The correlation study between MMP-3 rs35068180 and-8 rs11225395 gene polymorphism and premature rupture of membranes in Qinghai Han- and Hui-Chinese pregnant woman

BAI Yu-fang，LU Li，QI Cun-xiu，Liu Wen-cui，SHEN Guo-ping， GONG Hai-feng，ZHU Xue-ling，WANG Rong-hua

(1.Dept of Obstetrics，Qinghai University Affiliated Hospital； 2.Qinghai University Graduate School；3.Qinghai University Medical College； 4.Central Sterile Supply Department of Qinghai University Affiliated Hospital，Qinghai，810001)

白玉芳1，路 丽2，祁存秀2，刘文翠2，沈国平3，巩海凤2，朱雪玲2，王荣华4

(1.青海大学附属医院产科；2.青海大学研究生院；3.青海大学医学院； 4.青海大学附属医院消毒供应中心，810001 青海)

摘 要 目的 利用PCR-RFLP及测序法分析青海地区回、汉族胎膜早破孕妇MMP-3 rs35068180、-8 rs11225395基因多态性特点及是否具有民族差异性。方法 提取胎膜早破孕妇和对照组组织DNA，利用PCR-RFLP及琼脂糖凝胶电泳、测序法检测基因多态性，并行相关性分析。结果 MMP-3 rs35068180A/A、A/Del、Del/Del基因型频率在汉族对照组和胎膜早破组中分别是80.53%、17.70%、1.77%和100.00%、0.00%、0.00%，两组基因型频率分布具有显著差异性(P=0.000)；在回族中分别是81.61%、16.09%、2.30%和100.00%、0.00%、0.00%，两组基因型频率呈现显著差异性(P=0.003)。MMP-8 rs11225395三种基因型A/A、A/G、G/G在汉族对照组和胎膜早破组中基因型频率分别为17.14%、31.43%、51.43%和14.16%、57.52%、28.32%，两组相比，基因型频率分布具有显著性差异(P=0.002)；回族对照组和胎膜早破基因型频率分布分别为17.24%、32.76%、50.00%和14.94%、57.47%、27.59%，两组基因型频率相比具有显著性差异(P=0.009)。结论 MMP-3 rs35068180、-8 rs11225395基因多态性在青海汉族、回族胎膜早破者间无民族差异性。同时，青海汉族、回族对照组和胎膜早破组中MMP-3 rs35068180、-8 rs11225395基因频率分布差异无统计学意义。

ThecorrelationstudybetweenMMP-3rs35068180and-8rs11225395genepolymorphismandprematureruptureofmembranesinQinghaiHan-andHui-Chinesepregnantwoman

BAI Yu-fang1，LU Li2，QI Cun-xiu2，Liu Wen-cui2，SHEN Guo-ping3， GONG Hai-feng2，ZHU Xue-ling2，WANG Rong-hua4

(1.Dept of Obstetrics，Qinghai University Affiliated Hospital； 2.Qinghai University Graduate School；3.Qinghai University Medical College； 4.Central Sterile Supply Department of Qinghai University Affiliated Hospital，Qinghai，810001)

ObjectiveTo analyze the polymorphism of matrix metalloprotease-3(MMP-3)rs35068180 and-8 rs11225395 gene in pregnant women with premature rupture of membranes in Han- and Hui-Chinese in Qinghai province by PCR-RFLP and sequencing.MethodsGenome DNA was extracted from preterm premature rupture of membranes and control group.The polymorphism of the gene was analyzed by PCR-RFLP，agarose gel electrophoresis and sequencing.ResultsThe frequency of MMP-3 rs35068180A/A，A/Del，Del/Del genotype was 80.53%，17.70%，1.77% and 100.00%，0.00%，0.00%，respectively，in the Han control and the premature rupture of membranes groups，the genotype frequency was significantly different between two groups(P=0.000).Meanwhile the frequency of genotype was 81.61%，16.09%，2.30% and 100.00%，0.00%，0.00%，respectively，in the control and the premature rupture of membranes group in Hui Chinese，and the genotype frequency was obviously different between two groups(P=0.003).The frequency of MMP-8 rs11225395 A/A，A/G，G/G genotype was 17.14%，31.43%，51.43% and 14.16%，57.52%，28.32%，respectively，in the Han control and the premature rupture of membranes groups，the genotype frequency was significantly different between two groups(P=0.002).Meanwhile the frequency of genotype was 17.24%，32.76%，50.00% and 14.94%，57.47%，27.59%，respectively，in the control and the premature rupture of membranes group in Hui Chinese，and the genotype frequency was obviously different between two groups(P=0.009).ConclusionThe polymorphisms of MMP-3 rs35068180 and -8 rs11225395 may be related to the premature rupture of membranes in Qinghai Han and Hui Chinese pregnant women.Meanwhile，the allele frequencies of MMP-3 rs35068180 and -8 rs11225395 were not specific between Han and Hui nationality.

MMP Polymorphism Prematurerupture of membranes Qinghai Han Hui

白玉芳1，路 丽2，祁存秀2，刘文翠2，沈国平3，巩海凤2，朱雪玲2，王荣华4

(1.青海大学附属医院产科；2.青海大学研究生院；3.青海大学医学院； 4.青海大学附属医院消毒供应中心，810001 青海)

摘要目的利用PCR-RFLP及测序法分析青海地区回、汉族胎膜早破孕妇MMP-3 rs35068180、-8 rs11225395基因多态性特点及是否具有民族差异性。方法提取胎膜早破孕妇和对照组组织DNA，利用PCR-RFLP及琼脂糖凝胶电泳、测序法检测基因多态性，并行相关性分析。结果MMP-3 rs35068180A/A、A/Del、Del/Del基因型频率在汉族对照组和胎膜早破组中分别是80.53%、17.70%、1.77%和100.00%、0.00%、0.00%，两组基因型频率分布具有显著差异性(P=0.000)；在回族中分别是81.61%、16.09%、2.30%和100.00%、0.00%、0.00%，两组基因型频率呈现显著差异性(P=0.003)。MMP-8 rs11225395三种基因型A/A、A/G、G/G在汉族对照组和胎膜早破组中基因型频率分别为17.14%、31.43%、51.43%和14.16%、57.52%、28.32%，两组相比，基因型频率分布具有显著性差异(P=0.002)；回族对照组和胎膜早破基因型频率分布分别为17.24%、32.76%、50.00%和14.94%、57.47%、27.59%，两组基因型频率相比具有显著性差异(P=0.009)。结论MMP-3 rs35068180、-8 rs11225395基因多态性在青海汉族、回族胎膜早破者间无民族差异性。同时，青海汉族、回族对照组和胎膜早破组中MMP-3 rs35068180、-8 rs11225395基因频率分布差异无统计学意义。

Premature rupture of membranes referring to rupture of membranes before labor is a relatively high incidence of complications during middle and late pregnancy[1，2]，and the incidence rate is about 19.53%in mainland China[3].It has great effect on maternal and will be fetal.After the rupture of the membrane，the pathogenic bacteria in the vagina can be antidromically infected，and even cause intrauterine infection，severe cases can be complicated by septicemia[4，5]，intracranial infection，etc[6-9].The degree of infection is correlated with the time of rupture.Premature rupture of membranes is mostly premature，and premature infants peculiarly prone to respiratory distress syndrome，aspiration pneumonia，etc[6].Once a large number of outflow of amniotic fluid after ruptured of amniotic membrane may occur in the umbilical cord prolapse，umbilical cord compression caused fetal distress and even thus cause fetal death，neonatal intracranial hemorrhage.Premature rupture of membranes also can cause serious complications such as placental abruption，amniotic fluid embolism and postpartum hemorrhage.The perinatal mortality rate，maternal infection rate，neonatal morbidity and mortality were significantly increased[10，11].There are many causes of premature rupture of membranes.Recently，the study of its pathogenesis is mainly focused on the changes in the structure of the fetal membranes，which is mostly concentrated on the matrix metalloprotease(MMP)，the degradation of collagen was mediated by MMP[12-17].When the onset of labor，the activity of MMP in the fetal membrane increased significantly，which accelerated the degradation of interstitial collagen，affected the structure of the fetal membrane，so that the strength of the fetal membrane decreased，resulting in rupture of the fetal membranes[18-20].However，the relationship between the expression of MMP gene and the susceptibility to premature rupture of membranes in different nationalities in Qinghai province has not been studied.PCR and SNP genotyping technique were used in this study to analyze the polymorphism of MMP-3 and -8 gene and correlation between Han and Hui Chinese pregnant women in Qinghai province about premature rupture of membranes，in the meantime analyzed whether the expression of MMP gene polymorphism in premature rupture of the membranes with national specificity.Finally，it provides an important experimental basis for the molecular mechanism of premature rupture of membranes in Han- and Hui-Chinese pregnant women in Qinghai province.

1 Research subject and method

1.1 Subject and selection criteria

According to the requirements of the experiment，samples were collected from Affiliated Hospital of Qinghai University and other two hospitals.From January to December in 2016，among pregnant women of Hui nationality 58 cases of premature rupture of fetal membranes and 87 cases of normal pregnancy were collected，meanwhile 70 cases of premature rupture of fetal membranes and 113 cases of normal pregnancy were collected among Han nationality.The inclusion criteria was referred to eighth edition of “Obstetrics and Gynecology”published by people′s Medical Publishing House.

1.2 Research method

1.2.1 Specimens collection

After delivery of the placenta within 5 minutes，at rupture of fetal membranes closing to the cervical，2×2 cm membranes were sheared in a sterile environment，washed the amniotic fluid and blood on the membrane surface with physiological saline repeatedly，then it was put into the sterilized specimen collection tube，-80 degrees refrigerator freezing.

1.2.2 Reagents and equipments

absolute ethanol，agarose，electrophoretic buffer，genomic DNA extraction kit and primer(Biotechnology Corporation，Shanghai)，centrifugal machine(Eppendorf AG，Germany)，incubator(Lang GanCompany，Shanghai)，scroll device(IKA Laboratory Equipment，Holland)，electrophoresis apparatus trophoresis(Liuyi，Beijing)，Nanodrop 2000c spectrophotometer(Thermo Scientific，America)，Gel imaging system(Bio-Rad Laboratories，America).

1.2.3 Genome DNA extraction

Take the small membranes and put it into liquid nitrogen immediately，adding proteinase K solution mix them again.The mixture is placed in the incubator for 3 hours for the dissolution of the organization.According the manual of genomic DNA Extraction Kit，the genome DNA has been extracted and finally DNA is precipitated and purified by anhydrous ethanol and stored at -80 degrees for subsequent experiments.

1.2.4 PCR and SNP genotyping

The primers needed by PCR were synthesized by Shanghai Biotechnology Co.，Ltd(Table 1).PCR reaction include pre-denaturation at 99 ℃ for 94 s，and then 94 ℃ 45 s，anneal at 55 ℃～57 ℃ 45 s，extend at 72 ℃ 45 s，a total of 35 cycles.7 μL PCR amplification product was used on 1% agarose gel for electrophoresis at 100 voltage for 40 min，then the results was imaged by responsible system.Finally，the PCR amplification products were sent to Shanghai bio engineering Co.，Ltd.and sequenced by SNP technique.The genotype and gene frequencies are determined according to the results of sequencing.

Table 1 The primers of MMP-3 and -8

1.2.5 DNA agarose gel electrophoresis

Extracted genome DNA，put it on 1% agarose gel electrophoresis at 100 voltage for 30minutes.The results are preserved for analysis and verification after scanning by the gel imaging system.

1.2.6 Statistical analysis

SPSS17 was used for statistical analysis，and the results were analyzed by Fisher exact test and chi square test，P<0.05 was significant difference.

2 Results

2.1 Genomic DNA electrophoresis detection

Genome DNA was extracted from premature rupture of membranes and control group，detected by agarose gel electrophoresis and was analyzed by the gel imaging system camera(Figure 1).The results showed that genomic DNA was extracted successfully and the bands were very clear.

M：DNA Marker；1-3：DNA of fetal membranes in normal pregnant women；4-6：DNA of fetal membranes in premature rupture of membranes

Figure1GenomicDNAelectrophoresisresults

2.2 The detection of PCR product

M：DNAmarker；Left 1-8：MMP-3 primer PCR product；Right 1-8：MMP-8 primer PCR product

Figure2MMP-3andMMP-8partialspecimenPCRproductelectrophoresis

The amplification of PCR product was detected by DNA agarose gel electrophoresis.The results showed that PCR amplification was successful and the bands were clear.

2.3 Genotyping of SNP

By sequencing and genotyping，we found that the genotype of MMP-3 rs35068180 showed the changing from A/A into A/Del(Figure 3).In the gene MMP-8 rs11225395，it showed A/A genotype becoming to the A/G or G/G genotype(Figure 4).

The frequency of MMP-3 rs35068180A/A，A/Del，Del/Del genotype were 80.53%，17.70%，1.77% and 100.00%，0.00%，0.00%，respectively，in the control and the premature rupture of membranes groups of Han Chinese，and the genotype frequency was significantly different between two groups(P=0.000)after statistically analysis by Fisher′s exact test.Meanwhile the frequency of genotype were 81.61%，16.09%，2.30% and 100.00%，0.00%，0.00%，respectively，in the control and the premature rupture of membranes group in Hui Chinese，and the genotype frequency was obviously different between two groups(P<0.05)as well(Table 2).

Table 2 Genotype distribution and frequency analysis of MMP-3 rs35068180(%)

*：Applied by R，*：CFisher′s Exact Test.

Figure 3 MMP-3 rs35068180 gene type A/Abecoming to A/G

Figure 4 MMP-8 rs11225395 genotype A/A becoming to G/G or A/G

The frequency of MMP-8 rs11225395 A/A，A/G，G/G genotype were17.14%，31.43%，51.43% and 14.16%，57.52%，28.32%，respectively，in the control and the premature rupture of membranes groups of Han Chinese，and the genotype frequency was significantly different between two groups(P=0.002)，but there was no difference in gene frequency(allele frequency)distribution(P=0.055).Meanwhile the frequency of genotype were17.24%，32.76%，50.00% and 14.94%，57.47%，27.59%，respectively，in the control and the premature rupture of membranes group in Hui Chinese，and the genotype frequency was obviously different between two groups(P=0.009)，however，the gene frequency distribution was not statistically significant(P=0.086)(Table 3).

Table 3 Genotype distribution and frequency analysis of MMP-8 rs11225395(%)

By comparing the MMP-3 rs35068180 and MMP-8 rs11225395 genotypes frequency distribution among the Han and Hui control group or premature rupture of membranes，there found no difference between the groups(P>0.05).The results showed that the gene frequencies ofboth genes in Qinghai Han and Hui control group and premature rupture of membrane group had no national specificity.

3 Discussion

single-nucleotide polymorphism(SNP)is a DNA sequence polymorphism caused by the variation of the nucleotide level in the genome.The vast majority of SNPs itself is not a cause of susceptibility，However，in the whole genome，the SNP pattern between susceptible and non-susceptible populations can be displayed，and by comparison the structural characteristics of susceptible populations can be revealed.Research shows that the imbalance of the degradation of extracellular matrix(ECM)of fetal membranes is an important reason leading to the weak structure and easy rupture of fetal membranes，and MMPs plays an important role in the degradation of ECM.The main types of MMPs in placenta and fetal membranes are MMP-1，2，3，7，8，9，etc[13-17].MMP-8 can specifically degrade type I and type III collagen in fetal membranes，and showed different function in human diseases.MMP-8 does not appear in the amniotic fluid during normal pregnancy，but it occurs in cervical tissue，Kelly finds that cervical ripening is often accompanied by a large number of neutrophil invasion，and neutrophils can produce a large number of MMP-8，so as to maintain the hardness of the cervical collagen hydrolysis，impel cervical ripening.Clinical studies showed that MMP-8 rs1940475 SNP with AA genotype was associated with highest expression of tumor necrosis factor(TNF)and interleukin-6(IL-6)level after LPS stimulation[21]，and the MIP-1a(macrophage inflammatory protein-1a)higher expression was related with GG carriers in human endotoxemia[22，23].There was also evidence that showed MMP-8(-799)gene polymorphism with T allele was also related with high risk of developing tendinopathy[24]，and its also indicated that MMP-8 rs17099451 SNP was correlated with asthma development[16，21].Meanwhile，in Chinese Han population，it found that MMP-8 polymorphism is associated with osteonecrosis development[21].With the research results by Chen，it showed that the gene polymorphisms of MMP-3 and MMP-8 were also related with alcohol-induced osteonecrosis of the femoral head.MMP-7 is reported that in the promoter region exist A/G polymorphism，the A-G mutation of this site can increase the transcriptional activity of the gene.It also showed that the activity of MMP in amniotic fluid after rupture of membranes was significantly higher in the active phase of labor，especially in MMP-9[25-27].After the rupture of the membranes，it may cause the infection of amniotic cavity and even endanger the safety of mother and fetus.Therefore，these genes and their SNP types are closely related to premature rupture of membranes.

The present results show that the distribution of MMP-3 rs35068180 genotype frequencies and gene frequencies have significant differences in Qinghai Han and Hui Chinese control groups and premature rupture of membranes，and MMP-8 rs11225395 show only the genotype different between two groups，but the gene frequency has no significant difference between two groups.It suggests that the expression of SNP MMP-3 rs35068180 and MMP-8 rs11225395 distribution in control and premature rupture of membranes has certain correlation.After analyzing the frequency of genotype between Han and Hui-Chinese，it finds that there is no ethnic specificity in control groups and premature rupture of membranes.

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青海汉、回族胎膜早破与MMP-3 rs35068180、 -8 rs11225395基因多态性※

MMP 基因 基因多态性 胎膜早破 青海 回族 汉族

R394

A

2017-4-12

10.13452/j.cnki.jqmc.2017.03.003

※：青海省应用基础研究项目(2016-ZJ-762)；青海省卫计委项目(2015) 白玉芳(1972～)，女，青海籍，主任医师，硕导，教授