Introduction

There is no single factor that causes acute anterior cruciate ligament (ACL) injuries. ACL ruptures are therefore considered multifactorial conditions with evidence from both familial and case-control genetic association studies that DNA sequence variants play an important role in its etiology. A growing number of common variants within genes encoding proteins, such as collagens, fibrillins, and proteoglycans, involved in the structure and regulation of the formation of the basic building block of the ACL (namely, the collagen fibril), have been associated with ACL rupture. In addition, variants within genes coding for proteins involved in biological processes responsible for ligament remodeling, adaptation, and repair have also recently been associated with the risk of ACL rupture. Recently, variants within collagens and other genes encoding other ligament-associated proteins have also been associated with altered risk of the canine ACL via cranial cruciate ligament rupture, strengthening the hypothesis that DNA sequence variants play an important role in the etiology of ligament injuries.

This chapter will review our current understanding and future research directions in elucidating the genetic influences on ACL rupture. Molecular genetics is one of several scientific disciplines researchers can apply to explore the biological mechanisms of ACL ruptures. Finally, the clinical application of this area of investigation will briefly be reviewed in this chapter.

Molecular Genetics: Some Basic Concepts

The effective clinical management and prevention of ACL ruptures is most likely hinged on how rapidly we improve our current understanding of the dynamic relationship between the biomechanical and biological mechanisms underpinning ACL injury susceptibility. It is therefore imperative that clinicians and scientists understand both clinical and scientific terminology in order to facilitate the depth of research into the biological mechanisms of ACL ruptures. The next section will define some of the basic concepts in human molecular genetics.

Human beings are not identical; there are both visible and measurable normal biological variations among us, which contributes to each person being unique. Part of this biological variation is caused by common DNA sequence variations, known as polymorphisms, found within the three billion base pairs of the human genome. The human genome, which is 99.9% identical across all people, consists of approximately 19,000 coding genes (e.g., COL1A1 ) from which proteins (e.g., COL1A1 encodes for the α1(I) chain of type I collagen) are produced, representing the complete set of human genetic information, stored as DNA sequences ( Fig. 2.1 ). Although changes in the DNA sequence, usually referred to as mutations, can cause human disease, most DNA sequence changes are not considered deviations from the normal sequence, but rather are normal sequence alternatives. For example, recognised is also an acceptable alternative spelling for recognized . In this case, these polymorphisms do not cause severe diseases but potentially can modulate disease and/or injury susceptibility. Biologists have exploited the sequence differences noted between individuals (individual polymorphisms) to map susceptibility loci underlying common disease. Often, a combination of contiguous polymorphisms and their specific alleles are used to identify genetic intervals of inclusion or exclusion, and this is referred to as haplotype analyses (inheritance of a set of alleles representing a chromosomal position).

A schematic representation of the exon (vertical lines) and intron (horizontal lines) boundaries of the human COL1A1 genes. The DNA sequence flanking the functional G/T polymorphism (rs1800012) at nucleotide 1023 of intron 1 is indicated. Three genotype, namely GG ( white figure ), GT (red figure), and TT (blue figure), are produced from this single polymorphism. The relative contributions of the three genotypes within the East Asian (ASN), Nigerian and Kenyan (AFN), as well as European (EUR) populations are shown in the pie charts. The size of the gene in kilobases (kb) is indicated. Data used to construct this figure was obtained from Ensembl ( www.ensembl.org ).

Interindividual variations in physical characteristics (phenotypes), such as height and the response of maximal oxygen uptake to training, are determined in part by polymorphisms. It is therefore reasonable to propose that these polymorphisms also contribute to biological variation in the structure and function of tissues such as the ACL. The response of the ACL to load (training), susceptibility of the ACL to rupture, as well as the repair and healing of the ACL is therefore not identical among individuals. If we accept that polymorphisms contribute to a lesser or greater extent to normal biological variation, genes encoding proteins involved in ACL structure and function are therefore plausible candidates to be tested for association with risk of ACL rupture (as indicated in O’Connell et al.).

Although there are important limitations to the approach that need to be considered, all the published studies to date reporting an association of DNA sequence variants with ACL ruptures in humans have used a case-control candidate gene approach (reviewed in September et al.). A well-designed genetic association study should be sufficiently powered (sample size) and contain well-defined unrelated cases with similar environmental exposures and appropriately matched healthy and injury-free, unrelated controls. The diagnostic criterion used in the clinical diagnoses of the cases should be the same or very similar for all cases. Furthermore, the inclusion and exclusion criteria for the study need to be carefully considered and defined. For example, the mechanism of injury, such as noncontact or contact, is an important factor when recruiting participants with a history of an ACL rupture as cases. Those with no history of ligament injuries, the controls, should also be relatively healthy and need to be matched for ancestry (country of birth), age, weight, gender, level of sport participation (higher or equal), and other intrinsic and extrinsic factors known to associate with ACL rupture. The candidate gene selected for the association study should be based on an a priori hypothesis that the gene product (e.g., type I collagen) is directly involved in the etiology of ACL ruptures. DNA samples extracted from tissue (usually blood samples or buccal cells) donated by the cases and controls are genotyped for potentially informative polymorphisms (e.g., rs1800012) within the candidate gene (e.g., COL1A1 ) (see Fig. 2.1 ). The necessary experimental quality control measures need to be taken to ensure reliability and accuracy in the generation of the genotyping data and need to be reported. Finally, investigators will determine whether any of the genotypes (e.g., GG, GT, or TT for COL1A1 rs1800012) or alleles (G or T) are significantly associated with ACL ruptures using appropriate statistical analyses. The data should always be interpreted with caution, taking into account the limitations and confounders of the study design.

Genetic Risk Factors For Anterior Cruciate Ligament Injury

As summarized in Table 2.1 , a growing number of polymorphisms within genes encoding proteins involved in the structure and regulation of the formation of the collagen fibril, and biological processes within the ACL, have been associated with risk of ACL rupture. Sex-specific associations have also been reported for a subset of these polymorphisms.

The genes, which have been associated with ACL ruptures, encode for (1) collagens, (2) fibrillins and proteoglycans, (3) matrix metalloproteinases, or (4) cell signaling molecules. Since the majority of work has been performed investigating the association with collagen gene polymorphisms, and due to several replication studies having been performed within additional populations, the following section will focus on the reported association of variants within COL1A1 , COL3A1 , COL5A1 , and COL12A1 with ACL rupture.

COL1A1 encodes for the α1(I) chain of type I collagen, which is the most abundant structural protein within the collagen fibril. This collagen is a heterotrimer consisting of two α1(I) and one α2(I) chains; however, a homotrimer consisting of three α1(I) chains can also be produced in connective tissues. Rare mutations within COL1A1 have been shown to cause connective tissue disorders such as osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome (EDS), while common polymorphisms have been reported to be associated with several complex connective tissue disorders (reviewed in O’Connell et al.), suggesting that COL1A1 is a plausible candidate gene to test for association with ACL ruptures.

The functional polymorphism located in an Sp1 binding site (rs1800012, G/T) within the first intron of COL1A1 has been associated with ACL ruptures in four studies. The increased binding of the transcription factor, Sp1, to the rare T allele of this polymorphism has been reported to produce more α1(I) chains, which is proposed to and incorporation of the α1(I) ₃ type I collagen homotrimer into connective tissues. Similar genotype distributions, with an underrepresentation of the TT genotype within ACL rupture participants, was found in all except one previous study ( Fig. 2.2 ). The only exception was within a group of skiers sustaining ACL ruptures, where the GG genotype was significantly underrepresented in the ACL rupture group (see Fig. 2.2D ). Interestingly, the GG genotype has also been reported to be underrepresented in other multifactorial connective tissue disorders (reviewed in O’Connell et al.), such as stress urinary incontinence, highlighting that this polymorphism may affect the etiology of specific disorders differently. It is therefore possible that the association of the apposite genotype with skiing and non-skiing-related ACL injuries could be due to the difference in injury mechanisms and highlights the importance of considering the mechanism of the injury. Further research, where the mechanism of ACL injury is clearly documented, is however required to explore this possibility.

Relative genotype (GG, GT, and TT) frequency distributions of the COL1A1 rs1800012 G/T for the cruciate ligament (CL, blue bars) and anterior cruciate ligament (ACL, blue bars) groups, as well as the control (CON, red bars) groups in the previously published (A) Swedish patients, (B) South African patients, ,(C) Polish soccer players, and (D) Polish skiers. The Swedish and South African cohorts participated in mixed sports. All participants were of self-reported European ancestry. The significant differences ( P ) between the groups for a specific genotype are indicated. The total number ( n ) of participants in each group within each study is also indicated.

Ficek et al. also reported an interaction between this intronic polymorphism and a second functional polymorphism (rs1107946, G/T) at position –1997 within a regulatory element of the COL1A1 promoter in modulating risk of ACL ruptures. The haplotype constructed from the rs1800012 T to rs1107946 G alleles, both known to increase expression of COL1A1 , was significantly underrepresented in the individuals with ACL ruptures.

Although quantitatively less than type I collagen, the collagen fibril within the ligament also contains other collagen types, such as types III, V, and XII. Both types III and V collagen are homotrimers encoded by the COL3A1 and COL12A1 genes, respectively, while the major isoform of type V collagen is a heterotrimer, α1(V) ₂ α2(V), where the α1(V) chain is encoded by COL5A1 . Rare mutations in all three of these collagen genes cause Mendelian disorders, while common polymorphisms have also been implicated in modulating risk of multifactorial disorders (reviewed in O’Connell et al.). The association of a polymorphism (rs1800255, G/A) within exon 30 of the COL3A1 gene, which results in the substitution of the amino acid alanine at position 698 with a threonine and is proposed to affect collagen fiber assembly and tensile strength, has been investigated in three separate populations. Although not associated in a South African population, the AA genotype was significantly overrepresented with ACL rupture in two Polish cohorts with different mechanisms (skiing and nonskiing) of injury.

The association of the COL5A1 rs12722 (C/T) and/or other polymorphism within the functional 3′-untranslated region of the COL5A1 mRNA has been reported in several musculoskeletal soft tissue injuries and other exercise associated phenotypes (reviewed in O’Connell et al.). In agreement with the other injuries, the CC genotype of this polymorphism was originally reported to be associated with reduced risk of ACL ruptures (with T allele associated with increased risk), but only in females. Similarly the AA genotype of the COL12A1 polymorphism (A/G) within exon 65 (A9285G) was associated with an increased risk of ACL ruptures among female participants. In agreement, this COL12A1 polymorphism was not associated with ACL ruptures in Polish male soccer players. Although some of the sex-specific independent associations were not repeated, a significant interaction between the COL5A1 rs12722 (T/C) and COL12A1 rs970547 (A/G) polymorphisms was noted, where the T-A inferred allele pair was associated with increased risk of ACL rupture in females in two separate cohorts (South African and Polish).

Associations with single polymorphisms and/or haplotypes constructed from several polymorphisms within FBN2 , ACAN , BGN , the LUM-DCN gene cluster, the MMP10 – MMP1 – MMP3 – MMP12 gene cluster, VEGFA , and KDR in a single population have also been reported. These associations need to be repeated in larger independent studies.