The COL5A1 gene and Achilles tendon pathology

Abstract

Purpose: There is an increase in the incidence of Achilles tendon injuries as a result of the participation in physical activity. It has been suggested that some individuals have a genetic predisposition to Achilles tendon pathology (ATP). The aim of this study was to determine whether the α 1 type V collagen (COL5A1) gene, which encodes for a tendon protein, is associated with the symptoms of ATP.

Methods: One-hundred and eleven Caucasian subjects diagnosed with ATP and 129 Caucasian control (CON) subjects were genotyped for the BstUI and DpnII restriction fragment length polymorphisms (RFLPs) within the COL5A1 gene.

Results: There was a significant difference in the allele frequencies of the COL5A1 BstUI RFLP between the ATP and CON subjects (P=0.006). The frequency of the A2 allele was significantly higher in the CON group (29.8%) than in the ATP group (18.0%) (odds ratio of 1.9; 95% confidence interval (CI) 1.3–3.0; P=0.004). This allele had a stronger protective role when only the 72 patients diagnosed with chronic Achilles tendinopathy were analyzed (odds ratio of 2.6; 95% CI 1.5–4.5).

Conclusions: The COL5A1 BstUI RFLP is associated with ATP and more specifically, chronic Achilles tendinopathy. Individuals with an A2 allele of this gene are less likely of developing symptoms of chronic Achilles tendinopathy.

There is a reported increase in the incidence of injuries of the Achilles tendon in those who participate in competitive and recreational physical activity (Jozsa et al., 1989b). Although the causes of Achilles tendon injuries are poorly understood, both intrinsic and extrinsic factors have been implicated in its etiology. The relationship of these factors to Achilles tendon pathology (ATP) has recently been reviewed (Paavola et al., 2002; Riley, 2004). Specific intrinsic factors that have been identified include age, gender, body weight, vascular perfusion, nutrition, anatomical variants, joint laxity, muscular weakness or imbalance, the use of certain drugs, systemic disease and a history of a previous injury. Extrinsic factors include the type of activity, occupation, training, physical load, shoes and environmental conditions.

It has been proposed that, in addition to these non-genetic factors, certain genetic elements might, in part, be associated with an individual's susceptibility to Achilles tendon injuries. More specifically, several investigators have suggested that a gene(s) on the tip of chromosome 9q, closely linked to the ABO blood group gene, is associated with ATP (reviewed in Kannus & Natri, 1997). Jozsa et al. (1989a), Jozsa et al. (1989b), Kujala et al., (1992) and Kannus and Natri (1997) have shown that blood group O, and by implication the ABO gene, is associated with tendon injuries in a group of Hungarian or Finnish patients. But other studies have not shown an association between the ABO blood group with tendon pathology (Leppilahti et al., 1996; Maffulli et al., 2000). Recently, Årøen et al. also suggested, based on their findings that individuals who had ruptured an Achilles tendon had an increased risk of rupturing their contralateral tendon, the possible involvement of genetic elements in the etiology of ATP (Årøen et al., 2004).

Tendons have a highly ordered hierarchical structure made up of tightly packed protein bundles consisting predominantly of type I collagen fibers (reviewed in Silver et al., 2003). Trace amounts of other collagens, such as types III and V, form heterotypic fibers with the type I collagen found in tendons (reviewed in Birk, 2001; Silver et al., 2003). Increases in type V collagen content have been reported with age in the rabbit patellar tendon and in biopsy samples of degenerative tendons (Dressler et al., 2002; Goncalves-Neto et al., 2002).

The pro-α1(V) chain is found in most of the isoforms of type V collagen and is encoded by the α 1 type V collagen (COL5A1) gene, which has been mapped to the same locus as the ABO gene on chromosome 9q34 (Caridi et al., 1992). The gene was therefore identified as an ideal candidate genetic marker of ATP. Pro-α2(V) and pro-α3(V) chains are also found in some of the type V collagen isoforms. Neither of the genes that encode these two α-chains have, however, been mapped to human chromosome 9 and were therefore not investigated as candidate genes in this study (reviewed in Myllyharju & Kivirikko, 2001). In addition, several mutations within the COL5A1 and COL5A2 genes have been shown to cause more severe connective tissue disorders such as some of the Ehlers–Danlos syndromes (EDS), which have been shown to affect tendons (reviewed in Myllyharju & Kivirikko, 2001; Riley, 2004).

The COL5A1 gene contains a BstUI and a DpnII restriction fragment length polymorphisms (RFLPs) within its 3′-untranslated region (UTR) (Greenspan & Pasquinelli, 1994). To our knowledge, the influence of these polymorphisms on the expression of the COL5A1 gene and the ultimate function of type V collagen is unknown. The aim of the study therefore was to determine whether the BstUI and/or DpnII RFLPs within the 3′-UTR of the COL5A1 gene are associated with ATP.

Material and methods

Subjects

One-hundred and eleven Caucasian patients with a current or past clinical history of ATP were recruited from the Medical Practice at the Sports Science Institute of South Africa and other Clinical Practices within the Cape Town area in South Africa. The ATP patients included 72 with chronic tendinopathies (TEN) and 39 with either a complete (36 of 39) or partial (three of 39) rupture (RUP). They were all physically active prior to the development of symptoms. The clinical practices invited their eligible patients to participate in the study, by sending them a letter informing them about the study. Subjects were requested to contact the investigators if they were interested in volunteering for the study. An experienced clinician initially made the diagnosis of chronic Achilles tendinopathy (69 of 72) using clinical criteria. The diagnostic criteria for every subject were reviewed and confirmed by one of the investigators (M. S.). The clinical diagnostic criteria for chronic Achilles tendinopathy were gradual progressive pain over the posterior lower limb in the Achilles tendon area for greater than 6 months, together with at least one out of the following six criteria: (1) early morning pain over the Achilles tendon area, (2) early morning stiffness over the Achilles tendon area, (3) a history of swelling over the Achilles tendon area, (4) tenderness to palpation over the Achilles tendon, (5) palpable nodular thickening over the affected Achilles, or (6) movement of the painful area in the Achilles tendon with plantar-dorsi-flexion (positive “shift” test) (Kader et al., 2002; Paavola et al., 2002; Schepsis et al., 2002). In addition to these clinical diagnostic criteria, soft-tissue ultrasound examination was performed in a sub-group (22 of 72) of subjects to confirm the diagnosis of the affected Achilles tendon.

The diagnosis of Achilles tendon rupture was made clinically using standard validated criteria (Leppilahti & Orava, 1998; Schepsis et al., 2002; Maffulli et al., 2003) and confirmed in all cases by examination at the time of surgery (34 of 39) and/or by ultrasound imaging (five of 39), magnetic resonance imaging (MRI) (two of 39) or computerized tomography (CT) scan (one of 39).

One-hundred and twenty-nine apparently healthy physically active Caucasian control (CON) subjects without any history of ATP were also recruited for this study from various recreational sporting clubs. The subjects were matched for age and gender. To avoid any possible effects of population stratification, the ATP and CON groups were also similarly matched for their country of birth.

Approval for this study was obtained from the Research Ethics Committee of the Faculty of Health Sciences within the University of Cape Town. Once recruited, both the ATP and CON subjects were required to complete an informed consent form and personal particulars, physical activity and medical history questionnaires prior to participation. Subjects who had a history of current or past fluoroquinolone antibiotic use or previous local corticosteroids injection in the Achilles tendon or the area surrounding the Achilles tendon prior to the onset of symptoms were excluded from the study. This was necessary because of the known association between fluoroquinolone antibiotic (van der Linden et al., 2001) or possibly corticosteroids use, and an increased risk of Achilles tendon rupture (Leppilahti & Orava, 1998). Furthermore, ATP and CON subjects who had been diagnosed with any connective tissue disorders or any other systemic diseases believed to be associated with ATP, such as, but not limited to, EDS, benign hypermobility joint syndrome, rheumatoid arthritis, systemic lupus erythematosus, hyperparathyroidism, renal insufficiency, diabetes mellitus and familial hypercholesterolemia were also excluded from the study (Leppilahti & Orava, 1998).

Sample collection, total DNA extraction and blood grouping analysis

Approximately 4.5 mL of venous blood was collected from each subject into ethylenediaminetetraacetic acid (EDTA) vacutainer tubes by venipuncture of a forearm vein and stored at 4°C until total DNA extraction. Total DNA was extracted from the sample as described by Lahiri and Nurnberger (1991) with some modifications. Briefly, the blood samples were transferred to 15 mL polypropylene tubes, to which two volumes of TKM1 buffer (10 mM Tris-HCl pH 7.6, 10 mM KCl, 10 mM MgCl₂ and 2 mM EDTA) containing 2.5% Nonidet P-40 (Sigma, St. Louis, MO, USA) was added to lyse the red blood cells. After a 10 min incubation at room temperature, the white blood cells were pelleted by centrifugation at 1200 ×g at room temperature for 10 min and washed at least once with one volume of TKM1 buffer. The washed pellets were resuspended in 800 μL of TKM2 buffer (10 mM Tris-HCl pH 7.6, 10 mM KCl, 10 mM MgCl₂, 0.4 M NaCl₂ and 2 mM EDTA) containing 50 μL of 10% sodium dodecyl sulfate by incubation for at least 10 min at 55°C or until the pellets had dissolved. One hundred and fifty microlitres of 5 M NaClO₄ and 500 μL of chloroform was added to each sample, which was then mixed thoroughly by vortexing for 15–20 s. The samples were transferred to 1.5 mL microfuge tubes and the protein precipitated by centrifugation at 13 000 r.p.m. (15 000 × g) for 5 min at room temperature. Five hundred microlitres of the top aqueous phases were transferred to new microfuge tubes containing 1 ml of absolute ethanol, mixed and the DNA pelleted by centrifugation at 13 000 r.p.m. (15 000 × g) for 2 min at room temperature. The precipitated DNA was air dried for 30 min, resuspended in at least 100 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) either by incubation for 1 h at 65°C or over-night at room temperature, and stored at 4°C until polymerase chain reaction (PCR) analysis.

COL5A1 genotyping

A 667 bp fragment containing the BstUI and DpnII RFLPs within the 3′-UTR of the COL5A1 gene was PCR amplified as described by Greenspan and Pasquinelli (1994). The PCR was carried out in a total volume of 60 μL containing at least 100 ng DNA, 20 pmol of the forward and reverse primers, 20 mM Tris-HCl pH 8.4, 50 mM KCl, 1.5 mM MgCl₂, 0.2 mM of each dNTP (dATP, dTTP, dCTP and dGTP) and 2.5 U of DNA Taq polymerase (Invitrogen Life Technologies, Carlsbad, California, USA), using a PCR Express Thermal Cycler (Hybaid Limited, Middlesex, UK). The amplification was performed with an initial denaturing step at 94°C for 3 min, followed by 35 cycles of denaturing at 94°C for 1 min, annealing at 53°C for 1 min, extension at 72°C for 1.5 min, and a final extension step at 72°C for 8 min. The PCR products were digested with BstUI to produce 351 and 316 bp fragments for the A1 allele, 316, 271 and 80 bp fragments for the A2 allele and a 667 bp fragment for the A3 allele. The PCR products were also digested with DpnII to produce 418, 194, 40 and 15 bp fragments for the B1 allele and 612, 40 and 15 bp fragments for the B2 allele. The resulting fragments were separated, together with 100 bp DNA ladder known size markers (Promega Corporation, Madison, Wisconsin, USA), on 5% non-denaturing polyacrylamide gels and visualized by ethidium bromide staining. The gels were photographed under UV light using a Uvitec photodocumentation system (Uvitec Limited, Cambridge, UK) and the sizes of the DNA fragments determined (Fig. 1).

Figure 1.  Typical 5% non-denaturing polyacrylamide gels showing the common (a) BstUI and (b) DpnII restriction fragment length polymorphism (RFLP) genotypes. The sizes of the various DNA fragments are indicated on the left size of each gel. The 40 and 15 bp products produced during digestion of the polymerase chain reaction fragments with DpnII are not shown in panel (b). (Panel a) Lane 1 is an A1A1 genotype, while lane 2 is an A2A2 genotype for the BstUI RFLP. (Panel b) Lanes 1 and 2 are a B1B2 genotype, while lanes 3 and 4 are a B1B1 genotype of the DpnII RFLP.

Statistical analyses

The required sample size for this study was determined using QUANTO Version 0.5 (http://hydra.usc.edu/gxe) (Gauderman, 2002). Data were analyzed using the STATISTICA version 6.1 (StatSoft Inc., Tulsa, Oklahoma, USA) and GraphPad InStat version 2.05a (GraphPad Software, San Diego, California, USA) statistical programs. A one-way analysis of variance (ANOVA) was used to determine any significant differences between the characteristics of the ATP and CON groups, as well as the TEN and RUP sub-groups. When the overall F value was significant, a least significant difference post-hoc test was used to identify specific differences. Statistical significance was accepted when P<0.05. Where applicable, data are presented as means±standard deviations (SD) with the number of subjects in parentheses. Pearson's chi-square analysis was used to analyze differences in the genotype and allele frequencies between the ATP and CON groups. The genotype frequencies of the BstUI RFLP of the COL5A1 gene were analyzed using Monte Carlo simulations (CLUMP version 2.0 program) (Sham & Curtis, 1995).

Results

Subject characteristics

As shown in Table 1, the ATP, TEN, RUP and CON groups were similarly matched for age, height and country of birth. The ATP, TEN and CON groups were similarly matched for gender, while the RUP group (79.5%) contained significantly more male subjects than the CON group (61.7%, P=0.040). In addition, the ATP, TEN and RUP groups were significantly heavier with corresponding higher body mass indexes than the CON group.

Table 1.   Characteristics of the control (CON) and Achilles tendon pathology (ATP) groups, as well as the Achilles tendinopathy (TEN) and Achilles rupture (RUP) sub-groups

	CON	ATP	TEN	RUP
Values are expressed as mean±standard deviation or a frequency (%) where applicable. Number of subjects (n) is in parentheses. ^a,bCON vs ATP (P<0.001); ^c,dCON vs RUP (P<0.001); ^eCON vs TEN (P=0.006); ^fCON vs TEN (P=0.004); ^gCON vs RUP (P=0.040); ^h,iTEN vs RUP (P<0.001). ^* The age of the ATP group as well as the TEN and RUP sub-groups are the age of onset of the symptoms of Achilles tendon pathology.
Age (years)^*	40.3±11.0 (122)	40.1±14.0 (108)	39.7±15.3 (69)	40.8±11.3 (39)
Height (cm)	175±9 (121)	176±9 (108)	176±10 (69)	176±8 (39)
Weight (kg)	71.1±12.1 (125)^a,c,e	80.8±15.1 (108)^a	77.1±13.7 (69)^e,h	87.3±15.3 (39)^c,h
Body mass index (kg/cm^2)	23.2±2.7 (121)^b,d,f	25.9±3.9 (108)^b	24.7±3.3 (69)^f,i	28.1±4.1 (39)^d,i
Gender (% males)	61.7 (128)^g	73.0 (111)	69.4 (72)	79.5 (39)^g
Country of birth (% South Africa)	71.0 (124)	75.7 (107)	73.5 (68)	79.5 (39)

In the TEN group, the additional documented clinical criteria to confirm the diagnosis were tenderness to palpation (59 of 72), early morning stiffness (39 of 72), a history of swelling (24 of 72), early morning pain (15 of 72), palpable thickening (14 of 72) and a positive “shift” test (nine of 72). In 22 of the 72 subjects, the diagnosis was confirmed by soft-tissue ultrasound examination of the affected Achilles tendon. There were 29 of the 72 subjects with confirmed bilateral Achilles tendinopathy.

In the RUP group, 38 of 39 subjects experienced acute severe pain in the posterior lower leg as the main presenting symptom. The diagnosis was confirmed in all these subjects by either direct examination at the time of surgery (34 of 39) or by imaging (eight of 39) (soft-tissue ultrasound, MRI or CT). Four of the 39 subjects had confirmed bilateral ruptures of the Achilles tendon, and 16 of 39 subjects had a history of tendinopathy prior to rupture.

Range of motion and flexibility were assessed in 36 of the 129 CON and 65 of the 111 ATP subjects (data not shown). None of the subjects tested were hypermobile. In addition, none of the subjects included in this study had symptoms or signs (skin hyperextensibility, bruising, recurrent joint effusions, subluxations or dislocations, ocular manifestations or cardiovascular manifestations) of EDS or benign hypermobility joint syndrome when their medical examinations were reviewed by the investigating clinician (M. S.).

The activity resulting in injury in the majority of the ATP subjects was running (47 of 111, 42.3%) or playing squash (17 of 111, 15.3%), while the remaining injuries occurred as a result of participating in a variety of sports and activities.

As shown in Table 2, all the subject groups and sub-groups were matched for the number of years participated in running. Of the CON, ATP, TEN and RUP subjects, 74%, 56%, 61% and 46%, respectively, participated in running. Over the last 2 years, both the ATP and RUP groups, but not the TEN group, trained significantly less than the CON group. The three symptomatic Achilles pathology groups, however, participated for significantly more years in high-impact sports than the CON group. There were no significant differences in the hours of training in high-impact sports over the last 2 years between the groups. There were also no significant differences in years of participation and the level of training over the last 2 years when only the squash players were analyzed (data not shown).

Table 2.   Participation in physical activity and training of the control (CON) and Achilles tendon pathology (ATP) groups, as well as the Achilles tendinopathy (TEN) and Achilles rupture (RUP) sub-groups

	CON	ATP	TEN	RUP
Values are expressed as mean±standard deviation. Number of subjects (n) is in parentheses. ^aCON vs ATP (P=0.002); ^b,eCON vs RUP (P<0.001); ^cCON vs ATP (P<0.001); ^dCON vs TEN (P<0.001).
Running (years)	7.9±8.1 (129)	8.6±10.6 (111)	9.4±10.4 (72)	7.0±10.9 (39)
Running in the last 2 years (h/week)	3.3±2.9 (126)^a,b	2.1±2.6 (98)^a	2.6±2.8 (63)	1.2±1.9 (35)^b
High-impact sports (years)	11.5±8.5 (129)^c,d,e	20.2±13.7 (111)^c	18.5±14.1 (72)^d	23.5±12.4 (39)^e
High-impact sports in the last 2 years (h/week)	5.0±4.7 (129)	4.1±5.1 (111)	4.5±5.2 (72)	3.2±5.0 (39)

COL5A1 genotype and allele frequencies

There was a significant difference when the three COL5A1 BstUI RFLP alleles (A1, A2 and A3) of the CON subjects were compared with the ATP group (Pearson's χ²=10.3, P=0.006) [Fig. 2(a)]. The frequencies of the A1 and A3 alleles were higher in the ATP group (169 A1, 76.1% and 13 A3, 5.9%) than in the CON group (173 A1, 67.1% and eight A3, 3.1%), while the frequency of the A2 allele was higher in the CON subjects (77 A2, 29.8%) than in the ATP group (40 ATP, 18.0%) (odds ratio of 1.9; 95% confidence interval (CI) 1.3–3.0; P=0.004).

Figure 2.  Allele frequencies of the (a) BstUI and (b) DpnII restriction fragment length polymorphism (RFLP) within the α 1 type V collagen (COL5A1) gene of the asymptomatic control subjects (CON, solid bars), as well as the symptomatic Achilles tendon pathology (ATP, gray bars), chronic Achilles tendinopathy (TEN, hatched bars) and Achilles tendon rupture (RUP, clear bars) patients. (a) P=0.006, CON vs ATP; P=0.0009, CON vs TEN; P=0.578, CON vs RUP and (b) P=0.453, CON vs ATP; P=0.424, CON vs TEN; P=0.837, CON vs RUP.

When the ATP group was sub-divided into more homogeneous pathologies, namely, chronic TEN or RUP sub-groups, there was a stronger significant difference in the frequencies of the distribution of the A1, A2 and A3 alleles when the CON and TEN groups were compared (Pearson's χ²=14.0, P=0.0009) [Fig. 2(a)]. The frequencies of the A1 and A3 alleles were higher in the TEN group (115 A1, 79.9% and nine A3, 6.2%) than in the CON group. The frequency of the A2 allele was higher in the CON group than in the TEN group (20 A2, 13.9%) (odds ratio of 2.6; 95% CI 1.5–4.5; P=0.0005). Although it should be interpreted with caution because of the small sample size (78 alleles), there was no significant difference in the distribution of these three alleles between the CON and RUP group (54 A1, 69.2%; 20 A2, 25.7% and four A3, 5.1%) (Pearson's χ²=1.1, P=0.578) [Fig. 2(a)].

Figure 2(b) shows that there were no significant differences in the distribution of the two COL5A1 DpnII RFLP alleles (B1 and B2) between the CON group and either the entire ATP group (Pearson's χ²=0.6, P=0.453) or the TEN (Pearson's χ²=0.6, P=0.424) and RUP (Pearson's χ²=0.04, P=0.837) sub-groups.

There were no significant differences in the genotype frequencies of the COL5A1 BstUI (Table 3) and DpnII (Table 4) RFLPs between the CON and ATP (Pearson's χ² of the BstUI RFLP=5.2, P=0.169 and Pearson's χ² of the DpnII RFLP=3.7, P=0.160) groups, nor the CON and TEN (Pearson's χ² of the BstUI RFLP=7.7, P=0.057 and Pearson's χ² of the DpnII RFLP=2.8, P=0.243) or RUP (Pearson's χ² of the BstUI RFLP=6.6, P=0.089 and Pearson's χ² of the DpnII RFLP=1.6, P=0.460) sub-groups. Individuals with an A2A2 genotype were under-represented in the ATP (odds ratio of 2.1; 95% CI 1.1–4.1; P=0.035) and TEN (odds ratio of 2.9; 95% CI 1.2–6.6; P=0.018) subjects.

Table 3.   Relative frequencies of BstUI restriction fragment length polymorphism genotype of the COL5A1 gene within the control (CON) and Achilles tendon pathology (ATP) groups, as well as the Achilles tendinopathy (TEN) and Achilles rupture (RUP) sub-groups

Genotype	CON (n=129)	ATP (n=111)	TEN (n=72)	RUP (n=39)
The values are expressed as percentage with the number of subjects (n) in parentheses.
A1A1 (%)	62.8 (81)	70.3 (78)	76.4 (55)	59.0 (23)
A1A2 (%)	7.0 (9)	6.3 (7)	4.2 (3)	10.3 (4)
A1A3 (%)	1.6 (2)	5.4 (6)	2.8 (2)	10.3 (4)
A2A2 (%)	26.4 (34)	14.4 (16)	11.1 (8)	20.5 (8)
A2A3 (%)	0 (0)	0.9 (1)	1.4 (1)	0 (0)
A3A3 (%)	2.3 (3)	2.7 (3)	4.2 (3)	0 (0)

Table 4.   Relative frequencies of DpnII restriction fragment length polymorphism genotype of the COL5A1 gene within the control (CON) and Achilles tendon pathology (ATP) groups, as well as the Achilles tendinopathy (TEN) and Achilles rupture (RUP) sub-groups

Genotype	COn (n=129)	ATP (n=111)	TEN (n=72)	RUP (n=39)
The values are expressed as percentage with the number of subjects (n) in parentheses.
B1B1 (%)	58.1 (75)	58.6 (65)	59.7 (43)	56.4 (22)
B1B2 (%)	31.0 (40)	36.9 (41)	36.1 (26)	38.5 (15)
B2B2 (%)	10.9 (14)	4.5 (5)	4.2 (3)	5.1 (2)

Discussion

The main finding of this study was that the three alleles produced by the BstUI RFLP within the 3′-UTR of the COL5A1 gene were associated with ATP (P=0.006). There was a significant higher frequency of the A2 allele of this gene in the asymptomatic control subjects (CON 29.8% vs ATP 18.0%). Individuals with the A2 allele were therefore less likely of developing symptoms of tendon pathology (odds ratio=1.9; 95% CI 1.3–3.0; P=0.004).

It has previously been suggested that a gene(s) on the tip of the long arm of chromosome 9, closely linked to the ABO blood group gene, is associated with ATP (reviewed in Kannus & Natri, 1997). Since the ABO gene on chromosome 9q34 encodes for distinct transferases, some investigators have speculated that these enzymes, not only determine the structure of the glycoprotein antigens on red blood cells, but also the structure of some proteins making up the ground substance of tendons (Jozsa et al., 1989a; Bennett et al., 1995). It is however more likely that genes, such as COL5A1, which have also been mapped to chromosome 9q34 (Caridi et al., 1992), and known to encode for proteins involved in tendon structure, development and regeneration, are better candidate genes for ATP than the ABO gene.

The COL5A1 gene encodes for the pro-α1(V) chain which is found in most of the isoforms of type V collagen (reviewed in Ristiniemi & Oikarinen, 1989). The major isoform of type V collagen is a heterotrimer consisting of two pro-α1(V) chains and one pro-α2(V) chain. Trace amounts of type V collagen are found in tendons where it forms heterotypic fibers with the major structural collagen, namely type I collagen (reviewed in Birk, 2001; Silver et al., 2003). Although most investigators have speculated, based on the function of type V collagen in the cornea, that the protein plays an important role in regulating fibrillogenesis and modulating fibril growth in tendons, some investigators have suggested that the function of type V collagen in tendons, and other tissues where its content is low, is actually unknown (reviewed in Birk, 2001; Riley, 2004). Although there is no consensus about the function of type V collagen in tendons, Dressler et al. (2002) have reported an age-dependent increase in the content of the protein, together with a decrease in fibril diameter and the biomechanical properties in the rabbit patellar tendon. In addition, Goncalves-Neto et al. (2002) have shown an increase in types III and V collagen together with a reduction in the content of type I collagen in biopsy samples of degenerative tendons from patients with posterior tibial tendon dysfunction syndrome. Because of (i) its proximity to the ABO gene on chromosome 9 and (ii) the presence and proposed function of type V collagen in tendons, we propose that the COL5A1 gene is a better candidate genetic marker than the ABO gene for ATP.

The allele distributions of the COL5A1 DpnII RFLP within the control subjects and the various groups of subjects with symptoms of ATP were similar to those of previously reported values. In addition there was no significant difference in the allele distribution of the COL5A1 BstUI RFLP when the control subjects were compared with previously reported values (P=0.206) (Greenspan & Pasquinelli, 1994).

An additional finding of this study was that the alleles of the COL5A1 BstUI RFLP were strongly associated with chronic Achilles tendinopathy (P=0.0009), since individuals with the A2 allele were less likely to present with symptoms of tendinopathy (odds ratio of 2.6; 95% CI 1.5–4.5, P= 0.0005). This RFLP was however not associated with Achilles tendon ruptures in this study, suggesting that the etiology of ruptures and tendinopathies are distinct. These findings must however be interpreted with caution since only 78 alleles were analyzed.

Although the COL5A1 gene is an ideal marker for ATP and more specifically chronic Achilles tendinopathy, the findings of this study do not prove that type V collagen is involved in the etiology of tendon pathology. It is possible that another gene closely linked to the COL5A1 and ABO genes on the tip of the long arm of chromosome 9 encodes for a protein, which is directly involved in the pathogenesis of Achilles tendon injuries. One such gene, the tenascin-C (TNC) or hexabrachion (HXB) gene is expressed in tendons (Chiquet & Fambrough, 1984; Jarvinen et al., 1999). Since tenascin-C is able to bind to various components of the extracellular matrix and to cell receptors, it is believed to play an important role in regulating cell–matrix interactions (reviewed in Jones & Jones, 2000). In normal adult tendons, tenascin-C is localized predominantly in regions responsible for transmitting high levels of mechanical force such as the myotendinous and osteotendinous junctions (Chiquet & Fambrough, 1984; Jarvinen et al., 1999). The protein is also localized around the cells and the collagen fibers (Jarvinen et al., 2003). In addition, Jarvinen et al. (1999, 2003) have shown that expression of the TNC gene is regulated in a dose-dependent manner by mechanical loading in tendons. Mokone et al. (in press) have recently shown that the GT dinucleotide repeat polymorphism within intron 17 of the TNC gene is also associated with ATP. The possible role(s) of type V collagen and/or tenascin-C in the development of ATP needs to be investigated. Our results nevertheless suggest that both the COL5A1 and TNC genes are markers of ATP.

Finally, it is highly unlikely that a single gene or genes in the vicinity of chromosome 9q34 are exclusively associated with the development of the symptoms of ATP. It is perhaps more probable that this condition is polygenic in nature and that other genes which encode for important structural components of tendons are also associated with ATP.

In addition to the genetic factors identified in this study and others that may be identified in the future, several non-genetic intrinsic factors, such as, amongst others, age, gender, body weight and a history of a previous injury; as well as extrinsic factors, such as type of activity and training, have been implicated in the etiology of ATP (reviewed in Riley, 2004). Because the symptomatic subjects in this study were significantly heavier than the control subjects and had also participated for a significantly more years in high-impact sports, we cannot exclude the possibility that an interaction of weight and/or physical activity exposure with the COL5A1 gene was responsible for the development of symptoms of ATP. It should be noted however that because this was a retrospective study we could not record body weight of the subjects accurately at the time of injury. Anecdotally, many subjects reported increases in their body weight after injury as a result of a decrease in physical activity. Increased body weight has been documented as a risk factor for lower extremity injuries in some studies (Murphy et al., 2003), and therefore has also been suggested as an intrinsic risk factor for ATP (Paavola et al., 2002; Riley, 2004). However, to our knowledge no studies have shown that increased body weight is a specific risk factor for Achilles tendon injuries. It can however be noted that there is a reported interaction of obesity with the COL9A3 gene and lumber disc degeneration (Solovieva et al., 2002). Therefore, the possible interaction of non-genetic factors, such as body weight and exposure to physical activity, with the COL5A1 gene or any other gene needs to be investigated.

In conclusion, the BstUI RFLP within the COL5A1 gene is associated with ATP and more specifically chronic Achilles tendinopathy and that the A2 allele of this gene appears to protect individuals from developing symptoms.

Perspectives

Although a genetic predisposition to ATP has been suggested, no specific genes have been shown to be associated with this pathology. This is the first study to demonstrate an association between a polymorphism within a gene expressed in tendons, namely COL5A1, and symptoms of ATP in physically active individuals. The novel finding of this study is that the BstUI RFLP within the 3′-UTR of the COL5A1 gene is associated with ATP and more specifically chronic Achilles tendinopathy. The association of the COL5A1 gene with Achilles tendon injuries however does not prove that type V collagen, an important component of tendons, is directly involved in the development of this pathology. Perhaps other closely linked genes are involved. Besides the involvement of genetic factors, various non-genetic intrinsic and extrinsic factors have been implicated in the etiology of ATP. The interaction of these factors with an individual's genetic background in the development of ATP needs to be investigated. In conclusion, this study suggests that individuals with the A2 allele of the COL5A1 gene are less likely of developing symptoms of chronic Achilles tendinopathy.

Acknowledgements

This study was supported in part by funds from the University of Cape Town, the South African Medical Research Council and Discovery Health. G. Mokone was supported by fellowships from the Ministry of Health of the Botswana Government and the University of Cape Town. We thank Dr. A. September, Prof. J. Greenberg, Ms M. Gajjar and other colleagues from the Division of Human Genetics, Department of Clinical Laboratory Sciences, Faculty of Health Sciences, University of Cape Town for helpful comments on the project. Preliminary reports of this work have been published in abstract form: (1) M. Collins, G. G. Mokone, M. Gajjar, A. September, J. Greenberg, M. P. Schwellnus, T. D. Noakes (2003) The α 1 type V collagen (COL5A1) gene is associated with chronic Achilles tendinopathy. Medicine and Science in Sports and Exercise 35:S184 and (2) G. G. Mokone, M. Gajjar, A. September, M. P. Schwellnus, J. Greenberg, T. D. Noakes, M. Collins (2003) The BstUI RFLP within the COL5A1 gene is associated with chronic Achilles tendinopathy. The South African Journal of Sports Medicine 15:41.