We have shown that a common C to T single nucleotide polymorphism (SNP rs12722 or BstUI RFLP) within the COL5A1 gene 3'-untranslated region (UTR) (Fig. 1) is associated with a number of sports injury or performance-related phenotypes. The injury phenotypes include Achilles tendinopathy (25,33) and anterior cruciate ligament (ACL) ruptures in female subjects (31), whereas the performance-related phenotypes include joint range of motion (ROM) (4,7) and endurance running performance (3,30).
A SNP is a single nucleotide (A, T, C, or G) variation within the DNA sequence of an individual's genome. SNP rs12722 (one of many COL5A1 polymorphisms) produces two variants (or alleles) of the COL5A1 gene, namely, the COL5A1 rs12722 C- and T-alleles. Individuals either have two copies of one of these alleles or a single copy of each allele. Those with two copies of either the C or T alleles have a CC or TT genotype at SNP rs12722, respectively, whereas those with CT genotype have one copy each of the C and T alleles. Although SNP rs12722 is associated with injury and noninjury phenotypes, this single nucleotide DNA sequence variation is not necessarily the cause of these phenotypes. This SNP could be linked tightly to the as yet unknown phenotype-causing polymorphism(s) either within the COL5A1 gene or a neighboring gene. However, the results do indicate that SNP rs12722 is a representative genetic marker for the genetic region (locus) within or surrounding the COL5A1 gene, which potentially may cause these reported phenotypes.
The COL5A1 gene encodes the α1 chain of type V collagen (α1(V) chain), a minor fibrillar collagen. Although present in much smaller amounts than the other fibrillar collagens, type V collagen plays a critical role in the regulation of collagen fibril assembly and lateral growth (fibrillogenesis) (39). Therefore, type V collagen is an important structural component of tendons, ligaments, and other connective tissues.
We recently have reported that the COL5A1 3'-UTR SNP rs12722 is associated with altered COL5A1 messenger RNA (mRNA) stability (18). We therefore hypothesize that this variant results in an altered amount of type V collagen production, which may alter the fibril architecture and its mechanical properties.
Therefore, the purpose of this review is to present the evidence for the novel hypothesis we propose based on our published genetic association studies with injury and noninjury phenotypes and our more recent COL5A1 3'-UTR functional studies. A brief overview of the collagens, with a focus on type V collagen, within the connective tissue of musculoskeletal soft tissues is necessary to understand the model and therefore is included in this review. In addition, we briefly review the results from our previously published COL5A1 genetic association and functional studies. Finally, we develop an integrated model that explains how altered expression of the COL5A1 gene may associate with multifactorial injury and performance-related phenotypes as well as cause severe life-threatening diseases such as Ehlers-Danlos syndrome (EDS).
COLLAGENS: A BRIEF OVERVIEW
The collagens are structurally and functionally a heterogeneous superfamily of proteins located in the extracellular matrix (ECM) of almost all tissues (15). They maintain the structural integrity of tissues and regulate a variety of biological processes. The most abundant collagens (types I, II, and III) form elongated fibrils in fibrous connective tissues, such as tendons, ligaments, and cartilage, and in the ECM of other tissues such as skeletal muscle (Fig. 2) (15). Type II collagen is the basic building block of fibrils in cartilaginous tissue, whereas type I collagen fibril is found in noncartilaginous tissues, such as tendon, ligaments, and the connective tissue components of the skeletal muscle. Types I, II, and III collagens are considered to be the classical collagens and are referred to as the major fibrillar collagens. The fibrillar types V and XI collagens, which are referred to as the minor fibrillar collagens, are less abundant than the classical collagens but nevertheless are critical structural components of the types I and II fibrils, respectively (Fig. 2) (15). Because the focus of this review is type V collagen, the importance and biological function of only this collagen will be summarized in more detail in the next section. Although beyond the scope of this review, variants within other collagen and ECM genes have been associated previously with injury (8) and noninjury (27) phenotypes. These, as well as future findings, should be incorporated into our hypothesis.
Most of the remaining collagens do not form fibrils and are referred to collectively as the nonfibrillar collagens (15). A subgroup of these structurally related nonfibrillar collagens, known as the fibril-associated collagens with interrupted triple-helices (FACITs), are associated with the surface of the collagen fibrils (15). The FACITs (types IX, XXII, and XIV collagen) form interfibrillar connections and mediate fibril interaction with various components of the ECM and cell surface molecules (Fig. 2). The remaining collagens, which do not form part of the classical fibril, have a variety of shapes, sizes, and tissue distribution and are beyond the scope of this review.
Irrespective of their structure or tissue distribution, all collagens consist of three polypeptide (α) chains, which form one or more of the proteins' characteristic collagen triple helix domains. Within the triple helix domains, the α chains consist of repeating glycine-X-Y amino acid triplets, where the X and Y positions are frequently proline and hydroxyproline, respectively. Glycine at every third position is an absolute requirement for the formation of the triple helix. Proline and hydroxyproline also play an important role in the correct formation of the triple helix (15).
TYPE V COLLAGEN IS ESSENTIAL FOR LIFE
Type V collagen isoforms are heterotrimers made up of various combinations of the α1(V), α2(V), and α3(V) chains. The major isoform contains two α1(V) and one α2(V) chains, which are encoded by the COL5A1 and COL5A2 genes, respectively (23,39). Type V collagen is essential for life. This is illustrated by observations that col5a1 -/- mice die in utero (40). Furthermore, mutations within this gene and the COL5A2 gene cause the classic form (types I and II) of EDS (23).
As previously mentioned, type V collagen molecules intercalates with the type I collagen molecules to form heterotypic fibrils in noncartilaginous connective tissues, where it modulates fibrillogenesis (Fig. 2). Expression from both copies of the COL5A1 gene is required for normal fibrillogenesis. Most patients with classic EDS have disease-causing mutations within one copy of their COL5A1 genes, which result in a loss of function of the mutated gene and a 50% reduction in the production of type V collagen (haploinsufficiency) (23,40). A clinical feature of EDS is generalized joint hypermobility, which is related to the presence of large irregular collagen fribils in the connective tissue (38). Similar findings have been reported in col5a1 +/- mice (40).
THE ASSOCIATION OF THE COL5A1 GENE AND COMPLEX SPORTS INJURY AND PERFORMANCE PHENOTYPES
Initial evidence for the involvement of genes located on the tip of the long arm of chromosome 9 in the etiology of musculoskeletal soft tissue injuries arose from early reports of an association between the ABO blood group and Achilles tendon injuries (reviewed in (34)). The O blood group has been shown to be associated with an increased risk of Achilles and other tendon injuries (34). Because the ABO blood group is coded by a single gene, a mere 1.4 Mb upstream from the COL5A1 gene, we investigated the possible association between the COL5A1 gene and the risk of Achilles tendon injuries (25). We found that the SNP rs12722 within the COL5A1 3'-UTR (Fig. 1) was associated with Achilles tendinopathy in a physically active South African white population. The CC genotype of this variant was overrepresented significantly in the asymptomatic control participants of the study (25). We repeated this initial genetic association study in a second independent physically active Australian white population. The CC genotype of SNP rs12722 also was overrepresented in the asymptomatic control participants when compared with those with Achilles tendinopathy (33), thus providing further evidence that individuals with a wild-type CC genotype are "protected" against chronic degenerative changes in the Achilles tendon during running and other forms of physical activity (32). These findings imply that individuals with at least one copy of the T allele (CT or TT genotype) are at increased risk of chronic degenerative Achilles tendon injuries.
Because of the structural similarities of tendon and ligaments, our subsequent study further investigated this COL5A1 variant (SNP rs12722) as a risk factor for another musculoskeletal soft tissue injury, ACL rupture. In this study, we also found that the CC genotype of SNP rs12722 was significantly overrepresented among the controls, although only within the female subjects in the study (31). The reason for this sex-specific association remains largely unknown.
As COL5A1 haploinsufficiency (the common molecular mechanism underlying the classic form of EDS) results in joint hypermobility, we furthermore sought to determine if the COL5A1 rs12722 also was associated with individual joint ROM measurements. Our initial study confirmed this hypothesis. This SNP was associated with left and right leg straight leg raise and sit-and-reach (SR) measurements (7). A later study confirmed this association. We found that the COL5A1 CC genotype "protected" individuals against an age-related decline in ROM measurements. Within older (>35 yr) individuals, SR ROM was higher significantly in the CC genotype group (4). Simplified, this finding implies that older individuals with a wild-type COL5A1 CC genotype are more flexible. Collectively, our studies suggest that individuals with a CC genotype at SNP rs12722 are at reduced risk of musculoskeletal soft tissue injuries and have an increased ROM.
Furthermore, recent studies have shown that ROM measurements, such as the SR ROM test, have been shown to be associated with running economy (9,13,14). In these studies (9,13,14), reduced ROM was associated with improved running economy and/or performance. Because of this reported association, we hypothesized that the COL5A1 3'-UTR variant also was associated with endurance running ability. We determined the genotype of 313 participants who completed the 2006 or 2007 226-km South African Ironman triathlon. Our study found that the athletes with a TT genotype completed the 42.2-km running component of the Ironman triathlon significantly faster than the athletes with either a TC or CC genotype (30). There were no significant COL5A1 rs12722 genotype effects on performance during the nonweight-bearing swimming and cycling of the triathlon. This finding was repeated in a second study. In this study, the COL5A1 rs12722 TT genotype was significantly associated with improved endurance running performance during a 56-km ultra-endurance road run (3). Although we did not find a direct association between prerace SR ROM measurements (flexibility) and time to complete the 56-km ultra-marathon race, the COL5A1 T allele was significantly overrepresented in the "inflexible-fast" athletes, whereas the C allele was significantly overrepresented in the "flexible-slow" athletes (3). A plausible explanation for complex interaction between COL5A1 genotype, ROM, and endurance running performance has been discussed previously (3). The significance of these findings will be discussed in a subsequent section.
THE COL5A1 3'-UTR IS FUNCTIONAL
Although we have reported a genetic association of a sequence variant within the COL5A1 3'-UTR and several complex exercise-related phenotypes (3,4,25,30,31,33), the biological function of the COL5A1 3'-UTR initially was unknown. However, it is well documented that the 3'-UTR of eukaryotic genes contains important regulatory elements involved in the etiology of many diseases (24). We have reported recently an overall increase in COL5A1 mRNA stability in the tendinopathic phenotype, when the COL5A1 3'-UTR was cloned from participants with chronic Achilles tendinopathy or asympomatic controls and inserted upstream of a firefly-luciferase reporter gene and transiently transfected into HT1080 cells (18). We also identified two major functional forms, which contained a set of seven strongly linked sequence variants (Fig. 1) (18). These we termed the C- and T-functional forms of the COL5A1 3'-UTR. The C functional form, which corresponds to the wild-type sequence and includes the C allele of SNP rs12722, was identified in most of the clones generated from asymptomatic controls, whereas the T functional form, which includes the T allele of SNP rs12722 was identified predominantly in the Achilles tendinopathic patients. An overall increase in mRNA stability was associated with the T functional form of the COL5A1 3'-UTR. Furthermore, deletion constructs grossly mapped areas with potential regulatory elements to the region within the COL5A1 3'-UTR shown to be associated with the tendinopathic phenotype. This was the first study to investigate the biological function of the COL5A1 3'-UTR and a first attempt in validating our genetic association studies at the molecular level.
Because the C and T variants of SNP rs12722 were associated with the C and T functional forms of the COL5A1 3'-UTR, respectively, this SNP probably can be used to genetically distinguish between two major biologically functional forms of the COL5A1 3'-UTR (Fig. 1). In addition, because both copies of the COL5A1 gene are required for normal collagen fibril formation (23,40), it therefore is tempting to speculate that relatively small changes in COL5A1 mRNA stability within the normal physiological range (nonpathological) could result in interindividual variation in fibrillogenesis and susceptibility to musculoskeletal soft tissue injuries, as well as variations in flexibility and endurance running performance. This will be explored in the following section.
We have developed a novel hypothesis based on the associations between the COL5A1 gene and exercise-related phenotypes and the biological function of the associated region. We propose that there is an increased type V collagen production among individuals with a COL5A1 rs12722 TT genotype, resulting in structural and architectural changes within the collagen fibril. We further propose that these changes result in altered mechanical properties of musculoskeletal soft tissues, which in turn associates with increased risk of specific injuries, reduced joint ROM (flexibility), and increased endurance running ability. These reported associations are all consistent with our proposed hypothesis.
As previously discussed, our functional in situ studies have shown that the T functional form of the COL5A1 3'-UTR results in increased COL5A1 mRNA stability (18). Although further research is required, it is conceivable that this increased mRNA stability translates into increased mRNA levels and α1(V) chain production. As the α1(V) chain is the rate-limiting step for type V collagen trimer assembly (5), we speculate that the increased COL5A1 mRNA stability will have a direct effect on the amount of type V collagen incorporation into the collagen fibril (Fig. 3).
Tissues containing relatively large amounts of type V collagen, such as the chick cornea (roughly 20% type V collagen), have a characteristic architecture, characterized by a very narrow range of small diameter fibrils (2). Reduced fibril diameters also are observed when the relative levels of type V collagen are increased in in vitro assays (2). Because type V collagen also regulates the initiation of fibril assembly, col5a1 haploinsufficient models of classic EDS in mice have shown that reduced type V collagen is associated with a 50% reduction in fibril number and collagen content, thus leading to a reduced collagen fibril density. In addition, these mice also have poorly organized fibrils, a decreased tensile strength, decreased stiffness, and increased fibril diameter (40). Furthermore, recent genome-wide association studies have identified a variant within the COL5A1 gene, which associates with central corneal thickness (CCT) (37). Although the etiology of CCT is unknown, the change in type V collagen resulting from the associated SNP(s) was proposed to result in altered fibril diameter (37). Collectively, these observations suggest that an increase in type V collagen production from the T functional form of the COL5A1 3'-UTR may have a structural effect on the fibril architecture. The COL5A1 T 3'-UTR functional form therefore may affect the mechanical properties of musculoskeletal soft tissues. We speculate that common variants within the COL5A1 gene could be responsible partly for the normal interindividual variation in the mechanical properties of musculoskeletal soft tissues (Fig. 3).
Although structural and architectural changes to the collagen fibrils may result in a change to the mechanical properties of collagen-containing tissues, research describing the effect of the proposed changes is limited and contradictory (16). The mechanical properties of tissue may be described by the following: (i) the ultimate tensile strength, (ii) stiffness (the slope of a load-elongation curve), and (iii) creep (20).
Classically, it has been reported that an increased mean collagen fibril diameter will confer greater ultimate tensile strength, whereas smaller fibrils will inhibit creep (28,29). The density of intrafibrillar collagen molecule covalent cross-links is higher theoretically within the larger fibrils. These cross-links will confer greater ultimate tensile strength to the tissue (28). Conversely, the surface area per unit mass of smaller fibrils is higher (28). This increased surface area allows for increased electrostatic interactions between the collagen fibrils and ground substance (28). Although this mechanism may increase the stiffness of connective tissues, the reported relationship between mean fibril diameter and stiffness is complicated by the lack of appropriate models. Results may be contradictory because of the presence of immature or disused fibrils, which both have reduced diameter and stiffness (19,26).
The structure of the retained amino-terminal domain of type V collagen within the fibril also can explain why smaller fibrils, which result from increased type V collagen content, are creep resistant. The protruding type V collagen amino-terminal domains (Fig. 2) will inhibit slippage of collagen molecules during mechanical deformation, which also may result in increased stiffness (36).
The mechanical properties of the muscle tendon unit therefore explain our reported associations of COL5A1 with the following: (i) ROM, (ii) injury risk, and (iii) running performance. In this model, smaller more densely packed and organized collagen fibrils result in reduced ROM, improved endurance running performance, and an increased risk for specific musculoskeletal soft tissue injuries (Fig. 3).
Although the terms flexibility and stiffness often are used interchangeably, they remain separate entities (20). As previously discussed, stiffness is a structural property of tissue derived from a load-elongation curve, whereas flexibility as measured by the sit-and-reach test is the ROM across a joint(s). Although we are not aware of any associations between tendon-specific stiffness and ROM measurements, certain studies however have shown, as one would expect, an inverse relationship between hamstring ROM and hamstring stiffness (22). With our hypothesis as basis, we attribute the finding that the COL5A1 rs12722 TT genotype (a marker for two copies of the 3'-UTR T functional form) is associated with decreased flexibility among older individuals to altered mechanical properties of the musculoskeletal soft tissues. As we previously discussed, the TT genotype may result in increased stiffness and increased creep inhibition. An increase in both these mechanical properties may result in a decreased change in length in response to an applied force and, therefore, a decreased ROM (Fig. 3).
The association of the COL5A1 genotype and injury risk also is explained by our hypothesis. As larger fibrils have been shown to confer a greater ultimate tensile strength, the wild-type CC genotype (a marker for two copies of the COL5A1 3'-UTR C functional form) should be protective against specific musculoskeletal soft tissue injuries. In support, ruptured Achilles tendons contain a significantly increased proportion of smaller-sized fibrils (21). This supports the notion that smaller fibrils reduce the tensile strength of collagen-containing tissues. It also has been reported previously that increased stiffness results in increased peak forces experienced by the tendon, thereby leading to increased loading rates and increased shock (19). Regardless of the precise mechanism, these observations support the hypothesis that the COL5A1 CC genotype is protective against both acute ligament injuries, such as ACL ruptures, and chronic degenerative conditions, such as Achilles tendinopathy. It is interesting to note that investigators have proposed the use of antisense COL5A1 gene therapy to inhibit type V collagen production, thereby increasing fibril diameter, which will improve the quality of healing ligaments and tendons (35).
The association of the COL5A1 genotype and endurance running performance also may be explained by our hypothesis. Although the role of elasticity in muscle efficiency is controversial (11), the published associations between stiffness, flexibility, and running economy have been reasonably consistent. Runners with increased flexibility have been shown to have a decreased running economy (9,13,14). These findings may be due to inefficient energy storage within more compliant tissue or increased recruitment of stabilization muscles to maintain posture during running (14). In agreement, studies have shown an inverse relationship between running economy and muscle-tendon stiffness (1,10,12). Among trained runners, increased muscle-tendon stiffness is associated with increased running economy (1,10,12). This finding is contrary to what has been reported in stretch-shortening cycle muscle action, where more compliant and, thus, greater elastic energy storage may be preferred (17). However, it is proposed that during running, little prestretch of the muscle tendon unit occurs, thus favoring force transmission (a stiffer tendon) over elastic energy storage (Fig. 4) (12). Regardless of the specific mechanisms, these data support our findings that the COL5A1 TT genotype associates with increased endurance running performance.
Future work is required to test key components of this hypothesis. Although our initial studies have shown that the COL5A1 3'-UTR regulates COL5A1 gene expression and potentially plays a role in the etiology of musculoskeletal soft tissue injuries and other exercise-related phenotypes, future work is required to identify specific regulatory elements within the 3'-UTR and trans-acting factors involved in the regulation of COL5A1. The biologically functional polymorphisms within the COL5A1 3'-UTR, which are linked closely to SNP rs12722, also need to be identified. It is highly unlikely that only polymorphisms within the 3'-UTR regulate interindividual variation in COL5A1 gene expression. Therefore, potentially functional variants within the promoter, other regulatory regions, and the coding region of the gene need to be identified. The potential involvement of other genes, such as the genes encoding for types III, VI, and XII collagen, involved in musculoskeltal soft tissue biology also needs to be investigated.
Further key components of our hypothesis, which requires further investigation, include the influence of COL5A1 gene variants on the architecture of the fibril and the mechanical properties of the fibril. These future studies require the investigation of musculoskeletal soft tissue biopsy samples to determine if the COL5A1 genotype influences the following: (i) ratio of type I to type V collagen, (ii) fibril diameter, (iii) interfibrillar and intrafibrillar cross-links, (iv) fibril organization and density, (v) cross-section corrected ultimate tensile strength of collagen fibers, (vi) creep, and (vii) stiffness. The complex interaction of any of the above proposed COL5A1 genotype effects also require investigation. In adition, in vivo studies to determine if these tissue properties relate to functional changes in the mechanical properties of musculoskeletal soft tissue during muscle activation also are required.
Because the COL5A1 gene has been associated with risk of chronic Achilles tendinopathy and ACL ruptures in female subjects, this gene also may predispose to other musculoskeletal soft tissue injuries. In addition, the COL5A1 gene also may be associated with additional sports phenotypes. Further studies are required to investigate these relationships. The COL5A1 gene is an excellent candidate gene for other chronic injuries (i.e., patellar tendinopathy) or acute injuries (i.e., hamstring tears). Furthermore, based on our hypothesis, the COL5A1 gene also may be a candidate gene for any other endurance or power-based sports in which muscle tendon stiffness may be regarded as an important feature.
We have presented a logical biological argument for our hypothesis. However, we cannot exclude the possibility that the evidence we present may be explained by another mechanism. For example, the increased risk for injury in those athletes with a COL5A1 rs12722 TT or TC genotype merely could be directly a result of their improved running performance. Further work is required to test our or any other possible hypothesis.
APPLIED AND CLINICAL SIGNIFICANCE
SNP rs12722 within the 3'-UTR of the COL5A1 gene seems to be an important genetic marker for several injury and noninjury phenotypes. As previously reviewed (6), we have suggested that this genetic marker could be included in multifactorial models to explain these phenotypes. An understanding of the biological mechanisms for our reported COL5A1 genetic associations would strengthen the justification for the inclusion of SNP rs12722 or any other COL5A1 gene SNP in models that determine injury risk or endurance running performance. In addition, this hypothesis also may prove valuable in specific personalized training, injury prevention, treatment, and rehabilitation programs (6). However, it is important to note that injury and noninjury phenotypes all are multifactorial in nature. There is no single factor that causes any of these phenotypes. This review merely highlights one of the possible common biological processes involved in performance and injury phenotypes.
In summary, we have shown previously that a common C/T variant (rs12722) within the 3'-UTR of the COL5A1 gene is associated with several phenotypes that directly or indirectly are associated with the mechanical properties of musculoskeletal soft tissue. We hypothesize that the COL5A1 rs12722 TT genotype results in increased type V collagen production, decreased mean fibril diameter, and increased fibril density, resulting in changes to the mechanical properties of the musculoskeletal soft tissue. More specifically, we hypothesize that the changes in mechanical properties associated with the TT genotype result in the following: (i) a reduced tensile strength and (ii) increased creep inhibition and/or stiffness. However, future work is required to test this hypothesis.
This study was supported in part by the South African National Research Foundation, the South African Medical Research Council, and the University of Cape Town.
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