Are IL1B, IL6 and IL6R Gene Variants Associated with Anterior Cruciate Ligament Rupture Susceptibility?

Author:Lulinska-Kuklik, Ewelina
Position:Research article
 
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Introduction

The cellular and molecular mechanisms underlying the development of musculoskeletal soft tissue injuries (such as anterior cruciate ligament ruptures or tendinopathy) are complicated, but inflammatory mediators produced by connective tissue cells in response to repetitive mechanical loading may be an important factor for this pathology. Cytokines, such as interleukins, are crucial in regulating critical cell signaling pathways as well as being major contributors to inflammatory response which is upregulated during ligament and tendon injuries (Tsuzaki et al., 2003).

The inflammation processes involved during anterior cruciate ligament (ACL) injuries are accompanied by upregulation of interleukin-1[beta] (IL-1[beta], encoded by the IL1B human gene) production. IL-1[beta] is a potent pro-inflammatory cytokine produced mainly by macrophages in injured tissues and upregulates the expression of other inflammatory mediators (Newton and Covington, 1987), including interleukin-6 (IL-6, encoded by the IL6 gene), which is secreted after tendon and ligament injuries by human fibroblasts (Benazzo et al., 2008). IL-6 is a pleiotropic cytokine whose expression levels correlate with the severity of inflammation. IL-6 plays a crucial role in bone resorption (Ferrari et al., 2003) as well as apoptosis of cells (Legerlotz et al., 2012). In the context of ACL injuries, it is worth observing that IL6 expression increases after cyclic stretching of human tendon fibroblasts (Skutek et al., 2001) as well as in pathological conditions in tendons (Legerlotz et al., 2012; Millar et al., 2009) or ligaments (Cameron et al., 1994). IL-6 exerts its biological effect by binding to the interleukin-6 receptor (IL-6R), encoded by the IL6R gene (Galicia et al., 2004).

Taking these many factors into account, the genes encoding key interleukins, such as IL1B and IL6 as well as interleukin receptor IL6R, were chosen as candidate genes for association with soft tissues injuries. Thus, we have decided to test the hypothesis that sequence variants rs1143627, rs16944, rs1800795, rs2228145 in the IL1B, IL6 and IL6R genes are associated with ACL rupture susceptibility in the Polish population in a case study comparing ACL patients and healthy controls. Additionally, this study allows us to further explore the role of IL1B, IL6, IL6R polymorphisms in a risk model for ACL rupture.

Methods

Ethics committee

The procedures followed in the study were conducted ethically according to the principles of the World Medical Association Declaration of Helsinki and ethical standards in sport and exercise science research. The procedures followed in the study were approved by the Ethics Committee of the Pomeranian Medical University in Szczecin (approval number 09/KB/IV/2011). All participants were given a consent form and a written information sheet concerning the study, providing all pertinent information (purpose, procedures, risks, and benefits of participation). The potential participant had time to read the information sheet and the consent form. After ensuring that the participant had understood the information, every participant gave written informed consent (signed consent form) to genotyping with the understanding that it was anonymous and that the obtained results would be confidential. The experimental procedures were conducted in accordance with the set of guiding principles for reporting the results of genetic association studies defined by the Strengthening the Reporting of Genetic Association studies (STREGA) Statement (Little et al., 2009).

Participants

A total of 423 physically active, unrelated, self-reported Caucasian participants were recruited for the study between 2009 and 2016. The study group consisted of 229 (65 females and 164 males) individuals with surgically diagnosed primary ACL rupture who qualified for ligament reconstruction (ACLR group). All 229 participants from the ACLR group sustained their injury through non-contact mechanisms. The control group: 194 (85 females and 109 males) seemingly healthy participants without any history of ACL injuries (CON group).

The ACLR participants were soccer players from the 1st, 2nd, and 3rd division Polish soccer leagues (trained 11-14 hours per week). The control group were healthy, physically-active individuals, with the majority playing soccer as their main sport with no self-reported history of ligament or tendon injury. All the male participants (ACLR and CON groups) were from the same soccer teams, of the same ethnicity (all self-reported Polish, Eastern-Europeans for [greater than or equal to] 3 generations), of similar age (ACLR group = 26 [+ or -] 4 years, CON group = 25 [+ or -] 3 years), with a comparable level of exposure to risk of ACLR (same volume and intensity of training and match play). The ACLR female participants (mean age: 25 [+ or -] 4 years) consisted of soccer players from the 1st division Polish soccer league (trained 10-12 hours per week). The female control participants from the CON group (age 29 [+ or -] 2 years) were recruited from sports clubs and wellness centers and were self-reported as being physically active for a minimum of 7 hours per week.

Genetic analyses

The buccal cells donated by the subjects were collected in Resuspension Solution (GenElute Mammalian Genomic DNA Miniprep Kit, Sigma, Germany) with the use of sterile foam-tipped applicators (Puritan, USA). DNA was extracted from the buccal cells using a GenElute Mammalian Genomic DNA Miniprep Kit (Sigma, Germany) according to the manufacturer's protocol. All samples were genotyped in duplicate using an allelic discrimination assay on a StepOne Real-Time Polymerase Chain Reaction (RTPCR) instrument (Applied Biosystems, USA) with Taqman probes. To discriminate IL1Brs16944 and rs1143627 as well as IL6 rs1800795 and IL6R rs2228145 alleles, a TaqMan Pre-Designed SNP Genotyping Assay (Applied Biosystems, USA) (assay ID: C_1839943_10, C_1839944_10, C_1839697_20, C_16170664_10, respectively) were used, including primers and fluorescently labeled (FAM and VIC) MGB probes to detect both alleles. Every genotyping was carried out together with a no-template negative control, i.e. sample of appropriate Genotyping Assay mixed with distilled water without DNA, showing there is no contaminant in the reagents and that the fluorescence is not due to probe degradation. The positive controls containing genomic DNA samples of known genotypes (homozygous major and minor for each SNP) were used for all experiments to test that both assay probes function. The same genomic DNA samples have been used as positive controls for all PCR plates to check for consistency in genotype calls. Genotypes were assigned using all of the data from the study simultaneously.

Statistical analyses

Statistical analysis was conducted with SNPassoc package for R (version 3.4.0, The R Foundation for Statistical Computing, https://cran.r-project.org). A single locus analysis including pairwise SNP x SNP interaction was conducted using the SNPassoc (version 1.9.2) under the assumption of the four genetic models (codominant, dominant, recessive and overdominant). The models were constructed with respect to the minor allele. The p values for SNP x SNP interactions (epistasis) were computed using log-likelihood ratio test. The power was calculated using QUANTO (version 1.2.4, http://biostats.usc.edu) assuming a dominant genetic model, the incidence of ACL tears of 68 per 100,000 person-years (Sanders et al., 2016) and the risk allele frequencies taken from the HapMap in CEU population. The power for odds ratio ranging from 1.2 to 2.0 varied between 14% (OR=1.2) to 92% (OR=2.0). P values

Results

The measured genotype frequencies were not significantly different from the expectations of Hardy-Weinberg equilibrium in the control sample (p values range 0.083 to 0.859) as well as the pooled case-control sample (p values range 0.114 to 1.0). However, in the case sample, the observed rs1800795 (IL6) genotype frequencies differed significantly from expectations (p = 0.023) (Table 1).

Tables 2-5 summarize the results of association analysis between the single nucleotide polymorphism within the IL1B (Tables 2, 3), IL6 (Table 4), IL6R (Table 5) genes and predisposition to ACL rupture (ACLR). The IL6 gene polymorphism (rs1800795) was found to be the only SNP significantly associated with ACLR (Table 4). The associations were demonstrated for all genetic models except the dominant model (G/G vs C/G-C/C). The highest odds ratio (1.74, 95% CI 1.08-2.81, sex adjusted p = 0.032) was found for the recessive model (G/G-C/G vs C/C). IL6 rs1800795 heterozygosity was associated with an odds ratio for ACLR of 0.57 (95% CI 0.38-0.83, sex adjusted p = 0.007).

Gene-gene (only pairwise interactions were considered) interactions were investigated under the assumption of the same genetic models as for the single-gene analyses (except for the overdominant model). Only one IL1B SNP was used in the interaction analysis due to strong linkage disequilibrium between the two IL1B SNPs (D' = 0.98, [r.sup.2] = 0.96, p

Discussion

Correlation studies of selected gene variants that can be included in the group of genetic risk factors for musculoskeletal soft tissues, due to their ability to affect the structural development, function and mechanical properties of tendons or ligaments, are very common. In recent years, there have been a number of studies suggesting associations between ACL rupture and polymorphisms in the structural genes, possibly...

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