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The aim of writing this review is a pre-research of the further experiment which is focus on the function of genes implicated in myocardial infarction in Genome wide association study (GWAS) studies. This review will focus on the myocardial infarction, GWAS, genes loci, zebrafish and morpholino antisense. And list the three specific genes (PHACTR1, HHIPL1a and HHIPL1b) for further genetic loci study.
The main complication of coronary artery disease, myocardial infarction
Cardiovascular disease is the leading causes of death and disability, it significantly increased in North America and Western Europe recently (The et al., 2012) (Hansson, 2005). 38% of deaths in North America are caused by cardiovascular diseases, and it is also the most common causes of death in European men (under 65 years old). The most common diseases of cardiovascular disease is coronary artery disease (CAD) which is also the leading cause of death worldwide (Hansson, 2005).
CAD is an inflammatory disease that interact with the metabolic circle for arterial system (Hansson, 2005). Coronary artery disease is result from assembled atherosclerosis plaques (fatty materials and plaques) in the coronary arteries that supply the blood for the heart muscle (myocardium) (Hansson, 2005). Coronary spasm may one of reasons for infarction, but most common cases are the thrombus of the surface of plaque (Hansson, 2005). The major narrow of the artery was owe to the blocks of blood flow by plaque, when the atherosclerosis plaques completely obstruct the blood flow in coronary arteries and shortage of oxygen in myocardium, it can lead to myocardial infarction(MI) (Hansson, 2005).
Myocardial infarction is also known as a heart attack and irreversible necrosis of myocardial. In clinic, myocardial infarction can be classified into non-ST-segment elevation myocardial infarction (non-STEMI) and ST-segment elevation myocardial infarction (STEMI).The typical symptoms of myocardial infarction are sudden chest pain, nausea, vomiting, palpitations, sweating and anxiety, but most of time, the myocardial infarction patients without any symptoms. If amount of myocardium dead, it can leads to a severely heart failure. Normally psychological stress or physical exertion rates are believed link to myocardial Infarction. For the most important risk factors, (Yusuf et al., 2004) calculated the population attributable risks (PAR) of myocardial Infarction, and found that smoking and hypercholesterolemia account for about two-thirds of myocardial infarction. The dyslipidemia or hypercholesterolemia means the abnormal levels of lipoproteins in the blood, it express the low high-density lipoprotein, high low-density lipoprotein and high triglycerides. The increase risk factors of myocardial Infarction are hypertension, diabetes, abdominal obesity and diet (Prins BP, 2012; Scheffold et al., 2011; The et al., 2012).According to (Yusuf et al., 2004) research, ten risk factors account for 90% RAP in man and 94% in women affect the myocardial Infarction, there are smoking (PAR 35·7%), hypertension (PAR 17·9%), regular alcohol consumption (PAR 6·7%),diabetes (PAR 9·9%), hypertension (PAR 17·9%), psychosocial factors (PAR32·5%), history of abdominal obesity (PAR 20·1%), raised ApoB /ApoA1 ratio (PAR 49·2%), daily consumption of fruits and vegetables ( PAR 13·7%) and regular physical activity (PAR 12·2%). However, the first degree family history is strongly infect the risk of myocardial Infarction, it can increase 2 to 12 times risk compared with non-family history members. Myocardial Infarction is a complex disease with both environmental and genetic determinants (The et al., 2012). In (Scheffold et al., 2011) study, for the patients aged ≤ 65 years in genetic factors of onset of myocardial Infarction, the men has more higher risk of MI than women, probably because the female sex hormones and also can caused by the different frequency of classical risk factory (hypertension, diabetes and smoking habits).
Genome-wide association studies (GWAS)
Genome-wide association study (GWAS) is an investigation method of genetic variants by study the association of inheritance genetic in different individuals. It focus on the linkage of single-nucleotide polymorphisms (SNPs), complex diseases and traits(Chablais et al., 2011). In 2005, genome-wide association studies (GWAS) evident the specific DNA sequence differences can affect different people genetic susceptibility of over 40 diseases. Before GWAS, people use the genome-wide linkage study and linkage disequilibrium mapping as the methods. Linkage analysis, focus on association between illness and the alternative sequences of the markers which are closest to the disease-related genes (Psychiatric et al., 2009). GWAS Study Design a sample, by selected a well-defined heritable phenotype, it conclude ill subjects and control subjects (Mannucci et al., 2010; Psychiatric et al., 2009).
(Psychiatric et al., 2009) shown SNPs have different frequencies in different populations so we better choice the homogeneous samples. The genetic variants samples can be detected into four populations: Han Chinese, Japanese, Black African, and Caucasian (Mannucci et al., 2010; Prins et al., 2012). GWAS arrays major include common SNPs replace the rare variants. For the structure variation, a DNA segment variants can be deleted, rearranged or duplicated (Prins et al., 2012).
As (Mannucci et al., 2010) and (Psychiatric et al., 2009) mentioned, there are some principle of the GWAS we should considered. Firstly, not all of the diseases can be account ability to GWAS, such as a small samples or lower genotypic relatives SNPs, large-scale can be possible for GWAS. Secondly, current diagnostic methods may inadequate for large data collect. Then, the study may not correctly test the main effect SNPs caused by the error data interactions. Finally, there may have some unknown genetic mechanisms.
For the clinical field, the results of GWAS can be help to find the new drug of therapy and prediction of different diseases. The current knowledge of genetic risk variants is not enough to identify the heritability of common diseases. The future steps of genetic research will focus on two directions: one is successfully translation the GWAS results through development clinical techniques. The other one is by collecting amount of data for helping genetic basis diseases in future research (Mannucci et al., 2010).
Genetic loci in myocardial Infarction
The CAD gene database includes more than 300 genes. In 2002, the first genome-wide association study (GWAS) of myocardial Infarction researched 94 myocardial Infarction cases and 658 controls by using a genotyping array of >90,000 single nucleotide polymorphisms (SNPs) in Japanese group. And point out LTA and LGALS2 as the susceptible loci. After that, (Mannucci et al., 2010) research shown 13 loci genetic variation for the myocardial Infarction, and a locus on chromosome 9p21.3 identified successfully replicated in non-Caucasian populations lead to a high risk of myocardial Infarction (Prins et al., 2012) provided 32 genomic loci for the independent risk of myocardial Infarction.
Chromosome 1p13.3 is a second most important myocardial Infarction locus identified by GWAS (Erdmann et al., 2010; The et al., 2012).SNP rs646776 is located in 97 kb haplotype on 1p13.3. Chromosome 1p13.3 link to Low-Density Lipoprotein (LDL), total cholesterol, statin, Lp-PLA2 activity and progranulin level (Prins et al., 2012). The allele SNP rs599839 is associated with Low-Density Lipoprotein cholesterol (LDL-C) and region on four genes: proline /serine-rich coiled coil protein 1 (PSRC1), cadherin EGF LAG seven-pass G-type receptor 2 (CELSR2), myosin binding protein H-like (MYBPHL) and sortilin 1 (SORT1) (Coronary 2012;Kleberet al.2010).The liver mRNA expressions of PSRC1,CELSR2, and SORT1 has been shown to correlate with LDL-C plasma levels in cardiovascular diseases of mouse and in a human heart diseases(Erdmann et al., 2010). The CAD risk allele (A) was linked to a lower levels of CELSR2 and SORT1 expression and with higher levels of LDL-C expression. The (Kleber et al., 2010) shown the SNP rs599839 genotype can be partly responsible for the health of cardio in minor G allele.
The SNP rs17465637 located on chromosome 1q41 with a high risk of myocardial Infarction by C allele (Erdmann et al., 2010). This SNP rs17465637 on chromosome 1q41 is located in melanoma inhibitory activity family member 3 (MIA3) gene, which may leading a higher risk of plaque formation by promote the immigration of monocytic cells through fibrinogene or human microvascular endothelial cells . MIA3 gene is required for collagen VII (COL7A1) secretion by loading COL7A1 into transport member from the endoplasmic reticulum, and the MIA3 gene is impressed as a tumor suppressor of malignant melanoma (Wang et al., 2011a).
SNP rs6725887 on chromosome 2q33 is located in intron 12 of the WD repeat domain 12 (WDR12) gene, which is a member of WD repeat protein family (WD40). This gene family is linked to cell cycle progression, apoptosis, signal transduction and gene regulation (Erdmann et al., 2010).
Chromosome 3q22.3 SNP rs9818870 is located in the 3′ untranslated region of muscle RAS oncogene homolog (MRAS) gene which can widely expressed in all tissues, especially in cardiovascular system as asignificant role in adhesion signaling (Erdmann et al., 2010).
(Erdmann et al., 2010) shown that the SLC22A3-LPAL2-LPA gene cluster is strongly associated with myocardial Infarction, for chromosome 6P26-27, the four SNPs rs2048327 in SLC22A3 gene, rs7767084 in the LPA gene, rs3127599 in the LPAL2 gene and rs10755578 in the LPA gene are the high risk of myocardial Infarction. The chromosomes are linked to Lipoprotein (a) which is a main protein of coronary artery disease (CAD).
The chromosome 9p21 play an important role in myocardial Infarction, and play as a strongest common genetic factors linked to the risk of coronary artery disease (CAD) in European ancestry(Saleheen et al., 2010). (Scheffold et al., 2011) research shown the 240 kilobases (kb) at chromosome 9p21 (chr9:21936711-22176221) associated with the type 2 diabetes and myocardial infarction in 47 unrelated individuals from Northern and Western European. 635 high-confidence SNPs and 257 genotype sites are be identified at chromosome 9p21 by Using targeted sequencing (Shea et al., 2011). 21 variants on chromosome 9p21 are been identified associated with coronary artery disease by analysis of 38 250 CAD cases and 84 820 controls in Europeans (Shea et al., 2011). The nearby genes in three human tissues (liver, visceral fat and subcutaneous fat) are explored to relate with 9p21.3 SNP in (Myocardial Infarction Genetics et al., 2009) study. The (Scheffold et al., 2011) mentioned there are six SNPs represented on the chromosome 9p21.3 (rs1333049, rs1333040, rs10757274, rs10757278, rs2383206 and rs2383207). Among of them, two SNPs rs10757274 and rs2383206 are linked to myocardial infarction and located with a locus in 58 kb region on chromosome 9p21.3. For other three SNPS rs1333040, rs2383207 and rs10757278 on chromosome 9p21 are linked to myocardial infarction , This also shown in (Erdmann et al., 2010) search, each C alleles of SNP rs1333049 on chromosome 9p21 can increased 22% higher risk of myocardial Infarction or coronary artery disease. Variant studies shown the risk of chromosome 9p21 in different diseases. Two SNPs (rs1333049 and rs1412832) were associated with myocardial infarction Pakistanis (Shea et al., 2011).The same SNPs are reported to relevant the type 2 diabetes mellitus (T2DM), chromosome 9p21.3 is related to antisense non coding RNA in the INK4 locus ( ANRIL) for activity independently of T2DM. Chromosome 9p21 is also contribute to the risk of glioma (Erdmann et al., 2010). SNP rs4977574 on chromosome 9p21 with mRNA level of cyclin-dependent kinase inhibitor 2B (CDKN2B) are strongly relevant to intracranial aneurysm, abdominal aortic aneurysm and Type 2 diabetes (Prins et al., 2012). A variant at chromosome 9p21 adjacent to cyclin-dependent kinase inhibitor 2A and 2B (CDKN2A and CDKN2B) are related with cell carcinoma, and variant at 9p21 near methylthioadenosine phosphorylase( MTAP) and CDKN2A associated with familial melanoma (Erdmann et al., 2010). The (Saleheen et al., 2010) also identified the three near genes are in higher linkage disequilibrium (LD) and span 58 kb region. The three neighboring protein coding genes (CDKN2A, CDKN2B and MTAP) were relevant with cell cycle progress, cellular senescence, cellular proliferation and apoptosis. SNP rs4977574 was linked to level of CDKN2B transcript in visceral fat tissue that replace in human liver (Myocardial Infarction Genetics et al., 2009). SNP score is comparable in predictive value to plasma LDL cholesterol. Recent report shown the LTA4H haplotype associated with MI in Africans and TCF7L2 linked to type 2 diabetes mellitus in West Africans (Saleheen et al., 2010).
The SNP rs501120 located on chromosome 10q11.21 located in 5 region of the Stromal-derived factor 1(SDF1) gene in T allele, has shown a higher risk of myocardial infarction. SDF-1 gene is a member of the chemokine family which has related to atherosclerotic plaques. The CXCR4 receptor is express in endothelial and monocytes cells and SDF1 gene is the only ligand of the CXCR4 receptor. The effect of the SDF1 and CXCR4 receptor are regulate monocyte inflammatory response and endothelial progenitor cells. If the SDF1 and CXCR4 receptor over expressed, it will link to maintain atherosclerotic plaques by endothelial progenitor cell recruitment and inflammation (Erdmann et al., 2010). The (Coronary Artery Disease et al., 2009) shown the CXCL12 on SDF1 gene located on chromosome 10q11.21 is significant in stem cell, and promoting angiogenesis. In above genetic loci, men has higher risk of myocardial infarction than women.
The minor T allele of SNP rs9982601 on chromosome 21q22 increase the risk of myocardial infarction, this SNP is associated with MRPS6, SLC5A3 and KCNE2 as an intergenic region, KCNE2 is a inferior of potassium(K) channel and mutate of this encode can cause inherited arrhythmias. The SNP rs9982601 variants in noncoding transcript instead of change three protein coding genes. SNP rs2259816 on chromosome 21q22 located in intron 7 of the hepatocyte nuclear factor-1a (HNF1A) gene, the HNF1A encode a transcription factor binds to express variants of genes in liver, and effect plasma concentrate of C-reactive protein (Erdmann et al., 2010) .
PHACTR1, HHIPL1a and HHIPL1b
Phospjatase and actin regulator 1 (PHACTR1) is associated with a common C allele of SNP rs12526453 on chromosome 6p24 (Erdmann et al., 2010; Wang et al., 2011a). PHACTR1is an unidentified potential biomarker of endothelial cells (Patel et al., 2012).The PHACTR1 is an enzyme which dephosphorylates serine and threonine residues on a range of proteins, and an protein phosphatase 1 inhibitor. PP1 shown a increased in end stage heart failure in patients (Wang et al., 2011b). PHACTR1 is a member of the PHACTR/ Scapinin family which content four members (PHACTR-1 toPHACTR-4). PHACTR-3 transcripts are found in heart and in leukaemia, lung cancer and melanoma. A mutation of PHACTR-4 is responsible for serious disorder in the early development of the CNS by mouse. PHACTR-1 protein not expressed in brain, but highly found in the striatum, hippocampus and heart.PHACTR-1 is a VEGF-dependent gene which activity in human endothelial cells. VEGF is a significant factors for the blood vessel formation. For VEGFA165-dependent proteins play an important role in angiogenesis.In human endothelial cells, PHACTR-1-depleted HUVECs increased cell death, and reduced in tube formation. On PHACTR-1 knockdown, cytotoxicity and inhibition of tube formation related with the involvement of apoptosis. Apoptosis can be triggered into two pathways: an extrinsic pathway and an intrinsic pathway (the mitochondria-dependent pathway) (Patel et al., 2012). SNPs rs499818, rs1512411, rs507369 and rs9349379 are located in an intron of PHACTR1(Mehta, 2011). In more than 1000 participants in an independent GWAS for coronary artery, PHACTR1 has been identified be related to calcification(Erdmann et al., 2010). The OHACTR1 is phosphatase and actin regulator, the location of human is in chromosome 6:12717893-13288645:1. SNP rs9349379 in PHACTR1 with arterial stenosis is associated with the early onset of myocardial infarction, rs9349379 may a better marker than rs12526453. The relevant was highest in the left anterior descending artery and lowest in the left main artery if the specific main vessels were used as a alone outcome (Patel et al., 2012).
The chromosome 14q32 located in Hedgehog interacting protein like 1(HHIPL1) associated with myocardial infarction. HHIPL1 is an antagonist of the Hedgehog (HH) family of proteins, and HHIPL1 is major in normal vascular develop in vivo study. All of the variants in the 25kb CAD linkage obstruct at the 14q32 locus are situated within the intron of the HHIPL1 gene. HHIPL1a and HHIPL1b also linked to the function on angiogenesis, thrombosis and vascular permeability (Prins et al., 2012).
Figure 1: The adult zebrafish; Figure 2: The development of zebrafish in 48 hours; Figure 3: The inner organ of zebrafish.
The zebrafish (Danio rerio) is a small fresh water fish which covered with iridescent scales and black stripes, it is a tropical fish and popular in family aquarium in recent years (Quaife et al., 2012). The zebrafish is only 4cm in length and live up to 5 years. Zebrafish (Danio rerio) growth in the streams of Southeast Asia (Chico et al., 2008), the (Quaife et al., 2012) also mentioned it breed in India's Ganges River. Since 1990s, the zebrafish has been widely used in helping biomedical research to learn more about our hearts function and development in lab. An adult female zebrafish can produce about 300 eggs every week. If those eggs fertilized, it almost transparent immediately (Chico et al., 2008). These eggs developed rapidly, almost all major organs growth within 36 hours, after 24 hours a two chambered heart has developed, and the axial aorta and vein have been formed, after following days, the main vascular developed. And hatch will take place after 48 hours (Figure 3) (Chakraborty et al., 2009). After that the blood flow can been seen in all patent vessels with cardiac contraction commences in 24 hours (Quaife et al., 2012). The whole progress of zebrafish grown up will take about three months. Small molecular can be added into zebrafish by the aquatic environment of embryo's. The reason of using zebrafish to help research the function of heart diseases is not only caused by the no beating heart can survive and grow for several days even the embryos developing with severe malformation, and also caused of the rapid breeding, abroad available and cheap price. The early embryo can intake enough oxygenation that apply for its physiological needs through diffusion rather than circulation system. So the embryos stages are vital for phenotypes study in mammals without required cardiac contraction or blood flow(Nemtsas et al., 2010; Quaife et al., 2012).The zebrafish is a good example of vertebrate model in genetic, embryological and physiological studies. Although zebrafish only has two chambers, physiology of the zebrafish and the human heart are similar in many aspects (Nemtsas et al., 2010).
By compare with human and zebrafish hearts, both of their heart muscles are designed to pump oxygen carrying blood through the body, and they all have the valves to ensure the blood flows in the correct direction in heart chambers. The regular and rhythmic of heart beats are also the common character. The common between human and zebrafish are the QT interval even the zebrafish lack of pulmonary vascular, and both of them have the wrap (pericardial sac) of heart to buffer and reduce the friction. The bulbous arteriosus in zebrafish is similar to human aortic arch. For recycle blooding, the sinus venosus in zebrafish is similar as vena cava in human (Sun et al., 2009). Cause by that, the scientists using zebrafish to research the human heart disorder by locating mutant genetic loci of zebrafish. For the distinction of zebrafish and human heart is the electrical activity which shown on T type Ca2+ in ventricular and atrialmyocytes (Nemtsas et al., 2010).Angiogenesis and organogenesis is clearly show in zebrafish due to the transparency of embryo. The amount of fibrotic tissues will obstruct the contraction and relaxation of heart. After infarction of up to 20% ventricular resection, the zebrafish can regenerate their myocardium (Chablais et al., 2011). Recent research of assessment heart regeneration is use the molecular and histological methods(Chablais et al., 2011; Sun et al., 2009).The (Gonzalez-Rosa et al., 2011) research shown that after cryocauterization (CC), the zebrafish heart isable to completely regenerate 25% of coronary vasculature, myocardium and endocardium. In heart regeneration, the scar will not inhibit the proliferation of cardio myocyte, because the scar is reversible and can be gradually removed in zebrafish (Gonzalez-Rosa et al., 2011).
Morpholino antisense oligonucleotide
Morpholino antisense oligonucleotide is a useful technology to obstruct other molecules to specific sequences within nucleic acid. It provide a significant approach to investigate gene functions in zebrafish embryos (Corey and Abrams, 2001; Yuan and Sun, 2009).Antisense oligonucleotides, oligonucleotide analogs and other specific sequence block of the selected translation of messenger RNAs (mRNA).If the antisense oligo linked to targeted disease sequence, it can release the activity of target. By using antisense oligos can provide the prospect safety and effective therapeutics for several diseases(Corey and Abrams, 2001). The specific advantage of morpholino is that provide the resistance of nuclease, long-term activity and lower toxicity.For oligonucleotides cells, if want to reduce the regulation of gene expression, an antisense oligonucleotide must penetrate into the targeted cells. A major block of using antisense oligonucleotides is choosing a target sequence. Antisense oligonucleotides are able to form RNA-DNA hybrids, RNase H can promotes the mRNA target split through this hybrid. Morpholino antisense oligomers can be used for generating mutant phenotypes as a routine tool. Morpholino oligonucleotides has high success rate for inhibit gene expression. More than 40 oligonucleotides need to be tested for identified a efficiently inhibition of gene expression (Corey and Abrams, 2001). Over 20 years ago, it began to develop antisense RNA and stability of cell endogenous for inhibit the translation. In zebrafish, antisense RNA is widely used and affect sequence independent. In this century, the Morpholino oligonucleotides are used for targeted to knockdown specific gene expression in frog and zebrafish (Eisen and Smith, 2008). The zebrafish effectiveness and properly of interpret the link of morphant phenotype is an assessment of MO knockdown efficiency. Synthetic oligonucleotides, MOs, are chains composition which have a morpholine ring rather than a ribose ring. The resistant and stable of nucleases by MOs do not carry a negatively charged, it shown that they are less likely to interact non-specifically with other composed of the cells and less toxic. The luciferase assay system is a technology that can measure the knockdown efficiency of MOs in zebrafish embryos. Many experiments use MOs against the initiation codon of the target mRNA directly. This approach is activity in genome sequence unknown species, because one does not need to know the intron-exon structure of the target gene to inhibit its function (Eisen and Smith, 2008). Targeted the chordin (chd) gene encoding a BMP antagonist, by observed the joint effect between chd-MO1 and -MO2 we can known the inhibition of luciferase expression was very consistent in the chordin morphant embryos. The study shown the most efficient MOs cause more than 80% inhibition of translation and major knockdown effects in zebrafish embryos when used singly at 1-2 ng/embryo, by using double MO strategy can give over 90% decreased of protein expression in all of the cases. Oligonucleotides antisense also depends on temperature, cell line, structure and the oligonucleotide concentration.I n long-term therapeutic effect, associated virus-mediated production of antisense oligonucleotides (AO)has shown to be highly effective for targeted exon in both locally and systemically. This pathway has strongly benefit for synthetic AOs by eliminate the repeated injections needs. Morpholino oligomers(MO)and peptide nucleic acids (PNA) , be provided as specific splice switching agents to relate the process of pre-mRNA.
For micro injection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos, we have 8 processes in (Yuan and Sun, 2009). Firstly, preparation of micropipettes and micro injection chamber plates by heating and pulling glass tubes, and pouring 1.5% agar. Secondly, preparation of RNA. Perform an in vitro CAP RNA transcription reaction on transcript of interest RNA sample, concentrate the RNA preparation and inject the sample is what we should do in this step. Then, preparate of morpholino. After that, filling the micropipette with your working solution by the diatel end of the micropipette. At the fifth steps, calibrating the micropipette injection volume by adjust, inject and use of the pressure-pulsed micro injector apparatus. Sixth,preparing fertilized zebrafish embryos for micro injection. Zebrafish will randomly fertilized the first few hours, collect and shallow embryos. In seven steps, micro injection through the chorion, manipulate the embryos with the micropipette, penetrate the chorion and the yolk with micropipette for inject into the embryo, incubate the embryos and observe it. Finally, representative results(Yuan and Sun, 2009).
In recent year the GWAS identified dozens of genetic loci of increase risk of myocardial infarction, this review give a background of the gene loci in myocardial infarction in GWAS studies and introduce zebrafish as the attractive tool for identified the function of three specific genes, and give the reason of why we try to use morpholino antisense to knockdown genes in zebrafish embryos.