Study population

After obtaining institutional review board approval, Caucasian and AA women with stage > II POP (cases) and a control population with stage < II POP according to the POP Quantification system were prospectively recruited from the urogynecology and benign gynecology clinics of a study author (C.A.M.). Control subjects were recruited to match cases on the basis of age, race, menopausal status, smoking history, body mass index (BMI), and parity. Exclusion criteria for all study subjects included a personal history of a systemic collagen disorder such as Ehlers-Danlos or Marfan syndrome. Demographic data were obtained and recorded on data collection sheets at the time of written consent, including age, race, parity, menopausal status, smoking status, and BMI.

Genotyping

DNA samples were collected through buccal smears using buccal swabs (Simhelix; Boca Scientific, Boca Raton, FL), the extraction of which was performed using ChargeSwitch gDNA Buccal Cell Kit (Invitrogen, Carlsbad, CA). Three previously identified LAMC1 SNPs (rs10911193, rs20563, and/or rs20558) were evaluated in each subject. LAMC1 SNPs rs10911193, rs20563, and rs20558 were identified as NT_004487.18:g.33481002C>T , NM_002293.3:c.1372A>G , and NM_002293.3:c.2663T>C , respectively. Polymerase chain reaction (PCR) was performed on the samples using Taqman SNP Genotyping Assays (Applied BioSystems, Foster City, CA). Allelic PCR products were separated using the Applied Biosystems 7500 fast Sequence Detection System with SDS 1.3.1 software. Genotypes were auto-called by SDS 1.3.1 software with quality value set at 0.90.

To confirm rs10911193 C>T variant, we carried out PCR using a forward primer sequence of 5′-CACTGGCTGGTTACACTTTACCTCT-3′ and a reverse primer 5′-CCTTTTGAGTCCTAATGTCCAAGAC-3′ yielding a 200-base pair (bp) product. After initial denaturation at 95°C for 3 minutes, PCR was performed for 35 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds followed by a final 5-minute elongation at 72°C. The PCR products were used for restriction fragment length polymorphism analysis with the restriction endonuclease MaeIII (Roche, Indianapolis, IN) for genotyping. The “C” allele yielded 2 fragments (141 bp and 59 bp) and the “T” allele yielded 1 fragment with a size of 200 bp.

To confirm the rs20563 A>G variant, we carried out PCR using a forward primer sequence of 5′-AGGGCTGACTTGAAGAGTGG-3′ and a reverse primer 5′-GCCTTCGACATTGTCTTTGC-3′ yielding a 152-bp product. After initial denaturation at 94°C for 5 minutes, PCR was performed for 36 cycles of denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 30 seconds followed by a final 5-minute elongation at 72°C. The rs20563 A>G variant was confirmed by restriction fragment length polymorphism analysis with the restriction endonuclease SspI (New England Biolabs, Ipswich, MA). The “A” allele yielded 2 fragments (110 bp and 42 bp) and the “G” yielded 1 fragment with a size of 152 bp.

Statistical analysis

Descriptive statistics, including means and SD, for demographic information were conducted using software (SAS v9.2; SAS Institute, Cary, NC) and assuming an alpha level of 0.05. Fisher’s exact tests implemented in PLINK software ( http://pngu.mgh.harvard.edu/purcell/plink/ ) were used to test individual SNPs for allele frequency differences between races and for associations with POP within races. Inter-SNP linkage disequilibrium calculations for each race were performed in Haploview (Version 4.0; Harvard Broad Institute, Boston, MA). Haplotype frequencies were also generated in PLINK based on the 2 SNPs found to be linked using Haploview and haplotype-specific tests were performed in PLINK to test for frequency differences between races and for associations with POP within races.