Human Genome Project Using PCR - Dissertation Example

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Human Genome Project Using PCR Inserts Insert Grade Insert 25th April, Contents Annexin A5 Gene…………………………………………………………………………………...……………..3 Structural and functional characterization of the ANXA5 promoter haplotypes……….…………........................6 Functions of Extracellular Annexin A5 ………………………………………..………………………………….7 The Annexin family…………………………………………………….…………….…………………………….8 Structures of Annexin Protein Cores: The Conserved Membrane Binding Modules……………………………..10 Ca2+ Dependent Phospholipid Binding…………………………………………………………………………….12 Ca2+ Independent Phospholipid Binding……………………….……………………………………......................13 Annexin A5 interaction with cytoskeletal proteins………………………...………………………………………14 Annexin A5 and Intracellular Signaling…………………………...………………………………….....................14 Annexin A5 and Protein Kinase C (PKC…………………………………………………………………………...15 Annexin A5 and Human Disease……………………………………………………………………………………15 Recurrent Pregnancy Loss (RPL) ……………………………………………………………….………………….16 Venous Thromboembolism (VTE)………………………………………………………………………………….18 Heart Disease ……………………………………………………………………………………………………….19 Antiphospholipid (aPL) Syndrome …………………………………………………………………………………20 Carcinomas …………………………………………………………………………………………………………21 Cellular Aging ………………………………………………………………………………………………………22 Conclusion …………………………………………………………………………………………………………..23 References …………………………………………………………………………………………………………..24 Annexin 5 (ANXA5) Gene Annexin A5 (ANXA5) is a protein encoding gene which belongs to the annexin family of calcium-dependent phospholipid binding proteins. These proteins take part in endocytotic and exocytotic pathways as well as membrane-related events. Annexin 5 is shows inhibitory effects towards phospholipase A2 and protein kinase C (PKC). This gene is involved in numerous roles including signal transduction, growth, inflammation and differentiation of cells (Bogdanova, Horst, Chlystun, Croucher, Nebel, Bohring, et al. 2010). Additionally, annexin A5 has been reported to be a vascular anticoagulant-?, placental anticoagulant protein I, liporcotin V, endonexin II, anchorin CII and placental protein 4. The annexin A5 gene spans 29 kilobases and has 13 exons which encode a single transcript of approximately 1.6 kb. The protein product of this gene has a molecular weight of approximately 35 kDa (Carcedo, Iglesias, Bances, Morgan & Fernandez, 2011). Official name of the gene: annexin A5 Official symbol: ANXA5 Gene type: protein coding Other names: PP4, ANX5, ENX2, RPRGL3 Chromosomal location: 4q27 (http://www.ncbi.nlm.nih.gov). Annexin A5 is a classical member of the chordate annexin family. It displays essential tetra structure and calcium-dependent phospholipid binding. It is one of the few annexins that can be found within the extracellular matrix. Annexin A5 has been postulated to be an inhibitor of coagulation due to its ability to attach to anionic phospholipids exposed of surfaces of platelets (Gerke & Moss, 2010). Consequently, this important gene inhibits platelet aggregation and down regulates surface presentation of tissue factor. The annexin A5 gene covers approximately 9 kb of the human chromosome 4q27. It comprises of one non-translated exon and 12 coding exons. Little is known about the regulation of the annexin A5 gene expression. Annexin A5 is an abundantly and ubiquitously expressed protein with highest concentrations in the liver, placenta and kidney. The human annexin A5 gene produces numerous transcripts and has a complex promoter which is subject to intricate regulation mechanism (Gerke & Moss, 2010). Annexin A5 has been implicated in a wide range of disease phenotypes and etiology including recurrent pregnancy losses and cancer. It is a calcium and phospholipid protein localized within the surface of the placental syncytiotrophoblast layer. This protein performs vital anticoagulant function within the maternal blood and specifically at the intervillous space. Recently, it has been reported that polymorphisms in the promoter region of the annexin A5 gene can significantly be linked to recurrent pregnancy loss (RPL). Particularly, women who possess the M2 haplotype have more than two-fold higher risk of fetal loss compared to non-carriers (Bogdanova, Horst, Chlystun, Croucher, Nebel, Bohring, et al. 2010). Structural and functional characterization of the ANXA5 promoter haplotypes Comparisons of structures of annexin A5 orthologues derived form human, mouse, chick and rat have aided in the determination of conserved regulatory motifs as well as coding regions. Conversely, the functional active chains are yet to be characterized. The determination of important cis-acting genomic elements and localization of trans-acting protein factors have been useful. This approach is very crucial in comprehending the molecular modulation strategies of the gene. Additionally, this strategy may help in elucidating the involvement of the gene in cell growth and differentiation (Gerke & Moss, 2010). Annexin A5 and annexin A6 share almost similar ancestry. However, these two annexin genes have retained no evident homogeneity in non-coding sequences. Annexin A5 lacks a TATA box or initiator factor and it contains a high content of GC-rich residues that possess the putative Sp1 binding elements. Annexin A5 shows marked disparity compared to annexin A6 in it transcriptional initiation unit. This particular gene contains various multiple transcription initiation site hence its transcriptional regulatory mechanisms are not well understood (Carcedo, Iglesias, Bances, Morgan & Fernandez, 2011). Analysis of the entire exons, exon-intron boundaries and some 270 bp of the 5’ –untranslated region (5’-UTR) show a set of four consecutive nucleotide substitutions in the ANXA5 gene promoter. These single nucleotide substitutions include 19G - A, 1A - C, 27T - C and 76G – A. This numbering of the nucleotide refers to the first transcription start point of the gene, tsp1 (Carcedo, Iglesias, Bances, Morgan & Fernandez, 2011). Two of the four variants constitute the M1 haplotype, which essentially is the wild type and include 1A – C and 27T – C. On the hand, the M2 haplotype comprise four nucleotide substitutions exemplified by variations in 19G – A, 1A – C, 27T – C and 76G – A (Bogdanova, Horst, Chlystun, Croucher, Nebel, Bohring, et al. 2010).All these alterations in the bases changed a transcription factor consensus site or influenced the adjacent nucleotide. For instance, the 19G – A substitution adjoins a gGCCc consensus for the MTF-1 transcription factor in the location of zinc finger. The transcription start site, tsp1which is altered by the mutation on location 1A – C in both variant haplotypes appears in close propinquity to an HNF-3 consensus. The 27T – C alteration interrupts the restriction location of BamHI in the immediate surrounding of an AP4/MED-1 consensus. This effects has been reported to be indispensable for the promoter action of ANXA5 (Bogdanova, Horst, Chlystun, Croucher, Nebel, Bohring, et al. 2010). Figure 1: ANXA5 gene promoter variants. Exons are indicated by boxes and the coding region is denoted by a black box. Vertical arrows indicate the location of six common SNPs (thick arrows) and three rare SNPs (thin arrows). Triangles indicate the position recognized by primers used to amplify the promoter region. The horizontal short bar indicates the regions associated with the Taqman primers and probe. Functions of Extracellular Annexin A5 A number of roles have been put forward for extracellular annexin A5. Initially, this molecule was described as an anticoagulant protein and this action is probably dependent on the calcium ion modulated binding to negatively charged phospholipids. Such anionic phospholipids tend to be the ones that are expressed on the surface of stimulated thrombocytes or endothelial cells (Gerke & Moss, 2010). The association could hinder with the accessibility of such locations for coagulation factors hence blocking local stimulation. Recently, antibody-mediated hindrance of an anticoagulant feature of annexin A5 has been postulated to occur in recurrent pregnancy losses (RPL) in individuals with antiphospholipid syndrome (Rand, Wu, Quinn & Taatjes, 2010). Annexin A5 is able to interact and bind to the apical surface of placental synsytiotrophoblasts. In so doing, annexin A5 provides a protective role by shielding the coagulation factors. Essentially, this role is critical in the maintenance of flow of blood to the placenta during pregnancy. Antibodies of ant-annexin A5 have been isolated from patients suffering from antiphospholipid syndrome (Gerke & Moss, 2010). These protective proteins have also been found in the sera of patients suffering from systemic lupus erythematosus (SLE). These antibodies reduce the capacity of annexin A5 to form a barrier on the trophoblast surface leading to placental thrombosis. In vitro experiments have shown that annexin A5 bind to the amino terminal domain of polycystin I. the main protein influenced in autosomal dominant polycystic kidney disease. Furthermore, Annexin A5 is capable of binding to numerous extracellular matrix components such as types II and IV collagens. This binding has been postulated to have a bearing in the manner in which annexin affects calcium uptake from vesicles derived from chondrocyte matrix (Bogdanova, Baleva, Kremensky & Markoff, 2012). The Annexin family The body system has achieved capability of tightly regulating the intracellular calcium ions concentration. As a result, these essential molecules serve the purpose of acting as second messengers in a wide range of processes that require coupled signals from extracellular matrix, hence inducing cellular responses. Therefore, body systems that modulate calcium ions intracellularly are considered to possess an elaborate calcium ions signalling network (Lizarbe, Barrasa, Olmo, Gavilanes, Turnay, 2013). These systems are exemplified by energy dependent pumps and gated calcium ion channels usually found in plasma and organelle membranes. Nonetheless, the intracellular calcium ion binding proteins serve to modulate calcium ion buffers. Other subgroups of intracellular calcium ion binding proteins have an active participatory role in calcium ion signalling. This characteristic is displayed by such groups because of their modified properties in response to calcium ions assembly (Bogdanova, Horst, Chlystun, Croucher, Nebel, Bohring, et al 2010). Annexins can be categorized as a subgroup of intracellular calcium ion binding proteins. However, their accurate position in calcium ions signalling domain is still elusive. Furthermore, amassing knowledge of proof suggest that annexins can also play a role in their Ca2+ free conformation. Despite their unknown mechanism in this role, the overall effect increases the functional diversity of these proteins (Lizarbe, Barrasa, Olmo, Gavilanes, Turnay, 2013). The term annexin is derived from a Greek terminology, annex which essentially means to hold or bring together. This is in tandem with the description of all or at least a number of the main property of annexins which bind to and hold biological structures, specifically membranes. Initially, during their discovery in early 1970s and 1980s, annexins were given several terminologies. These include synexin (granule aggregating protein), calcimedins (proteins mediating Ca2+ signals). Other names included chromobindins (proteins binding chromatic granules), calpactins (proteins that bind to phospholipids, Ca2+ and actin) as well as lipocortins (lipase inhibitors that are induced by steroids). Following thorough research on the protein, biochemistry and cDNA sequencing, these proteins were a multigene entity. This is because these proteins had similar properties biochemically as well as common structure of genes and sequence features (Carcedo, Iglesias, Bances, Morgan & Fernandez, 2011). In designation, an annexin protein requires two attributes for it to qualify as one. First, the annexin must have the capability of linking in a Ca2+ dependent method to negatively charged phospholipids. Secondly, the molecule must possess a conserved structural element referred to as the annexin repeat (Lizarbe, Barrasa, Olmo, Gavilanes, Turnay, 2013). Essentially this element is a segment of about 70 amino acids residues. Continued research in this important gene family has seen the advancement of knowledge in magnificent rates. Recently, knock-out representations both at animal and cellular levels have provided insights direct strategies in functional analyses of the proteins. This has also been boosted by the invention and use of dominant-negative mutant proteins. Such methods have underpinned the theory of functional diversity observed within the annexin family. Moreover, in the recent past, it has been made clear that specific dysregulations in expression of annexins and their activity can lead to various pathogenesis (Grandone, Tiscia, Colaizzo, Chinni, Pisanelli, Bafunno, et al. 2010). Structures of Annexin Protein Cores: The Conserved Membrane Binding Modules Each annexin is comprised of two main chains or domains namely; the conserved COOH (carboxyl) terminal protein core and the divergent NH2 (amino) terminal head. The carboxy terminal possesses the sites for calcium ion and membrane binding. It is also responsible for playing an intermediary role in the standard membrane binding properties. The annexin core consists of between for to eight segments of interannexin and internal homology depending on the annexin type. These segments can be easily identified in a linear sequence arrangement. The core is represented by a highly ?-helix and a rigidly grouped disk having a slight curvature as well as two major sides. The side having a convex sort of shape contains unique types for calcium ions binding sites, specifically those referred to as types II and III. This particular side also faces the membrane when the annexin is linked peripherally with phospholipids. The concave surface points away from the membrane. It appears easily reachable for interactions with the amino terminal chain and probably cytoplasmic binding associations (Carcedo, Iglesias, Bances, Morgan & Fernandez, 2011). Annexin A5 was the first member of the multigene family whose structural core was solved. Over the past years, researchers have showed more than 10 crystal structures of annexin cores. These structural studies have described the core as having an outstanding conservation of the overall 3-dimensional fold. Annexins that have been studied most recently and whose molecular elucidation has been described include those from lower eukaryotic organisms and plants. An incredible advancement in structural studies was the introduction of benzothiazepine and benzodiazepine derivatives as annexin ligands. Additionally, these benzyl derivatives were shown to co-crystallize with the annexins. The benzothiazepine K201 was initially reported to interact with annexin A5 and hinder Ca2+ action. Probably, this inhibition was attributed to the holding back of a hinge motion of the two Annexin A5 units formed by annexin repeats II/III and I/IV. The pharmacological roles of such interactions have not yet been reported (Carcedo, Iglesias, Bances, Morgan & Fernandez, 2011). Characterizing the contributions made by certain amino acid residues to the overall annexin structure through crystal structure elucidation and biochemical methods have been significant. Preserved arginine residues that occur in the so called endonexin fold of each homology section have been shown stabilize the tertiary structure of annexin A5. In contrast, when this amino acid residue is replaced by another residue such as alanine, the membrane binding property of the annexin is altered. This underpins the importance of these residues in arbitrating the intermolecular activities such as annexin-phospholipid contacts. Furthermore, site-directed mutagenesis has shown that the aspartate residue at position 226 of annexin A5 plays a role of a molecular switch. This alteration produces calcium ion and pH dependent conformation. Annexins are also substrates of protein kinase C (PKC) and in vitro studies of several gene families have indicated changes in conformation due to phosphorylation (Kheifets, Bright, Inagaki, Schechtman & Mochly-Rosen, 2009). Apart from crystallization techniques, different methods have been introduced to examine in specificity structural components of annexins particularly when they are bound to phospholipids and membranes. These techniques include atomic force microscopy (AFM) and cryoelectron microscopy. These techniques have enabled the elucidation of highly complex junctions of different annexins and high resolution analyses. Infrared spectroscopy of the membrane bound annexin A5 have indicated that a new ?- structure possessing interstrand hydrogen bonds is formed with interaction of a lipid monolayer (Irman, Skarabot, Musevic, Rozman & Bozic, 2011). Ca2+ Dependent Phospholipid Binding In biochemical reference, annexins can be described as soluble, hydrophobic proteins which interact with negatively charged phospholipids in a fashion that is dependent on calcium ions (Ca2+). This association with the phospholipid and calcium ions molecules can be reversed. Thus, getting rid of calcium ions through the use of Ca2+ chelating agents can lead to detachment of the annexins (Lizarbe, Barrasa, Olmo, Gavilanes, Turnay, 2013). The binding of negatively charged phospholipids to annexins has been hypothesized to resemble the interaction of cellular membranes and organelle membranes. This unique feature of annexins is inherent within the cores with the conserved annexin units representing the building blocks for peripheral binding. Despite having a common feature of phospholipid binding, each member of the annexin family show significant disparities towards Ca2+ sensitivity and phospholipid head group specificity. Annexin cores display specificity with regard to their membrane binding in living cellular components. However, there exists an additional level of such specificity and is related to unique amino terminal domains. Although properly articulated by in vitro experiments, the physiological relevance of Ca2+ dependent phospholipid binding is still not yet comprehensive (Kheifets, Bright, Inagaki, Schechtman & Mochly-Rosen, 2009). Interesting representations have been put forward to allocate functions to peripherally related and abundant membrane binding protein exemplified by annexin A5. In situ, annexin A5 is able to form two-dimensional crystals on a planar lipid bilayer which has negatively charged phospholipids. This conformation of crystalline structure shows impacts on the fluidity, rigidity and lipid segregation. Consequently, such a conformation allows the molecule to take part in the modulation and stabilization of membrane chains. This proposition has been illustrated by electron paramagnetic resonance spectroscopy which shows that Ca2+ dependent binding to annexin A5 parallels the rigidification aspect (Lizarbe, Barrasa, Olmo, Gavilanes, Turnay, 2013). Additionally, this technique has displayed that the binding of annexin A5 to T-cells surface delays programmed cell death a process called apoptosis. However, the mechanism for such an event remains elusive. It is thought that there is generation of a membrane constraint that hinders the release of CD4+ particles from the T-cell membranes. Moreover, this binding also affects the annexin protein because annexin A5 is thermodynamically a minor stable protein. Therefore, it is protected to a considerable degree from thermal destruction or inactivation by calcium ion dependent phospholipid interaction (Grandone, Tiscia, Colaizzo, Chinni, Pisanelli, Bafunno, et al. 2010). Ca2+ Independent Phospholipid Binding There exists another mechanism for binding of annexins to calcium ions- dependent phospholipid despite the criterion in biochemical characterizing of this multigene family. This atypical mechanism is Ca2+ independent lipid interaction has been reported recently by various studies. Just like the Ca2+ dependent phospholipid binding, this interaction shows disparities among the various annexins (Lizarbe, Barrasa, Olmo, Gavilanes, Turnay, 2013). Currently, studies have indicated that the single most determining factor in the Ca2+ independent binding of lipids is the pH value selected in the analysis of the interaction. Annexin A5 interacts and binds and infiltrates the bilayer of phosphatidylserine (PS) vesicles at a pH 4. However, at pH 5 it triggers a leakage of phosphatidylserine vesicles. Both of these actions are detected in the absence of calcium ions. At neutral potential difference the binding of Ca2+ to protein is a requirement for lipid binding. In most cases, it is thought that the change in properties is linked to a conformational change in annexin A5. This conformational change has been shown to occur at pH 4.6 and pH 4 when the acid triggered unfolding of the annexin A5 was examined. This alteration is characterized by solvent exposure of a distinct tryptophan residue (Trp-187) within the annexin A5. Thus, it is suggestive of a calcium ion stimulated exposure of the same tryptophan at neutral pH (Carcedo, Iglesias, Bances, Morgan & Fernandez, 2011). Annexin A5 interaction with cytoskeletal proteins Several annexins have been reported as cytoskeleton and specifically F-actin binding proteins. It has been postulated that various members of the multigene family may have a participatory role in modulating the membrane-cytoskeleton dynamics. Annexin A5 has been studied and shown to relocate towards the cortical membrane cytoskeleton following stimulation of platelets. This displacement appears to engage the interaction to the plasma membrane and to a particular isoform of actin, ?-actin. It is also paralleled through a linking with the thrombocyte membrane of cytosolic phospholipase A2 hence suggesting an association between this phospholipase and annexin A5 (Bogdanova, Baleva, Kremensky & Markoff, 2012). Annexin A5 and Intracellular Signaling Annexins play the role of intracelluar sensors hence differentiating the signals coming which have an effect on the response of eukaryotic cells with regard to the alterations in the environment. Annexins are able to associate with membranes at partcicular mcrochains whichj are charactertized by structure an lipid composition. Preferentially, this multigene family interact with phosphatidylserine (PS) although they can bind to and interact with PE, PI, PIP2 as well as PA. A majority of these biomolecules take part in lipoid mediated signalling pathways which modulate important cellular processes. Annexin A5 and Protein Kinase C (PKC) A range of serine and threonine kinases have been reported to phosphorylate annexins. Phosphorylation sites usually are located within the amino terminal domain of the annexins and modifications in some circumstances have been shown to influence biochemical properties. This effect is usually displayed in the specific affinity for calcium ion phospholipid binding. Protein kinase c (PKC) is capable of phosphorylating a number of annexins with annexin A5 being the most outstanding molecule. Annexin A5 can act as potent inhibitor of protein kinase C (PKC) (Kheifets, Bright, Inagaki, Schechtman & Mochly-Rosen, 2009). Annexin A5 and Human Disease There have been none or minimal reports on specific human diseases in which a mutation in the annexin A5 gene is the principal causative agent. Perhaps the only exception a disease phenotype due to annexin A5 gene mutation is myocardial disease of the heart. Nonetheless, there is substantive proof that due to changes in the expression, features or localization of annexins various disease pathophysiologies can result. Generally, the most profound disease phenotypes due to annexins have been referred to as annexinopathies. Such diseases have been characterized by dysregulation of what may be the typical anticoagulant properties of extracellular annexins (Rand, 2009). Recurrent Pregnancy Loss (RPL) Recurrent pregnancy loss (RPL) is a complicated condition having multiple factors as contributors and having a polygenic setting. Principal candidates for the molecular etiology of RPL include numerous inherited hypercoagulation disorders which enhance blood aggregation. These conditions are generally referred to as thrombophilias. The occurrence of circulating maternal antiphospholipid antibodies (aPL) is one of the critical risk factor in recurrent pregnancy loss (Rand, Wu, Quinn & Taatjes, 2010). Increased incidences of recurrent pregnancy loss have been reported in high risk as well as low risk pregnancies in the presence of aPL. Antiphospholipid antibodies have been postulated to cause fetal loss through platelet aggregation in placental vessels. Due to its anatomic positioning, annexin A5 has the ability of enhancing fluidity of maternal flowing blood. It is able to accomplish this though constant circulation within the intervillous space hence making sure that nutrients are exchanged and the fetus is viable (Bogdanova, Horst, Chlystun, Croucher, Nebel, Bohring, et al 2010). Annexin A5 has been postulated to be an antithrombotic agent just like annexin A2. However, this specific function becomes compromised in disease. In contrast, whereas annexin A2 is depicted to function as an intermediate in the fibrinolytic pathway, annexin A5 is thought to have direct role. This immediate and active role of annexin A5 is that the molecule forms a molecular barrier or shield (Rogenhofer, Engels, Bogdanova, Tuttelmann, Markoff & Thaler, 2012). The shield insulates/cushions the apical planes of placental villi form actions of circulating coagulating factors. Anticoagulant action is an attribute of all annexins that bind to calcium ions in a dependent manner. This activity can be elaborated by the simple calcium ions dependent sequestration of the phospholipid matrix with which procoagulant factors associate with. With this regard, annexins act in precisely similar manner as when initially described as inhibitors of PLA2 (Grandone, Tiscia, Colaizzo, Chinni, Pisanelli, Bafunno, et al. 2010). This analog is true because the enzyme also needs both calcium ions and phospholipid as co-factor and substrate respectively. Sufficient evidence in current research show that this anticoagulant activity of annexin A5 is of biological significance in recurrent pregnancy losses. This condition is more predominant in patients with antiphospholipid syndrome. Diagnostic examination among patients with antiphospholipid syndrome against a range of phospholipids and proteins depict this proposition clearly. Displacing annexin A5 shield by anti-annexin A5 antibodies is the main cause of creation of a thrombogenic environment resulting in fetal loss. For instance the shifting of the annexin A5 from the syncytiotrophoblast surface with chelating agents of calcium or specific antisera can lead to drastic coagulation of plasma (Bogdanova, Baleva, Kremensky & Markoff, 2012). Thrombophilias have an important role in the etiology of recurrent pregnancy loss (RPL). In most individuals with thrombophilic conditions, the main hereditary lesion is the alteration of a blood coagulating factor gene. This factor referred to as Factor V Leiden (FVL) or prothrombin (PTm). However, in 2007, a novel hereditary factor for recurrent pregnancy loss was identified and was linked to thrombophilia obstetric complications. This factor is exemplified by the common M2 haplotype which is composed of four consecutive nucleotide substitutions within the core promoter of ANXA5. Commonly this haplotype is responsible for the decrease in annexin A5 gene expression levels in the placentae. In addition to its anatomical location, the ability of annexin A5 to structure lateral aggregates on cell membranes has enaaabled ANXA5 to be used asa model in protective shielding. In this representation, the protein applies its anticoagulant role which is essential for hemodynamic balance (Rogenhofer, Engels, Bogdanova, Tuttelmann, Markoff & Thaler, 2012). This balance is attributed to occupation of PS determinants, attachment and stimulation of coagulation factors cascade. Therefore, inefficient shileding numerous pathologies due to thrombophilia arise and include pre-eclampsia, gestational hypertensionn, fetal growth restriction (FGR) and RPL (Sifakis, Soufla, Koukoura, Soulitzis, Koutroulakis, Maiz, et al. 2010). It has been reported that abortion risk among mothers having the M2 haplotype in ANXA5 is increased by 2.4 fold. This is in comparison to the general polpulation. In Central Europe for instance, the M2 haplotype has been documented as a pregnancy loss factor with a percentage of 15%. A slightly lesser incidence trate but with similar risk of pregnancy loss has been reported among Japanese women (Miyamura, Nishizawa, Ota, Suzuki, Inagaki, Egusa, et al. 2011). Venous Thromboembolism (VTE) Another closely pregnancy related condition is venous thromboembolism (VTE) which is popular among the whites. This condition has been strongly linked to pulmonary embolism (PE), a leading cause of maternal deaths in devloped nations (Grandone, Tiscia, Colaizzo, Chinni, Pisanelli, Bafunno et al. 2010). Generally,pregnancy is a risk factor for venous thromboembolism and this predisposition can be promoted by hereditary or acquired thrombophilia. In fact, women who possess thrombiphilia have elevated risk factors of developing VTE. Again, annexin A5 gene remains a risk factor in the development of venous thromboembolism which consequently may lead to fetal loss (Grandone, Tiscia, Colaizzo, Chinni, Pisanelli, Bafunno et al. 2010). Variation in the placental anticoagualant annexin A5 affects the risk of RPL via thrombotic influences within the placenta. Specifically, variation at the promoter haplotype due to mutations can lead to progreesion of fetal loss in recurrent pregnancy loss (Markoff, Gerdes, Feldner, Bogdanova, Gerke & Grandone, 2010). This has been shown through in vitro studies in which reporter gene assays have indicated that the haplotype M2 reduces the action of ANXA5 promoter. Direct correlation between decresed annexin A5 level of expression and prothrombotic environment in placenta can lead to fetal growth hindrance especially in pre-eclamptic patients. Such analyses are indicative of dimished expressionlevels of annexin A5 can be responsible for hemostatic and immunological circumstances that lead to loss of foetuses (Markoff, Gerdes, Feldner, Bogdanova, Gerke & Grandone, 2010). Heart Disease Calcium ions play a significant role in maintaining homeostasis in the heart. This is aggravated by the rich and ubiquitous annexin A5 in cardiomyocytes as well as the supporting cellular morphology. Consequently, there has been considerable interest in understanding the roles of annexin A5 in cardiac disease (Lizarbe, Barrasa, Olmo, Gavilanes, Turnay, 2013). Studies involving immunocytochemical assays have shown that annexin A5 occur in myocytes and non-myocytes and such analyses have reported concentrations in sarcolemma and Z lines in cardiomyocytes. Moreover, annexin A5 has been described to be present within the nuclear membrane and nucleus of differentiating myocytes of neonates. The annexin is only localized in sarcolemma of a terminally differentiated adult cell. Even though the functional role of cardiac annexin A5 is not clearly known, the benzothiazepine derivative has been shown to synergize ANXA5 role (Ravassa, Gonzalez, Lopez, Beaumont, Querejeta, Larman, et al. 2010). Benzothiazepine is a potent blocker of the calcium ion channel action of annexin A5. In vitro studies have indicated that it may have a protective role on myocardium from cytotoxic effects of Ca2+ linked to reperfusion injury and ischemia. Examination of annexin A5 gene expression in end stage heart failure have depicted that this important molecule expression levels significantly increase. However, in cases of ventricular hypertrophy, annexin A5 expression levels are insignificantly altered. The significance of such observations has not been elucidated. However, it is thought that overexpression annexin A5 increases the contractility of cardiomyocytes (Ravassa, Gonzalez, Lopez, Beaumont, Querejeta, Larman, et al. 2010). Antiphospholipid (aPL) Syndrome Antiphospholipid (aPL) syndrome is essentially a disease caued by immune disorder hence it is an autoimmune disorder. It affects individuals who possess antibodies that act antagonistically towards negatively charged phospholipd–protein complexes (Rand, Wu, Quinn & Taatjes, 2010). This disorder is symptomized as vascular thromoembolism or as reccureent pregnancy losses. Initially, it was categorized as a primary disease when it appeared without any other autoimmune disoder such as SLE. Additionally, this disorder was also classified as secondary when it occurred in conjuction with other autoimmune disorders. Annexin A5 has been shown to have potent anticoagulant attributes in vitro (Rand, Wu, Quinn & Taatjes, 2010). This molecule delays the phospholipid dependentcoagulation processes. The anti-aggreating effect is because annexin A5 protein is able to displace the coagulation proteins from phospholipid surfaces. Sufficient evidence have provided the hypothesis that annexin A5 forms a two-dimensional structures in clusters on the phospholipid surface. Consequently, there is creation of a barrier hence annexin A5 prevents coagulation proteins from interacting with the phospholipid by decreasing their availability as well as lateral movement (Rand, Wu, Quinn & Taatjes, 2010). Carcinomas Annexin A5 has been implicated in the carcinogenesis of numerous carcinomas due to its putative role in tumor development and differentiation. Colorectal cancer is the fourth most prevalent cancer with about one million reported cases per year in the entire world. It therefore remains a major public health and clinical problem with raised mortality and morbidity across all nations. The most essential prognostic markers for individuals with colorectal cancer are majorly predictive or tumor stage. Viable candidates for such markers include tumor enzymes, blood antigens, gene expression and flowing tumor cells. pharmacodynamic endpoints are also used as tumor stage predictive indicators (Xue, Hao, Ding, Mei, Huang, Fu, et al. 2010). Nevertheless, helpful prognostic dynamics that interrelate to tumor stage and clinical outcome in such patients are not clearly understood. Therefore, there is a critical need to investigate novel cancer-related genes to serve as the diagnostic indicators for colorectal cancer. Annxin as descride initailly is a well known a potent protein kinase C (PKC) inhibitor. Changes within the ANXA5 gene expression have been reported in numerous tumors although the biological significance of such alterations have not been elucidated. Research conducted by Karube have indicated that there is a decline of ANXA5 in carcinomas of the uterine cervix as well as endometrial cancer. Additionally, Mulla documented that the expression of ANXA5 in pituitary carcinomas is more inconsistent. With these reports taken into consideration, ANXA5 is a candidate gene in relation to colorectal cancer (Xue, Hao, Ding, Mei, Huang, Fu, et al. 2010). However, the clinopathologic importance of ANXA5 has not been well elucidated in clinical samples of colorectal cancer. Annexin A5 has traditionally been known in the detection of apoptosis base on its phosphatidylserine-binding activity. A still elusive case is whether annexin A5 can provide a prospective target for therapy. On the basis of structural traits, annexins have many physiologic functions and have been reported to take part in many physiologic roles (Xue, Hao, Ding, Mei, Huang, Fu, et al. 2010). Cell apoptosis is one of the functions implicated in tumor development and differentiation. It has been reported that washout of extracellular ANXA5 or anti-ANXA5 antibodies is able to diminish apoptosis at the upstream stage during stimulation in mitochondria. Annexin A5 invilvement in apoptosis occurs via two mechanisms; the first one being the typical calcium ion dependent binding. The second mechanism, can be through cooperation with the ?v?5-integrin and protein kinase C (Xue, Hao, Ding, Mei, Huang, Fu, et al. 2010). Cellular Aging Cellular senescence is a condition of irreversible growth arrest in which somatic cells enter due to replicative exhaustion via telomere erosion or as result of stress from oncogenes. Senescent cells do not leave the cell cycle but rather go through a state of active arrest at progressed points in G1, G1/S and G2 phases. The general strategy of cellular senescence has been postulated to be triggered by a number of stresses such as DNA damage, oxidative damage, telomere dysfunction and chemotherapeutic drugs (Klement, Melle, Murzik, Diekmann, Norgauer & Hemmerich, 2012). The application of new biomarkers for determination of cellular senescent cells may be useful in the diagnosis of aging cells. The accumulation of annexin A5 at the nuclear envelope during replicative and drug-triggered cellular senescence in primary human fibrolblast can be used as a biomarker for aging. This is attributed to the aging phenotype called SA-ANX5 (senescence associated accumulation at the nuclear envelope of annexin A5) (Klement, Melle, Murzik, Diekmann, Norgauer & Hemmerich, 2012). Conclusion There has been significant developments in molecular studies of annexin A5 and the outcomes of such analyses have indiccated that this protein plays roles in physiologic modulation of bllod aggregation. Additionally, these studies have alluded that abnomalies resulting from gene expression especially at the promoter region due to nucleotide substitutions has been associated to various clinical diseases. Annexin has been implicated in diseases such as myocardial infarction, recurrent pregnancy loss, carcinomas, venous thromboembolism and antiphospholipid syndrome. Annexin A5 levels are reduced on placental trophoblasts and on endothelial cells in aPL syndrome. This effect is mediated by antidobies and has been the major etiological cause of pregnancy losses and platelet aggregation. Therefore, annexin A5 can be utilized as useful biomarker in numerous disease conditions (Waerzeggers, Monfared, Viel, Faust, Kopka, Schafers, et al. 2011). References Bogdanova, N., Baleva, M., Kremensky, I., & Markoff, A. (2012). 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