2-1. Patients and sample collection
The blood of 101 children of congenital heart defects will be recruited in this study of Taipei and Hsin-Chu Maykay memorial hospital from the outpatient base in one year period.
All the patients of congenital heart defects are enrolled in study when they are already
documented by 2-D echocardiography (Figure 2). The 152 control subjects contain no genetic diseases patients or the people who did physical examination in hospital, besides, the plasma of 12 health people were classified as control group in MMPs activity assays.
The blood samples will be obtained by nontraumatic needle aspiration from the
antecubital vein, with no hemolysis occurring in any of the samples when they receive clinical follow-up at 0, 6, and 12 months. The plasma and white blood cells were separated by
centrifuge 2,000 rpm for 10 min, and stored to -80°C.
2-2. Genomic DNA extraction
Genomic DNA for PCR-RFLP genotyping was isolated from the blood of 101 children of congenital heart defects using the genomic DNA Mini kit. Up to 200 L of whole blood add 30 L of Proteinase K (10 mg/mL) and mix briefly. After that, incubate the mixture at 60°C for 15 min. Following then add 200 L of GB Buffer, mix by shaking vigorously and incubate the mixture in a 60°C water bath for 15 min. Add 200 L of absolute ethanol to the sample lysate and immediately mix by shaking vigorously. Transfer the entire mixture (including any precipitate) to the GD Column. Centrifuge at 14,000 x g for 5 min. The collections add 400 L of W1 Buffer to the GD Column and centrifuge at 14,000 x g for 1 min. Then add 600 L of Wash Buffer (ethanol added) to the GD Column and centrifuge at
14,000 x g for 1 min. Displace into new collection tube and centrifuge again at 14,000 x g for 3 min to dry the column matrix. Finally, add 30 L of pre-heated Elution Buffer (10 mM Tris-HCl, pH 8.5) to the matrix and last for 5 min; after that, centrifuge at 14,000 x g for 1 min to elute the purified DNA. The maximum yield is up to 50 μg. Purified genomic DNA were Stored in the -80°C.
2-3. Genetic analysis
2-3-1. MMP-2 -735C>T polymorphism
To analyze the -735C>T polymorphism, we amplified a region of the MMP-2 gene with the primers pair were shown in Table 2. The target sequence was amplified in a 50 µL reaction volume containing 1 μL of genomic DNA, 1 µL each primer (10µM), 5 µL of 10X PCR buffer, 4 µL of 2.5 mM dNTPs, 0.5 µ L of Taq Polymerase (Promega, Madison, WI, USA), and 37.5 µL distilled water. The PCR started with an initial activation step of 94°C for 2 min followed by 40 cycles of denaturation at 94°C for 30 sec, annealing at 60°C for 30 second, and extension at 72°C for 30 sec, and a final step at 72°C for 7 min. The 297-bp fragment was purified with a purification kit (Invitrogen, Grand Island, NY, USA). Purified PCR products were then digested with Sau96 I (New England BioLabs, Ipswich, MA, USA) at 37°C overnight and separated on a 2% agarose gel. Sau96 I does not digest the T allele but generates 202-bp and 95-bp fragments for the C allele.
2-3-2. MMP-9 -1562C>T polymorphism
The primers used to detect this polymorphism were shown in table, which generated a 435-bp fragment containing the -1562C>T site in a 50 µL reaction volume, and the annealing temperature were shown in Table 2. The PCR product was then digested with Sph I (New
England BioLabs) at 37°C overnight and separated on a 2% agarose gel. Sph I does not digest the C allele but generates 247-bp and 188-bp fragments for the T allele.
2-3-3. MMP-9 R279Q polymorphism
The primers used to detect this polymorphism were sense 5’-GGT GGG TGC TTC CTT TAA CA-3’ and antisense 5’ -GCG TTA GAG ACG TTG GAA CC-3’ (Ikeda et al., 2008), which generated a 467-bp fragment containing the R279Q site in a 50 µL reaction volume, and the annealing temperature were shown in Table 2. The PCR product was then digested with Sma I (New England BioLabs) at 25°C overnight and separated on a 2% agarose gel.
Sma I does not digest the A allele but generates 296-bp and 171-bp fragments for the G allele.
2-3-4. MMP-9 R574P polymorphism
The primers used to detect this polymorphism were shown in Table 2, which generated a 246-bp fragment containing the R574P site in a 50 µL reaction volume, and the annealing temperature were shown in table. The PCR product was then digested with Nla IV (New England BioLabs) at 25°C overnight and separated on a 2% agarose gel. Three genotypes could be distinguished: GG (246-bp), GC (246, 182 and 65-bp) and CC (182 and 65-bp).
2-4. Gelatin zymography assay
The activity of MMP-2 and MMP-9 in the plasma was measured by gelatin gel zymography as previously has described (Chen et al., 2008). Plasma was mixed with 2×
zymography sample buffer (0.125 M Tris-HCl, pH 6.8, 20% [v/v] glycerol, 4% [w/v] SDS, 0.005% bromophenol blue) incubated for 10 min at room temperature, and then loaded into SDS-PAGE which was performed in 7% acrylamide gels containing 0.1% (w/v) gelatin (Sigma-Aldrich, St. Louis, MO, USA). After electrophoresis under power supply of 100 V,
the gel was washed twice for 30 min in zymography renaturing buffer (2.5% Triton X-100) with gentle shake at room temperature to remove SDS, then incubated 18 h at 37°C in reaction buffer (50 mM Tris-HCl, pH 7.4, 200 mM NaCl, and 5 mM CaCl2). The gels were then stained with Coomossie blue for 30 min prior to destain with destain buffer (50%
methanol, 10% acetic acid, and 40% ddH2O). The presence of enzyme activity was evident by clear or unstained zones, indicating the action of the enzyme on the gelatin substrate
(Stawowy et al., 2004). Gelatinase activities in the gel slabs were quantified by Scion Image software (NIH, Bethesda, MD, USA), which quantifies the area of bands hydrolyzed by gelatinase. A MMP-2 or MMP-9 positive controls (Chemicon, Temecula, CA, USA) was contained in each gel as a standard inensity value to normalize smaple intensityand express in arbitrary units.
2-5. Statistical analysis
Data analysis was performed using GraphPad Prism 5 statistical software. Continuous variables were expressed as mean ± standard deviation (SD), and categorical variables were shown as frequencies and percentages. All polymorphisms were tested for confirmation with Hardy–Weinberg expectations in both groups separately. Differences between patients with and without VSDs were examined with the χ2 test for categorical variables or the Student's t-test and analysis of covariance (ANCOVA) for continuous variables. The association between the MMPs polymorphisms and the risk of VSDs was estimated by odds ratios (ORs) and their 95% confidence intervals (CIs), which were calculated by binary logistic regression models. A 2-tailed p < 0.05 was considered statistically significant.