of Birmingham Variations of collagen-encoding genes are associated with exercise-induced muscle damage

Variations of collagen-encoding genes are associated with exercise-induced muscle damage. Physiol Genomics investigated whether single nucleotide polymorphisms (SNPs) within genes encoding the alpha-1 chain of type I ( COL1A1 , rs2249492; rs1800012), type II ( COL2A1 , rs2070739), and type V (COL5A1 , rs12722) collagen were associated with the variable response to exercise-induced muscle damage (EIMD). Knee extensor muscle strength and soreness were assessed pre-, post-, and 48 h post-EIMD (120 maximal eccentric knee exten- sor contractions) in 65 young healthy participants, who were genotyped for the aforementioned SNPs. We found that COL1A1 (minor) T-allele carriers (rs1800012) and (major) T-allele homozygotes (rs2249492) were generally weaker ( P (cid:2) 0.019); and (minor) A-allele carriers of COL2A1 ( P (cid:2) 0.002) and (major) T-allele carriers of COL5A1 ( P (cid:2) 0.004) SNPs reported greater muscle soreness, all compared with their respective major (rs1800012; rs2070739) and minor (rs2249492; rs12722) allele homozygote counterparts. To con-clude, the risk alleles of these four SNPs appear to negatively inﬂuence muscle strength and post-EIMD recovery, possibly via a dysregulated collagen network affecting the muscle’s mechanical properties.


PG SNPs
Variations of collagen-encoding genes are associated with exercise-induced muscle damage X P. Baumert, 1 G-REX Consortium, C. E. Stewart, 1 M. J. Lake, 1 B. Drust, 1  -We investigated whether single nucleotide polymorphisms (SNPs) within genes encoding the alpha-1 chain of type I (COL1A1, rs2249492; rs1800012), type II (COL2A1, rs2070739), and type V (COL5A1, rs12722) collagen were associated with the variable response to exercise-induced muscle damage (EIMD). Knee extensor muscle strength and soreness were assessed pre-, post-, and 48 h post-EIMD (120 maximal eccentric knee extensor contractions) in 65 young healthy participants, who were genotyped for the aforementioned SNPs. We found that COL1A1 (minor) T-allele carriers (rs1800012) and (major) T-allele homozygotes (rs2249492) were generally weaker (P Յ 0.019); and (minor) A-allele carriers of COL2A1 (P ϭ 0.002) and (major) T-allele carriers of COL5A1 (P ϭ 0.004) SNPs reported greater muscle soreness, all compared with their respective major (rs1800012; rs2070739) and minor (rs2249492; rs12722) allele homozygote counterparts. To conclude, the risk alleles of these four SNPs appear to negatively influence muscle strength and post-EIMD recovery, possibly via a dysregulated collagen network affecting the muscle's mechanical properties.

BACKGROUND/MOTIVATION FOR THE STUDY
Unaccustomed strenuous exercise involving eccentric muscle contractions (i.e., when the muscle is active while being forcibly lengthened) induces ultrastructural muscle damage. However, the changes in muscle damage-related biomarkers, e.g., loss of strength and delayed-onset muscle soreness, differ considerably between people (1).
Tendon and the extracellular matrix (ECM), which surrounds the contractile elements of the muscle, are composed of different types of collagen. These influence the mechanical properties of the muscle-tendon unit (MTU), ultimately influencing the transmission of force from the muscle to the bone. The ECM enables muscle force to be transmitted both longitudinally and laterally to the tendon, and, during eccentric contractions, external force can be transmitted laterally to the sarcomeres, causing MTU damage (5).
Single nucleotide polymorphisms (SNPs) of collagen genes have been associated with MTU mechanical properties (4) and injury risk (2), possibly by affecting RNA stability and hence collagen abundance. Furthermore, resistance exercise increases collagen gene expression (3), but no study has investigated whether SNPs of collagen-encoding genes are associated with the response to exercise-induced muscle damage (EIMD). Therefore, we aimed to investigate if intron SNPs within the COL1A1 (T/C, rs2249492; G/T rs1800012) and in COL5A1 (T/C, rs12722) genes, and a missense SNP within the COL2A1 (G/A, rs2070739) gene, which encode the alpha-1 chain of type I, V and II collagens, respectively, were associated with the skeletal muscle response to EIMD in untrained young men and women.
Cohort details. Sixty-five untrained, healthy (identified by physical activity and health questionnaires) female (n ϭ 39) and male (n ϭ 26) Caucasians (mean Ϯ SD; age ϭ 22.5 Ϯ 4.0 yr; height ϭ 1.71 Ϯ 0.09 m; body mass ϭ 70.9 Ϯ 14.4 kg) were recruited in this population cohort study. All recruits gave written, informed consent to participate in the study, which was approved by the Liverpool John Moores University Ethics Committee in accordance with the Declaration of Helsinki. Inclusion criteria included age between 18 and 35 yr, and potential participants were excluded if they 1) had performed leg resistance training within the last 6 mo, and 2) suffered a muscle-tendon injury in the last 12 mo and/or bone fracture in the lower limbs. Participants were instructed to maintain their normal routine, to refrain from drinking alcohol, and to avoid any exercise 48 h before each testing session and throughout the study.
Type of study. Purification of DNA was performed using QIAamp DNA Blood Kit (Qiagen, Crawley, UK) and was prepared according to manufacturer protocols for this candidate SNP study. Each participant was genotyped for COL1A1 (T Ͼ C, rs2249492; GϾT rs1800012), COL2A1 (GϾA, rs2070739), and COL5A1 (T Ͼ C, rs12722) SNPs via real-time polymerase chain reaction (PCR) with a Rotor-Gene Q (Rotor-Disk) Instrument (Qiagen). The 10 l reaction volume included 5 l Genotyping Master Mix (Applied Biosystems, Foster City, CA), 0.5 l genotyping assay (Applied Biosystems), 3.5 l nuclease-free H 2 O (Qiagen), and 1 l DNA. For control wells, 1 l nuclease-free H 2 O replaced the DNA template. Reactions were incubated in a 72-well optical plate at 92°C for 15 s (denaturation), followed by annealing and extension at 60°C for 1 min (60 cycles). Each sample was run in duplicate, and there was 100% agreement between samples from the same participant. Genotype calls were performed with Rotor-Gene Q Software 2.3.1 (Qiagen). Analysis model. The violations of Hardy-Weinberg equilibrium (HWE) for each SNP were examined using 2 tests. All parameters were normally distributed according to the Shapiro-Wilk test and by inspection of the Q-Q plots. Genotype associations with isometric and isokinetic kneeextension MVC torque, and muscle soreness over time were investigated via two-way mixed ANCOVAs [within-subjects factor: time (pre-, post-, and 48 h post); betweensubjects factor: genotype; covariate: sex). Recessive models were used when n Յ 2 for one genotype, and false discovery rate (FDR) was performed to account for multiple testing (FDR set at Ͻ20%; for references, see Appendix 3). All results were expressed as means Ϯ SD. MVC data were analyzed with AcqKnowledge software 4.4 (Biopac-Systems), and statistical analyses was performed using SPSS 23 (IBM, Armonk, NY).

INTERPRETATION
The T-alleles for both COL1A1 SNPs were associated with lower maximum strength compared with their respective major (G, rs1800012) and minor (C, rs2249492) alleles. COL2A1 A (minor) allele carriers and COL5A1 T (major) allele carriers recovered more slowly from EIMD than their respective COL2A1 G (major) allele and COL5A1 C (minor) allele homozygote counterparts. Further work is required to 1) validate the results in a larger independent cohort and 2) investigate LD between these SNPs and SNPs of other genes to precisely map the causal variants and genes influencing the phenotypic outcome. However, our results suggest that these four SNPs within three collagenencoding genes affect muscle strength and recovery following EIMD, possibly by influencing RNA stability, potentially causing dysregulation of the collagen network, thus negatively affecting the mechanical properties of the MTU.