Keywords
anterior cruciate ligament - athletes - athletic injuries - polymorphism, single nucleotide
- rupture
Introduction
Anterior cruciate ligament rupture (ACLR) is a frequent disabling injury among athletes,
that causes knee joint instability. In the USA, estimated incidence of ACLR ranges
between 100,000 and 200,000 / year.[1] Reconstructive surgery allows sports practice and a better quality of life. In Italy,
the incidence of ACL reconstruction surgery is 21-33 rate per year in 100,000 people,
with an incidence between 0.16 and 2.04 procedures per 100,000 individuals younger
than 15 years.[2]
[3]
Despite surgical ligament reconstruction, about 79% of these patients develop knee
osteoarthritis and 20% suffer a new injury within the next 2 years.[4]
Sport ACLRs can be contact injuries (with a teammate or opponent), more frequent in
contact sports such as soccer or basketball,[5] or non-contact injuries. In a 10-year period of observation, Majewski et al.[6] noted that 60% of sport knee injuries treated in their hospital were volleyball-related
ACL injuries. Agel et al.[7] found that 14% of injuries in volleyball occur via non-contact.
Over a 16-year period of collegiate injuries registration, Hootman et al.[8] reported an injury rate per 1000 athlete-exposures of 0.07 and 0.09 for male basket
and soccer players. They report 0.23, 0.28 and 0.09 for female basket, soccer and
volleyball players respectively.
The risk factors for ACL injury are classified as exogenous (e.g. laying surface)
and endogenous like genetic factors.[9] In inheritable collagen disorders, genetics influence laxity of tissues[10]: generalized joint laxity and hyperextension were found to significantly increase
the risk for ACL injury in female athletes.[11] Mutations within the COL5A1 gene cause the classic form of Ehlers-Danlos syndrome
(EDS) characterized by joint hypermobility involved in sprains, dislocation / subluxation,
and early osteoarthritis.[12] As reported by Mokone et al.[13], the COL5A1 gene contains RFLP of BstUI (rs 12722) and DpnII (rs13946) within its
3'-untranslated region (UTR). The functional COL5A1 specificity protein 1 (Sp1) binding
site polymorphism - BstUI RFLP C / T is positively correlated with tendon and ligament
injuries, especially in Caucasian subjects.[14]
[15] Accordingly, a study on soccer players found that the T/T genotype of the COL5A1
BstUI RFLP showed a trend toward a higher severity of musculoskeletal injuries with
respect to the individual carriers of the CC genotype.[15]
The aim of this study was to correlate the COL5A1 gene BstUI RFLP and DpnII RFLP polymorphisms
with ACLR in volleyball, basketball and soccer athletes.
Materials and Methods
Subjects
68 Caucasic players (n = 36 women and n = 32 men) with ACLR occurred during sport
practices (ACLR Group) and 42 healthy players (n = 20 women and n = 22 men) (Control
Group) participated to the study. All of them practiced team-sports (Volleyball, Basketball,
and Soccer) in Italian teams. Subjects gave informed consent to participate. To obtain
demographic information and data regarding sport practice; each athlete completed
a self-administered questionnaire. The ACLR Group (32 athletes practiced Volleyball,
19 Basketball and 17 Soccer) provided information on the mechanisms and date of anterior
cruciate ligament injury.
The study was approved by the Institutional Review Board of the University of Cagliari
(Prot. PG/2017/1700) and was carried out in accordance with the ethical and humane
principles of the research.
DNA Analysis
Genomic DNA was extracted from buccal swab, performed on the oral mucosa of the cheek,
through salting out method, and amplified by standard PCR following the protocol suggested
by Galasso et al.[16]
PCR products were subjected to two enzymatic digestions: digestion with BstUI produced
two fragments for the T allele (351 and 316 bp) and three fragments for C allele (316,
271, and 80 bp); digestion with DpnII produced a unique fragment for B2 (612 bp) and
two fragments (412 and 194 bp) for B1. The fragments obtained were separated through
an 8% polyacrylamide gels for BstUI and a 2% agarose gel for DpnII and visualized
with Syber Safe staining.
Statistical Analysis
All data were initially entered into an Excel database (Microsoft, Redmond, Washington
– United States) and the analysis was performed using the Statistical Package for
the Social Sciences Windows, version 15.0 (SPSS, Chicago, Illinois, USA). Descriptive
statistics consisted of the mean ± standard deviation (SD) for parameters with gaussian
distributions (after confirmation with histograms and the Kolgomorov-Smirnov test).
Comparison among groups was performed with the ANOVA one-way for continuous parametric
variables or the Chi-square test or Fisher's exact test (if cells < 5) for frequencies
variables.
HWE tests were conducted considering population frequencies (p2 + 2pq + q2 = 1) and
then reported in SPSS, Chi-square nonparametric test. Similarly, the G-square test
(G2 test) was performed. A p value of < 0.05 was considered statistically significant.
Results
No significant difference has been found between ACRL and Control groups in age, height,
weight body mass index, sport practice (hours/week) and gender distribution among
the different team-sports). Control Group athletes have longer sport careers (p< 0.005) ([Table 1]).
Table 1
|
ACLR
|
Control
|
p
|
|
Number
|
68
|
42
|
|
|
Age (years)
|
27.3 ± 6.4
|
28.5 ± 8.5
|
0.406*
|
|
Height (cm)
|
173.0 ± 9.5
|
174.0 ± 11.4
|
0.588*
|
|
Weight (kg)
|
70.0 ± 11.8
|
70.2 ± 12.3
|
0.945*
|
|
Body mass index (kg/cm2)
|
23.30 ± 3.30
|
23.16 ± 2.99
|
0.782*
|
|
Gender (n°males/n°females)
|
32 / 36
|
22 / 20
|
0.200^
|
|
Sports (Volleyball; Basketball; Soccer)
|
32; 19; 17
|
26; 8; 8
|
0.312^
|
|
SPORT (years)
|
10.8 ± 4.9
|
16.8 ± 8.5
|
0.001*
|
|
SPORT (hours/week)
|
7.7 ± 3.2
|
7.8 ± 4.0
|
0.799*
|
The genotype and allele frequencies of COL5A1 BstUI RFLPC/T and COL5A1 DpnII RFLP
B1/B2 are shown in [Table 2]. Genotype frequency distributions of COL5A1 DpnII and COL5A1 BstUI RFLP nucleotide
polymorphisms meet the HWE in both groups (p. value > 0.05).
Table 2
|
BstU
|
ACLR
|
Control
|
|
CC
|
58.90
|
22.22
|
|
CT
|
34.25
|
44.44
|
|
TT
|
6.85
|
33.33
|
|
MAF (T)
|
0.2397
|
0.5556
|
|
p
|
0.3881
|
0.3274*
|
|
DpnII
|
ACL
|
Control
|
|
B1B1
|
71.23
|
75.56
|
|
B1B2
|
26.03
|
20.00
|
|
B2B2
|
2.74
|
4.44
|
|
MAF (B2)
|
0.15
|
0.1444
|
|
p
|
0.556
|
0.2060*
|
Linkage disequilibrium was tested with LDlink (Machiela and Chanock, 2015)[17] using data from 1000 genomes (1000 Genomes Project Consortium, 2015). The two single
nucleotide polymorphisms (SNPs) resulted in a linkage disequilibrium for European
populations.
The distribution of the C/C genotype and the C allele of the COL5A1 BstUI RFLP were
lower in the ACLR Group compared with the Control Group (pG2 test= 0.001-[Table 3]). No significant differences have been found in the distribution of the DpnII RFLP
polymorphism between ACLR Group and controls ([Table 3]). Finally, the combination of CC + B1B1 genotypes was more frequent in the controls
than in the ACLR group, and associated with a protective effect (OR = 83.3 / 16.7 = 5).
The TT, B2B2 genotypes were absent among participants.
Table 3
|
ACLR
|
|
Control
|
p
|
|
COL5A1 BstUI% (CC; CT; TT)
|
23,8;61,0; 79,2 (0.954)
|
76,2; 39,0; 20,8 (0.368)
|
0.001°
|
|
COL5A1 DpnII% (B1B1; B1B2; B2B2)
|
60,8;66,7; 50,0 (0.990)
|
39,2;33, 3; 50,0 (0.497)
|
0.952°
|
Discussion
Although the biological changes underlying the increased risk of ACL sports injury
have not yet been find out, the family predisposition of athletes to ACL ruptures
is a consolidated knowledge.[18]
[19]
The COL5A1 gene, located at 9q34.2-q34.3, contains 66 exons distributed over 150 kb
of gDNA, and it encodes the 1 chain of type V collagen.[20]
The COL5A1 gene codes for a protein chain in type V collagen, which is found in ligaments
and tendons, and mutations within it are indicated as a cause responsible for the
increased risk of ACL rupture.[19]
[21]
We examined the association between COL5A1 rs12722 C / T (BstUI RFLP) and COL5A1 rs13946
B1/B2 (DpnII) polymorphisms individually and as haplotypes with risk of anterior cruciate
ligament rupture in male and female athletes competing in contact / noncontact team
sports.
Our results show a lower frequency of the COL5A1 BstUI C/C genotype in the ACLR group
compared with the Control Group. No significant differences in genotype distribution
or allele frequencies of COL5A1 DpnII was observed.
Posthumus et al.[21] found an underrepresented C/C genotype of COL5A1 BstUI RFLP in Caucasian females
but not in males with surgically diagnosed ACL ruptures recruited from sports and
recreational clubs.
In male recreational skiers, Stępień-Słodkowska et al.[22] noted no significant differences in genotype distribution or allele frequencies
of COL5A1 BstUI RFLP C/T (rs 12722) and COL5A1 DpnII RFLP C/T (rs 13946) polymorphisms
between the ACLR group and control group. These authors found an underrepresentation
tendency of the C-T haplotype in the ACLR group compared to controls.
Luliska-Kuklik et al.[23] found a significant decrease in frequency in the dominant model of the C/C genotype
for the COL5A1 rs13946 gene in male professional soccer players with surgically diagnosed
primary ACL rupture.
In our study, athletes within the ACLR and control groups were matched for age, height,
body weight, BMI, sport, and length of their sport career. The longer is the sport
career the higher is the risk of trauma exposure.
In addition to having a higher frequency of the C/C genotype, the athletes in the
Control Group have a longer sports career than those of the ACLR Group: with caution
due to the small number of subjects, we hypothesize a protective effect of the C/C
genotype, especially if associated with B1B1 (CC, B1B1 OR protective = 83.3 / 16.7 = 5).
Likewise, Posthumus et al.[24] found a significant age dependent increase in the distribution of the COL5A1 BstUI
RFLP C/C genotype in a group of physical activity male, asymptomatic for musculoskeletal
soft-tissue injuries (without a reported history of tendon injuries). According to
these authors, prolonging exposure time to extrinsic risk of injury selects individuals
who are genetically low risk of injury, who will be found in greater numbers among
older asymptomatic subjects than in the younger asymptomatic subjects.
The COL5A1 BstUI RFLP C/C polymorphism may play a role in increasing the length of
the sports career of athletes.
It is unclear which phenotypic expression of COL5A1 gene polymorphisms may be associated
with an increased risk of ACL rupture.
In EDS, several COL5A1 gene mutations occur concurrently with specific clinical features.[10] The connective tissue of these patients shows structural changes and ligamentous
laxity causing different degrees of joint hypermobility.
Hypermobility has been implicated in ACL injury,[25] and lower limb joint proprioception is reduced in those with benign joint hypermobility
syndrome[26]: no correlation has been described between the COL5A1 BstUI RFLP and COL5A1 DpnII
polymorphisms with an increase in ligamentous laxity. Future investigations should
include the search for an association between changes in joint mobility or ligament
tension and the presence of these polymorphisms.
In a retrospective genetic case-control association study, O'Connell et al.[27] found the COL5A1 C/C genotype significantly overrepresented in triathlon and ultra-marathon
athlete without a history of exercise-associated muscle cramping (EAMC) compared with
athletes with a history of EAMC. The efficiency of the knee flexor muscles, the abductors
muscles of the hip and trunk stabilizers muscles efficiency prevent ACL injuries.[28]
According to Collins and Posthumus,[29] the COL5A1 rs12722 TT genotype is associated with a surplus V collagen production
with a higher risk of some musculoskeletal injuries due to collagen fibril structural
changes in collagen fibrils and changes in the properties of soft tissues.
Laguette et al.[30] demonstrated greater stability of the COL5A1 mRNA (encoding for α1(V)) in C/T genotype
and speculated that small changes in COL5A1 mRNA stability, even if within the normal
physiological range, could result in inter-individual variation in fibrillogenesis;
therefore, a different vulnerability to musculoskeletal lesions COL5A1-dependent.
We theorize that, in the study group (ACLR Group), the frequent exposure of the ACL
to abnormal stresses during team sport practice, reveals the relative susceptibility
of the ACL of the athletes with C/T genotype.
As reported by Smith et al.,[19] tree more genetic factors may be associated with an increase in ligament fragility:
1) TT genotype of the COL1A1 Sp1, binding site polymorphism. The COL1A1 gene encodes
a protein chain within type I collagen, a major structural component of ligaments;
2) the AA genotype of the COL12A1 AluI polymorphism (only in women). This gene encodes
for protein chains in type XII collagen, which is believed to regulate fibril diameter
in ligaments; 3) the chromosomal region 11q22, where several matrix metalloproteinase
genes of physiologic mediators of collagen cleavage and removal are located.
Multiple genetic dependency of ACL susceptibility to rupture may suggest scanning
changes in multiple genes.
The main limitation of the present work is represented by the small number of athletes
included in the study, which has precluded the possibility of analyzing any differences
between genders. The development of clinical or laboratory tools which identify subjects
at great risk to this common musculoskeletal injury, would ease a greater people awareness
of their own exposure to an ACL rupture. Besides it would be useful in settle the
best therapeutic choice after primary ACL rupture diagnosis.
Conclusions
The COL5A1 gene may be one of the genetic factors associated with ACLR in team-sport.
COL5A1 C/C polymorphisms provides a protective effect against the ACLR in both sex.
Longer sport career linked to an increased frequency of COL5A1 BstUI RFLP C/C.
