The role of rs267606943 polymorphism in the prolidase gene and plasma prolidase in polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a relatively common reproductive/endocrine problem [1]. Altered levels of reproductive hormones are the main biochemical feature of PCOS, and if untreated, long-term effects include increased risk for dysfunctional uterine bleeding, infertility, endometrial cancer, hypertension, type II diabetes, cardiovascular disease, stress and depression [2–6]. Polycystic ovaries arise due to alteration in folliculogenesis, normally a highly regulated process in which various endocrine, paracrine and autocrine factors act in a spatial and temporal manner, and in which the extra-cellular matrix (ECM) is believed to play an essential role in regulation. The ECM of the ovarian theca and granulosa cells are chiefly made up of collagens type I and IV, whilst matrix metalloproteinases are thought to play an important role in matrix remodelling, wherein, collagen molecules degrade, and new molecules of collagen are formed. Prolidase, EC 3.4. 13.9, also known as Xaa-pro dipeptidase, a cytosolic exopeptidase, catalyses the final step of collagen degradation and releases proline for the synthesis of new collagen molecules, thereby acting as a rate limiting step of collagen metabolism [7]. In this way prolidase is thought to regulate the process of remodelling and, ultimately, cyst formation [8]. A number of studies have characterised and purified prolidase in different expression systems, and levels are associated with disorders related to reproduction, collagen metabolism and various cancers [9,10]. A small study (33 cases, 28 controls) reported raised levels of prolidase in PCOS [11]. Polymorphism rs267606943 is a substitution missense mutation found in exon 8 of the prolidase gene, PEPD, located on chromosome 19 [12]. As this position is highly conserved in protein structure, substitution of their position might affect the physiological levels of protein. Therefore, we hypothesised a link between rs267606943 polymorphism and the presence of PCOS and with plasma levels of prolidase. A case control study was carried out on 200 PCOS patients and 200 healthy controls aged 16–40. Diagnosis of PCOS was by the Rotterdam criteria [13]. Subjects were considered obese if the body mass index (BMI) was ≥23 kg/m2, or normal if BMI was between 18.5 and 22.9 kg/m2. The study was approved by the Institutional Ethical Committee and carried out according to the principles of the Declaration of Helsinki. Written informed consent was obtained for all patients and controls. Venous blood, anticoagulated with EDTA, remained at room temperature (3–4 h) before plasma separation, whereupon prolidase levels were measured by the method of Myara et al. [14]. Briefly, plasma was incubated at 37 °C for one hour with Mn2+, then mixed with glycine– proline followed by incubation at 37 °C for another hour. The reaction was stopped by 0.45 mol/L trichloroacetic acid. A mixture of glacial acetic acid, orthophosphoric acid and ninhydrin was added followed by incubation at 90 °C for 10 min. Absorbance was taken at 515 nm and prolidase activity was calculated by using a proline standard. The intra-assay coefficient of variation (CV) was 3.7%. Fasting glucose, HDL, LDL, CRP, TSH, LH, SHBG, testosterone and prolactin were measured by standard routine pathology laboratory techniques. Genomic DNA was isolated by the Genetix MiniPrep DNA isolation kit from blood. The sequence of exon 8 carrying the S202F polymorphism was amplified by PCR (Applied Biosystems, Foster, U.S.A.) using 2 μl of genomic DNA with 1 μl of both the forward and reverse primers, 2 μl of 25 mM MgCl2, 2 μl of dNTPs and 1 μl of Taq polymerase with 5 μl of buffer supplied with Taq polymerase. The aliquot was brought to 50 μl with nuclease free water. Primers used for amplification were: forward primer 5′-GCAGCAGCTCTGCTCCCTGC-3′ and reverse primer 5′-GTGTCCACGACTGCATGATG-3′ respectively with annealing temperature at 66 °C for 30 s. Amplified PCR products were visualized on 2% agarose gel. The PCR