Influence of the MCT1 rs1049434 on Indirect Muscle Disorders/Injuries in Elite Football Players

Participants

One hundred seventy-three male elite football players (age 19.4?±?5.2 years; height
177.1?±?7.4 cm; weight 69.3?±?8.8 kg) participated in the study. All participants
have played at the Official National Football Championship (Serie A, Primavera, Allievi
and Giovanissimi) during five seasons (2009–2014). Participants were all Italian (Caucasian)
for ?3 generations. The participants trained for ~8 weeks (25.0?±?10.3 h per week)
in the pre-season period and ~38 weeks during the competitive season (10.0?±?7.4 h
per week). Players who join/left the cohort were included/excluded from the date when
they join/left the team. All participants provided informed written consent, and the
study protocol was approved by the Ethics Committee of the club and it was in accordance
with Declaration of Helsinki for Human Research of 1974 (last modified in 2000).

Study Design

The study design followed the consensus on definitions and data collection procedures
in studies of football injuries outlined in the consensus document 21] and by UEFA 22].

Injuries Data Collection

Data was collected from 25 players (14.4 %) during the 2009–2010 season, 15 players
(8.6 %) during the 2009–2011 season, 14 players (8.0 %) during the 2009–2013 season,
67 players (38.7 %) during the 2012–2013 season, and 53 players (30.6 %) during 2013–2014
season. The follow-up time for injuries was between 1 and 5 years. Injury data of
players who left the team during the season, for example, due to a moving to another
team, were included only for the period they were part of the roster. Players with
an existing injury were not excluded from the study; however, their existing injuries
were recorded and were not included in the study.

An indirect muscle injury was defined as any physical complaint occurring during practice
that prevented a player from participating in training or match play for at least
1 day after the day of the onset 21]. Injuries were categorized under four degrees of severity based on the number of
days’ absence: minimal (code 1; 1–3 days), mild (code 2; 4–7 days), moderate (code
3; 8–28 days), and severe (code 4; 28 days) 21]. The registration of a muscle injury was based on a clinical examination by the team
medical staff. Time loss due to injury was recorded on a weekly basis by the team’s
medical staff (physicians and coaches) using a standardized injury report form during
the preseason and during the regular season.

Ultrasound and magnetic resonance imaging scans were used to morphologically classify
the injuries. Muscle injuries included in the study were classified according to a
scheme devised by Muller-Wohlfarth 20] derived from a recent consensus statement on sports injuries in relation to indirect
mechanisms of injury (Table 1).

Table 1. Classification of indirect muscle disorders and injuries

Participants with direct muscle injury (contusion and laceration) were excluded from
the study.

Injury incidence was calculated per 1000 h of training exposure ((? number of muscle
injuries/? h training exposure)×1000). Training exposure was defined as any team-based
or individual physical activity, conducted under the control or guidance of the team’s
coaching and fitness staff that was aimed at maintaining or improving players’ football
skills or physical condition. Matches between teams were considered to be training
exposure. Any match activity that was part of a player’s rehabilitation from injury
was not recorded as a match exposure. The follow-up time was accounted in the injury
incident calculation.

DNA Analysis

Genomic DNA was extracted from buccal swab using a QIAamp DNA Minikit (QIAGEN, Hilden,
Germany). Concentration of extracted DNA was determined by fluorometric method (through
Qubit by Invitrogen). On average, the DNA concentration registered was 20 ?g/ml, which
is sufficient for performing PCR.

Following performing the PCR, an electrophoresis on agarose gel was carried out as
quality control to verify the purity of the PCR products. Genomic DNA from the subjects
was analyzed by polymerase chain reaction (PCR) following the protocol previously
published 9]. The primers used for amplification were as follows 8]: sense primer 5?-ACACATACTGGGCATGTGGC-3? (1455–1474); antisense primer 5?-AAA TCCCATCAA
TGA ACAACTGGTATGATTTCCAC-3? (1807–1841). Using the primers and the missense polymorphism
(rs1049434) described by Cupeiro et al. 9], we searched the sequence on gene bank. Through the Neb Cutter tool (http://tools.neb.com/NEBcutter2/index.php), we individuated the restriction enzyme BccI that allows to discriminate the presence
or absence of missense polymorphism A1470T. This enzyme recognizes the sequence 3?-GGTAG-5?
and produces three fragments in the mutate sequence (TT: 14,171, and 202 bp), whilst
only two fragments (AA: 14 and 373 bp) are generated in the wild sequence. Heterozigotes
AT were individuated by four fragments: 14, 171, 202, and 373 bp.

Statistical Analysis

Levene’s tests were performed to verify the homogeneity of variances. ANOVA was used
to examine the differences between genotype (or allele) groups and continuous data.
Where appropriate, a Fisher LSD post hoc analysis was used to determine which of the
three genotypes were significantly different from each other.

Hardy-Weinberg equilibrium was calculated using Genepop version 4.0.10 (http://genepop.curtin.edu.au). The P value for significance was set at P??0.05.