Balkan Journal of Medical Genetics

DOES THE A9285G POLYMORPHISM IN COLLAGEN TYPE XII α1 GENE ASSOCIATE WITH THE RISK OF ANTERIOR CRUCIATE LIGAMENT RUPTURES?
Ficek K, Stepien-Slodkowska M, Kaczmarczyk M, Maciejewska-Karlowska A, Sawczuk M, Cholewinski J, Leonska-Duniec A, Zarebska A, Cieszczyk P, Zmijewski P,
*Corresponding Author: Piotr Zmijewski, Ph.D., Institute of Sport, Department of Physiology, Trylogii 2/16, 01-982 Warsaw, Poland. Tel.: +48-228340812. Fax: +480228350977. E-mail: piotr.zmijewski@insp.waw.pl
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MATERIALS AND METHODS

Study Subjects. A total of 91 male football players (23 ± 3 years) with surgically diagnosed primary ACL ruptures who qualified for ligament reconstruction, were recruited for this study through Galen Orthopaedics, Bieruń, Poland. The control group comprised 143 apparently healthy, male football players (25 ± 2.6 years), of the same ethnicity, a similar age category, and a comparable level of exposure to ACL injury, who were without any selfreported history of ligament or tendon injury. Ethics Committee. The study was conducted in accordance with the ethical standards as described by Kruk [19]. Additionally, the Pomeranian Medical University (Szczecin, Poland) Ethics Committee approved the details of this study and all related informational and consent documentation before any data collection. In accordance with the Pomeranian Medical University Ethics Committee’s guidelines, the investigator informed all the subjects as to the benefits and possible risks associated with participation in the study, and all subjects signed a written informed consent document indicating their voluntary participation. Genotyping. Genomic DNA was extracted from the oral epithelial cells using GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich Chemie, Steinheim, Germany) according to manufacturer’s protocol. Allelic discrimination of the A9285G COL12A1 (rs970547) polymorphic site was performed using a TaqMan Pre-Designed SNP Genotyping Assays (Applied Biosystems, Foster City, CA, USA), including primers and fluorescently labelled (FAM and VIC) MGB probes for the detection of the alleles. All samples were genotyped on a Rotor-Gene real-time polymerase chain reaction (Re-Ti-PCR) instrument (Corbett Research, Sydney, NSW, Australia). Thermal cycler conditions were as follows: an initial step at 95 °C for 5 min., followed by 45 cycles of denaturation at 94 °C for 15 seconds and annealing/extension at 60 °C for 1 min. Statistical Analysis. Any differences in genotype and allele frequency were analyzed using χ2 tests (or Fisher exact tests). Odds ratios (OR) with 95% confidence intervals (95% CI) were calculated. All calculations were performed using Statistica (StatSoft Inc., Tulsa, OK, USA; 2011). STATISTICA (data analysis software system, version 10, www.statsoft.com) was used for computing statistics, except Hardy-Weinberg equilibrium which was tested with the programming language and environment R (http://www.r-project.org) and the test for linear trend, which was performed using the STATCALC module in Epi Info (http://wwwn.cdc.gov/ epiinfo). The p <0.05 values were considered to be statistically significant.

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