|Year : 2018 | Volume
| Issue : 3 | Page : 363-368
Matrix metalloproteinase-9 gene variants and pregnancy-induced hypertension in Egyptian women: a lack of association
Neveen A Abdulhafeez1, Eman R Abd-Elgawad1, Ahmed W.A Morad2, Ola S El-Shimi1, Rana A Khashaba1
1 Department of Clinical and Chemical Pathology, Faculty of Medicine, Benha University, Benha, Egypt
2 Department of Gynecology and Obstetrics, Faculty of Medicine, Benha University, Benha, Egypt
|Date of Submission||01-May-2018|
|Date of Acceptance||26-Jul-2018|
|Date of Web Publication||07-Jan-2019|
Dr. Rana A Khashaba
Department of Clinical and Chemical Pathology, Faculty of Medicine, Benha University, Benha 13511
Source of Support: None, Conflict of Interest: None
Background Gestational hypertension (GH) is a common disorder during pregnancy that can progress to preeclampsia (PE). PE and its complications have become the leading cause of maternal and fetal morbidity and mortality worldwide. The development of PE is unpredictable and thus challenging to prevent and manage clinically. A cluster of enzymes, called matrix metalloproteinases (MMPs), have been reported to be involved in the pathophysiology of hypertensive states of pregnancy.
Aim We aimed to investigate the association of two functional polymorphisms [−1562C/T and −90(CA)13–25] in the MMP-9 gene and their haplotypes with PE and/or GH in a group of Egyptian patients.
Methods A total of 150 pregnant women; 50 healthy (control), 50 with GH, and 50 with PE were enrolled and genotyped for −1562C/T and −90(CA)13–25 polymorphisms by PCR-RFLP and end-point PCR correspondingly.
Results For both studied polymorphisms, no significant differences were found in genotype, allele, and haplotype frequencies when PE or GH groups were compared with control group.
Conclusions Although MMP-9 −1562C/T and −90(CA)13-25 polymorphisms and their haplotypes were not associated with either GH or PE Egyptian patients, the role of MMP-9 and its genetic variants cannot be ruled out in the pathophysiology of different hypertensive states of pregnancy.
Keywords: Egypt, gestational hypertension, matrix metalloproteinase-9, preeclampsia
|How to cite this article:|
Abdulhafeez NA, Abd-Elgawad ER, Morad AW, El-Shimi OS, Khashaba RA. Matrix metalloproteinase-9 gene variants and pregnancy-induced hypertension in Egyptian women: a lack of association. Benha Med J 2018;35:363-8
|How to cite this URL:|
Abdulhafeez NA, Abd-Elgawad ER, Morad AW, El-Shimi OS, Khashaba RA. Matrix metalloproteinase-9 gene variants and pregnancy-induced hypertension in Egyptian women: a lack of association. Benha Med J [serial online] 2018 [cited 2020 Aug 6];35:363-8. Available from: http://www.bmfj.eg.net/text.asp?2018/35/3/363/249433
| Introduction|| |
Hypertensive disorders of pregnancy (HDP) are a significant health problem for women and their offspring worldwide. HDP are classified into (a) preeclampsia (PE)-eclampsia, (b) chronic hypertension (of any cause), (c) chronic hypertension with superimposed PE, and (d) gestational hypertension (GH) . GH − a major precursor of PE − occurs in 5–10% of pregnancies, though its complications are less severe . However, PE complicates 5–10% of all pregnancies worldwide . PE and its complications have become a leading cause of maternal and fetal morbidity and mortality, responsible for ∼40% of births delivered at early gestation . The development of PE is hardly predictable, therefore challenging to prevent and manage clinically . Normally in early pregnancy, trophoblasts invade maternal vessels resulting in extracellular matrix remodeling, which leads to high uteroplacental vessel dispensability to accommodate the increased blood flow . However, in PE, this trophoblastic invasion is reduced, causing inadequate modification of maternal spiral arteries and thus decreases the placental perfusion . Matrix metalloproteinases (MMPs) are a group of zinc-dependent proteolytic enzymes with various functions and tissue distribution . MMPs target extracellular matrix components during development and morphogenesis . Of them, MMP-2 and MMP-9 are involved in remodeling of placental and uterine arteries, and their abnormal expression have been described in HDP . Gene polymorphisms can potentially affect the expression levels of the corresponding genes. Thus, the functional MMP-9 polymorphisms, −1562C/T substitution (rs3918242) and the microsatellite −90(CA)13–25 (rs3222264), have been reported with many diseases, including cardiovascular diseases . The aim of the present study was to investigate the 2 functional polymorphisms [−90(CA)13–25 and −1562C/T] in the MMP-9 gene either alone or combined within haplotypes that are associated with GH and/or PE in a group of Egyptian patients from Kalyobia Governorate.
| Participants and methods|| |
This randomized case–controlled study was conducted on 150 pregnant females attending the Gynecology and Obstetrics Outpatient Clinic and Department, Benha University hospitals. They were divided into three groups: group I included 50 pregnant women with PE with mean age 26.24±2.55 years, group II included 50 pregnant women with GH with mean age 25.64±2.84 years, and group III included 50 apparently healthy age-matched pregnant women as a control group with a mean age of 25.48±3.15 years with no past or family history of GH or PE. Informed consent was obtained from each participant, and the study was approved by local ethics committee of Faculty of Medicine, Benha University. For group I, patients were diagnosed as PE after 20 weeks of gestation when the blood pressure was higher than 140/90 mmHg at two separate occasions, 6 h apart, along with significant proteinuria by dipstick reading of greater than 2+ on a voided random urine sample. For Group II, patients were diagnosed as GH after 20 weeks of gestation, when the blood pressure was higher than 140/90 mmHg at two separate occasions, 6 h apart. Any individual complicated by clinical chorioamnionitis or any infectious disorder or having urinary tract infection or any other cause of proteinuria was excluded from the study.
Genotyping the MMP-9 polymorphisms
Genomic DNA was extracted from peripheral blood leukocytes employing Gene Purelink Whole Blood Genomic DNA Purification Mini Kit QIAamp (QIAGEN, Hilden, Germany) according to the manufacture’s protocol. Two PCR reactions were performed separately using 2× DreamTaq Green PCR Master Mix (Thermo Scientific, Waltham, MA, USA), using the following primer sets: for MMP-9 (−1562C/T) (rs3918242), forward primer was 5′-GCCTGGCACATAGTAGGCCC-3′ and reverse primer was 3′-CTTCCTAGCCAGCCGGCATC-5′. For MMP-9 (−90(CA)13–25) (rs3222264), forward primer was 5′-GACTTGGCAGTGGAGACTGCGGGCA-3′ and reverse primer was 5′-GACCCCACCCCTCCTTGACAGGCAA-3′. The reactions were performed in PikoReal 24 (Thermo Scientific), in the following steps: for rs3918242, initial denaturation at 95°C for 3 min; then 35 cycles of denaturation (95°C for 30 s), annealing (60°C for 30 s) and extension (72°C for 60 s); then a final extension step for 15 min at 72°C. For rs3222264, initial denaturation at 95°C for 3 min; then 35 cycles of denaturation (95°C for 30 s), annealing (65°C for 30 s), and extension (72°C for 60 s); and then a final extension step for 15 min at 72°C. The amplified product of the first SNP was subsequently digested by SphI restriction enzyme (Invitrogen, Carlsbad, CA, USA) at 37°C overnight. The enzyme digested the 435bp PCR product in the presence of T allele into 247bp and 188bp fragments, whereas the C allele remained un-cleaved, which was detected on 1% agarose gel ([Figure 1]). In rs3222264, two sizes of tandem repeats were detected on 2% polyacrylamide gel mixed with urea to discriminate a high tandem repeat (H) (25 repeats) at 168bp from a low tandem repeat (L) (13 repeats) at 144bp ([Figure 2]).
|Figure 1 Genotyping of the MMP-9 (rs3918242) by electrophoresis on 1% agarose gel. M: a 100bp marker, Lanes 1,7 TT genotype, Lanes 2,3,5,6 CC genotype and Lanes 4,8 CT genotype. MMP, matrix metalloproteinase.|
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|Figure 2 Genotyping of the MMP-9 (rs3222264) by electrophoresis on 2% polyacrylamide gel mixed with urea. M: a 50bp marker, Lanes 4,5 HH genotype, Lanes 2,3 LL genotype and Lanes 1,6,7 HL genotype. MMP, matrix metalloproteinase.|
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Statistical analysis was run on IBM statistical package for the social sciences program (SPSS; SPSS Inc., Chicago, IL, USA) version 20. Qualitative data were presented as frequency and percentage. χ2-Test or Fischer’s exact test were used to compare groups. Quantitative data are summarized as mean±SD. Comparisons between groups were done using Student’s t-test. Deviations from Hardy–Weinberg equilibrium expectations were determined using the χ2-test. Odds ratio and 95% confidence interval were calculated. The HaploView program (version 4.2) was applied to estimate the haplotypes and linkage disequilibrium, which uses the expectation maximization algorithm . Tests are considered statistically significant if P value less than 0.05 at 95% confidence interval.
| Results|| |
The anthropometric measures along with the clinical and laboratory characteristics of the included groups are represented in [Table 1]. Healthy pregnant (control), GH and PE women were matched by age, gestational age, % primigravida, and hemoglobin concentration (P>0.05 each). As expected, PE and GH presented higher systolic and diastolic blood pressures compared with the control group (P<0.001 each). Significant proteinuria was found in patients with PE only when compared with both control and patients with GH (P<0.001 each). The distribution of genotypes for the two studied polymorphisms showed no deviation from Hardy–Weinberg equilibrium (P>0.05 each).
|Table 1 Anthropometric measures and clinical and laboratory characteristics of the studied groups|
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Results of genetic analysis of MMP-9 rs3918242
The genotype CC was the common genotype among the three studied groups (36/50 in control and 34/50 in GH and 38/50 in PE groups), with no significant differences (P>0.05). There were no significant differences among all groups regarding the allele frequency. The T allele frequency in the studied population (patients and controls) was 0.160, compared with the Global Minor Allele Frequency: 0.155 ([Table 2]). No statistically significant association was found between the MMP-9 rs3918242 genotypes and the mean age, primigravida, and mean hemoglobin concentration. Regarding gestational age, the CC genotype was significantly associated with patients with GH and PE when compared with the control group (P=0.008 and 0.002, respectively). However, for systolic and diastolic blood pressure, both CC and CT genotypes were significantly associated with patients with GH and PE when compared with control group (P<0.001 each) (data not shown).
|Table 2 Frequency distribution of matrix metalloproteinase-9 rs3918242 and rs3222264 genotypes and alleles among the studied groups|
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Results of genetic analysis of MMP-9 rs3222264
The heterozygous genotype HL was the common genotype among the three studied groups (50/50 in control and 24/50 in GH and 20/50 in PE groups), with no significant differences (P>0.05). There were no significant differences among all groups regarding the allele frequency (P>0.05) ([Table 2]). No statistically significant association was found between the MMP9 rs3222264 genotypes and the mean age, primigravida, and mean hemoglobin concentration. Regarding gestational age, the LL genotype was significantly associated with patients with GH and PE when compared with control group (P=0.016 and 0.009, respectively). Regarding systolic and diastolic blood pressures, all genotypes were significantly associated with patients with GH and PE when compared with the control group (P<0.001 each) (data not shown).
Comparing the possible haplotypes of both MMP-9 studied variants among all groups did not show any significant association (P>0.05 each) ([Table 3]). However, the percentages of linkage disequilibrium between rs3918242 and rs3222264 were 71% in control group of normal pregnancy, 72% in patients with GH, and 100% in patients with PE ([Figure 3]).
|Table 3 Comparison of rs3918242 and rs3222264 haplotype frequencies among all studied groups|
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|Figure 3 Linkage disequilibrium between rs3918242 and rs3222264 in the studied groups.|
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| Discussion|| |
PE is a pregnancy-specific multisystem disease , which represents the second leading direct cause of maternal morbidity especially in developing countries . Although exact pathogenesis of PE is not fully known, the inadequate placental perfusion that leads to extensive maternal endothelial dysfunction is suggested to be a major mechanism . MMPs are proved to affect the vascular function and have a key role in vascular alterations occurs in PE and other cardiovascular diseases . MMP-9 might be included in remodeling placental and uterine blood vessel and in controlling the vascular tone . In the present study, we analyzed the genotype and allele frequencies for two functional MMP-9 polymorphisms [−90(CA)13–25 and −1562C/T] in pregnant women with PE and GH.
Serum MMP-9 level has been extensively studied in hypertensive states of pregnancy. It was found to be higher in patients with GH , but not with PE, when compared with normotensive pregnant women , suggesting that MMP-9 plays a role in the pathophysiology of GH and that HDP have different pathophysiological mechanisms. Moreover, the in-vitro genetic studies of MMP-9 showed that the ‘C’ to ‘T’ substitution at −1562 position is associated with increased MMP-9 expression . Various studies revealed that the (CA)14 allele causes a 50% reduction in MMP-9 promoter activity as compared with the (CA)21 ,. The elevated MMP9 concentrations reported to be associated with the −1562T allele might be essential for the development of an adequate maternal-fetal interface early in pregnancy by facilitating trophoblast apoptosis and degradation . From our results, both MMP-9 −1562C/T and MMP-9 −90(CA)13–25 SNPs failed to show any significant association with either PE or GH. Our findings confirmed previous results that did not find any significant association between MMP-9 −1562 (C>T) SNP and PE ,. These results were also reported by Gong et al.  in their meta-analysis, which revealed no evidence for significant association between MMP-9 −1562C/T polymorphism and risk of PE. Although we did not find any association between the minor allele T of MMP-9 −1562C/T polymorphism and neither GH nor PE, previous studies reported a significant association of the T allele with the risk of GH but not PE, which might explain the higher plasma MMP-9 levels previously reported in GH compared with HP ,. However, Coolman et al.  found a lower prevalence of the rare T allele in women with PE (odds ratio: 0.48, 95% confidence interval: 0.25–0.90), and they suggested that the MMP-9 −1562T allele is associated with a reduced risk of PE and therefore may protect against maladaptation of the spiral arteries and decreased decidual degradation. Regarding genotypes, neither CT nor TT genotype was associated with the studied groups. This was on the contrary of previous findings by Rahimi et al.  who observed a significantly higher frequency of CT genotype in both mild and severe preeclamptic women compared with controls. They found that the presence of CT+TT genotype was significantly higher in early-onset rather than late-onset PE. Moreover, the presence of CT+TT genotype increased the risk of severe PE by 2.37-fold, which led them to suggest the role of MMP-9 variant as a useful biomarker of susceptibility to severe PE and early-onset severe PE . From our results, we could not detect any significant association of MMP-9 −90(CA)13–25 with GH or PE. However, in a previous study, no significant differences were found in genotype and allele distributions when PE or GH groups were compared with control group . Although Palei et al.  had we found increased MMP-9 levels in patients with GH with the LH genotype for the −90(CA)13–25 polymorphism. In our haplotype analysis, we could not find any significant association between any of the possible four haplotypes and GH or PE. These results confirmed the findings of Palei et al.  who found no significant differences in overall distributions of haplotype frequencies when the GH or the PE group was compared with the HP group. However, in another study although no differences were found when haplotypes were compared between PE and healthy pregnant women, the distribution of MMP-9 TH haplotype was more commonly found in the GH group than in the healthy pregnant women .
In conclusion, our limited study on a small group of Egyptian population demonstrated that MMP-9 −1562C/T and −90(CA)13–25 polymorphisms in addition to their haplotypes did not show significant deviation in patients with GH or PE compared with normotensive pregnant women controls. More studies with larger sample size and multiple subgroups are needed to further explore the association between them. This will help to predict the risk of PE and take measures to avoid the maternal and fetal morbidity and mortality.
The authors thank all patients and controls for their participation in this study.
N.A.A., E.R.A., and A.W.A.M. designed the study and defined the intellectual content; O.S.E. and R.A.K. searched the literature and performed clinical and experimental studies, data acquisition, data analysis, statistical analysis, manuscript preparation, and manuscript editing; N.A.A., E.R.A., A.W.A.M., O.S.E., and R.A.K. reviewed the manuscript.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
ACOG Hypertension in pregnancy. Report of the American College of Obstetricians and Gynecologists’ Task Force on Hypertension in Pregnancy.Obstet Gynecol 2013; 122:1122–1131.
Garovic VD, Hayman SR. Hypertension in pregnancy: an emerging risk factor for cardiovascular disease. Nat Clin Pract Nephrol 2007; 3:613–622.
Cindrova-Davies T. The therapeutic potential of antioxidants, ER chaperones, NO and H2S donors, and statins for treatment of preeclampsia. Front Pharmacol 2014; 5:119.
Sanchez-Aranguren LC, Prada CE, Riano-Medina CE, Lopez M. Endothelial dysfunction and preeclampsia: role of oxidative stress. Front Physiol 2014; 5:37210.
Damsky CH, Librach C, Lim KH, Fitzgerald ML, McMaster MT, Janatpour M et al.
Integrin switching regulates normal trophoblast invasion. Development 1994; 120:3657–3666.
Roberts JM, Gammill HS. Preeclampsia: recent insights. Hypertension 2005; 46:1243–1249.
Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res 2003; 92:827–839.
Palei ACT, Granger JP, Tanus-Santos JE. Matrix metalloproteinases as drug targets in preeclampsia. Curr Drug Targets. 2013; 14:325–334.
Isaka K, Usuda S, Ito H, Sagawa Y, Nakamura H, Nishi H et al.
Expression and activity of matrix metalloproteinase 2 and 9 in human trophoblasts. Placenta 2003; 24:53–64.
Zhang B, Ye S, Herrmann SM, Eriksson P, de Maat M, Evans A et al.
Functional polymorphism in the regulatory region of gelatinase B gene in relation to severity of coronary atherosclerosis. Circulation 1999; 99:1788–1794.
Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 2005; 21:263–265.
Steegers EA, von Dadelszen P, Duvekot JJ, Pijnenborg R. Pre-eclampsia. Lancet 2010; 376:631–644.
von Dadelszen P, Magee LA. Pre-eclampsia: an update. Curr Hypertens Rep 2014; 16:454.
Lalu MM, Xu H, Davidge ST. Matrix metalloproteinases: control of vascular function and their potential role in preeclampsia. Front Biosci 2007; 12:2484–2493.
de Jager CA, Linton EA, Spyropoulou I, Sargent IL, Redman CWG. Matrix metalloprotease-9, placental syncytiotrophoblast and the endothelial dysfunction of pre-eclampsia. Placenta 2003; 24:84–91.
Ab Hamid J, Mohtarrudin N, Osman M, Andi Asri AA, Wan Hassan WH, Aziz R. Matrix metalloproteinase-9 and tissue inhibitors of metalloproteinases 1 and 2 as potential biomarkers for gestational hypertension. Singapore Med J. 2012; 53:681–683.
Palei AC, Sandrim VC, Cavalli RC, Tanus-Santos JE. Comparative assessment of matrix metalloproteinase (MMP)-2 and MMP-9, and their inhibitors, tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 in preeclampsia and gestational hypertension. Clin Biochem 2008; 41:875–880.
Peters DG, Kassam Ast, Jean PL, Yonas H, Ferrell RE. Functional polymorphism in the matrix metalloproteinase-9 promoter as a potential risk factor for intracranial aneurysm. Stroke 1999; 30:2612–2616.
Shimajiri S, Arima N, Tanimoto A, Murata Y, Hamada T, Wang KY et al.
Shortened microsatellite d(CA)21 sequence down-regulates promoter activity of matrix metalloproteinase 9 gene. FEBS Lett 1999; 455:70–74.
Coolman M, de Maat M, Van Heerde WL, Felida L, Schoormans S, Steegers EA et al.
Matrix metalloproteinase-9 gene −1562C/T polymorphism mitigates preeclampsia. Placenta 2007; 28:709–713.
Fraser R, Walker JJ, Ekbote UV, Martin KL, McShane P, Orsi NM. Interleukin-4 −590 (C>T), toll-like receptor-2 +2258 (G>A) and matrix metalloproteinase-9 −1562 (C>T) polymorphisms in pre-eclampsia. BJOG 2008; 115:1052–1056.
Leonardo DP, Albuquerque DM, Lanaro C, Baptista LC, Cecatti JG, Surita FG et al.
Association of Nitric Oxide Synthase and Matrix Metalloprotease Single Nucleotide Polymorphisms with Preeclampsia and Its Complications. PLoS One 2015; 10:e0136693.
Gong LL, Liu H, Liu LH. Lack of association between matrix metalloproteinase-9 gene-1562C/T polymorphism and preeclampsia: a meta-analysis. Hypertens Pregnancy 2014; 33:389–394.
Palei AC, Sandrim VC, Duarte G, Cavalli RC, Gerlach RF, Tanus-Santos JE. Matrix metalloproteinase (MMP)-9 genotypes and haplotypes in preeclampsia and gestational hypertension. Clin Chim Acta 2010; 411:874–877.
Palei AC, Sandrim VC, Amaral LM, Machado JS, Cavalli RC, Lacchini R et al.
Matrix metalloproteinase-9 polymorphisms affect plasma MMP-9 levels and antihypertensive therapy responsiveness in hypertensive disorders of pregnancy. The Pharmacogenomics J 2012; 12:489–498.
Rahimi Z, Shahsavandi MO, Bidoki K, Rezaei M. MMP-9 (-1562C:T) polymorphism as a biomarker of susceptibility to severe pre-eclampsia. Biomark Med 2013; 7:93–98.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]