIM - Sickle Cell Anemia, MIM# 603903, 2011).
Genetic strategies have also been tested to treat the diseases associated with mutations and deletions of the human HBB gene. These methods include: 1) additions of an “HBB like gene” to haematopoietic stem cells to use for bone marrow transplantation, 2) of the endogenous HBG gene (gamma globin) using oligonucleotides
instead of chemicals, 3) targeting the mutant beta globin transcript by strategies, such as RNA interference and 4) gene repair or addition at the HBB gene by homologous recombination (Mansilla-Soto et al,
2011).
The most promising strategy to be used in clinical trials has been shown to be the transduction of the HBB like gene into haematopoietic stem cells (HSC- stems cells that give rise to all types of blood cells) (Mansilla-Soto et al, 2011). However, these studies have demonstrated that there are possible side effects and risks. Insertional oncogenesis has occurred with the use of retroviral vectors which can result in the development of leukemia. The patient may also reject the HSC transplantation. Further studies need to be completed to make transduction of the HBB like gene safer by using lentiviral vectors, instead of retroviruses. This vector would provide a more specific gene insertion and less risks resulting in oncogenesis (Mansilla-Soto et al, 2011). Furthermore, studies using pluripotent stems cells may be a more effective area of study, providing they will be approved for clinical use (Mansilla-Soto et al, 2011).
Medicines and Genetic
Treatments
To combat sickle cell disease, medicines are being tested to increase the fetal hemoglobin levels of gamma globin molecules found in the beta globin gene cluster. Chemicals, such as hydroxyurea (aka hyroxycarbamide) and Azacytamide, have been shown to boost the levels of gamma globin and in turn also prevent polymerization of the HbS protein subunits and hence sickling of the blood cells (OMIM - Sickle Cell Anemia, MIM# 603903, 2011).
Genetic strategies have also been tested to treat the diseases associated with mutations and deletions of the human HBB gene. These methods include: 1) additions of an “HBB like gene” to haematopoietic stem cells to use for bone marrow transplantation, 2) of the endogenous HBG gene (gamma globin) using oligonucleotides
instead of chemicals, 3) targeting the mutant beta globin transcript by strategies, such as RNA interference and 4) gene repair or addition at the HBB gene by homologous recombination (Mansilla-Soto et al,
2011).
The most promising strategy to be used in clinical trials has been shown to be the transduction of the HBB like gene into haematopoietic stem cells (HSC- stems cells that give rise to all types of blood cells) (Mansilla-Soto et al, 2011). However, these studies have demonstrated that there are possible side effects and risks. Insertional oncogenesis has occurred with the use of retroviral vectors which can result in the development of leukemia. The patient may also reject the HSC transplantation. Further studies need to be completed to make transduction of the HBB like gene safer by using lentiviral vectors, instead of retroviruses. This vector would provide a more specific gene insertion and less risks resulting in oncogenesis (Mansilla-Soto et al, 2011). Furthermore, studies using pluripotent stems cells may be a more effective area of study, providing they will be approved for clinical use (Mansilla-Soto et al, 2011).
Globin Gene Family
The HBB gene is classified in the globin gene family. The Globin gene family consists of proteins which contain 8 alpha helical segments which bind heme and are associated with reversible oxygen binding and transport (Pfam Globin PF00042). There are 2 main types of globins which span the three kingdoms of life (Bacteria, Archaea and Eukaryotes): single domain globins and two types of chimeric globins (Vinogradov et al., 2006; Vinogradov et al., 2007). Flavohaemoglobins and globin coupled sensors comprise the types of globin molecules found in the chimeric globins group. Bacteria utilize all 3 types of globins, while organisms in the archaea group use single domain globins and globin coupled sensors. Eukaryotes make use of the Flavohaemoglobins and single domain globins(Vinogradov et al., 2006).Flavohaemoglobins are said to give unicellular organisms protection again nitric oxide and other reactive nitrogen species (Mukai et al., 2001). Globin coupled sensors are said to be chimeric two domain regulators containing heme that were found to be involved in gene or aerotactic regulation (Freitas et al, 2005). Proteoglobins are a group of globin coupled sensors that have been proposed as the ancestral globin molecule(Vinogradov et al., 2006).