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IV. Results
4-1. The information of patients and Patient grouping by VSD diameter/diameter of aortic root (Ao) ratio
Age and gender distribution of the 95 patients and 120 control children having MMP-2/-9 genetic polymorphisms analysis in this study were presented in Table 2-1. The mean age of all patients is 4.23 ± 2.51 (mean ± SD) years old, with no significant difference from the control group (5.53 ± 2.87 years) (Table 4-1).
All patients enrolled in this study were examined by two cardiologists using 2-D and Doppler echocardiography and got the average. The diameter of VSD was defined as VSD size, and ratio of VSD diameter and diameter of aortic root (Ao) was measured. According to the VSD/ Ao ratio, patients were classified into three groups, Trivial (VSD/Ao ratio = 0.14 ± 0.03), Small (0.25 ± 0.04) and Median (0.43 ± 0.10). Twelve control children having plasma MMP-2/-9 activities analysis and each subgroup (Trivial, Small and Median) are comprised of 12, 47, 29 and 19 patients, respectively (Table 4-2).
4-2. Genotype distribution and allele frequency of MMP-2 -735C>T
The genomic organization of the human MMP-2 gene and localization of our target SNPs were depicted in Fig. A1A. In this study, -735C>T polymorphism of MMP-2 was analyzed by PCR-RFLP. The sequence of the human MMP-2 gene and the position of the PCR primers that were used to detect MMP-2 -735C>T was shown in Fig. A1B. Restriction enzyme digestion using the restriction enzyme Sau96 I generated three different length bands among different genotypes (Fig. A1A). We also used direct sequencing to detect MMP-2 polymorphism in some patients, and there are three genotypes of MMP-2 -735C>T shown in
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Fig. A2B.
The genotypes and allele frequencies of MMP-2 -735C>T were recorded in Table 4-3.
The frequencies of MMP-2 -735C>T genotypes and allele frequency in the VSD patients and control subjects should not conform to the Hardy-Weinberg equilibrium if there exist
differences. As showed in Table 4-3, the allele frequencies of C and T allele are 66.3% and 33.7%; however, the results were similar to control group (p = 0.919). The genotype frequencies of MMP-2 -735C>T were CC: 47.4%, CT 37.9%, TT 14.7%. No significant difference was found in the patient and control groups (p = 0.143).
4-3. 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 Fig. A3, A5, A7. 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
4-3-1. MMP-9 -1562C>T polymorphism
The sequence of the human MMP-9 gene and positions of the PCR primers that were used to detect MMP-9 -1562C>T was shown in Fig. A3B. PCR products were digested by restriction enzyme Sph I and generated two different length bands between different genotypes (Fig. A4A). There are two genotypes of direct sequencing maps for MMP-9 -1562C>T shown in Fig. A4B.
The distribution of the MMP-9 -1562C>T genotypes were described in Table 4-4. Only 2 genotypes, CC and CT, of the MMP-9 polymorphism -1562C>T were detected in both the
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VSD and control groups. Genotype distribution and allele frequency were analyzed by using the chi-square test and the results showed that there was no significant difference in the genotypes and allele frequencies between the VSD and control groups.
4-3-2. MMP-9 R279Q polymorphism
The sequence of the human MMP-9 gene and positions of the PCR primers for MMP-9 R279Q were shown in Fig. A5B. PCR products were digested by restriction enzyme Sma I and generated three different bands (AA genotype: 467-bp, AG genotype: 467-bp, 296-bp, and 171-bp, GG genotype: 296-bp and 171-bp) (Fig. A6A). Fig. A6B shows the direct sequencing map of three genotypes of MMP-9 R279Q.
The genotypes and allele frequencies of MMP-9 R279Q between VSD and control group were summarized in Table 4-5. With regard to the genotype distribution and allele frequency of the MMP-9 polymorphism R279Q, the genotypes G/G, G/A, and A/A were detected in 41.1%, 53.7%, and 5.3% of subjects in the VSD group, respectively, and in 57.5%, 35%, and 7.5% of subjects in the control group, respectively. The genotype distribution of the MMP-9 polymorphism R279Q in the VSD group was significantly different compared to the control group (p =0.023); however, there was no significant difference in the allele frequency between the VSD (G: 67.9% and A: 32.1%) and the control (G: 75% and A: 25%) groups.
4-3-3. MMP-9 R574P polymorphism
The sequence of the human MMP-9 gene and positions of the PCR primers that were used to detect MMP-9 R574P are shown in Fig. A7B. PCR products were digested by restriction enzyme Nla IV and generated three different length bands among different
genotypes (Fig. A8A). There are three genotypes of MMP-9 R574Q in the direct sequencing maps (Fig. A8B).
The genotypes and allele frequencies of MMP-9 R574P are reported in Table 4-6. The
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results show that the homozygous C/C, heterozygous C/G and homozygous G/G genotypes of MMP-9 R574P are 52.5%, 38.3% and 9.2% in the control group. Otherwise, the patient groups are 48.4 %, 47.4 % and 4.2 %, respectively. There are no significant differences in genotypes and allele frequencies between two conditions.
4-4. Plasma MMPs activity in the VSD children with different MMP polymorphisms
In order to investigate the differences of MMPs activities in VSD patients of different MMP polymorphisms, we used the gelatin zymography to detect MMP-2 and MMP-9 activity in plasma.
4-4-1. MMP-2 activity in genotypes of -735C>T
The activities of MMP-2 enzyme in the different genotypes of MMP-2 -735C>T
polymorphism were compared with the Control group in Fig. 4-1 [CC, CT, TT genotypes vs.
Control group: 808 (642–980), 806(617–996), 869 (581–1044) ng/mL vs. 554 (407–689) ng/mL]. CC; CT; and TT genotypes of MMP-2 -735 C>T alleles contain 45, 36, and 14 samples, respectively. We used standard MMP-2 as positive control (Chemicon, Temecula, CA, USA) to calibrate MMP-2 activity of each group. MMP-2 activity has no significant difference in three genotypes of MMP-2 -735 C>T polymorphism. All three genotypes have higher activities level than control group. (p = 0.01)
4-4-2. MMP-9 activity in genotypes of -1562C>T, R279Q and R574P
We also investigated the MMP-9 activities of VSD patients in different MMP-9 SNPs, including MMP-9 -1562C>T, MMP-9 R279Q and MMP-9 R574P.
The activities of MMP-9 enzyme in the different genotypes of MMP-9 -1562 C>T polymorphism were compared with the Control group in Fig. 4-2A [CC, CT genotypes vs.
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Control group: 31.8 (24.6–38.2), 33.9(24.6–42.8) ng/mL vs. 29 (25–32) ng/mL]. These two genotypes of MMP-9 -1562 C>T include 72 and 23 patients, respectively. We test the data through Student’s t-test and there is no significant difference of MMP-9 enzyme activities between different MMP-9 -1562C>T genotypes.
The results of MMP-9 polymorphism R279Q were shown in Fig. 4-2B. Each group of GG, GA, and AA has 40, 50, 5 samples and activities of them are 32.5 (25.8 – 39.0), 31.7 (24.1 - 39.1), 37.5(31.7– 45.6) ng/mL. Fig. 4-2C shows the part of MMP-9 R574P. Three groups of CC, CG, GG contain 47, 44, 4 samples and the results are 32.5 (24.6 – 41.5), 31.7 (24.2 – 38.2), 37.2(27.4 – 46.0) ng/mL, respectively. We used standard MMP-9 as positive control (Chemicon, Temecula, CA, USA) to calibrate MMP-9 activity of each group. As the data shown, MMP-9 activity had no significant difference in these MMP-9 genotypes of these two polymorphisms.
4-5. Relationship between plasma MMPs activity and VSD severity
Fig. 4-3 shows the echocardiograms of a normal people and a perimembranous VSD patient. In clinically, large VSD patients with poor medical control are mandatory to surgery.
So, our patients conducted in this study were brought into focus on the VSD patients without extreme large defects. First, we used VSD and VSD/Ao ration as the independent variables and determined the relationships between circulating MMP-2 and MMP-9 activities and VSD defects defined by VSD size and VSD/Ao ratio. According to the definition of VSD/aortic root (Ao) ratio, the patients were further classified into three groups: VSD/Ao ratio ≦ 0.2 is trivial group; 0.2 < VSD/Ao ≦ 0.3 is small group; and 0.3 < VSD/Ao ≦ 0.66 is considered to median group. Control group and each subgroup contain 12, 47, 29 and 19 patients,
respectively. The MMPs activity was detected by zymography, and the results are shown in
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next two paragraphs.
4-5-1. Relationships between MMPs activities and VSD defects
For the plasma MMP-2 activity, it did not show significant correlations with VSD size and VSD/Ao ratio (Fig. 4-4A and 4-4B). Whereas, positive correlations between MMP-9 activity and VSD size (r2 = 0.073, p < 0.05) as well as VSD/Ao ratio (r2 = 0.170, p < 0.05) were evaluated (Fig. 4-4C and 4-4D).
4-5-2. Relationships between MMPs activities and VSD defects grouping MMP-2 activity in different VSD size groups
The MMP-2 activities in children with normal and different VSD groups were
determined by gelatin zymography. The activities of MMP-2 enzyme in the VSD groups were compared with the Control group in Figure 13A (Median, Small, Trivial groups vs. Control group: 845 (710 – 945), 840 (618 – 1,050), 787 (604 – 935) ng/mL vs. 554 (407 – 689) ng/mL). The results revealed that the circulating MMP-2 activity was significantly increased in the VSD patients. Compared with the Control group, the MMP-2 activity of Trivial, Small and Median groups were enhanced by 1.4 (p < 0.01), 1.5 (p < 0.01) and 1.5 (p < 0.01) folds, respectively (Fig. 4-5A). However, differences in the plasma MMP-2 activity among the VSD groups were insignificantly.
4-5-3. MMP-9 activity in different size groups
The activities of plasma MMP-9 was also determined by gelatin zymography and shown in Fig. 4-5B (Median, Small, Trivial groups vs. Control group: 38 (32 – 43), 35 (26 – 43), 28 (19 – 35) ng/mL vs. 29 (25 – 32) ng/mL). A trend of increased MMP-9 activity in the VSD groups was found. However, only the MMP-9 activity in Median group was
significantly higher than that in Control group (p < 0.01).
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4-6. Correlations between plasma MMP-2, MMP-9 activity, concentrations of TIMP-3 and BNP in VSD patients
4-6-1. TIMP-3 in different VSD patients and MMPs polymorphisms
The concentrations of plasma TIMP-3 were also determined by ELISA, and similar to MMP-9 (Fig. 4-6A), there seemed to be a trend for a correlation between TIMP-3
concentration and VSD/Ao ratio; however, the result did not reach the significance threshold (r2 = 0.04, p = 0.054, shown in Fig. 4-6C). The TIMP-3 concentrations are no significant difference among VSD grouped by VSD/Ao ratio. [Median, Small, Trivial groups vs. Control group: 1853 (1222 – 2019), 1748 (1092 – 1827), 1800 (1135 – 1859) pg/mL vs. 1230 (1045 – 1462) pg/mL].
The TIMP-3 ELISA assay showed that the concentration of circulating TIMP-3 was higher concentration in subjects of the CC genotypes of MMP-2 -735 C>T and the QQ genotype than in those with other genotypes of the MMP-9 R279Q polymorphism (Fig. 4-7)
4-6-2. BNP in different VSD patients and MMPs polymorphisms
The plasma BNP concentration was determined by an immunoassay. There was a positive trend between plasma BNP concentration and VSD/Ao ratio (r2 = 0.36, p < 0.001 shown in Fig. 4-6B), similar to MMP-9. BNP concentrations were not significantly different among the different MMP-2, and MMP-9 polymorphisms investigated in this study, as shown in Fig. 4-8B, although each genotypes of MMP-2/-9 had higher BNP concentration than control group.
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4-7. Pearson’s correlation between MMP-2/-9, TIMP-3, and BNP
Pearson’s correlation coefficient showed that there was correlation between plasma activity of MMP-9 and TIMP-3 concentration (r = 0.567, p < 0.001) (Fig. 4-9A) and also of MMP-9 and BNP (r = 0.463, p < 0.001) (Fig. 4-9B). In the contrast, there was no correlation between MMP-2 and TIMP-3 (r= 0.178, p = 0.139) (Fig. 4-9C); MMP-2 and BNP (r= 0.185, p = 0.07) (Fig. 4-9D). As shown in Fig.4-9E, there was no correlation between TIMP-3 and BNP concentration (r = 0.151, p = 0.130).
4-8. Relationship between MMPs activity and spontaneous closure rate of VSD
Patient groups were followed one year later by echocardiography. Totally 82 patients returned to the outpatient department for the programmed echocardiographic examinations.
After one-year follow-up, a total of 14 spontaneous closures occurred (17%) and 68 patients remained their defects. The plasma MMP-2 activity of VSD patients in the spontaneous closure group was compared with non-closure group, and no significant difference was noted [791 (617 – 945) ng/mL vs. 808 (624 – 990) ng/mL, p = 0.825] (Fig. 4-10A). In contrast, the plasma MMP-9 activity of VSD patients in the spontaneous closure group was significantly higher than non-closure group [42 (32 – 48) ng/mL vs. 31 (25 – 38) ng/mL, p < 0.01] (Fig.
4-10B).
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Table 4-1. VSD patients’ age and control group’ age, gender, VSD sizes, and VSD/Ao ratio
VSD group (n = 95)
Control group (n = 120)
Male: Female 42 : 53 62 : 58
Age (years) 4.23 ± 2.51 5.53 ± 2.87
BNP (pg/mL) 35.5 ± 21.7 15.7 ± 9.5
VSD size (mm) 3.98 ± 1.40
Ao (mm) 18.92 ± 5.29
VSD/Ao ratio 0.23 ± 0.12
Data are expressed as mean ± SD; BNP, B-type natriuretic peptide; VSD, ventricular septal defect; VSD size, diameter of the VSD; Ao, diameter of the aortic root; VSD/Ao ratio, ratio of the diameter of the VSD and the diameter of the aortic root
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Table 4-2. Patient grouping by VSD diameter/diameter of aortic root (Ao) ratio Control
group (n=12)
Trivial group (n=47)
Small group (n=29)
Median group (n=19)
Male: Female 6:6 21:26 14:15 7:12
Age (years) 4.76 ± 2.74 4.06 ± 2.26 4.16 ± 2.66 4.84 ± 2.96
VSD size (mm) - 3.06 ± 0.88 4.30 ± 0.85 5.75 ± 1.21
VSD/Ao ratio - 0.14 ± 0.03 0.25 ± 0.04 0.43 ± 0.10
Data were expressed as mean SD; VSD, ventricular septal defect; Ao, aortic root diameter
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Table 4-3. Genotype distribution and allele frequency of MMP-2 -735C>T polymorphism
SNP
Control (n = 120)
VSD
(n = 95) OR (95% CI) p value
n (%) n (%)
MMP-2 -735C>T
CC 48 (40.0) 45 (47.4) Reference
CT 60 (50.0) 36 (37.9) 0.6 (0.16-1.14) 0.143
TT 12 (10.0) 14 (14.7) 1.24 (0.52-2.58) 0.662
C allele 156 (65.0) 126 (66.3) 0.96 (0.64–1.43) 0.919
T allele 84 (35.0) 64 (33.7)
VSD, ventricular septal defect; SNP, single-nucleotide polymorphism; OR, odds ratio
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Table 4-4. Genotype distribution and allele frequency of MMP-9 -1562C>T polymorphism
SNP
Control (n = 120)
VSD
(n = 95) OR (95% CI) p value
n (%) n (%)
MMP-9 -1562C>T
CC 94 (78.3) 73 (76.8) 1.09 (0.57–2.08) 0.79
CT 26 (21.7) 22 (23.2)
TT 0 0
C allele 214 (89.2) 166 (88.3) 1.09 (0.60–1.99) 0.78
T allele 26 (10.8) 22 (11.7)
VSD, ventricular septal defect; SNP, single-nucleotide polymorphism; OR, odds ratio
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Table 4-5. Genotype distribution and allele frequency of MMP-9 R279Q polymorphism
SNP
Control (n = 120)
VSD
(n = 95) OR (95% CI) p value
n (%) n (%)
MMP-9 R279Q
GG 69 (57.5) 39 (41.1) Reference
GA 42 (35) 51 (53.7) 0.47 (0.26–0.82) 0.023
AA 9 (7.5) 5 (5.3) 0.98 (0.31–3.14) 0.98
A allele 60 (25) 61 (32.1) 1.41 (0.92–2.15) 0.11
G allele 180 (75) 129 (67.9)
VSD, ventricular septal defect; SNP, single-nucleotide polymorphism; OR, odds ratio
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Table 4-6. Genotype distribution and allele frequency of MMP-9 R574P polymorphism
SNP
Control (n = 120)
VSD
(n = 95) OR (95% CI) p value
n (%) n (%)
MMP-9 R574P
CC 63 (52.5) 46 (48.4) Reference
CG 46 (38.3) 45 (47.4) 1.34 (0.77–2.35) 0.31
GG 11 (9.2) 4 (4.2) 0.50 (0.15–1.66) 0.25
C allele 172 (71.7) 136 (72.3) 0.97 (0.63–1.48) 0.89 G allele 68 (28.3) 52 (27.7)
VSD, ventricular septal defect; SNP, single-nucleotide polymorphism; OR, odds ratio
A. B. C.
Figure 4-2. Plasma MMP-9 activities in the VSD children of -1562C>T, R279Q and R574P. (A) MMP-9 -1562C>T; (B) MMP-9 R279Q; (C) MMP-9 574P. As data shown, MMP-9 activities have no significant difference between VSD patients in these three subgroups of MMP-9 polymorphisms.
A.
B.
A. B.
C. D.
A. B.
C. D.
A. B.
C. D. E.
A. B.
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