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Human Molecular Genetics Advance Access originally published online on September 14, 2004
Human Molecular Genetics 2004 13(21):2659-2669; doi:10.1093/hmg/ddh287
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Human Molecular Genetics, Vol. 13, No. 21 © Oxford University Press 2004; all rights reserved

Functionally significant SNP MMP8 promoter haplotypes and preterm premature rupture of membranes (PPROM)

Hongyan Wang1, Samuel Parry1, George Macones1, Mary D. Sammel2, Pedro E. Ferrand1, Helena Kuivaniemi3, Gerard Tromp3, Indrani Halder4, Mark D. Shriver4, Roberto Romero3 and Jerome F. Strauss, III1,*

1Center for Research on Reproduction and Women's Health and 2Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania, Philadelphia, PA 19104, USA, 3Perinatology Research Branch, NICHD, Hutzel Hospital, Detroit, MI 48201, USA and 4Department of Anthropology, The Pennsylvania State University, University Park, PA 16802, USA

Received July 23, 2004; Accepted September 3, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Matrix metalloproteinase 8 (MMP8), an enzyme that degrades fibrillar collagens imparting strength to the fetal membranes, is expressed by leukocytes and chorionic cytotrophoblast cells. We identified three single nucleotide polymorphisms (SNPs) at –799C/T, –381A/G and +17C/G from the major transcription start site in the MMP8 gene, and determined the functional significance of these SNPs by analyzing their impact upon MMP8 promoter activity and their association with preterm premature rupture of membranes (PPROM). The minor alleles +17 (G) and –381 (G) were in complete linkage disequilibrium. A promoter fragment containing the three minor alleles had 3-fold greater activity in chorion-like trophoblast cells (BeWo, JEG-3 and HTR-8/SVneo) compared with the major allele promoter construct. Electrophoretic mobility shift assays revealed differences in BeWo nuclear protein binding to oligonucleotides representing the –381 and –799 SNPs, suggesting that the minor alleles have reduced transcription factor binding. A case–control study of African-American neonates using allele-specific primers revealed a statistically significant association between the three minor allele haplotype, which displays the highest MMP8 promoter activity in trophoblast cells, with PPROM with an odds ratio (OR) of 4.63 (P<0.0001), whereas the major allele promoter appeared to be protective (OR=0.52, P<0.0002). None of the minor alleles were individually associated with PPROM. These findings demonstrate the functional significance of SNP haplotypes in the MMP8 gene and associations with obstetrical outcomes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Extensive extracellular matrix (ECM) remodeling is an important process in several phases of human parturition, including cervical ripening, fetal membrane rupture and placental detachment from the uterus (1–3). The fetal membranes are a complex multilaminate tissue composed of the amnion and chorion. These two closely adherent tissues consist of several cell types, including epithelial cells, mesenchymal cells and cytotrophoblast cells. The tensile strength of the fetal membranes depends on the integrity of these cells and their associated ECM (4–6). Preterm premature rupture of the membranes (PPROM), defined as spontaneous rupture of the membranes before 37 weeks of gestation, is a significant obstetrical complication associated with increased risk for intrauterine infection (7). PPROM is the leading identifiable cause of preterm delivery and its complications (8). Historically, obstetricians have attributed fetal membrane rupture to physical stress. However, familial clustering and ethnic differences in the incidence of PPROM (9), which cannot be accounted for by socioeconomic status (10), suggest that genetic factors contribute to the risk of PPROM.

The matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade the main protein components of the ECM (11). Consequently, MMPs are widely assumed to play a central role in the remodeling of the cervical and fetal membrane ECM throughout gestation and preceding parturition. Also, we previously reported that polymorphisms in the MMP1 and MMP9 promoters that increase promoter activity are associated with PPROM (12,13), suggesting that genetic variation in MMP genes contributes to the risk of adverse obstetrical outcomes.

MMP8 (also known as collagenase-2 or neutrophil collagenase) is a member of the MMP family. It is a glycoprotein that is synthesized as a zymogen. Activation of MMP8 requires autolytic removal of 80 amino acids from the N-terminus (14,15). MMP8 cleaves fibrillar collagens, such as collagen types I, II, III, V and XI, as well as non-fibrillar collagens, including collagen types IX, XII and XIV (16). Cleavage of the triple helical collagen molecule by MMP8 changes the stability and solubility properties of the collagen, resulting in denaturation in cleavage products that are subsequently degraded by other MMPs (so-called gelatinases) (17–21).

MMP8 is released from leukocytes during chemotactic stimulation in vitro and in response to inflammatory conditions in vivo (22). Although MMP8 was thought to be expressed exclusively by neutrophils, its expression has recently been detected in a number of other cell types, including chondrocytes, fibroblasts, corneal epithelial cells, endothelial cells, smooth muscle cells and cytotrophoblasts of the chorion (23–29), which raises the possibility of its involvement in fetal membrane rupture. Indeed, MMP8 concentrations are high in the amniotic fluid of patients with intra-amniotic infection, preterm labor and PPROM (30,31). Elevated amniotic fluid MMP8 levels are also strongly associated with neonatal death and other adverse neonatal outcomes (32).

We undertook the present study to determine whether there is genetic polymorphism in the MMP8 promoter, whether the polymorphism has functional significance and whether it is associated with PPROM. The association study was conducted in an African-American population because of the higher incidence of PPROM in this ethnic group (8–10). In this report, we demonstrate for the first time that there are functionally significant polymorphisms in the MMP8 gene; that the impact of these variants on MMP8 promoter function is influenced by cell context and that there is an association between the minor allele haplotype with increased promoter activity in chorion-like cytotrophoblast cells and PPROM in an African-American population.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of the MMP8 transcription start site
5' RACE analysis indicated that the major transcription start site in the MMP8 gene is at –2 relative to the transcription start site designated in the sequence deposited in GenBank (accession no. AF059679). Of seven sequenced PCR products, six ended at nucleotide –2 and one at +1. Transcription start sites do not share extensive sequence homology, but in ~50% of transcription initiation sites, the first base of the mRNA is an A flanked on either side by pyrimidines. The start site for the MMP8 mRNA identified in our 5' RACE is at an A flanked 3' by a C. Most promoters have a TATA box, usually located about 25 bp upstream from the transcription start point. A TATA-like sequence, TTTAAA, is located 26–31 bp upstream of the putative transcript start site. A CAAT box, another characteristic promoter feature, is located at base pair –49 to –52. The structure of the MMP8 promoter is shown in Figure 1. In the process of performing the 5' RACE, we identified a novel MMP8 mRNA splice variant, which is the result of the insertion of a 67 bp sequence from a cryptic exon (exon 1a) located in the 2 kb intron between exons 1 and 2 (see later).



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  		Figure 1. Genomic structure of the promoter and the adjacent sequences of the human MMP8 gene. The promoter of the gene is depicted as a thick line, the exons as striped boxes and the introns as thin lines. SNPs C/T (–799), A/G (–381) and C/G (+17), as well as CAAT box (–49 to –52), putative TATA box (TTTAAA) (–28 to –33) and the transcription start site are shown. The translation start site is indicated in exon 1. Between exons 1 (ends at +198) and 2 (begins at +199), there are two other potential exons of 91 bp (close to exon 2) and 67 bp (next to exon 1) within intron 1.

 
Identification of SNPs in the MMP8 promoter
Analysis of promoter fragments amplified by PCR from 32 unrelated African-Americans identified single nucleotide polymorphisms (SNPs) at positions –799C/T (minor ‘T’ allele detected in 11 subjects), –381A/G (minor ‘G’ allele detected in seven subjects) and +17C/G (minor ‘G’ allele detected in seven subjects) with an allele frequency >5%. The allele frequencies and carrier rates of SNP haplotypes were determined from genotypes of 216 African-American controls (Table 1). The –381 and +17 alleles appeared to be in complete linkage disequilibrium. When a subject had the minor ‘G’ allele at –381, the +17 position was invariably the minor ‘G’ allele. Thus, there are four possible SNP haplotypes including the major allele promoter –799C/–381A/+17C, one minor allele promoter –799T/–381A/+17C, two minor alleles promoter –799C/–381G/+17G and three minor alleles promoter –799T/–381G/+17G.


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  		Table 1. MMP8 promoter SNP allele frequencies
 
Functional significance of the MMP8 promoter alleles
We examined the activities of MMP8 promoter fragments containing different experimentally detected SNP haplotypes transfected into different host cells that express the MMP8 gene endogenously (Table 2). Each promoter fragment demonstrated activity that was substantially greater than the pGL3 empty vector. Moreover, when the promoter fragments were cloned into the pGL3 vector in the opposite orientation, all promoter function was lost and relative luciferase values were similar to those found with the empty vector (data not shown). Cell host-dependent differences in MMP8 promoter activity were observed. A promoter fragment containing the three minor alleles (–799T/–381G/+17G) showed 2–3-fold greater activity in the three chorion-like cytotrophoblast cells, BeWo, HTR-8/Svneo and JEG-3 cells, compared with the major allele promoter construct (–799C/–381A/+17C) and the other promoters with one or two minor alleles. However, in U937 leukocytes cells, the minor allele promoter had ~34% of the activity of the major allele promoter. These promoters had similar activities in THP-1 monocytes/macrophage cells.


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  		Table 2. Genotype-dependent MMP8 promoter activities
 
We analyzed the binding of nuclear extract (NE) proteins prepared from BeWo, THP-1 and U937 cells to oligonucleotides representing the different –381 and –799 alleles by electrophoretic mobility shift assays (EMSA) (Fig. 2). Specific binding of proteins was identified through cross-competition using unlabeled major allele, minor allele and non-specific oligonucleotide probes. The binding pattern for proteins from the different cell lines to the –381A/G and –799C/T oligonucleotides differed. NE from BeWo cells produced one complex (arrow) with the –381A probe, and showed significantly less binding to the minor allele –381G probe. Similarly, decreased binding was observed with NE from U937 cells when we compared the binding to the major –381A allele probe (arrow) and the minor –381G allele probe (arrow). Weak binding to the common –381A allele probe (arrow) and little binding to the minor –381G allele probe was found with THP-1 cell NE.



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  		Figure 2. EMSA of nuclear proteins from different cell types: (A) BeWo; (B) THP-1; (C) U937 to the oligonucleotides containing MMP8 –381A/G alleles (upper pictures) and –799C/T alleles (lower pictures). Every left seven lanes show NE binding with wild-type –381 (A) or –799 (C) probe; every right seven lanes show NE binding with mutant –381 (G) or –799 (T) probe; NE: nuclear extract; +: present; –: empty; {ddh28706}: increased amount of competitor or cross-competitor probes.

 
NE from BeWo cells produced two complexes on binding to the common –799C allele probe and the minor –799T allele probe (Fig. 2). The binding to the lower mobility complex was equivalent (arrow), but the higher mobility complex showed higher affinity for the common –799C allele probe (arrow). Only one complex was formed by extracts from U937 cells with similar binding (arrow) to the common –799C and the minor –799T allele probes. No specific binding was observed with NE prepared from THP-1 cells. Collectively, the EMSA results revealed differences in nuclear protein binding to the –381A/G and –799C/T oligonucleotides, suggesting that the minor alleles have reduced BeWo cell transcription factor binding. The nature of the proteins binding to the different oligonucleotides remains to be determined.

Identification of a new MMP8 mRNA splice variant and its expression pattern
The nucleotide sequence of the MMP8 cDNA (GenBank accession no. NM_002424) encodes a protein of 467 amino acids, with a secretory signal sequence of 20 residues followed by the prodomain of 80 residues. In the course of performing 5' RACE to identify the MMP8 transcription start site, we identified three different cDNAs from THP-1 and U937 cells by RT–PCR using an antisense primer corresponding to bp –392 to –413 of the MMP8 cDNA sequence deposited in GenBank together with the universal primer from the Clontech SMART RACE kit as the sense primer. A number of PCR products were recovered and sequenced. Besides fragments matching the MMP8 cDNA sequence in GenBank, and a previously reported splice variant of the MMP8 transcript with an additional 91 bp insertion encoded by a cryptic exon in the intron between exons 1 and 2 (33), we found a new splice variant containing a 67 bp insertion, also resulting from a cryptic exon in the intron between exons 1 and 2 (Fig. 3). The +91 and +67 bp insertions separate bp 173 and 174 of the coding sequence and both inserted sequences in the first intron are flanked by donor and acceptor splice junctions that obey the GT/AG rule (33,34). The +91 and +67 bp splice variants encode a short peptide that starts with the reference sequence initiation codon and contains the leader sequence of the reference MMP8 protein. A new open reading frame beginning with Met 85 of the reference sequence would yield a protein lacking the signal sequence, and thus would be expected to be an intracellular enzyme (33).



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  		Figure 3. Nucleotide sequence and genomic structure of MMP8 around the 67 bp insertion (exon 1a). (A) Nucleotide sequence of the 5' 350 bp of the MMP8 cDNA and the sequence of the 67 bp insertion. The nucleotide sequence of the 67 bp insertion is shown in the box. Numbering follows the published sequence. The potential stop codon TGA is indicated. (B) Genomic organization of MMP8 gene encodes three alternatively spliced transcripts. The schematic shows splicing patterns that generate MMP8 and MMP8 with 67 bp insertion transcripts. Open boxes represent exons and lines represent introns. Numbers indicate the positions in the MMP8 cDNA. Consensus splice donor/acceptor sites are underlined.

 
Quantitative RT–PCR was used together with primer pairs for amplification of each splice variant to define the MMP8 mRNA expression patterns in human chorion, BeWo cells and the U937 and THP-1 cell lines (Fig. 4). Chorion was relatively enriched with all the MMP8 splice variants, but the 0 bp form predominated. BeWo cells express equivalent levels of the 0 bp variant and +67 bp variant without detectable +91 bp splice variant. The MMP8 mRNA variants were expressed in a similar pattern in THP-1 cells, whereas in U937 cells the 0 bp insertion predominated.



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  		Figure 4. Quantitative RT–PCR amplification of different splice variants of human MMP8 gene with exclusive primer pairs in chorion tissue and following cell types. (A) THP-1 cells; (B) U937 cells; (C) BeWo cells; (D) human chorion; real-time PCR (red line: +91 bp; green line: +67 bp; yellow line: 0 bp insert) are shown above and the gel analysis of PCR products below.

 
To determine whether the +67 and +91 bp splice variants are translated yielding detectable amounts of protein, we performed western blot analysis on chorion tissue extracts and the extracts from BeWo, U937 and THP-1 cells (Fig. 5). The MMP8 proenzyme has a molecular weight of ~85 kDa and the active enzyme is 64 kDa. The protein encoded by the insertion splice variants is theoretically 9 kDa smaller than the proenzyme. When equivalent amount of total protein from amnion, chorion, BeWo cells, THP-1 cells and U937 cells were loaded on gels with amnion extract as a negative control, chorion samples contained abundant MMP8 proenzyme; BeWo cells contained active MMP8 with no detectable proenzyme; and THP-1 and U937 cells had only trace amounts of active form (Fig. 5). In no case was a 76 kDa immunoreactive protein detected. These findings demonstrate that chorion is enriched in MMP8 and that the proteins encoded by +91 and +67 bp splice variants are either not very abundant or rapidly processed to active enzyme.



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  		Figure 5. Western blot analysis of MMP8 in amnion tissue, chorion tissue, BeWo cells, THP-1 cells and U937 cells. (A) Shorter exposure time; (B) longer exposure time; (C) ß-actin protein to assess equality of protein loading. No detectable MMP8 protein in amnion samples (negative control). Chorion samples contain abundant 85 kDa (latent form) and trace amount of 64 kDa (active form) MMP8 protein. BeWo cells contain 64 kDa protein but no detectable 85 kDa protein. THP-1 and U937 cells have similar trace amounts of 64 kDa protein only. A non-specific band appears at 60 kD in amnion and, to a lesser extent, chorion.

 
The minor MMP8 promoter allele haplotype is associated with risk of PPROM and the common allele MMP8 haplotype is protective
To determine if the functional differences in MMP8 promoter activity for the different SNP haplotypes could contribute to differences in ECM degradation, we performed a case–control study to test the association between the MMP8 SNPs and PPROM. The study was conducted in an African-American population because PPROM is 2–4 times more prevalent in this ethnic group (8–10). The study was also focused on the genotype of the offspring based on the hypothesis that the genotype of the extraembryonic tissues (fetal membranes) represents the primary determinant of risk of premature rupture of the membranes.

The demographic characteristics of the 216 controls, neonates born at term from normal pregnancies, and the 168 cases, neonates from pregnancies complicated by PPROM, are shown in Table 3. There were no significant differences in maternal age, gravidity and parity, but the length of gestation and birth weight were significantly lower in the PPROM group, as expected.


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  		Table 3. Demographic characteristics and clinical outcomes of index pregnancies
 
Our case–control study of neonatal genotypes and PPROM revealed no significant association between the individual SNPs and PPROM (Table 1). However, an analysis based on carriage of the three minor allele haplotype (–799T, –381G, +17G) that confers increased MMP8 promoter activity in cytotrophoblasts demonstrated that it was significantly more frequent in PPROM (8.04% of cases compared with 1.85% of controls) with an odds ratio (OR)=4.63 [P<0.0001; 95% confidence intervals (CI) 2.01, 11.94] (Table 4). Conversely, homozygosity for the major allele MMP8 promoter haplotype appeared to be protective against PPROM (P<0.0002; CI: 0.362, 0.751; Table 4).


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  		Table 4. Frequencies of the triple minor and triple major SNP MMP8 promoter haplotypes and PPROM
 
As the urban African-American population from which our subjects were drawn is heterogeneous, we performed analyses to determine if population stratification could have affected our findings. The impact of population admixture was also considered for subjects for whom genotypes were available to evaluate (171 cases of PPROM and 208 controls). For this analysis, estimates of ancestry [proportion/probability African-American (Nigeria, Sierra Leone and Central African Republic) versus European (combined British, Irish, German and Spanish populations)] were made for cases and controls and for each subject, and the impact of the genetic admixture was evaluated using multiple logistic regression analysis. For the cases and controls, there was no significant difference in ancestry using a dihybrid model (% African ancestry: cases, 0.847±0.141; controls, 0.827±0.149, P=0.487). There was also no evidence of confounding due to ancestry in the multiple logistic regression analysis, as the OR estimates decreased by at most 3% for the association for the three minor SNP haplotype and PPROM (adjusted OR=4.48; CI: 1.90, 11.75).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The ECM is recognized as a key regulatory component in cellular physiology, providing an environment for cell division, differentiation, migration and invasion, and in some cases, cell survival and cell death (35). ECM turnover and homeostasis are highly regulated and the catabolism is due, in part, to the action of a specific class of proteolytic enzymes known as MMPs. Currently, the MMP family encompasses four broad classes: collagenases, gelatinases, stromelysins and membrane-type enzymes. Collagenases, which degrade the fibrillar collagens, include MMP1, MMP8 and MMP13. The ECM of fetal membranes and uterus (cervix and lower uterine segment), in particular collagen and elastin, is degraded in preparation for delivery. Each of the three collagenases may participate in this process with the enzymes originating from different cell types. In the amnion, epithelial and mesenchymal cells are sources of MMP1 and MMP13, whereas the cytotrophoblast of the chorion and invading leukocytes are the primary source of MMP8. Chorion tissue, as shown here and in our previous work, is a rich source of MMP8. Because the chorion is adherent to the amnion, chorion-derived MMP8 would presumably have ready access to the fibrillar collagens of the amnion.

The significance of the different MMP8 mRNA splice variants with respect to ECM catabolism remains to be clarified. The splice variants containing insertions of cryptic exons encoded in the intron between exons 1 and 2 appear to be intracellular proteins, on the basis of the absence of a signal sequence, but their role in intracellular proteolysis is a matter of speculation (33). Our findings demonstrate that in the chorion, the MMP8 transcript encoding the enzyme with an intact leader sequence predominates, which is consistent with a role for cytrophoblast-derived MMP8 in fetal membrane ECM metabolism.

Because of the important role of MMPs in ECM turnover, we have been interested in the question of whether genetic variation in MMP promoter regions could contribute to familial clustering of preterm birth as well as differences in the incidence of preterm birth among various ethnic groups. Our past investigations have indicated that specific polymorphisms in the MMP1 and MMP9 promoters are associated with risk of PPROM in African-Americans (12,13). The present study suggests that the haplotype with three minor alleles of the MMP8 promoter is also associated with PPROM. Interestingly, this haplotype conferred greater promoter activity only when the constructs were introduced into cells resembling the chorion cytotrophoblasts. In U937 and THP-1 cells, this haplotype did not show increased promoter activity, and it was in fact reduced in U937 cells. The basis for the cell-specific responses is not entirely clear. However, EMSA with NE derived from BeWo cells did reveal a complex with the –799C/T oligonucleotide that was not detected in EMSAs with NE from U937 and THP-1 cells. The minor allele oligonucleotide was bound with lower affinity, suggesting that the protein producing this higher mobility complex could be a transcription factor whose binding determines promoter function. Because the mutant allele appeared to have lower affinity for this protein, it is possible that the protein is a transcription repressor and that reduced binding results in increased promoter activity. Notably, the haplotype with minor alleles of the three SNPs was significantly more common in PPROM. This raises the possibility of interactions among different cis elements. Unfortunately, there have been no prior functional analyses of the human MMP8 promoter, and the cis elements important for MMP8 transcription and the transcription factors that bind to them have not been identified. This is, to the best of our knowledge, the first exploration of human MMP8 promoter function.

To determine the possible identities of the proteins responsible for the high mobility complex seen only with BeWo cell NE, we searched various databases for transcription factor binding sites. This search indicated that the –799 oligonucleotide with the SNP in the middle contains a potential binding site for CBG-02 protein (36), whereas –381 oligonucleotide contains a potential binding site for GATA-1 (http://www.genomatix.de and http://www.ifti.org). However, at this juncture it is a matter of speculation as to whether CBG-02, GATA-1 or other factors play roles in modulating MMP8 transcription, especially because the two transcription factors previously noted have not yet been reported to be expressed in chorion trophoblast cells.

There are a number of important caveats that must be kept in mind when interpreting the results of association studies. First, the findings may be influenced by population stratification. This is a particularly important concern when there is heterogeneity in the population under investigation, as is the case with African-Americans. Recruitment of cases and controls from the same communities and recruiting a large number of subjects mitigates this concern to some extent. Moreover, an analysis of potential population stratification using 29 markers selected for significant differences among African and European populations revealed no evidence for stratification influencing the major conclusions of this study. Second, association does not mean linkage and it is possible that the MMP8 SNP haplotype found to be associated with PPROM is also in linkage disequilibrium with another SNP or mutation in a gene other than MMP8. MMP8 lies on chromosome 11 in a cluster of other MMP genes including MMP1, 3, 7, 10, 12, 13, 20 and 26. Thus, it is possible that our genetic study implicates an MMP other than MMP8. As we had previously studied a polymorphism in the MMP1 promoter and PPROM, we examined the possibility that the –1607 2G polymorphism associated with PPROM was in linkage disequilibrium with the MMP8 minor allele haplotype. It was not, making it unlikely that the significant association we observed between the MMP8 minor allele promoter haplotype and PPROM is due to a MMP1 promoter variation. Third, our findings in an African-American population may not be generalizable to other ethnic groups.

In conclusion, we have identified three SNPs in the MMP8 gene 5' region and a haplotype that confers increased MMP8 promoter activity in chorion-like cytotrophoblast-like cell lines. This haplotype is associated with increased risk of PPROM. Collectively, these findings suggest that genetic variation at the MMP8 locus can contribute to adverse events linked to ECM breakdown.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Subjects
Subjects in this study were African-American women and their neonates receiving obstetrical care at the Hospital of the University of Pennsylvania, Philadelphia, and Hutzel Hospital, Detroit. The study was approved by the respective Institutional Review Boards, and written informed consent was obtained from mothers before collection of the samples. Control samples (n=216) were obtained from singleton pregnancies delivered at term of mothers with no prior history of PPROM or preterm labor. Cases of PPROM (n=168) were defined as neonates from pregnancies complicated by rupture of membranes prior to 37 weeks of gestation. The diagnosis of membrane rupture was based on pooling of amniotic fluid in the vagina, amniotic fluid ferning patterns and a positive nitrazine test. Patients with multiple gestations, fetal anomalies, trauma, connective tissue diseases and medical complications of pregnancy requiring induction of labor were excluded. The chi-square test was used to determine the significance of the association between MMP8 promoter alleles and PPROM. The OR and 95% CI were also determined.

Mapping of the MMP8 transcription start site
Mapping of the transcription start site of the MMP8 gene was a prerequisite for studies of MMP8 promoter function. We employed SMART technology (37) for mapping the transcription start site, based on direct sequencing of the SMARTTM (switching mechanism at 5' end of RNA template) (38) RACE products. Total RNA or mRNA was prepared from cultured THP-1 and U937 cells with TRIzol® or using the poly(A) tract. Using 5' RACE CDS primer and PowerScriptTM reverse transcriptase from the SMARTTM RACE cDNA amplification kit (Promega), cDNA was synthesized in a 10 µl volume according to the manufacturer's instructions. The cDNA was then diluted with Tricine–EDTA buffer into 100 µl, and 2.5 µl of the diluted sample was used as a template for a 50 µl PCR in the presence of 5 µl 0.4 µM universal primer mix (from the kit) and 1 µl of 20 µM gene-specific primer (5'-AGGTCAAGTTAGTGCGTTCCCAC-3'). Advantage® 2 enzyme mixture and reagents (Clontech) were used. Amplification was performed in a model 9600 thermal cycler (Applied Biosystems) as follows: five cycles of 5 s at 95°C and 3 min at 72°C, and then five cycles of 5 s at 94°C, 10 s at 70°C and 3 min at 72°C, followed by 27 cycles of 5 s at 94°C 10 s at 68°C and 3 min at 72°C. PCR products were purified on QIAquickTM spin columns (QIAgen) and subjected to sequencing using the gene-specific primer as a sequencing primer. The position of the transcription start site was detected at the point of abrupt loss of sequence identity between the RACE product and the 5' genomic sequence.

Identification of SNPs and genotyping of the MMP8 promoter
DNA was isolated from umbilical cords, cord blood or neonatal cheek swabs by digestion with proteinase K and extraction with conventional reagents or by an alternative isolation method using the BioRobot 9604 (QIAgen) (39). On the basis of the human MMP8 promoter sequence deposited in GenBank (accession no. AF059679), a promoter fragment was amplified by PCR with a forward primer sequence of 5'-CTGTTGAAGGCCTAGAGCTGCTGCTCC-3' (corresponding to bp –872 to –846) and a reverse primer 5'-CATCTTCTCTTCAAACTCTACCC-3' (corresponding to bp +74 to +96) yielding a 968 bp product. PCR was performed in a 50 µl reaction volume containing 100 ng genomic DNA, 0.5 pmol of each primer, 0.2 mM dNTPs, 1xreaction buffer, 2.5 mM MgCl2 and 2.5 units AmpliTaq GoldTM DNA polymerase (Perkin–Elmer) in a 9600 Gene Amp PCR thermal cycler (Perkin–Elmer Life Sciences). After initial denaturation at 94°C for 5 min, PCR was performed for 35 cycles of denaturation at 94°C for 45 s annealing at 56°C for 45 s and extension at 72°C for 1 min followed by a final 10 min elongation at 72°C.

MMP8 promoter fragments were amplified from 32 unrelated individuals and subjected to DNA sequence analysis to identify polymorphisms. The PCR products were cloned into the TOPO TA cloning vector (PCR2.1-TOPO, Invitrogen) and four to five separate clones derived from each subject's PCR amplification were sequenced. Three SNPs (–799C/T, –381A/G and +17C/G) were identified in more than two subjects in this screen. These SNPs were subjected to further analysis.

Genotypes of the MMP8 promoter were determined by two different methods: restriction fragment length polymorphism (RFLP) and GeneScan analysis. For RFLP, the PCR products were digested with either restriction endonuclease SfcI (New England Biolabs) for genotyping of the –799C/T SNP or restriction endonuclease DdeI (New England Biolabs) for the +17C/G SNP. The digested PCR products were separated in 1% agarose gels. The –799C allele is digested by SfcI yielding fragments 894 and 74 bp; whereas the –799T allele is not cleaved by SfcI. The +17C allele is digested by DdeI yielding fragments 799, 90 and 79 bp; whereas the +17G allele yields fragments of 878 and 90 bp.

For GeneScan, the purified 968 bp PCR products were analyzed with an ABI 3100 genetic analyzer using GeneScan software in order to genotype the SNP at –381A/T and to verify the results of RFLP analysis. We used HPLC-purified primers with a sequence of 5'-AGCCAGAGACTCAAGTGGGAGACTACCATGCAGAGCC-3' (36 nt) for genotyping the –799C/T SNP; 5'-CTCCACATACAATGAGGGAGG-3' (21 nt) for –381A/G SNP; and 5'-GCTGTGAGTGACACATGATGCTGTGAAC-3' (28 nt) for +17C/G SNP. The RFLP results for the –799C/T and +17C/G SNPs were completely concordant with the GeneScan analysis.

We also designed allele-specific primers based on the –799 and +17 SNPs to directly determine the haplotypes of the study population. The two forward primers (A and B) are close to –799 SNP and only varied at the last nucleotide with either the major –799 ‘C’ or the minor –799 ‘T’: 5'-AGTGGGAGACTACCATGCAGAGCC/T-3'. The two reverse primers (C and D) are close to +17 SNP and only varied at the last nucleotide with either the major +17 ‘C’ or the minor +17 ‘G’: 5'-TTCCCTGGCGAGCACCCTGAC/G-3'. We used one set of four pairs of primers (AC, AD, BC and BD) to amplify DNA samples using conditions: 5 min denaturation at 94°C, followed by 24 cycles of 94°C, 1 min; 65°C, 1 min and 72°C, 1 min, and finally 10 min elongation at 72°C.

Assessment of population structure
As many African-American populations have substantial admixture and this admixture is not evenly distributed throughout the population (40), there is the chance that some of the observed association could be the result of admixture stratification. To control for the possibility that admixture stratification may be the source of the association, we have typed 29 ancestry informative markers (AIMs), which are particularly useful for calculating gene flow between West African and West European populations (41). These markers were then used to calculate the individual biogeographical ancestry (BGA) levels of the persons in the study in the context of the two primary parental populations (West African and West European) using parental allele frequencies (42) and the maximum likelihood as first described in Hanis et al. (43). These BGA estimates were then used as conditioning variables in the logistic regression analyses to control for any effects that admixture stratification could be having on the phenotype. This secondary analysis was conducted using subject level data, where the subject was considered to have the MMP8 haplotype of interest (exposed) if they were heterozygous or homozygous for the haplotype. Next, using logistic regression methods, the unadjusted OR for the MMP8, PPROM association was estimated. Next, adjusted OR estimates were computed by incorporating the admixture estimates into the model. The resulting OR is an average of the MMP8, PPROM association, over subjects with like genetic profiles.

Construction of promoter-reporter plasmids
To determine whether the –799C/T, –381A/T and +17C/G SNPs influence transcription of the MMP8 gene, we obtained a 968 bp fragment from –872 to +96 bp of the MMP8 promoter, amplified using forward and reverse primers with the indicated sequences (forward primer: 5'-CTGTTGAAGGCCTAGAGCTGCTGCTCC-3' reverse primer: 5'-CATCTTCTCTTCAAACTCTACCC-3'). A mutagenesis kit (Stratagene) was used to create the targeted genotypes with a uniform backbone sequence. Promoter fragments containing each of the haplotypes that we identified (major allele, –799C/–381A/+17C; one minor allele, –799T/–381A/+17C; two minor alleles, –799C/–381G/+17G and the three minor alleles: –799T/–381G/+17G) were cloned into the pGL3 vector (Promega), which contains the firefly luciferase gene as a reporter. The DNA sequences of the promoter constructs were confirmed prior to use and three different plasmid preparations for each construct were tested.

Cell culture and transfection
BeWo, HTR-8/SVneo and JEG-3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) and THP-1 and U937 cells were cultured in RPMI 1640 medium. The media were supplemented with 10% fetal bovine serum and antibiotics (100 IU/ml penicillin G, 100 IU/ml streptomycin sulfate, 0.25 µg/ml amphotericin B; Gibco/BRL). All cells were maintained at 37°C in a water-saturated atmosphere under 5% CO2 in air.

For transfection, 10x105 BeWo cells, 50x105 HTR-8/SVneo cells, 30x105 JEG-3 cells, 80x105 THP-1 cells and 5x105 U937 cells were seeded in individual wells of a 12-well culture plate. Cells were transfected using FuGENETM 6 transfection reagent (Roche) with 0.5 µg of the pGL3 vector containing a 968 bp MMP8 promoter fragment coupled to the firefly luciferase reporter gene. In each transfection, 25 ng pRL-TK (Promega), a control plasmid expressing Renilla reniformis luciferase, was used to correct for transfection efficiency. We changed the medium 12 h post-transfection and continued the culture for an additional 24–36 h before collecting cells for the luciferase assays.

Luciferase assay and statistical analysis
After 36–48 h culture, the transfected cells were broken using lysis buffer and 20 µl aliquots of supernatant were then assayed for luciferase activity using the Dual-Luciferase Reporter Assay System (Promega) in a luminometer (Lumat LB 9507, Berthold). Promoter activities were expressed as the ratio between Photinus luciferase and Renilla luciferase activities. Significant differences in activities among the different promoter constructs were evaluated using the Tukey–Kramer test with P<0.05 considered as significant.

Preparation of NE and EMSA
Nuclear proteins were extracted as described previously (44). The following double-stranded oligonucleotide probes (SNP identified in bold) were constructed: –381A sense: 5'-ACAATGAGGGAGGATAAGTACAGAG-3'; –381A antisense: 5'-CTCTGTACTTATCCTCCCTCATTGT-3'; –381G sense: 5'-ACAATGAGGGAGGGTAAGTACAGAG-3'; –381G antisense: 5'-CTCTGTACTTACCCTCCCTCATTGT-3'; –799C sense: 5'-CCATGCAGAGCCTATAGTAGCTCC-3' –799C antisense: 5'-GGAGCTACTATAGGCTCTGCATGG-3'; –799T sense: 5'-CCATGCAGAGCTTATAGTAGCTCC-3'; –799T antisense: 5'-GGAGCTACTATAAGCTCTGCATGG-3'; sense unrelated competitor: 5'-ATGCTGTGAACCTCAGGGTGCTCG-3'; antisense unrelated competitor: 5'-CGAGCACCCTGAGGTTCACAGCAT-3'. The double-stranded synthetic oligonucleotides were labeled with T4 polynucleotide kinase (Invitrogen) and [{gamma}–32P]-ATP. The EMSA binding reaction was mixed in 1xbinding buffer (Promega) with 10 µg of nuclear protein, 1x105 c.p.m of 32P-labeled double-stranded oligonucleotide probe (1 ng) with or without unlabeled competitor probe in a total volume of 10 µl. Reaction mixtures were incubated at room temperature for 30 min and then subjected to 8% PAGE at 250 V for 4.5 h. The dried gels were then exposed to X-ray film.

Quantitative real-time PCR for MMP8 mRNA splice variants
Total RNA was extracted from the cultured BeWo cells, THP-1 cells and U937 cells using TRIzoL reagent (Invitrogen). PolyATtract® mRNA Isolation System (Promega) was used to isolate mRNA from total RNA according to the manufacturer's instructions. Two micrograms of total RNA or 150 ng mRNA was reverse transcribed to single-strand cDNA using oligo (dT) primer, RNasin inhibitor and Moloney murine leukemia virus reverse transcriptase (Clontech) as described by the manufacturer. Quantitative real-time PCR was performed to compare the levels of the MMP8 mRNA splice variants with +91, +67 and 0 bp insertions from cryptic exons in the intron between exons 1 and 2 using primers designed with the Primer Express software package that accompanies the Applied Biosystems 7700 sequence detector (Perkin–Elmer Life Sciences). The forward primer 5'-AAGATCATGTTCTCCCTGAAGACG-3' was shared by the +91 and +67 bp variants. The reverse primer for the +91 bp variant was selected within the extra +91 bp region (5'-GCATCAGTGCAGTTCCTCTTTTT-3'). The reverse primer for the +67 bp variant (5'-GGCTGGGAAGTCCAAGATCAG-3') was selected from sequence in the +67 bp insertion. The size of PCR product for +91 bp variant was 170 bp, and 167 bp for the +67 bp variant. We designed the forward primer (5'-GAGGACAGAAAGAAAGCCAGGAG-3') and reverse primer (5'-AACTTTTCCAGGTAGTCCTGAA-3') to amplify a 165 bp PCR product for the 0 bp variant. All reverse primers, of which the one for +0 bp variant spanned exons 1 and 2, gave exclusive amplification of the +91, +67 and +0 bp variants. The real-time PCR used a 300 nM concentration of each primer and 12.5 µl of 2x SYBR Green PCR Master Mix (Applied Biosystems). The PCR products were also analyzed on 1% agarose gels to display the 170, 167 or 165 bp products.

Western blotting for MMP8 protein
RIPA buffer (45) was used for lysis of freshly frozen human chorion samples, amnion tissue samples, cultured BeWo cells, THP-1 cells and U937 cells. Western blotting was carried out as previously described (46). We loaded 66 µg protein from each sample and detected MMP8 with an anti-human MMP8 monoclonal antibody (Research Diagnostics, Inc.), which recognizes both latent (85 kDa) and active (64 kDa) forms of the enzyme. The blots were also probed for ß-actin to assess protein loading.


   ACKNOWLEDGEMENTS
 
This work was supported by HD34612 (J.F.S.) TW006197 (P.E.F.) and the Bill and Melinda Gates Foundation (J.F.S.).


   FOOTNOTES
 
* To whom correspondence should be addressed at: Center for Research on Reproduction and Women's Health, University of Pennsylvania, 1354 BRB II/III, 421 Curie Boulevard, Philadelphia, PA 19104, USA. Tel: +1 2158980147; Fax: +1 2155735408; Email: jfs3{at}mail.med.upenn.edu


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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