基質金屬蛋白酶基因多型性及酵素活性在兒童心室中膈缺損的相關性

Abstract

中 文 摘 要 先天性心臟病佔了總出生人口比例約 1–3%，而其中心室中膈缺損(ventricular septal defect ,VSD)又是最為常見的先天性心臟病之一。比率可以佔所有先天性心臟病的40%，部分病童的中膈缺損會隨著年紀增長而逐漸變小或完全閉合，其餘在臨床追蹤時則依然保持缺陷。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 polymorphisms, SNPs)被指出可能是造成VSD疾病主要原因之一。 基質金屬蛋白酶 (matrix metalloproteinases, MMPs)是一類酵素, 其活性依賴於Zn2+等金屬離子, 參與降解細胞外基質(extracellular matrix, ECM)，並精密調控著組織中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，文獻探討中可以發現許多心臟疾病也與MMPs的基因多型性有關。據此，我們檢測VSD的病患的MMPs基因SNPs與其血漿中MMPs活性，來探討MMPs基因SNPs和血液中MMPs活性與VSD之嚴重程度。 自2010年9月起，我們收集了95 位VSD病童的血液樣品，使用聚合酶連鎖反應-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基因SNPs包括：MMP -2 -735C>T，以及 MMP-9 -1562C>T、R279Q及R574。並使用明膠酶電泳(gelatin zymography)用以測定其血漿中MMP-2和MMP-9活性。 在所檢測的四個SNPs中，結果顯示MMP-9 R279Q基因頻率在控制組與病例組分布比例有統計上的顯著差異性 (p < 0.05)。而檢測的MMP-2/-9的不同單核苷酸基因多型性基因型別中的MMP-2及MMP-9活性並無不同。另一方面，我們依病患超音波檢查之VSD直徑/主動脈主動脈直徑 (aortic root)比值分類病童VSD程度，將之分成輕微缺損(VSD/Ao ≤ 0.2)、輕度缺損(0.2 < VSD/Ao ≤ 0.3)及中度缺損(0.3 < VSD/Ao ≤ 0.66) 三組。明膠酶電泳結果顯示，特別在中度以上缺損病童分組中，皆可測得較高活性之MMP-2與MMP-9並顯著高於控制組。而分析結果也指出，隨著VSD趨於嚴重時，其MMP-2與MMP-9的活性皆有增加的趨勢。經過一年的追蹤，我們發現VSD的病患癒合與其血液中的MMP-9活性有顯著相關 (p < 0.01)。 另外我們也研究了其血液中第三型金屬蛋白酶抑制因子(TIMP-3)以及B型利鈉肽(BNP)的濃度和VSD 病童血液中MMP-2/-9活性的關聯性。分析結果發現，TIMP-3在MMP-2 -735 C>T 的CC以及MMP-9 R279Q 的QQ對偶基因型病人的濃度是較高，並與其他對偶基因有統計的顯著性差異; 並且TIMP-3和MMP-9活性、BNP血中濃度都有正相關。此外，我們也發現MMP-9活性、TIMP-3及BNP血中濃度都與VSD 的嚴重度呈現正相關。 據本研究之結果，我們推測MMPs的活性可能對於VSD 病童其患病與否、及嚴重程度、或其癒合比率上有一定的角色，因此值得未來進一步探討MMPs 與VSD病理機轉之間的關係。

Abstract Congenital heart disease (CHD) account for 1–3% population, and ventricular septal defect (VSD) is the most common form of CHD that accounts for approximately 40% of total patients. Some of children with VSD can have spontaneous closure with aging, but the others don’t. Single nucleotide polymorphism (SNP) in specific gene is considered as one of the main factors of VSD formation. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases involved in breakdown, and physiological homeostasis of extracellular matrix. MMPs play important roles in embryonic development, cardiomyocyte differentiation and causing cardiac tissue remodeling. The literature review shows many cardiac diseases are related to the SNPs of MMPs gene. Therefore, we aim to examine the SNPs of MMPs gene in patients with VSD and their plasma MMPs activities, in order to find out the relationship among gene SNPs, plasma MMPs activities and the severity of VSD. Since September 2010, 95 children with perimembranous VSD were enrolled in this study and blood samples were collected. 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 activities were also confirmed by gelatin zymography. From the results of four SNPs assay in this study, there is a significant difference of MMP-9 R279Q SNP frequency between VSD patients and control group (p < 0.05). There is no difference between MMP-2 or MMP-9 genotypes among all the four polymorphisms. Children with VSD according to the ratio of VSD diameter/diameter of aortic root (Ao), were categorized into different groups: Trivial group (VSD/Ao ratio ≤ 0.2), Mild group (0.2 < VSD/Ao ≤ 0.3), and Median group (0.3< VSD/Ao ≤ 0.66). According to the results of gelatin zymography, there is significant higher MMP-2/-9 activity in each groups compared with control group. After one year follow-up, we also found that there are significant differences of MMP-9 activities between VSD spontaneous closure group and non-closure group (p < 0.01). The concentrations of plasma tissue inhibitor of metalloproteinase-3 (TIMP-3) and B-type natriuretic peptide (BNP) in VSD children, and the correlations with MMP-2/-9 activities in VSD patients with different severities were also investigated. The data showed that the TIMP-3 concentration is significantly higher in carriers of the CC genotype of MMP-2 -735 C>T and QQ genotype of the MMP-9 R279Q polymorphisms than other genotypes. We also found that TIMP-3 correlated with MMP-9 activity and BNP concentration. Overall, MMP-9, TIMP-3, and BNP all show a similar positive trend with regard to the severity of VSD. In conclusion, we found the relation between the severity of VSD and activities of MMPs. Our data might hint that MMPs expression may play a role in the pathogenesis of VSD. It is worth to further investigate the correlation between MMPs and the mechanism of VSD formation or closure.