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Posterior tibial tendon is particularly vulnerable and is responsible for much morbidity in sportspersons. Some patients have a predisposition without a clinically recognized cause, suggesting that individual characteristics, inclusive genetic inheritance, play an important role in tendinopathy. Matrix metalloproteinase (MMP)-8 is a proteinase capable of degrading a large amount of extracellular proteins, and influence degradation and remodeling of collagen. To determine whether the −799 polymorphism in the promoter of MMP-8 gene is associated with tendinopathy in posterior tibial tendon, 50 patients undergoing surgical procedures and anatomopathological diagnosis of degenerative lesions of the posterior tibial tendon and 100 control patients with posterior tibial tendon integrity and without signs of degeneration in magnetic resonance imaging were evaluated for the −799 MMP-8 polymorphism. There was a significant difference in the presence of the different alleles (P = 0.001) and genotype (P = 0.003) between the control group and the test group for the MMP-8 gene. The polymorphism at position −799 of the gene for MMP-8 is associated with tendinopathy primary posterior tibial tendon in the population studied. The results suggest that individuals with the T allele are at greater risk of developing tendinopathy.
The posterior tibial tendon insufficiency is recognized as the main cause of adult-acquired flat foot, causing significant functional loss, pain, and secondary osteoarthritis of the hindfoot joints (Pomeroy et al., 1999).
Posterior tibial tendon insufficiency is three times more common in women. The average age of onset is 40 years, with peak incidence at 55 years. It is more common in Caucasians, and in the obese and hypertensive population (Deland et al., 2005). The study shows a prevalence rate of 3.3% in British female population (Kohls-Gatzoulis et al., 2009).
Several etiological factors are proposed to explain the syndrome: the presence of congenital flat foot (Kettelkamp & Alexander, 1969; Jahss, 1982; Mann, 1982; Johnson, 1983; Funk et al., 1986; Dyal, Feder, Deland, Thompson, 1987), the impact on the osteofibrous tunnel (Jahss, 1991), the presence of accessory navicular bone (Kidner, 1933), the tendon hypovascular region (Frey et al., 1990; Holmes & Mann, 1992), and increased mechanical demands (Pomeroy et al., 1999). However, some questions about the physiopathology of primary degeneration of the posterior tibial tendon remain open.
Biochemical investigations reveal that the posterior tibial tendon is typically composed of more than 95% of type I collagen fibers and relatively small amounts of fibers of collagen types III, IV, and V (Goncalves-Neto et al., 2002; Satomi et al., 2008).
The matrix metalloproteinase (MMP)-8, also known as collagenase-2, was previously discovered as an exclusive product of neutrophils, but was subsequently shown to be expressed by a variety of other cell types, such as endothelial, macrophages and polymorphonuclear leukocytes, gingival fibroblasts, keratinocytes, chondrocytes, and odontoblasts as well as cells oral cancer (Moilanen et al., 2002).
The MMP-8 degrades type I collagen, contributing to degradation and tissue remodeling (Galis et al., 1994) and some out-of-the-matrix components, such as angiotensin I (Laxton et al., 2009). It is an important mediator of destruction in several inflammatory diseases and is related to cardiovascular disease (Herman et al., 2001), bronchiectasis (Lee et al., 2007), pulmonary insufficiency (Roderfeld et al., 2009), melamonas (Vihinen et al., 2008), cancer of the head (Köhrmann et al., 2009), and healing of diabetic (Kumar et al., 2006).
Collins and Raleigh (2009) show that individual characteristics may predispose to injuries of the locomotor system, suggesting that intrinsic factors play an important etiologic patient.
Although there has been previous speculation about the possible role of MMP genes in tendinopathy (September et al., 2007; Magra & Maffulli, 2008; Raleigh et al., 2009), little is known about the influence of MMP polymorphism in tendon degeneration.
The purpose of this study was to investigate the frequencies of the −799 polymorphism in the promoter of MMP-8 gene in patients undergoing surgical procedures and pathological diagnosis of degenerative lesions of the posterior tibial tendon so as to verify if this polymorphism can be risk factors to tendinopathy.
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The volunteers were recruited for study from the female patient pool at the Foot and Ankle Group, Department of Orthopedics and Traumatology Hospital of the University of São Paulo, Brazil. After explanation of the work, as recommended by the Research Ethics Committee, written consent was obtained from each participant.
The study protocol was approved by the institutional ethics committee (Scientific Committee of the Institute of Orthopedics and Traumatology, University of São Paulo and the National Ethics Committee).
Patients were identified by age, gender, body mass index (BMI), diagnosis of disease, medication use, personal history of systemic diseases, infectious diseases, inflammatory diseases, and prior information about the presence of flat foot.
All subjects were in good general health and did not have any of the following exclusion criteria: (a) rheumatic diseases; (b) immunological diseases; (c) diabetes; (d) hepatitis; (e) obesity higher than the grade I; and (f) prior or current infection in the topography of the foot and ankle.
Respected the criteria for inclusion and exclusion, the sample was obtained by convenience and composed of 150 women.
The test group was composed 50 patients undergoing surgical procedures and have anatomopathological diagnosis of degenerative lesions of the posterior tibial tendon.
The control group was composed 100 patients with posterior tibial tendon integrity and without signs of degeneration in magnetic resonance imaging.
Sampling and DNA extraction
DNA from epithelial buccal cells was extracted using the procedure described by Aidar and Line (2007). DNA was estimated by measurements of optical density 260/280.
Polymerase chain reaction (PCR) and restriction endonucleases digestion
The MMP-8 genotype was determined using the PCR–restriction fragment length polymorphism assay. The PCR primers used for amplifying the MMP-8 polymorphism were: forward primer 5′-CAGAGACTCAAGTGGGA-3′ and reverse primer 5′-TTTCATTTGTGGAGGGGC-3′. PCR were carried out in a total volume of 10 μL containing 400 ng genomic DNA, 5 μL Taq Green Jumpstart Taq Ready Mix (Amersham Pharmacia-Biotech, Uppsala, Sweden), and 200 nmol of each primer. A 6-μL aliquot of PCR products was then digested with one unit of SfcI enzyme at 37°C overnight.
The total amount aliquot of the digest was electrophoresed on a 10% vertical non-denaturing polyacrylamide gel at 20 mA. The gel was stained by ethidium bromide.
A one-way analysis of variance was used to determine any significant differences between the characteristics of the both groups. The significance of the differences in observed frequencies of polymorphism in both groups was assessed by the chi-square test. P < 0.05 was considered statistically significant.
The program ARLEQUIN (v. 2.0 – Schneider et al., 2000) was used to verify the Hardy–Weinberg equilibrium in the studied sample.
This study obtained a bank of DNA from 150 individuals, 50 patients with degenerative lesions of the posterior tibial tendon and 100 with posterior tibial tendon integrity.
This database is the result of strict criteria to obtain the sample in order to reduce the influence of systemic factors that may mask or increase the real role of genetic polymorphisms in the pathogenesis of posterior tibial tendon tendinopathy.
The age of the sample groups of patients, considered as the independent variable and continues with non-normal distribution, was analyzed using the Mann–Whitney U-test, which identified no significant difference (P = 0.0839) between the two groups.
Data on BMI in both groups were identified according to the classification system in normal individuals (18.5–24.9) or individual class I obesity (25.0–29.9). Considered as an independent variable and categorical with non-normal distribution, was analyzed using the Fisher's exact test, which identified no significant difference (P = 0.721) between the two groups. The mean age and distribution of BMI of the sample are shown in Table 1.
Table 1. Mean age and distribution of the BMI of the sample
|Parameters||Control group (n = 100)||Test group (n = 50)|
|Mean age||51.76 years (47–56)||53.06 years (48–56)|
|BMI||61 (normal)/39(grade I obesity)||33(normal)/17(grade I obesity)|
Regarding questions about the presence of flat foot from the skeletal maturity – in their late teens – the response was positive in 30% of subjects in test group and 32% of subjects in the control group.
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The mechanisms of tendinopathy are complex, and involve mechanical stress, degenerative changes in the tendon tissue, and disorganized healing, along with a contribution from inflammatory processes, although it is unclear (Sharma & Maffulli, 2006; Fredberg & Stengaard-Pedersen, 2008).
The question arises why some individuals are more susceptible to developing tendinopathy compared with others who have a similar level of physical activity and clinical conditions. It is possible that an interaction between the various intrinsic and extrinsic factors with the genetic make up of a given individual increases the likelihood of that individual developing tendinopathy.
Some authors already show the influence of genetic polymorphisms in tendinopathy. Mokone and collaborators (2006) demonstrated that a polymorphism in the alpha 1 type V collagen (COL5A1) is associated with symptoms of pathologies of the Achilles tendon, where the A2 allele seems to exert a protective role. September and collaborators (2009) showed association of this same polymorphism with chronic Achilles tendinopathy in a second population and suggested that a region within the COL5A1 untranslated region may predispose individuals to an increased risk of developing chronic Achilles tendinopathy.
The literature also demonstrated the role of tenascin-C in this process (Mokone et al., 2005). Analyzing a polymorphism in the gene for tenascin-C is characterized by repeating guanine–thymine (GT)n, the authors show that individuals with 12 and 14 GT repeats in the tenascin-C gene have six times higher risk to develop lesions in the tendon Achilles (Mokone et al., 2005).
A polymorphism in the alpha 1 chain of type I collagen (G/T) was associated with anterior cruciate ligament rupture (Posthumus et al., 2009a), but not with Achilles tendon injuries (Posthumus et al., 2009b). Raleigh and collaborators (2009) suggested that variants within the MMP-3 gene are associated with Achilles tendinopathy but not Achilles tendon rupture.
The posterior tibial tendon has some intrinsic and extrinsic factors associated with its degeneration described in the literature (Kettelkamp & Alexander, 1969; Jahss, 1982, 1991; Mann, 1982; Johnson, 1983; Funk et al., 1986; Dyal et al., 1987; Frey et al., 1990; Holmes & Mann, 1992; Pomeroy et al., 1999; Deland et al., 2005). However, a significant number of patients diagnosed with posterior tibial tendon insufficiency have no history of factors associated with pathogenesis.
The participants in this study are similarly matched in the control and test groups, minimizing the interaction of age, BMI, and systemic condition in the pathogenesis of degeneration of the posterior tibial tendon.
It is highly unlikely that a single gene is exclusively associated with the development of the symptoms of tendinopathy. Genetic polymorphisms probably influence the tendinopathy process through the accumulated effect of multiple polymorphisms.
To understand the importance of polymorphisms of each allele, it is important to analyze the relative contribution of each polymorphism to the disease phenotype.
In this study, the C allele was observed in most of the control group while the T allele was more frequent in the test group. The T allele may contribute to the knowledge of the molecular basis for a more intense degradation of the extracellular matrix, which may indicate an increased susceptibility to injury in the posterior tibial tendon.
The polymorphism at position −799 of the gene for MMP-8 is associated with tendinopathy primary posterior tibial tendon in the population studied. The results suggest that individuals with the T allele are at greater risk of developing tendinopathy.