汉族人群MMP-9基因单核苷酸多态性与PACG的相关性
蔡 晖1,石华宗1,杨豫湘2
作者单位:1( 435000)中国湖北省黄石市第二医院眼科;2(435005)中国湖北省黄石市第五医院眼科
作者简介:蔡晖,本科,副主任医师,研究方向:青光眼、白内障的临床及基础研究
通讯作者:蔡晖.caihui2015yk@ 163.com
收稿日期:2018-05 -29 修回日期:2018-09-04
Correlation between extracellular MMP-9 polymorphism and genetic susceptibility of PACG in a Han Chinese population
Hui Cai', Hua-Zong Shi', Yu-Xiang Yang2
Department of Ophthalmology, Second Hospital of Huangshi,Huangshi 435000, Hubei Province, China; 2Depafiment of Ophthalmology, Huangshi No. 5 Hospital, Huangshi 435005,Hubei Province, China
Correspondence to: Hui Cai. Department of Ophthalmology,Second Hospital of Huangshi, Huangshi 435000, Hubei Province,China. caihui2015yk@ 163. com.
Received :2018 -05 -29 Accepted :2018 -09 -04
Abstract
.AIM: To analyze the association of extracellular matrix metalloprotease 9 ( MMP - 9 ) single nucleotide polymorphism ( SNP) with genetic susceptibility of primary angle - closure glaucoma ( PACG) in a Han Chinese population.
.METHODS: Totally 200 PACG patients ( PACG group) and 200 healthy people ( normal control group) were collected in our hospital from January 2014 to December 2016. Peripheral venous blood was collected and extracted for genomic DNA, the polymerase chain reaction - restriction fragment length polymorphism technique ( PCR-RFLP) was applied to detect the alleles and genotypes of rs2250889, rs2274755 and rs2664538 sites in MMP-9 gene. The frequency distribution of alleles and genotypes in the two groups were calculated by chi-square test, and its association with genetic susceptibility of PACG was analyzed.
. RESULTS: There were no statistical differences in age,gender, body mass index, diastolic blood pressure and systolic blood pressure in the two groups ( P>0.05). The genotype frequencies of rs2250889, rs2274755 and rs2664538 sites in MMP-9 gene were in line with Hardy-Weinberg equilibrium. The genotype and allele frequency distribution of rs2250889 and rs2664538 sites were significantly different between the PACG group and the normal control group ( P0. 05). The subjects whose rs2250889 site carrying the CC genotype was susceptible to PACG. Similarly, the rs2664538 site carrying the GG genotype was susceptible to PACG.
* CONCLUSION: The rs2250889 and rs2664538 polymorphisms of MMM-9 are correlated with the risk of PACG in a Han Chinese population, while the rs2274755
polymorphism is not related to genetic susceptibility of PACG.
. KEYWORDS: primary angle- closure glaucoma; matrix metalloprotease 9; single nucleotide polymorphism; susceptibility; rs2250889; rs2664538
Citation: Cai H, Shi HZ, Yang YX. Correlation between extracellular MMP-9 polymorphism and genetic susc:eptibility of PACG in a Han Chinese population. Guoji Yanke Zazhi ( Int Eye Sci) 2018 ;18 (10) :1859-1862
摘要
目的:探讨基质金属蛋白酶9( extracellular matrix metalloprotease 9.MMP -9)基因单核苷酸多态性(single nucleotide polymorphism,SNP)与汉族人群原发性闭角型青光眼( primary angle closure glaucoma,PACG)易感性的关联。
方法:选取我院2014 - 01/2016 - 12收治的200例汉族PACG患者作为PACG组,同时招募200例汉族体检健康者作为正常对照组。常规外周静脉采集,提取全血基因组DNA,采用聚合酶链反应一限制性片段长度多态性技术(polymerase chain reaction - restriction fragment lengthpolymorphism.PCR-RFLP)检测两组受检者MMP-9基因rs2250889、rs2274755及rs2664538位点等位基因及基因型,分析等位基因和基因型频率分布及其与PACG易感性之间的关系。
结果:两组受检者年龄、性别、体质量指数、血压等一般资料差异均无统计学意义(P>0.05)。MMP -9基因rs2250889、rs2274755及rs2664538位点基因型频率均符合哈迪一温伯格平衡(Hardy - Weinberg equilibrium)。两组受检者rs2250889和rs2664538位点基因型及等位基因频率分布差异均有统计学意义(P<0.05),而rs2274755位点基因型及等位基因频率分布差异均无统计学意义(P>0.05)。rs2250889位点中携带CC基因型人群为PACG的易感人群。同样,rs2664538位点中携带GG基因型人群为PACG的易感人群。
结论:汉族人群中,MMP-9基因rs2250889和rs2664538位点多态性与PACG的发病存在一定关联,而rs2274755位点多态性与PACG的易感性无关。
关键词:原发性闭角型青光眼;基质金属蛋白酶9;基因单核苷酸多态性;易感性;rs2250889;rs2664538
引用:蔡晖,石华宗,杨豫湘.汉族人群MMP-9基因单核苷酸多态性与PACG的相关性.国际眼科杂志2018;18( 10):1859 -1862
O引言
青光眼( glaucoma)是全球第二大致盲眼病,我国常见的青光眼类型是原发性闭角型青光眼(primary angle-closure glaucoma,PACG)。PACG是一种不可逆性致盲眼病,以特征性视神经萎缩及视野缺损为主要临床特征。流行病学研究发现,PACG的发病与种族、年龄、性别及眼解剖结构等因素密切相关。研究表明,50岁及以上人群PACG患病率较50岁以下人群明显升高,且患病率随年龄的增长而逐渐升高。新研究发现,PACG的发生与小梁网组织细胞外基质的结构和功能及代谢密切相关[5-6]。基质金属蛋白酶9( extracellular matrix metalloprotease 9.MMP-9)又称明胶酶B,是基质金属蛋白酶家族( matrix metalloproteinases,MMPs)成员之一,参与维持正常细胞外基质的结构和功能门1。目前,关于MMP -9基因单核苷酸多态性( single nucleotidepolymorphism,SNP)与PACG遗传易感性的关系尚未见报道。本研究选取我院2014 -01/2016 -12收治的200例汉族PACG患者作为PACG组,同时招募200例汉族体检健康者作为正常对照组,拟通过聚合酶链反应一限制性片段长度多态性技术( polymerase chain reaction - restrictionfragment length polymorphism,PCR-RFLP)检测两组受检者MMP-9基因rs2250889、rs2274755及rs2664538位点等位基因及基因型,探讨其与PACG遗传易感性之间的关系。
1对象和方法
1.1对象 选取我院2014 - 01/2016 - 12收治的汉族
PACG患者200例作为PACG组,同时招募汉族体检健康者200例作为正常对照组。PACG组纳入标准:(1)年龄>30周岁;(2)典型青光眼视野缺损;(3)典型青光眼视神经损害;(4)眼压>21mmHg;(5)窄房角,≥180。房周的房角关闭;(6)排除各种继发性青光眼,如由葡萄膜炎、眼外伤、晶状体半脱位等引起的青光眼。正常对照组纳入标准:(1)年龄>30周岁;(2)无青光眼的症状和体征;(3)无青光眼病史及家族史;(4)无其它眼内疾病。两组受检者的一般临床资料比较,差异均无统计学意义(P>0. 05),见表1。本研究已获得所有患者知情同意,并得到本院伦理委员会批准。
1.2方法
1.2.1样本采集所有研究对象均于清晨空腹采集外周静脉血SmL,使用乙二胺四乙酸钠盐抗凝,颠倒摇匀,将所得血细胞置于-80℃超低温冰箱保存。
1.2.2全血基因DNA提取 参照Takara公司全血基因组DNA提取试剂盒说明书提取基因组DNA,采用NanoDrop 2000超微量分光光度计测定样本的DNA浓度,随后将DNA放置于-80℃保存备用。
1.2.3 PCR-RFLP检测MMP-9基因位点 参照Takara公司EmeraldAmp PCR Master Mix试剂盒进行普通PCR反应。PCR扩增引物由Takara公司合成,引物序列见表2。PCR反应体系:PCR Master Mix 101JL,正向和反向引物各0.5肛L,去离子水8¨L。PCR扩增条件:98℃预变性2min,98℃变性10s,55℃退火30s,72℃延伸Imin,共37个循环,72℃延伸10min,4℃冷却30min。随后采用新英格兰NEB公司限制性内切酶切割PCR产物,2.0010琼脂糖电泳检测产物大小。
统计学分析:采用SPSS21.0统计软件包和UNPHASED 3.1.5软件进行统计学分析。计量资料以均数±标准差(x+s)表示,组间比较采用独立样本f检验。基因型频率、等位基因频率等计数资料的组间比较采用卡方检验。以P<o. p="" 05为差异有统计学意义。
2结果
2.1 MMP-9基因位点检测结果 本组受试者PCR -RFLP检测结果显示,rs2250889位点未变异,可被BsaA I内切酶识别,GG基因型产生2个片段,GC基因型则产生3个片段,而CC基因型只有1个片段;rs2274755位点未变异,可被Bcl I内切酶识别,GG基因型产生2个片段,GT基因型则产生3个片段,而TT基因型只有1个片段;rs2664538位点未变异,可被CviAⅡ内切酶识别,AA基因型产生2个片段,AG基因型则产生3个片段,而CG基因型只有1个片段,见图1。
700 bp
500 hp
400 hp
300 bp
2∞ bp
IOO bp
rs2250889位点 rs2274755 j,7.点 rs26645 38化点
Marker GG GC CC TT GG GT GO AG AA
图1 本组受试者PCR-RFLP检测结果。
2.2 MMP-9基因位点的多态性分析 采用哈迪一温伯格平衡( Hardy-Weinberg equilibrium)对基因型频率进行检验,结果表明MMP -9基因rs2250889、rs2274755及rs2664538位点基因型频率均符合哈迪一温伯格平衡。两组受检者rs2250889位点基因型和等位基因频率分布比较,差异均有统计学意义(X2= 11. 34、14. 66,均P<o. p="" 03、0.01,表5)。
2.3 MMP -9基因rs2250889和rs2664538位点与PACG易感性分析 rs2250889位点中PACG组CC基因型人群比例显著高于正常对照组,故携带CC基因型人群为PACG的易感人群。同样,rs2664538位点中PACG组GG基因型人群比例显著高于正常对照组,故携带GG基因型人群为PACG的易感人群。SNP位点 ;I物类型 引物序列(5’-3’)
表1 两组受检者的一般临床资料比较 | |||
血压(x+s,mmHg) | |||
组别 例数 女/男(例)
| 年龄(x+s,岁)
| 体质量指数(x+s,kg/rT12),
|
舒张压 收缩压 |
正常对照组 200 114/86 | 57. 4+5.1 | 21. 8+3.1 | 78. 5+9. 9 113. 2+15. 6 |
PACG组 200 121/79 | 55. 5+6.2 | 22. 7+2.8 | 75. 4+7. 6 115. 4+17. 8 |
t/X2 2. 52 | 1. 93 | 1. 46 | 2. 21 3.57 |
JP 0. 98 | 0. 76 | 0. 52 | 0. 54 0.69 |
注:正常对照组:汉族体检健康者。 | |||
表2引物序列 |
正常对照组 200 106(53.0) 67(33.5) 27(13.5) 279( 69.8) 121(30.2)
PACG组 200 78(39.0) 62(31.0) 60(30.0) 218 (54.5) 182(45.5)
rs2250889 | 正向引物 | TTCACTGAGAAGCTTAGGGGAGCAG |
反向引物 | AGAGGGAGCACGTACTCCTAGAACG | |
rs2274755 | 正向引物 | GGAAAATCCAAGAGACCTGGGCGGG |
反向引物 | GAATTGCAGGCCACACAGCACTAGT | |
rs2664538 | 正向引物 | C'ITCGAGGGCCGCTCCTACTCTGCC |
反向引物 | GGCGGAGTCACGGTCGTCCTGAGCA | |
表3两组受检者 | rs2250889位点基因型和等位基因频率分布比较 | 例(%) |
组别 | 基因型 扁I{阱 | 等位基因
|
y,IJ,烈 GG GC |
CC G C |
注:正常对照组:汉族体检健康者。基因型分布比较:OR(95%可信区间):1. 86(1.39~2.36);等位基因频率分布比较:OR(95%可信区间):1. 91(1.43~2.55)。
表4两组受检者rs2274755位点基因型和等位基因频率分布的比较 例(%)
组别 | 例数 | 基因型 | 等位基因 | |||
GG | GT | TT | G | T | ||
正常对照组 | 200 | 113(56.5) | 50(25.0) | 37(18. 5) | 276( 69. | 0) 124(31. 0) |
PACG组 | 200 | 119(59. 5) | 53(26.5) | 28 (14.0) | 291( 72. | 8) 109(27. 2) |
注:正常对照组:汉族体检健康者。基因型分布比较:OR( 95%可信区间):0. 52(0.40~1.08);等位基因频率分布比较:OR(95 010可信区间):0. 83(0.61~1.13)。
表5两组受检者rs2664538位点基因型和等位基因频率分布比较 例(%)
组别 | 例数 | 基因型 | 等位基冈 | |||
AA | AG | GG | A | G | ||
正常对照组 | 200 | 128 (64. 0) | 54 (27. 0) | 18f9.0) | 310f 77. | 5) 90(22.5) |
PACG组 | 200 | 115 (57.5) | 48(24.0) | 37(18.5) | 278f 69. | 5) 122(30.5) |
注:正常对照组:汉族体检健康者。基因型分布比较:OR(95%可信区间):1. 26(1.04~1.85);等位基因频率分布比较:OR(95%可信区间):1. 51(1. 10~2.08)。
3讨论
青光眼是临床中一种常见的眼病,其致盲率仅次于白内障,具有较高的遗传患病风险。PACG是青光眼中发病率较高的一种类型,我国成年人PACG的患病率约为1. 4%,女性患者数量显著高于男性[8-9]。PACG的发病与虹膜、晶状体及房角之间的解剖空间密切相关[10]。
PACG发病主要是由于虹膜和小梁网粘连,导致房水在小梁网处的外流通道堵塞所致。研究显示,短眼轴、前房较浅及晶状体较厚的人群更容易发生PACG[1l]。PACG的患病率随年龄的增加而升高,可能是因为晶状体增厚及向前移动导致房角狭窄所致。
基质金属蛋白酶家族是一类细胞外基质成分,是具有特异性降解作用的锌依赖蛋白水解酶家族。MMP-9又称明胶酶B,是基质金属蛋白酶家族成员之一,可参与细胞外基质成分的降解、重构、血管生成,肿瘤细胞迁移及炎性反应等各种生理和病理过程。研究发现,血清中MMP-9的高表达与冠状动脉斑块不稳定、冠状动脉粥样硬化及支架内再狭窄密切相关[14];MMP-9活性升高与心肌缺血再灌注损伤中心肌顿抑有关[15];MMP-9在胃癌、肺癌、肾癌和子宫内膜癌等组织呈高表达,且与肿瘤预后密切相关[16]。此外,高表达的MMP-9可促进肿瘤新生血管生成,从而加速肿瘤的生长和侵袭17。
MMP-9基因位于人类20号染色体长臂1区3带12亚带,由13个外显子组成,全长7 654个核苷酸。截至目前,共发现155个MMP -9基因SNP位点,其中以rs6558394、rs3918242、rs2274755、rs2250889及rs2664538位点的研究为广泛。现已证实,汉族人群中,rs3918242位点多态性与慢性心力衰竭的发病风险密切相关18];rs2274755位点多态性与原发性开角型青光眼易感性相关联[19];rs2664538位点多态性与新加坡人群PACG无明显相关性,而与台湾人群PACG发病存在一定相关性[20-21]。
本研究选取我院200例汉族PACG患者作为PACG组,同时招募200例汉族体检健康者作为正常对照组,采用PCR - RFLP技术检测两组受检者中MMP -9基因rs2250889、rs2274755及rs2664538位点等位基因及基因型。结果发现,rs2250889和rs2664538位点基因型及等位基因频率分布在PACG组和正常对照组差异显著,提示汉族人群中rs2250889和rs2664538位点多态性与PACC遗传易感性相关,这与既往文献报道的台湾人群21结果一致,而与新加坡人群汹1不一致,亦表明PACG发病与种族密切相关。本研究两组受试者中rs2274755位点基因型及等位基因频率分布无统计学差异,这与既往研究19结果一致。我们进一步分析MMP -9基因rs2250889和rs2664538位点与PACG易感性的关系,发现rs2250889位点中携带CC基因型人群为PACG的易感人群。同样,rs2664538位点中携带GG基因型人群为PACG的易感人群。
综上所述,本研究表明汉族人群中MMP -9基因rs2250889和rs2664538位点多态性与PACC的发病风险存在一定关联,而rs2274755位点多态性与PACG的遗传易感性无关。本研究不足之处在于临床样本量偏少,后期仍需大规模人群流行病学研究论证上述结论的可靠性。
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