Associations of matrix metalloproteinase-2 and -9 gene polymorphisms and enzyme activity in the children with ventricular septal defects
|關鍵字:||心室中膈缺損;基質金屬蛋白酶-2;基質金屬蛋白酶-9;單核苷酸基因多型性;ventricular septal defect;matrix metalloproteinases-2;matrix metalloproteinases-9;single nucleotide polymorphism|
|摘要:||心室中膈缺損(ventricular septal defect ,VSD)為常見的先天性心臟病之一。其VSDs佔所有先天性心臟病的20%，多數病童的中膈缺損會隨著年紀增長而逐漸變小或完全閉合。目前有許多因素被認為與VSD形成有關，其中特定基因的單核苷酸基因多型性(single nucleotide polymorphism, SNP)被指出是造成VSD疾病主要原因之一。基質金屬蛋白酶 (matrix metalloproteinases, MMPs)是一類活性依賴於Zn2+等金屬離子且參與降解細胞外基質(extracellular matrix, ECM)的蛋白酶家族，其精密調控著組織中ECM的代謝平衡。MMPs已被報導在胚胎發育、心肌分化，以及心臟組織的重塑扮演著重要的角色，且許多心臟疾病也與MMPs的基因多型性有關。據此，我們檢測VSDs的病患的MMPs基因SNPs與其血漿中MMPs活性，來探討MMPs基因SNP和血液中MMPs活性與VSDs之嚴重程度。
自2010年9月起，我們收集了101位7.4 □ 3.8歲VSDs病童的血液樣品，自白血球萃取其基因組DNA，利用聚合酶連鎖反應-限制酶片段長度多型性(polymerase chain reaction-restriction fragment length polymorphism, PCR-RFLP)來檢測這些病童的MMP-9 SNPs，包括 -1562C>T、R279Q及R574P，也檢測MMP-2 -735C>T SNP。我們亦分離病童的血漿，以明膠酶電泳(gelatin zymography)測定其MMP-2和MMP-9活性。實驗結果顯示，在所檢測的四個SNPs中，僅有MMP-9 R279Q基因型的分布與VSD的發生有統計上的顯著意義(p < 0.05)，其VSD組中GA基因型的發生頻率明顯高於控制組(分別為52.2%及37.2%)。另一方面，我們依病患超音波檢查之VSD/aortic root (Ao)比值判定病童VSD嚴重程度，我們將之分成輕微缺損(VS；VSD/Ao □ 0.2)、中度缺損(VM；0.2 < VSD/Ao □ 0.3)及嚴重缺損(VL；VSD/Ao > 0.3) 三組。Gelatin zymography分析結果顯示，在VSD病童血漿中皆可測得較高活性之MMP-2與MMP-9，特別在VSD嚴重缺損病童中，與控制組相較，其MMP-2與MMP-9活性表現分別顯著提升60% (p < 0.001) 與50% (p < 0.01)。
本研究結果指出MMP-9 SNP R279Q可能與VSD的發生有關，而隨著VSD趨於嚴重時，其血液中MMP-2與MMP-9的活性有增加的趨勢。我們推測MMPs的活性可能影響VSD的嚴重程度或其癒合率，此提供未來繼續探討MMPs影響VSD病理機制十分有價值的線索。|
Ventricular septal defect (VSD) is the most common form of congenital heart disease. Approximately 20% of patients in congenital heart disease registries have VSD as a solitary lesion. Rates of spontaneous closure for membranous and muscular VSDs in infant and children were 37% and 50%, respectively, during a mean follow-up of 12 months. Single nucleotide polymorphism (SNP) is considered one of the main factors to VSD formation. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases involved in breakdown, and physiological homeostasis of extracellular matrix. MMPs have been reported that play the important roles in embryonic development, cardiomyocyte differentiation and causing cardiac tissue remodeling. Also, many cardiac related diseases are associated to the SNP of MMPs gene. Therefore, we tried to determine the SNPs of MMPs genes and plasma MMPs activity in the patients with VSD and investigate the relation between MMPs and VSD in the aspect of incidence and severe level. Since September 2010, 101 children with VSD aging from 8 to 13 years old were studied and sampled the blood for genomic DNA preparation. The SNPs of MMP-2 (-735C>T) and MMP-9 (-1562C>T, R279Q, and R574P) were determined using polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) analysis. Plasma MMP-2 and MMP-9 activity were detected by gelatin zymography. From the four SNPs assay in this study, only the MMP-9 R279Q SNP showed a statistically significant correlation with VSD formation (p < 0.05). The frequency of GA genotype in the VSD group was higher than Control group (52.2% and 37.2%, respectively). On the other hand, according to the definition of VSD/aortic root (Ao) ratio, the patients were classified into three groups: VS (VSD/Ao ratio □ 0.2), VM (0.2 < VSD/Ao □ 0.3), and VL (VSD/Ao > 0.3) which stand for small, medium and large defect, respectively. The MMP-2 activity in the VS, VM and VL groups is about 40%, 50 and 60% higher than that in the Control group, respectively. MMP-9 has the similar trend of MMP-2. MMP-9 activity of VL group is about 50% higher than that in Control group (p < 0.01). In conclusion, MMP-9 R279Q may participate VSD pathogenesis; however, there is need to study further for clarifying the relationship between MMP-9 R279Q polymorphism and VSD. The most valuable found out in this research is that MMP-2 and MMP-9 enzyme activity increases with the defect serious level of VSD increase. Our results hint that MMPs expression may affect the severe level of VSD. It is very valuable to further investigate the exact mechanism of MMPs cause effect on VSD in the future.