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I. Literature Review
1-1. Single-nucleotide polymorphisms
Polymorphisms represent natural sequence variants (alleles), which may appear in at least 1% of a population and are considered biologically normal. Approximately 90% of DNA polymorphisms are single-nucleotide polymorphisms (SNPs) due to a single base exchange (Collins et al., 1998). A SNP represents an alternative nucleotide in a given and defined genetic location. This definition does not include other types of genetic variability like insertions and deletions, and variability in copy number of repeated sequences. They occur one in every 300 nucleotides on average. Although the majority of DNA polymorphisms are probably functionally neutral, a proportion of them can exert allele-specific effects on the regulation of gene expression or function of the coded protein, then cause individual differences in various biological processes or in susceptibility to diseases (Brookes, 1999).
A SNP in which both alleles produce the same polypeptide sequence is called a
synonymous polymorphism (sometimes called a silent mutation); which produce a different polypeptide sequence is called a non-synonymous polymorphism (replacement
polymorphism). Mutations found in known diseases over half of all come from
non-synonymous polymorphisms (Stenson et al., 2009). When gene expression is affected by a SNP, this SNP type is referred to as an eSNP (expression SNP) and might be upstream or downstream from the gene.
Analytical methods to discover novel SNPs and detect known SNPs include DNA sequencing, restriction fragment length polymorphism (RFLP), capillary electrophoresis, mass spectrometry, single-strand conformation polymorphism (SSCP), electrochemical analysis, denaturing HPLC, gel electrophoresis and hybridization analysis (Kwok and Chen,
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2003). All of the background reference and information of SNPs in this study were obtained from National Center for Biotechnology Information (NCBI)-SNP database
(http://www.ncbi.nlm.nih.gov/snp/).
1-2. Ventricular septal defect
Ventricular septal defect (VSD) is the most common form of congenital heart diseases which account for 40% of the patients having congenital heart diseases (Hoffman, 1995). The defect can be in any portion of the ventricular septum, and the physiologic consequences can range from trivial to severe. Although historically the incidence of VSD is cited as
approximately 1.5 to 3.5 per 1,000 term infants and 4.5 to 7 per 1,000 premature infants (Moe and Guntheroth, 1987), recent studies demonstrated an incidence of VSD in newborns to be 5 to 50 per 1,000 (Tikanoja, 1995). Sometimes, VSD might be not only an isolated cardiac malformation but also an intrinsic component of several complex malformations such as tetralogy of Fallot. However, patients whose cardiac malformation is predominately caused by a VSD will be focus on in this proposal.
There are many systems to classify the different VSD types. One of the systems can classify VSD according to their location, either within the muscular septum (muscular defects) or at its margins. VSD at the margins of the muscular septum can be related to hinge-points of the leaflets of the atrioventricular valves (perimembranous type), those of the arterial valves (juxta-arterial or subarterial type), or both (Fig. 1-1) (Penny and Vick, 2011). Symptoms, natural history and management of VSD depend on size, anatomical characteristics, and patients’ variances.
Rates of spontaneous closure for membranous and muscular VSD in infant and children were 37% and 50%, respectively during a mean follow-up of 12 months (Moe and Guntheroth,
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1987). The others have no such a fortunate clinical course and their VSD persist. The
development of echocardiography had provided a good tool to observe the natural processes of spontaneous VSD closure (Murphy et al., 1986); Perimembranous defects often close by the development of a saccular pouch or aneurysm derived from tissue from the septal leaflet of the tricuspid valve. Muscular defects appear to close by progressive growth of tissue from the right ventricular side of the circumference of the defect. Neither the detail molecular insight of spontaneous VSD closure nor the study of association between extracellular matrix (ECM) and spontaneous VSD closure has been clarified.
1-3. Matrix metalloproteinase
1-3-1. Structures and functions of matrix metalloproteinases
Matrix metalloproteinases (MMPs) is a family of extracellular zinc-dependent neutral endopeptidases (Lombard et al., 2005), capable of degrading essentially all ECM components including fibrillar and non-fibrillar collagens, fibronectin, laminin and basement membrane glycoproteins (Fedarko et al., 2004). MMPs usually play an important role in ECM
remodeling in physiologic situations, such as embryonal development, tissue regeneration, and wound repair. In addition, there are more and more researchers turn their focus on
relationships of MMP proteins function and different pathological conditions, because of their potent degradative capacities. Some articles indicate the change of nucleotides and the SNPs of MMPs and tissue inhibitors of matrix metalloproteinases (TIMPs) correlated with many different diseases like coronary artery disease (Renko et al., 2004), Kawasaki disease (Ikeda et al., 2008), and cancer (Liu et al., 2011) (Table 1-1).
The others show serum levels of MMPs are altered in various diseases, and have been
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considered as potential clinical markers of disease activity. For example, serum MMP-2 level has been reported to be increased in the patients with liver cirrhosis (El-Gindy et al., 2003), and endometriosis (Huang et al., 2004); Serum MMP-9 level appears to increase in patients with congestive heart failure (Abou-Raya et al., 2004), stroke (Lynch et al., 2004; Reynolds et al., 2003), or myocardial infarction (Renko et al., 2004).
The MMP family currently consists of 28 enzymes with somewhat different capacities.
The members are generally divided into si× groups according to either structure or preferred substrates and referred to as interstitial collagenases (MMP-1, -8, and -13), gelatinases (MMP-2 and MMP-9), stromelysins (MMP-3, -10, -11, and -12), matrilysins (MMP-7 and MMP-26), membrane-type MMPs (MMP-14, -15, -16, -17, -24, and -25) and others (Bode, 2003; Maskos and Bode, 2003; Visse and Nagase, 2003). Although the groupings of MMPs are based on their abilities to degrade various proteins of the ECM, they also play other important roles such as the activation of cell surface receptors and chemokines (Stefanidakis and Koivunen, 2006). In addition, MMP-2 has proteolytic activity to specific targets within the cell to cause acute, reversible contractile dysfunction in cardiac disease (Schulz, 2007).
The basic structures of MMPs can be approximately divided into three structurally well-preserved domain motifs, including a catalytic domain, an N-terminal domain and a C-terminal domain (Fig. 1-2A). Zinc-dependent catalytic domain of MMP is similar with subtle structural differences among the substrate specific groups (Nagase and Woessner, 1999).
The N-terminal domain (propeptide domain) contains a unique PRCG(V/N)PD sequence in which the cysteine residue interacts with the catalytic zinc atom in the active site, prohibiting activities of the MMPs. Thus, the interaction has to be disrupted to “open” the cysteine switch in the process of MMPs activation (Van Wart and Birkedal-Hansen, 1990), which is a critical step that leads to ECM breakdown (Carmeli et al., 2004). The C-terminal hemopexin domain of metalloproteinases has a four-bladed propeller structure and contributes to substrate
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specificity (Fig. 1-2B) (Wallon and Overall, 1997). In membrane-type MMPs, the hemopexin domain contains a transmembrane domain for anchoring the protein in the membrane; besides, the hemopexin domain in MMP-2 also has a function in the activation of the enzyme
(Morgunova et al., 1999).
The regulations of MMPs occur at many levels, including transcription (the major one), post-transcriptional modulation of mRNA stability, secretion, localization, zymogen
(proenzyme) activation and inhibition of activity by natural inhibitors of MMPs, TIMPs (Brew et al., 2000).
Cell migration and tissue remodeling are two fundamental processes of embryogenesis.
One group of MMPs in particular, gelatinases, are implicated in tissue remodeling and in enabling cell migration and invasiveness. Two forms of gelatinases have been extensively studied in cells, and both can degrade type IV collagen and interstitial collagens as well as many other ECM molecules.
1-3-2. Gelatinase A (MMP-2, Type II collagenase)
In 1978, Sellers et al. was the first team to separate a gelatinase activity from collagenase and stromelysin in culture medium from rabbit bone (Sellers et al., 1978). Next year, a similar enzyme, acting on basement membrane type IV collagen, was reported by Liotta et al (1979).
Gelatinase was purified from human skin, mouse tumor cells, rabbit bone, and human gingiva.
Human and mouse MMP-2 are secreted as 72 kDa proenzymes of 631 and 662 amino acids, respectively. The removal of the pro-domain can be initiated by serine proteases such as thrombin and activated protein C. The resulting mature and active enzyme consists of a catalytic domain which is interrupted by three contiguous fibronectin type II-like domains, participating in binding to the gelatin substrates of the enzyme, and a C-terminal,
hemopexin-like domain (Lee et al., 1997) (Fig. 1-2B). The fibronectin type II-like domain is a
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feature of gelatinase. The complete sequence of the human MMP-2 was published by Collier et al. (2001)
1-3-3. Gelatinase B (MMP-9, Type V collagenase)
Krane (1994) detected a gelatinase activity in rheumatoid synovial fluid. Sopata and Wize (1979) described a gelatinase from human polymorphonuclear leukocytes . Purification was achieved in 1983 and sequencing of the cDNA in 1989. Human neutrophil MMP-9 commonly occurs as a complex with lipocalin, and this complex protects this extracellular matrix remodeling enzyme from autodegradation (Fernandez et al., 2005).
MMP-9 is secreted as a 92 kDa zymogen. Cleavage of pro-MMP-9 at near residue 87 results in activation of enzyme with a mass of approximately 82 kDa. Pro-MMP-9 is secreted by monocytes, macrophages, neutrophils, keratinocytes, fibroblasts, osteoclasts, chondrocytes, skeletal muscle satellite cells, endothelial cells, and various tumor cells. Pro-MMP-9 can be activated by MMP-3 or by certain bacterial proteinases. MMP-9 has prodomain, three
fibronectin type II motifs in catalytic domain, and a hemopexin-like domain just like MMP-2.
MMP-9 is inhibited by α2-macroglobulin or by TIMP-1. An interesting phenomenon, still not fully understood, is the binding of TIMP-1 to proMMP-9 to form a complex before secretion out of the cell (Roderfeld et al., 2007).
1-3-4. Roles of MMPs in cardiac development
MMP-2 is the earliest MMP known to be expressed during heart development. The process of cardiac looping converts the single, straight tubular heart into a S-shaped tube and transforms the primitive heart chambers into their adult anatomical positions before cardiac septation is complete. During the process of building a single heart tube, MMP-2 is expressed in the endocardium, early differentiating cardiomyocytes, and dorsal mesocardium but is soon lost within the myocardium (Cai et al., 2000).
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Cell proliferation is more pronounced within the left splanchnic mesoderm and left dorsal mesocardium. Blocking MMP-2 activity not only disrupts this asymmetric pattern of proliferation, it also randomizes the direction of cardiac looping and increases the incidence of dextrocardia (i.e., right-sided heart). Blocking MMP-2 activity also prevents midline fusion of the primitive heart tubes leading to cardiac bifida (Linask et al., 2005).
Some experiments support the postulate that MMP-2 has an important functional role in early cardiogenesis, cardiac cushion migration, and remodeling of the direction of cardiac looping (Cai et al., 2000; Linask et al., 2005).
Defects in cardiogenesis during the first three weeks of gestation are usually fatal;
however, embryo could survive with anomalous developmental events occurring in later stage.
These anomalies often manifest as great vessels or cardiac septal defects in neonates. The septation of the atria and ventricles and division of the cardiac outflow tract into the aorta and pulmonary artery requires the migration, proliferation, and differentiation of two distinct mesenchymal populations, i.e. endocardial-derived cushion cells and cardiac neural crest (NC) cells (Creazzo et al., 1998).
MMPs have been implicated in regulating epithelial-to-mesenchymal transitions (EMTs) which responsible for forming both populations of cells. MMP-2 is expressed by endocardial cells prior to and during the EMT of the endocardium and the neural crest cells detach from the neural epithelium. Perturbed MMP-2 expressions in these studies disturb the migration and tissue remodeling of cushion cells and NC cells. Both of them are important and
participate in formation of atrioventricular and outflow tract regions of the developing heart (Alexander et al., 1997; Cai et al., 2000)..
Hyaluronan is abundant in the ECM of the pre-mesenchymal heart (Fig. 1-3A).
Hyaluronan is an essential mediator of cell migration and invasion for proper heart
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development. Hyaluronan relates to regulate the expression of multiple MMPs in several cell types and the transformation of endothelial cells to mesenchymal cells. Therefore, MMPs might play pivotal roles and involve in the endocardial and cardiac septation, and cardiac developing (Camenisch et al., 2002).
1-4. Tissue inhibitor of metalloproteinases
1-4-1. Structures and functions of tissue inhibitor of metalloproteinases
As the variable and complex functions of MMPs, one of the important mechanism for the regulation of the activity of MMPs is via binding to a family of homologous proteins referred to as the tissue inhibitors of metalloproteinases (TIMP-1 toTIMP-4). TIMPs can form 1:1 enzyme-inhibitor complexes to inhibit matrixins, and each member of TIMPs can inhibit almost every member of the MMP family. The two-domain TIMPs are of relatively small size, yet have been found to exhibit several biochemical and physiological/biological functions, including inhibition of active MMPs, proMMP activation, cell growth promotion, matrix binding, inhibition of angiogenesis and the induction of apoptosis (Woessner, 2001).
The TIMPs have the shape of an elongated contiguous wedge consisting of an N-terminal segment, an all-β-structure left-hand part, an all-helical center, and a β-turn structure to the right. The N- and the C-terminal halves of the polypeptide chain form two opposing subdomains, each domain being stabilized by three disulfide bonds (Bode et al., 1999; Brew et al., 2000) (Fig. 1-3B).
The N-terminal subdomain exhibits a so-called OB-fold, known for a number of oligosaccharide/oligonucleotide binding proteins. This region consists of a five-stranded β-pleated sheet of Greek-key topology rolled into a closed β-barrel of elliptical cross-section.
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The narrower opening of this barrel is bounded by the sB-sC loop, while its wider exit is (in contrast to other OB-fold proteins) covered by an extended segment connecting strands sC and sD, designated as “connector.” After leaving the barrel, the polypeptide chain passes two helices, forms a two-stranded β-sheet, runs through a wide multiple-turn loop, and terminates in a β-hairpin sheet. The last C-terminal residues do not exhibit a defined conformation and presumably form a flexible tail on the TIMP surface (Fig. 1-3B)
Although the TIMPs are similar to each other to the extent of 35-40% amino acids identity, these key similarities suggest a significant structural conservation. For example, all TIMPs contain 12 cysteine residues at conserved locations, and in the case of TIMP-1, it has been shown that these participate in the formation of six intrachain disulfide bonds and then stabilize the whole structure. The N-terminal halves of the TIMPs share the most amino acid identity, suggesting that this region may underlie the common property of MMP binding and inhibition; It is possible that the more variant C-terminal half of the TIMPs may subserve distinctive properties of each TIMP and in the case of TIMP-3 the C-terminal half contains the ECM-binding domain (Anand-Apte et al., 1996).
1-4-2. Tissue inhibitor of metalloproteinase-3
In 1983, TIMP-3 was first isolated as a transiently expressed 21 kDa protein in the ECM of transforming chick fibroblast cultures (Blenis and Hawkes, 1983). Peptide sequencing subsequently demonstrated homology to the TIMPs and its identity as a novel member of the family was confirmed by cloning of the chicken cDNA and demonstration of MMP inhibition (Staskus et al., 1991). Subsequently, cDNAs of human TIMP-3 had been isolated (Apte et al., 1994).
TIMP-3 is an effective inhibitor of MMPS, proved by gelatin reverse zymography assays (Staskus et al., 1991), and in assays using radiolabeled ECM substrates (Apte et al., 1995).
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These assays have shown that TIMP-3 and TIMP-1 are equipotent in their ability to inhibit MMP-1, MMP-2, MMP-3, and MMP-9. Otherwise, TIMP-3 has poor aqueous solubility and a specific localization in the ECM in contrast to TIMP-1,2,and 4 (Pavloff et al., 1992). The ECM ligand of TIMP-3 has been suggested being the hyaluronic acid (Yang and Hawkes, 1992).The affinity of TIMP-3 for ECM and its expression in a number of epithelia led some researchers to propose that TIMP-3 might be a component of basement membrane.
TIMP-3 also has unique function of playing an role in inhibiting the endothelial cell responses to physiologically relevant antigenic agents such as VEGF and bFGF, inhibiting migration and preventing invasion and tube formation by endothelial cells in a collagen matrix (Qi et al., 2003). TIMP-3 is the only TIMP known to be related to a disease: mutation of certain cysteine residues to serine results in early blindness, a condition known as Sorsby’s fundus dystrophy (Felbor et al., 1995).
1-5. B-type natriuretic peptide
Over the recent decades, one group of neurohormonal markers, including atrial
natriuretic peptide (ANP), N-terminal proBNP (NT-proBNP), and B-type natriuretic peptide (BNP), has generated much interest in the evaluation and management of cardiovascular disease. C-type natriuretic peptide is produced by endothelial cells and macrophages, whereas ANP and BNP are derived from cardiac muscle. ANP and BNP act through the natriuretic A-type receptor. Each of the natriuretic peptides is cleared by the natriuretic C-type receptor and degraded by neutral endopeptidases. ANP is produced from a 126 amino acid precursor (pro-ANP) whilst BNP is synthesized as a 134 amino acid pre-pro form that is secreted as a 108 amino acid pro-BNP precursor. Proteolytic cleavage of these pro-natriuretic peptide precursors produces active ANP and BNP (Fig 1-4) as well as other fragments. The
N-terminal end of pro-BNP (NT-pro BNP), which comprises residues 1–76, lacks biological
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activity, but the cleavage products of pro-ANP are active (Goetze, 2012).
Both the ANP and BNP have beneficial compensatory actions including vasodilation, natriuresis, growth suppression and inhibition of both the sympathetic nervous system and the renin-angiotensin-aldosterone axis (Ruskoaho, 2003). ANP is derived from atrial as well as ventricular myocytes, and significant amounts can be released from cytoplasmic granules in response to relatively minor stimuli; on the other hand, nearly all circulating BNP is derived from ventricular myocytes, and its pulsatile synthesis and release is predominantly in
response to ventricular volume, pressure and wall tension (Sullivan et al., 2005). Besides the above advantage, BNP also has longer half-life than ANP and B-type natriuretic peptide (BNP) has better diagnostic, prognostic, and therapeutic values in heart failure and other heart
diseases (Ruskoaho, 2003).
As it is successfully applied in adult heart diseases, BNP has attracted increasing interests as a biomarker of VSD in the field of pediatric cardiology (Ozhan et al., 2007;
Rademaker and Richards, 2005). Some researchers have stated that MMP-9 serves as potential biomarker of heart remodeling (Halade et al., 2013; Lopez et al., 2010). These findings also drew our attention to the correlations among levels of plasma BNP in VSD patients.
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Table 1-1. Outlines of the information of MMPs and TIMPs SNPs defined and relationship with particular diseases Targeting gene (position, dbSNP
rs# cluster ID) Genotypes Relationship with particular diseases References
MMP-1 (-1607, rs1799750)
G GG
Patients who had the MMP-1 2G/2G genotype had a 1.71-fold increased risk of lung cancer (95% confidence interval,
1.22-fold to 2.41-fold increased risk) compared with patients who had the 1G/1G genotype.
(Liu et al., 2011)
MMP-2 (-1059, rs17859821)
GG AG AA
MMP-2 rs17859821 A allele carriers had lower all cause death rate, cardiac death rate and MACE rate than did GG genotype carriers (OR = 0.655, 0.580, 0.705; p = 0.030, 0.008, 0.011).
The allele and genotype frequencies of MMP-13-77A>G showed significant differences between Kawasaki disease patients with coronary artery lesions and without coronary artery lesions (p = 0.00989 and p = 0.00551, respectively).
(Ikeda et al., 2008)
MMP-9 (-1562, rs3918242)
CC CT TT
The presence of MMP-9 1562C>T allele was found to be associated with early-onset coronary artery disease (OR = 3.2, p = 0.001). The ECAD patients with MMP-9 1562C>T allele had higher MMP-9 activity (p = 0.001).
(Saedi et al., 2012)
There are significant differences in the genotype distributions for rs2250889 between diffferant groups, suggest that the G allele of MMP-9 polymorphism rs2250889 is overrepresented in patients with histologically confirmed Giant cell arteritis.
(Rodriguez-Pla et al., 2008)
MMP-9 (-1562, rs3918242)
CC CT TT
The -1562C>T polymorphism of MMP-9 gene is significantly associated with atrial fibrillation risk in Chinese Han patients with hypertensive heart disease. The -1562T allele which is associated with increased expression of MMP-9 might be a genetic risk for the development of AF in this cohort.
(Gai et al., 2009)
MMP-9 (exon 6, rs17576)
GG AG AA
The MMP-9 rs17576 genotype AG and GG appeared to be significant ‘at-risk’ genotypes for Pelvic organ prolapse (OR:
5.41, 95% CI: 1.17– 25.04, p = 0.031; OR: 5.77, 95% CI:
1.29–25.86, p = 0.0219).
(Chen et al., 2010)
A.
B.
A.
B.
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