3-1. The information of patients
The 101 congenital heart defects patients contained 44 boys and 57 girls were studied at mean age of 8.2 ± 5.0 years old. The blood of CHD patients recruited from hospital
containing 90 patients of type 2 VSD, 6 patients of other type VSD, and 5 patients of ASD.
All the congenital heart defects patients enrolled in this study were already documented by 2-D echocardiography.
3-2. Genotype distribution and allele frequency of MMP-2 -735C>T
The genomic organization of the human MMP-2 gene and localization of the studied SNPs were shown in Figure 3A. In this study, -735C>T polymorphism of MMP-2 was analyzed by PCR-RFLP. The sequence of the human MMP-2 gene and position of the PCR primers which were used to detect MMP-2 -735C>T were shown in Figure 3B. Restriction enzyme digestion was performed on MMP-2 -735C>T PCR products using the restriction enzyme Sau96 I and generate three different length bands between different genotype (Figure 4A). We also used direct sequencing to detect MMP-2 polymorphism in patients with VSD, and there were three genotypes of direct sequencing map for MMP-2 -735C>T shown in Figure 4B.
The genotype and allele frequencies of MMP-2 -735C>T between groups were reported in Table 3. The frequencies of MMP-2 -735C>T genotypes and alleles in the VSD, and control subjects conformed to the Hardy-Weinberg equilibrium. As showed in Table, the allele frequencies of C and T allele were 66.3% and 33.7%; however, the results were similar to control group (p = 0.92). The genotype frequency of MMP-2 -735C>T were CC: 47.4%, CT
37.9%, TT 14.7%, but no significant differences was found in the genotype frequencies between the VSDs and the control group (p = 0.1554).
3-3. Genotype distribution and allele frequency of MMP-9 -1562C>T, R279Q and R574P
Localizations of the studied SNPs of human MMP-9 gene were shown in Figure 5A, 7A and 9A. MMP-9 gene contains 13 exons. MMP-9 -1562C>T, R279Q and R574P
polymorphisms of MMP-9 are located on promoter region, exon 6 and exon 10 of MMP-9 gene, respectively. PCR-restriction fragment length polymorphism analysis was used for the genotyping of the MMP-9 -1562C>T, R279Q and R574P polymorphism in this study.
3-3-1. MMP-9 -1562C>T polymorphism
The sequence of the human MMP-9 gene and position of the PCR primers which were used to detect MMP-9 -1562C>T were shown in Figure 5B. PCR product digestion by restriction enzyme Sph I and generate two different length bands between different genotype (Figure 6A). And there were two genotypes of direct sequencing map for MMP-9 -1562C>T were shown in Figure 6B.
The distribution of the MMP-9 -1562C>T genotype were described in Table 4. However, no MMP-9 T/T genotype was detected in patients with VSD and control group. The
distribution of the genotype and allelic status were analyzed by the χ2 value test.
3-3-2. MMP-9 R279Q polymorphism
The sequence of the human MMP-9 gene and position of the PCR primers which use to detect MMP-9 R279Q were shown in Figure 7B. PCR product digestion by restriction
enzyme Sma I and generate three different length bands (AA genotype: 467-bp, AG genotype:
467-bp, 296-bp, and 171-bp, GG genotype: 296-bp and 171-bp) between different genotype (Figure 8A). And there were three genotypes of direct sequencing map for MMP-9 R279Q were shown in Figure 8B.
The genotype and allele frequencies of MMP-9 R279Q between VSD and control group were summarized in Table 5. As shown in Table 5, the genotype frequency of MMP-9 R279Q was GG: 45.6%, GA: 52.2% and AA: 2.2%. The results indicate that the genotype distribution MMP-9 R279Q have statistically significant differences compared to control group which the samples of patients without VSD (p = 0.0361); however, there were no significant differences was found in the genotype frequencies between the VSD and the control group (p = 0.6635).
3-3-3. MMP-9 R574P polymorphism
The sequence of the human MMP-9 gene and position of the PCR primers which were used to detect MMP-9 R574P were shown in Figure 9B. PCR product digestion by restriction enzyme Nla IV and generate three different length bands between different genotype (Figure 10A). And there were three genotypes of direct sequencing map for MMP-9 R574P were shown in Figure 10B. The genotypes and allele frequencies of MMP-9 R574P that found in patients with VSD were reported in Table 6. The results shows that the homozygous C/C, heterozygous C/G and homozygous G/G genotypes of MMP-9 R574P were 52.0%, 44.9%
and 3.1%, respectively, in the VSD group and 53.2%, 37.6% and 9.2%, respectively, in the control group. There were no significant differences in genotypes (p = 0.1533) and allele frequencies (p = 0.1664) between groups.
3-4. Plasma MMPs activity in the VSD children with different MMP polymorphisms
In order to investigate the differences of each MMPs activity in VSD patients that have different genotype of each MMPs polymorphism, we used the gelatin zymography to detect MMP-2 and MMP-9 activity in plasma from congenital heart defects children.
3-4-1. MMP-2 activity
MMP-2 activity in sample plasma was detected by gelatin zymography and samples were classified according to genotype of patients on MMP-2-735, which has CC, CT, TT genotype and each group contained 45, 39, and 13 samples, respectively. Used standard MMP-2 as positive controls (Chemicon, Temecula, CA, USA) to calculate MMP-2 activity of each group and collected the statistic as shown in Figure 11. MMP-2 activities have no significant difference in MMP-2 subgroups, but TT allele has the highest MMP-2 activity in MMP-2 -735C>T group (p = 0.052).
3-4-2. MMP-9 activity
For investigating the relation of MMP-9 activity in the VSDs children and MMP-9 SNP, three SNP were chosen, MMP-9 -1562C>T, MMP-9 R279Q and MMP-9 R574P. According to different genotype of patients underwent classification. Figure 12A shows the part of MMP-9 R279Q and is departed in GG, GA, AA and each group have 42, 50, 2 samples, sequentially.
Figure 12B shows the part of MMP-9 R574P and is departed in CC, CG, GG and recruit 51, 44, 3 samples, respectively. Figure 12C shows the part of MMP-9 -1562C>T and is classified in CC, CT and includes 69 and 21 patients, respectively. We used standard MMP-9 as positive controls (Chemicon, Temecula, CA, USA) to calculate MMP-9 activity of each group. As the data showed, MMP-9 activities have no significant difference in the subgroups of MMP-9 genotypes.
3-5. The relationship between plasma MMPs activity and VSD severity
Anatomically, VSDs are classified in to three defect levels: large, medium and small. The large defect is defined as the size is larger than two-thirds of the aortic root diameter; the medium size is in between two-thirds and one-third of the aortic root diameter; and small if the size is less than one-third of the aortic root diameter (Hornberger et al., 1989; Kabra and Srivastava, 2010). Figure 13 shows the Echocardiograms of normal, small defect and medium defect. The sample of patients with VSD conducted in this study are relative not severe
population, which the severe population means surgery needed. According to the definition of VSD/aortic root (Ao) ratio, the patients were classified into three groups: VSD/Ao ratio 0.2 is VS group; 0.2 < VSD/Ao 0.3 is VM group; and VSD/Ao > 0.3 is considered to VL group.
The VS, VM and VL stand for small, medium and large defect, respectively; each group includes 12, 45, 25 and 15 patients, respectively. The MMPs activity was detected by zymography, and the results were shown in Figure 14.
3-5-1. MMP-2 activity
According to figure Figure 14A, the MMP-2 activity of VS group is 1.4 fold higher than control group (p < 0.01), besides, VM and VL group MMP-2 activity are 1.6 fold higher than control group (p < 0.01, p < 0.001, respectively).
3-5-2. MMP-9 activity
In the part of MMP-9 activity, the patients also were separately assigned into VS, VM and VL group, and three of them were compared to control group (no VSD). Figure 14B shows that there is no significantly difference between MMP-9 activity of VS defect level and control group; but when defect level rises to VM defect level, the MMP-9 activity
significantly increases (p < 0.05); as defect level considered large also performs same trend which MMP-9 activity of VL group is about 1.5 fold higher than control group.
3-6. Investigating the relationship between MMPs activity and spontaneous closure rate of VSD
The patients with VSD were total diagnosed with echocardiographic examination twice with the interval of six months. Patients were initially assigned by first echocardiographic examination result based on t VSD/Ao ratio and then all samples were classified into VS, VM and VL groups. For the purpose of investigating the relation between MMPs activity and spontaneous closure rate of VSD, the ratio of VSD to aortic root from second
echocardiographic examination result were compared to first ratio. If the compared ratio value were above 15 % , then the patient with VSD were viewed as happened spontaneous closure of VSD; if the value below 15%, then the patient with VSD were considered as happened no spontaneous closure or personal equation. There total were 73 patients conducted in this experiment, the statistic table of spontaneous closure classification were shown at Table 7. If the patient with VSD were operated or had only one time echocardiographic examination, then will not include in this study. The relative MMPs activity was defined as the spontaneous closure MMPs activity to the basic of non-spontaneous closure MMPs activity.
Figure 15A denotes that the MMP-2 activity in spontaneous closure subgroup of VS group is significantly lower than non-spontaneous closure subgroup (p < 0.05), indicating that the mechanism of spontaneous closure might suppress the activity of MMP-2. However, the unusual point is that the phenomenon presented in VM and VL subgroups are opposite to VS, which non-spontaneous closure subgroup owns not significantly but higher MMP-2 activity
than spontaneous closure subgroup. The study of MMP-9 also demonstrates same trend in MMP-2 as showed in Figure 15B.