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Matrix metalloproteinase-3 gene promoter polymorphisms: A potential risk factor for pelvic organ prolapse

View Article: PubMed Central - PubMed

ABSTRACT

Pelvic organ prolapse (POP) is a common multifactorial condition. Matrix metalloproteinases (MMPs) are enzymes capable of breaking down various connective tissue elements. Single-nucleotide polymorphisms (SNPs) in regulatory areas of MMP-encoding genes can alter their transcription rate, and therefore the possible effect on pelvic floor supporting structures. The insertion of an adenine (A) base in the promoter of the MMP-3 gene at position −1612/−1617 produces a sequence of six adenines (6A), whereas the other allele has five (5A). The aim of the present study was to investigate the possible association of MMP-3 gene promoter SNPs with the risk of POP. The patient group comprised 80 women with clinically significant POP [Stage II, III or IV; POP quantification (POP-Q) system]. The control group consisted of 80 females without any or important pelvic floor support defects (Stages 0 or I; POP-Q system). All the participants underwent the same preoperative evaluation. SNP detection was determined with whole blood sample DNA analysis by quantitative polymerase chain reaction (PCR) in LightCycler® PCR platforms, using the technique of sequence-specific hybridization probe-binding assays and melting temperature curve analysis. The results showed there was no statistically significant difference between 5A/5A, 5A/6A and 6A/6A MMP-3 gene promoter variants in the two study groups (P=0.4758). Therefore, MMP-3 gene promoter SNPs alone is insufficient to increase the genetic susceptibility to POP development.

No MeSH data available.


Melting temperature (Tm) peak curve analysis.
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f1-br-0-0-736: Melting temperature (Tm) peak curve analysis.

Mentions: The MMP-3 gene promoter variants were determined using hot-start quantitative polymerase chain reaction (qPCR) in LightCycler® PCR platforms, using the technique of sequence-specific hybridization probe binding assays. LightSNiP® assays (TIB MOLBIOL GmbH, Berlin, Germany) and LightCycler® FastStart DNA Master HybProbe (Roche Diagnostics GmbH) were used for the reaction. The former is a special mixture of primers and fluorescent probes (primer 1, primer 2, 3′-FL HybProbe and 5′-LC HybProbe), which is used for the identification of point mutations, such as SNPs, provided that the reference SNP ID number (rs) of the SNP is known. The rs for the insertion of an adenine (A) deoxyribonucleotide in the promoter of the MMP-3 gene at position −1612/−1617, upstream from the beginning of the transcription frame was 3025058 (28). The latter was a hot-start reaction mix for sensitive PCR applications in LightCycler® capillaries, using HybProbe probes as a detection format. It is a master mix for performing SNP detection. This kit contains all necessary reagents [LightCycler® FastStart enzyme, LightCycler® FastStart reaction mix HybProbe, MgCl2 stock solution (25 mM) and water (PCR grade)]. However, the exact sequences of primers and exact sequences of the probes was unavailable, due to patent and marketing policies of TIB MOLBIOL GmbH. Further technical details for the preparation of PCR are provided in Tables I and II. Determination of each woman's genotype was based on the fact that each DNA fragment's melting Tm was specific for only one of the MMP-3 gene promoter variants (Fig. 1). As a result, 5A/5A women had a slightly higher melting Tm peak than the 6A/6A women, while heterozygotes (5A/6A) had a bimodal (i.e., with two peaks) melting Tm curve.


Matrix metalloproteinase-3 gene promoter polymorphisms: A potential risk factor for pelvic organ prolapse
Melting temperature (Tm) peak curve analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4997987&req=5

f1-br-0-0-736: Melting temperature (Tm) peak curve analysis.
Mentions: The MMP-3 gene promoter variants were determined using hot-start quantitative polymerase chain reaction (qPCR) in LightCycler® PCR platforms, using the technique of sequence-specific hybridization probe binding assays. LightSNiP® assays (TIB MOLBIOL GmbH, Berlin, Germany) and LightCycler® FastStart DNA Master HybProbe (Roche Diagnostics GmbH) were used for the reaction. The former is a special mixture of primers and fluorescent probes (primer 1, primer 2, 3′-FL HybProbe and 5′-LC HybProbe), which is used for the identification of point mutations, such as SNPs, provided that the reference SNP ID number (rs) of the SNP is known. The rs for the insertion of an adenine (A) deoxyribonucleotide in the promoter of the MMP-3 gene at position −1612/−1617, upstream from the beginning of the transcription frame was 3025058 (28). The latter was a hot-start reaction mix for sensitive PCR applications in LightCycler® capillaries, using HybProbe probes as a detection format. It is a master mix for performing SNP detection. This kit contains all necessary reagents [LightCycler® FastStart enzyme, LightCycler® FastStart reaction mix HybProbe, MgCl2 stock solution (25 mM) and water (PCR grade)]. However, the exact sequences of primers and exact sequences of the probes was unavailable, due to patent and marketing policies of TIB MOLBIOL GmbH. Further technical details for the preparation of PCR are provided in Tables I and II. Determination of each woman's genotype was based on the fact that each DNA fragment's melting Tm was specific for only one of the MMP-3 gene promoter variants (Fig. 1). As a result, 5A/5A women had a slightly higher melting Tm peak than the 6A/6A women, while heterozygotes (5A/6A) had a bimodal (i.e., with two peaks) melting Tm curve.

View Article: PubMed Central - PubMed

ABSTRACT

Pelvic organ prolapse (POP) is a common multifactorial condition. Matrix metalloproteinases (MMPs) are enzymes capable of breaking down various connective tissue elements. Single-nucleotide polymorphisms (SNPs) in regulatory areas of MMP-encoding genes can alter their transcription rate, and therefore the possible effect on pelvic floor supporting structures. The insertion of an adenine (A) base in the promoter of the MMP-3 gene at position −1612/−1617 produces a sequence of six adenines (6A), whereas the other allele has five (5A). The aim of the present study was to investigate the possible association of MMP-3 gene promoter SNPs with the risk of POP. The patient group comprised 80 women with clinically significant POP [Stage II, III or IV; POP quantification (POP-Q) system]. The control group consisted of 80 females without any or important pelvic floor support defects (Stages 0 or I; POP-Q system). All the participants underwent the same preoperative evaluation. SNP detection was determined with whole blood sample DNA analysis by quantitative polymerase chain reaction (PCR) in LightCycler® PCR platforms, using the technique of sequence-specific hybridization probe-binding assays and melting temperature curve analysis. The results showed there was no statistically significant difference between 5A/5A, 5A/6A and 6A/6A MMP-3 gene promoter variants in the two study groups (P=0.4758). Therefore, MMP-3 gene promoter SNPs alone is insufficient to increase the genetic susceptibility to POP development.

No MeSH data available.