WO2010146437A1

WO2010146437A1 - Screening methods of genetic polymorphisms associated to the immune response for the evaluation of athletic predisposition and relative kits

Info

Publication number: WO2010146437A1
Application number: PCT/IB2010/001435
Authority: WO
Inventors: Giuseppe Banfi; Sabina Cauci
Original assignee: Istituto Ortopedico Galeazzi S.P.A.
Priority date: 2009-06-15
Filing date: 2010-06-15
Publication date: 2010-12-23
Also published as: ITMI20091054A1

Abstract

The present invention relates to methods for the screening of athletic predisposition by detecting polymorphisms at the level of the gene of the receptor antagonist of interleukin IL-I (IL-lra) IL-IRN and the relative kits of the genetic or immunologic type.

Description

SCREENING METHODS OF GENETIC POLYMORPHISMS ASSOCIATED TO THE IMMUNE RESPONSE FOR THE EVALUATION OF ATHLETIC PREDISPOSITION AND RELATIVE KITS

The present invention relates to methods for the screening of athletic predisposition by the detection of polymorphisms at the level of the gene of the receptor antagonist of interleukin IL-I (IL- Ira) IL-IRN and the relative kits of the genetic or immunologic type. So far, factors which influence athletic predisposition have only been partially defined. In this context, there is a growing interest towards objective biomarkers which could effectively support phenotypic observations and induce the development of training programs aimed at precociously selecting top athletes, also preventing subjects with great potentiality from being overlooked or not being sufficiently encouraged.

Various data suggest that both pro- and anti- inflammatory responses can play an important role in physical activity and in athletic performance. Prolonged exercise can in fact induce the activation of the acute phase response with some similarities to sepsis [1] . The induction of a systemic response mediated by cytokines, characterized by high plasmatic levels of interleukins (IL) such as IL-lra, IL-6, IL-8, IL-10, and G-CSF (granulocyte colony-stimulating factor) , following intense physical activity, has been well documented ( [2] - [5] ) .

IL- Ira is a member of the family of IL-I cytokines

(which comprises, in addition to the well-known cytokines IL-lα, IL-lβ, IL-18, also cytokines recently discovered as IL-IF factors from 5 to 11) . The main role of IL- Ira is to regulate the activity of IL-I (α and β) , by preventing the signal induced by the IL-I cell receptor of type I, IL-IRl. IL-lα acts as agonist of IL-IRl, it has a pro- inflammatory activity, but IL- lα acts primarily as an intracellular transcriptional regulator. IL-lβ, on the other hand, can be secreted outside the cells, it generally acts synergically with TNF-α, it activates pro- inflammatory responses in many types of cells and promotes the acute phase response. It has been assumed that as IL-lβ plays a very important role in regulating the acute inflammatory response, it also has an important role in modulating the immune response associated with physical exercise. In reality, however, numerous studies have demonstrated that the circulating concentration of IL-lβ remains unvaried after exercise, or shows minimum, and delayed variations [1] . In spite of this, various studies have indicated an increase in the local levels of IL-lβ after exercise, probably associated with microlesions at the level of the skeletal muscle caused by intense physical activity [6] . In addition, Mahoney et al . [7] have recently demonstrated that an increased gene expression of the receptor IL-IRl is induced in the skeletal muscle after intense physical exercise. These observations support an important role of cytokines of the family IL-I in the post-traumatic period. It should be noted that inflammatory mediators and chemoattractants are capable of promoting the phagocytosis of cellular debris induced by traumas by means of macrophages and it has been demonstrated that these cells continue to secrete IL- lβ even 5 days after the trauma [6] . Among other things, this accumulation and activation of macrophages resident in the muscle is a rich source of growth factors able to stimulate biogenesis [8] . The inflammation can therefore be involved in muscular hypertrophy mechanisms.

Genetic associations which characterize the athletic phenotype are at present only partially known. Various genetic studies have been focalized on some candidate genes (about 200) which are mainly involved in cardiovascular functionality, including the enzyme which converts the angiotensin (ACE) and proteins involved in the activity of the skeletal muscle such as α-actinins ( [9] , [10] , [11] ) .

Very few studies have so far examined the association between polymorphisms of genes relating to cytokines and physical activity. A study has examined polymorphisms at the level of the gene of the α receptor for IL-15 (IL- 15RA) and identified a polymorphism with a single nucleotide (SNP, single nucleotide polymorphism) strongly associated with muscular hypertrophy in young subjects of mixed ethnic origins who performed physical resistance exercises [12] .

Two studies performed in non-athletes have shown that the variant -174G/C in the IL- 6 gene affected the response to exercise by modulating changes in serum concentrations of IL-6 [13] and high-density lipoprotein cholesterol (HDL-C) [14] . However, Walston et al . [15] identify no significant correlation between IL-6 SNPs or IL-6 haplotypes with the serum concentration of IL-6 or physical strength or frailty in 463 older women.

US2006/0121478 [11] describes methods for evaluating athletic predisposition based on the determination of the genotype ACTN3 (the gene which encodes α-actinin-3) and various polymorphisms. According to what is described in US2006/0121478 , top athletes who practise sprint sports (such as track-and field sports, swimming and track cycling) prove to be less frequently homozygotes (6% vs. 18% of Australian Caucasian controls) for the alleles 577X of the gene ACTN3 , which encodes a premature stop codon and therefore causes a deficiency of α-actinin-3, a protein of the skeletal muscle only expressed in type 2 fibres (fast fibres) , important for the anchorage of actin and which exerts a regulatory function in the coordination of the contraction of the muscular fibre. Top athletes who practise endurance sports (such as rowers, tri- athletes, track cyclists and marathoners) are, instead, more frequently 577X/X homozygotes (25% vs. 18% of Australian Caucasian controls) . Many sports activities, however, do not exactly fall either within the sprint or endurance category, for example volley ball, rugby, football are so-called alternating aerobic/anaerobic activities, which involve both sprint and endurance attributes and consequently for all these sports activities the determination of the ACTN3 genotype remains of an undefined utility. Curiously, in US2006/0121478, track cyclists are classified as both endurance athletes (36.4% 577X/X homozygotes) , and sprint athletes (12.5% 577X/X homozygotes), however these athletes frequently participate in both types of track racing.

These methods, moreover, are limited by the fact that the polymorphisms which influence the gene ACTN3 can have different effects among male and female individuals and this can influence the accuracy of the result to be determined. A very recent study [16] , has in fact demonstrated that only women, but not men, with a 577X/X genotype (corresponding to the α-actinin-3 deficiency) show a reduced muscular force of the knee extensors and a smaller fat-free mass.

The effects of the polymorphism ACTN3 R577X in subjects who are not elite athletes, are generally not well-defined. Intense physical activity can cause microlesions at a skeletal muscular level, and it is believed that cytokines such as IL- lβ and TNF-α can trigger and regulate the repair process. IL-lβ is capable of inducing the secretion of various inflammatory factors, such as IL- 6, IL-8, TNF-α, and GM-CSF, from different cell types including myoblasts, smooth and skeletal muscular cells, fibroblasts, macrophages, peripheral blood mononucleated cells (PBMC) and endothelial cells ( [17] - [19] ) . IL-lβ binds the receptor of IL-I of type I present on the surface of a variety of cells and initiates a cascade of events which lead to the recruitment and activation of macrophages and neutrophils. However, inflammation must be finely regulated to avoid secondary tissue damage and myopathies .

The activity of IL- lβ is modulated mainly by the receptor antagonist for IL-I (IL- Ira), that specifically inhibits the action of IL-I by competition for its receptor and inhibition of the intracellular signal mediated by IL-IRl ( [20] - [22] ) . In healthy individuals, IL- Ira can be easily detected in the plasma (concentrations of hundreds of pg/mL) , whereas the levels of IL-lβ cannot normally be revealed (very few or less than pg/mL) ( [22] - [23] ) . The levels of both IL-lβ and IL-lra, generally increase in the body after an insult, and thus, the plasma levels of these cytokines are positively correlated [24] . However, the final inflammatory activity of IL-lβ is considered to depend on the actual ratio of IL-lβ over IL-lra ( [20] - [21]) .

Among the polymorphisms at the level of the single nucleotide (SNP) in the gene of IL-lβ (IL-IB) , two have been particularly studied for the predisposition to diseases, that in position -511 in the region of the promoter [25] and another in position +3954 in the hexon 5 ( Taql restriction site polymorphism, indicated as +3953 in old scientific literature) [26] . The gene of the cytokine IL-lra (IL-IRN) has a penta-allelic polymorphic site in intron 2, consisting in a variable number of 86 identical tandem repeats (VNTR) , which has been actively studied in relation to various pathological conditions [27] . A study recently carried out on 24 sedentary subjects (of unspecified ethnicity) selected on the basis of their haplotype pattern in the IL-I gene cluster (based on specific combinations of +4845 IL-IA₁ +3954 IL-IB, -511 IL-IB₁ and -3737 IL-IB polymorphisms) showed that a particular IL-I genotype (subjects C/C for IL-IB +3954 carriers of allele 2 at IL-IRN +2018) is associated with inflammation of the skeletal muscle, following acute physical resistance exercise [28] . These authors suggest that the genotype of IL-I can influence adaptation to resistance to chronic physical exercise. The pre- and post-exercise inflammatory factor levels, however, show a considerable variability among different persons and this is at least partly influenced by genetic variability ( [28] - [29] ) . On the basis of what is indicated above, the necessity is evident for availability of new screening methods and relative kits, which allow transversally high-level athletic predisposition to be reliably evaluated regardless of the sex of the athlete and type of physical activity.

The authors of the present invention have now found that some polymorphisms of genes of the interleukin- 1 family (IL-I) are associated with the athletic phenotype in white subjects belonging to the Caucasian population, regardless of the sex and type of physical activity.

In particular, the study was carried out starting from the genomic DNA of 205 athletic subjects and 458 non-athletic controls.

The objective of the study was: to verify whether the frequencies of the genotypes of -511 and +3954 SNPs of IL-IB and of the VNTR in intron 2 of IL-IRN (which are the most widely studied polymorphisms of the cytokines IL- lβ and IL- lra) vary between athletes and non-athletes in the Caucasian population: to verify whether the polymorphisms of these genes are associated with being a professional or non- professional athlete.

The analysis was carried out on a relatively homogeneous ethnic group as the associations between the polymorphisms in genes correlated to the immune system can be confused by differences in the allelic frequency among ethnic groups ( [30] , [31] ) .

Two diallelic polymorphisms were evaluated in the gene of the cytokine IL-lβ [IL-IB) consisting in a C/T transition of bases in positions -511 and +3954, and a variable number of tandem repeats (VNTR) in intron 2 of the gene of the cytokine IL- Ira (IL-IRN) . With respect to the non-athlete controls, in athletes a frequency of the genotype VNTR IL-IRN 1/2 almost double was found

(41.0% vs. 26.4%, OR = 1.93, 95% CI = 1.37 - 2.74), and, on the other hand, a lower frequency of the genotype 1/1 (43.9% vs. 58.5%, OR = 0.55, 95% CI =

0.40-0.77) was detected. Whereas the frequency of the genotype IL-IRN 2/2 did not differ between athletes and non-athlete controls. Furthermore, significant differences between athletes and controls were not found for the polymorphisms -511 and +3954 IL-IB. The haplotype (-511) C- ( +3954) T- CVNTR) 2, however, proved to be 3 times more frequent in athletes with respect to non-athletes (OR = 3.02, 95% CI = 1.16-7.87) . This indicates that the variants at the level of the locus of the gene of IL- Ira are predisposing for an athletic status, with respect to the other subjects.

An object of the present invention therefore relates to a method for determining the athletic predisposition in the Caucasian population comprising the following steps: a) genotyping of a subject for the presence of at least one polymorphic site located in the IL-IRN gene (Gene Bank, ID: 3557) encoding for the receptor antagonist of interleukin IL-I (IL-lra), alone or in association with at least one polymorphic site located in the IL-lβ gene, IL-IB (Gene Bank, ID: 3553) ; b) predicting the athletic phenotype on the basis of the presence of the one or more polymorphic sites. According to preferred embodiments, said genotyping takes place by means of allele-specific amplification, DNA sequencing, allele-specific hybridisation, and/or RT-PCR analysis. Said polymorphic site of the IL-IRN gene preferably consists in the pentaallelic polymorphic site at the level of intron 2 containing a variable number of 86 tandem repeats (VNTR) and said polymorphic site of at the level of the IL- lβ gene is selected from the transition of C/T bases at position -511 of the promoter or +3954 of exon 5 of IL-IB.

According to a preferred embodiment of the invention, the presence of allele 2 at the level of intron 2 containing VNTR of the IL-IRN gene or genotype 1/2 IL-IRN VNTR, or the absence of allele 1 IL-IRN VNTR or genotype 1/1 IL-IRN VNTR is positively associated with the athletic phenotype . In other words, the presence of the genotype 1/1 IL-IRN VNTR is negatively associated with the athletic phenotype. Alternatively, the presence of the haplotype in three sites ( -511) C- (+3954) T- (VNTR) 2 is highly associated with the athletic phenotype (i.e. positively associated with a high athletic predisposition) / the presence of the haplotype in two sites (+3954) T- (VNTR) 2 or the haplotype in three sites ( -511) T- (+3954) T- (VNTR) 2 is associated with a medium-high athletic predisposition; the presence of the haplotype in two sites (-51I)C- (VNTR) 2 or ( -511) T- (VNTR) 2 is associated with a medium athletic predisposition,- the presence of the haplotype in two sites (+3954) C- (VNTR) 2 is associated with a moderate athletic predisposition.

Alternatively, said polymorphic site of the IL-IRN gene consists in polymorphic sites in linkage disequilibrium with the pentaallelic polymorphic site at the level of intron 2 containing a variable number of 86 tandem repeats (VNTR) , such as +2018 IL-IRN SNP.

A further object of the present invention relates to a kit for determining athletic predisposition in the Caucasian population comprising a pair of primers for the amplification of the pentaallelic polymorphism at the level of intron 2 containing a variable number of 86 tandem repeats (VNTR) .

According to a preferred embodiment in which the haplotype is to be determined, the kit for determining athletic predisposition in the Caucasian population comprises the following components: a) pair of primers for the amplification of the pentaallelic polymorphism at the level of intron 2 containing a variable number of 86 tandem repeats (VNTR) Of IL-IRN; b) pair of primers for the amplification of the polymorphism consisting in the transition of C/T bases in position -511 of the promoter of IL-IB; c) pair of primers for the amplification of the polymorphism consisting in the transition of C/T bases in position +3954 in exon 5 of IL-IB.

The pair of primers a) for the amplification of the pentaallelic polymorphism at the level of intron 2 containing a variable number of 86 tandem repeats (VNTR) used in the kit preferably consists in:

- 5' -CTCAGCAACACTCCTAT- 3' (SEQ ID No:l) and

- 5' -TCCTGGTCTGCAGGTAA-S' (SEQ ID No: 2) ; and the pair of primers b) for the amplification of the polymorphism consisting in the transition of C/T bases in position -511 of the promoter of IL-IB consists in:

- 5' -TGGCATTGATCTGGTTCATC-S' (SEQ ID No: 3) and

- 5' -GTTTAGGAATCTTCCCACTT-S' (SEQ ID No: 4) ; and the pair of primers c) for the amplification of the polymorphism consisting in the transition of C/T bases in position +3954 in exon 5 of IL-IB consists in:

- 5' -GTTGTCATCAGACTTTGACC-S' (SEQ ID No: 5) and

- 5' -TTCAGTTCATATGGACCAGA- 3' (SEQ ID No : 6 ) . A further object of the invention relates to an immunologic kit for determining athletic predisposition in the Caucasian population comprising at least one monoclonal or polyclonal antibody specific for the protein IL-lra. The invention also relates to the use of monoclonal or polyclonal antibodies specific for the protein IL- lra for the quantification of the levels of said protein for detecting athletic predisposition in the Caucasian population. Finally, an object of the invention relates to the use of allele 2 IL-IRN or the genotype 1/2 IL-IRN or the absence of allele 1 IL-IRN or the genotype 1/1 IL- IRN or alternatively the use of the haplotype in three sites (-51I)C- (+3954)T- (VNTR) 2 as a high-level athletic predisposition marker.

The present invention is now described for illustrative but non- limiting purposes, according to its preferred embodiments in the following examples. EXAMPLES

MATERIALS AND METHODS Subjects and biological samples

The population examined consisted in subjects attending the Galeazzi Orthopaedic Hospital in Milan for routine blood analyses. The exclusion criteria were age younger than 18 years and presence of an acute or chronic pathological condition such as diabetes, autoimmune diseases, cardiovascular pathologies and malignancies. The subjects included in the group of athletes were enrolled from teams of Italian professional and non-professional athletes during routine physical examinations. The control subjects were enrolled in the general Italian population of unrelated subjects after verifying that they had never been athletes and had not participated in agonistic competitions .

The study was approved by the ASL Ethic Committee of Milan. The methods used in the present experimentation were in accordance with the Helsinki Declaration of 1975, as revised in 1996. All eligible participants were enrolled after signing an informed consent .

Of the 688 subjects enrolled, a total of 25 were excluded from the study as they did not fall within the inclusion criteria (14 subjects) or because their laboratory data were not interpretable (11 subjects) . Consequently, the final analysis was carried out on 663 subjects, which included 205 athletes (30.9%) and 458 (69.1%) non-athlete controls. The population under examination of 205 white athletes included 53 professionals (50 current and 3 who had been professional athletes in the past) and 152 nonprofessional athletes who participated in agonistic competitions at a regional level. Among the professional athletes, the soccer players played in the Italian championship in Third Division, whereas all the other professional athletes competed at a National level. In the group of athletes there was a total of 139 women and 66 men. The age of the athletes ranged from 18 to 53 years. A total of 458 healthy white Italian non-athlete subjects selected at random of both sexes within an age range of 18-53 years (average 33.6 ± 9.23 years) formed the control group. The blood samples were obtained in the morning from seated and fasting subjects, the sample being taken from the antecubital vein with test-tubes containing EDTA (Vacutainer Tubes, Becton-Dickinson, Franklin Lakes, NJ, USA) . The samples were centrifuged at 2,000 g for 10 minutes at 4°C and the cellular pellet was processed for DNA extraction. Personnel isolating and processing the DNA did not have access to the demographic characteristics of the subjects. Identification of the gene polymorphisms IL-IB and IL- IRN

The genomic DNA was extracted from the blood pellet fraction according to the standard proteinase-K digestion and ethanol extraction method. The extracted DNA was preserved at -20⁰C until further analysis.

In order to examine the -511 single nucleotide polymorphism, the promoter region of IL-IB was amplified by means of PCR, using the primers 5 ' -TGGCATTGATCTGGTTCATC- 3 ' (SEQ ID No : 3 ) and 5' -GTTTAGGAATCTTCCCACTT-S' (SEQ ID NO:4) as described in Cauci et al . [32] and Di Giovine et al . [25] . The protocol included 35 cycles at 94°C for 1 minute, 55⁰C for 30 s, and 72°C for 30 s, and a final extension at 72⁰C for 5 minutes. At the end of the procedure, the amplicons were digested with Aval at 37°C for 3 hours. The fragments were analyzed by an electrophoresis run on acrylamide gel at 10% and visualized with ethidium bromide. This provides products of 190 bp and 114 bp (allele C) or 304 bp (allele T) as described in Cauci et al . [32] . Among the 663 subjects examined, the data relating to the genotype -511 of 4 individuals were not available.

In order to determine the +3954 single nucleotide polymorphism, the polymorphic region containing the Taql restriction site (indicated as +3953 SNP in older literature) was amplified using the following primers: 5' -GTTGTCATCAGACTTTGACC- 3' (SEQ ID No : 5 ) and 5'- TTCAGTTCATATGGACCAGA- 3' (SEQ ID No: 6) as described by Bioque et al . [33] . The products of 249 bp were digested with Taql at 65°C for 1 hour, resulting in fragments which remained intact (allele T) or were digested into two fragments of 135 and 114 bp (C allele) . The restriction fragments were analyzed by electrophoresis on 10% acrylamide gel and visualized with ethidium bromide ( [32] ) .

The VNTR polymorphism of intron 2 of IL-IRN was analyzed using 5 ' - CTCAGCAACACTCCTAT - 3 ' (SEQ ID No:l) and 5' -TCCTGGTCTGCAGGTAA-S' (SEQ ID No: 2) as primers [27] . The PCR products of 410 bp (allele 1 = 4 repetitions of the 86 bp region) , 240 bp (allele 2 = 2 repetitions) , 500 bp (allele 3 = 5 repetitions) , 325 bp (allele 4 = 3 repetitions) , 595 bp (allele 5 = 6 repetitions) were analyzed by electrophoresis on 8% acrylamide gel stained with ethidium bromide ( [32] ) . A blinded quality control was performed in a random subgroup of 20 samples, which were seguenced by use of an ABI Prism 310 genetic analyzer (Applied Biosystems) . The sample consisted of 11 subjects with the genotype IL-IRN VNTR 1/1, 6 subjects with the genotype 1/2, and 3 subjects with the genotype 2/2; a 100% correspondence with gel data was obtained. Statistical analysis

The genotype frequencies for each polymorphism were compared by means of Fisher's exact test or Pearson's chi-square test (as appropriate) , using STATA statistical software version 10, College Station, TX, USA and Powermarker [34] . The odds ratio (OR) values and the confidence interval at 95% (CI) were calculated to evaluate the genotype effects of each genotype against all others. The Hardy-Weinberg equilibrium

(HWE) was estimated by means of Powermarker using a chi- square distribution in cases and controls separately for each SNP. Haplotype analysis was carried out with the Unphased program [35] . Haplotypes estimated to be below 1% in frequency were not reported. Due to the low allelic frequencies of alleles

3, 4 and 5 of IL-IRN, the intron 2 VNTR genotypes were grouped into short allele (S, allele 2) and long alleles (L, alleles 1, 3, 4 and 5) . The differences in the distribution of the haplotypes between cases and controls were determined by means of a probability ratio test. The reference distribution to which all the other haplotypes were compared was that of the most common haplotype. The OR value was considered significant if the CI did not cross the unit (1.00) . The P value for each haplotype was determined by comparing the haplotype against all others. Therefore, a P value may be slightly significant even if the OR value is not. RESULTS

The population examined consisted in 205 white athletes; of these, 53 were professionals (high grade, National level) and 152 were non-professional athletes (medium level, not paid for participating in Regional level competitions) who regularly participated in athletic races. The controls were 458 healthy white non-athlete subjects. The demographic characteristics and sports activities practised by the 205 athletes examined are indicated in the following Table 1.

Table 1. Demographic characteristics and sports activities practised by 205 athletes under examination

4 were professional athletes b 48 were professional athletes c 22 were professional athletes d 23 were professional athletes e 8 were professional athletes A total of 663 subjects was genotyped for the three gene loci (IL-IB in positions -511 and +3954; and IL- IRN intron 2 VNTR) .

Neither cases nor controls deviated from Hardy Weinberg equilibrium (HWE) at site -511 or at site +3954. The controls deviated from HWE at the VNTR in IL-IRN (P = 0.001); while athletes only marginally deviated from HWE (P = 0.097) . Athletes and controls did not differ in the distribution of the genotypes - 511 of IL-IB; 38.8% of athletes vs. 44.8% of controls were CC homozygotes, 48.8% of athletes vs. 45.9% of controls were CT heterozygotes, and 12.4% of athletes vs. 9.4% of controls were TT homozygotes. Neither did the two groups significantly differ in the allelic frequency (P = 0.11) .

Table 2. Genotype frequencies of IL-IB promoter at position -511 in 659 white subjects and comparison between 201 athletes and 458 non-athlete controls .

The distribution of the genotypes and alleles of the +3954 polymorphism of the gene IL-IB are shown in Table 3. Athletes and controls did not differ; in particular, 60.0% of athletes vs. 62.2% of controls were CC homozygotes, 36.1% of athletes vs. 32.3% of controls were CT heterozygotes , and 3.9% of athletes vs. 5.5% of controls were TT homozygotes. The two groups did not differ significantly in allele frequency [ P = 0 . 891

Table 3. Genotype frequencies of IL-IB exon 5 at position +3954 in 663 white subjects and comparison between 205 athletes and 458 non-athlete controls.

For the polymorphism IL-IRN "VNTR three alleles (alleles 1, 2 and 3) were common in the population included in the study; whereas allele 4 was observed in only 3 subjects (one genotype 1/4 and two genotypes 2/4, all in the control group), finally allele 5 was detected in only 1 subject (one genotype 1/5, in the group of athletes) . The most common allele, the allele IL-IRN*!, was less frequent in athletes than in controls (65.4% vs. 74.0%, P = 0.001) , whereas the second most common allele, IL-1RN*2, had greater allele frequency in athletes than in controls (32.2% vs. 22.9%, P < 0.001); finally, IL-1RN*3 had similar allele frequency in athletes and controls (P = 0.57) (overall alleles, P = 0.001) . Specifically, athletes were less likely to have the 1/1 genotype (OR = 0.55; 95% CI 0.40-0.77), and 2-fold more likely to have the 1/2 genotype (OR = 1.93; 95% CI 1.37-2.74) . Athletes and controls did not differ in frequency of the 2/2 genotype. However, increased frequencies of cumulative genotypes 1/2, 2/2, 2/3 and 2/4 were found in athletes (53.7%) with respect to controls (36.7%), OR = 2.00

(95% CI = 1.43-2.79), this suggests a dominant effect of the 2 allele. Genotypes which included allele 3 did not differ between athletes and controls.

Table 4. Genotype frequencies of IL-IRN VNTR in 663 white subjects and comparison between 205 athletes and 458 non-athlete controls .

In a further analysis, IL-IRN genotypes were compared among 53 professional athletes, 152 nonprofessional athletes and 458 non-athlete controls. The frequencies of the IL-IRN genotypes are shown in Table 5 (below) . It is interesting to note that the "VNTR 1/2 genotype was almost 2 -fold more frequent in professional athletes with respect to non-professional athletes (OR = 1.92, 95% CI = 1.02-3.61) . No other significant differences were observed between professional and non-professional athletes.

A comparison between professional and non-athletes showed that the 1/2 VNTR genotype was three times more frequent in high-grade athletes (OR = 3.12; 95% CI = 1.75-5.56) (see Table 5) . The same genotype, on the other hand, was 1.6 times more frequent in nonprofessional athletes with respect to non-athletes (OR = 1.62; 95% CI = 1.10-2.40) (Table 5) . The 1/1 genotype was 2.5 times more frequent in non-athlete controls with respect to professional athletes (OR = 2.52; 95% CI = 1.40-4.56), but 1.6 times more frequent in non- athletes with respect to non-professional athletes (OR = 1.61; 95% CI = 1.11-2.33) . Overall, the 1/1 genotype was 1.8 times more frequent in non-athlete controls than in all athletes (OR = 1.80, 95%; CI = 1.29-2.51) . Table 6 (below) shows the results of the analyses of the haplotype in athletes and controls, using all three polymorphism sites.

Table 6. Haplotype frequencies in athletes and non- athlete controls.

a The IL-IRN VNTR genotypes were grouped into the short allele S, allele 2) and long alleles (L, alleles 1, 3, 4, and 5) due to the low allelic frequency of alleles 3 , 4 and 5. The three sites haplotype was differently distributed between athletes and controls (P = 0.012) . Only one haplotype was associated with the athlete status. Specifically, ( -511) C- (+3954 ) T- (VNTR) 2 was three times more common in athletes than in non- athletes (OR = 3.02; 95%CI = 1.15-7.87) .

The three haplotypes with two sites were also analyzed and it was found that only those which included VNTR were significant, indicating that the association was correlated to VNTR or to a marker in linkage disequilibrium with it.

These observations were significant in male and female athletes when analyzed separately as shown by the results illustrated in the following Tables 7-9.

Table 7 A and B. Genotype Frequencies at position -511 of the IL-IB promoter in female athletes (A) and male athletes (B) .

Table 8 A and B. Frequencies of the genotype at position +3954 in exon 5 of IL-IB in female athletes (A) and male athletes (B) .

Table 9 A and B. Frequencies of the genotype IL-IRN VNTR in female athletes (A) and male athletes (B) .

The results show that the genotypes -511 and +3954 of the IL-IB gene do not influence the athletic phenotype (either in males or females) . This seems to agree with the observation of various authors according to whom, in vivo, neither the levels of circulating IL- lβ, nor those of IL-lβ mRNA are significantly altered by physical activity ( [7] , [1] , [36] ) . It is demonstrated, on the contrary, that the specific genotype of the IL- Ira gene, 1/2 VNTR IL-IRN is at least 2-fold more frequent in athletes than in non-athletes. Furthermore, a dose-effect relation was also observed, as the genotype 1/2 IL-IRN is twice more frequent in professional athletes than in nonprofessional athletes, and three times more frequent in professional athletes than in non-athletes. Therefore, the study indicates that the frequency of the genotype 1/2 IL-IRN increases parallelly with the athletic level. Importantly, no effect was observed, with respect to the gender, which could limit the applicability of the method according to the invention.

The polymorphism VNTR IL-IRN could favour the repair mechanisms of the muscle and muscular hypertrophy in athletes ( [8] , [12] ) . This could occur through a possible influence on the circulating or local levels of IL-lra in human beings ( [37] - [39] ) .

In the study, athletes were more frequently heterozygous, but non-homozygous for allele 2. As in vitro the VNTR allele 2 in intron 2 of IL-IRN was associated with an increase in the production of IL- lβ, in a dose -dependant manner, these results suggest that the presence of only one allele 2 IL-IRN can favour athletic performances through a moderate inflammatory activation, modulated by the levels of IL-Iβ and/or IL- lra, but not an excessive activation as in the case of the genotype 2/2 IL-IRN.

Coherently with what is indicated above, it is specified in the literature that the 2/2 IL-IRN genotype is more frequently associated with pathological conditions with an inflammatory or autoimmune nature with respect to the 1/2 IL-IRN genotype ( [33] , [40] - [46] ) , with particular reference to gastric cancer ( [42] , [46] ) .

It is known that there are different genetic profiles in the cluster of the IL-I gene among different ethnic groups, in particular between white and black populations [30] . Consequently, in the present study the results refer to a homogeneous population representative of the Caucasian ethnic group .

BIBLIOGRAPHY

[I] Petersen AM, Pedersen BK. J Appl Physiol 98: 1154-1162, 2005.

[2] Gleeson M. J Appl Physiol 103: 693-699, 2007. [3] Nieman DC, Henson DA, Smith LL, Utter AC, Vinci DM, Davis JM, Kaminsky DE, Shute M. J Appl Physiol 91: 109-114, 2001.

[4] Peake JM, Suzuki K, Hordern M, Wilson G, Nosaka K, Coombes JS. Eur J Appl Physiol 95: 514-521, 2005. [5] Tidball JG . Am J Physiol Regul Integr Comp Physiol 288: R345-353, 2005.

[6] Fielding RA, Manfredi TJ, Ding W, Piatarone MA, Evans WJ, Cannon JG. Am J Physiol 265: R166-172, 1993. [7] Mahoney DJ, Safdar A, Parise G, Melov S, Fu M, MacNeil L, Kaczor J, Payne ET, Tarnopolsky MA. Am J Physiol Regul Integr Comp Physiol 294: R1901-1910, 2008.

[8J Robertson TA, Maley MA, Grounds MD, Papadimitriou JM. Exp Cell Res 207: 321-331, 1993.

[9] Macarthur DG, North KN. Hum Genet 116: 331-339, 2005. [10] Rankinen T, Bray MS, Hagberg JM, Perusse L, Roth SM, Wolfarth B, Bouchard C. Med Sci Sports Exerc 38: 1863-1888, 2006.

[II] US2006/0121478

[12] Riechman SE, Balasekaran G, Roth SM, Ferrell RE. J Appl Physiol 97: 2214-2219, 2004.

[13] Oberbach A, Lehmann S, Kirsch K, Krist J, Sonnabend M, Linke A, Toenjes A, Stumvoll M, Bluher M, Kovacs P. Eur J Endocrinol 159: 129-136, 2008.

[14] Halverstadt A, Phares DA, Roth S, Ferrell RE, Goldberg AP, Hagberg JM. Biochim Biophys Acta 1734: 143-151, 2005.

[15] Walston J, Arking DE, Fallin D, Li T, Beamer B, Xue Q, Ferrucci L, Fried LP, Chakravarti A. Exp Gerontol 40: 344- 352, 2005. [16] Walsh S, Liu D, Metter EJ, Ferrucci L, Roth SM. J Appl Physiol 105: 1486-1491; 2008.

[17] Dinarello CA. Blood 87: 2095-2147, 1996.

[18] Gallucci S, Provenzano C, Mazzarelli P, Scuderi F, Bartoccioni E. Int Immunol 10: 267-273, 1998. [19] Dewberry R, Holden H, Crossman D, Francis S. Arterioscler Thromb Vase Biol 20: 2394-2400, 2000.

[20] Arend WP. Cytokine Growth Factor Rev 13: 323-340, 2002.

[21] Dinarello CA. N Engl J Med 343: 732-734, 2000.

[22] Dinarello CA. J Exp Med 201: 1355-1359, 2005. [23] Hurme M, Santtila S. Eur J Immunol 28: 2598-2602, 1998.

[24] Buchs N, di Giovine FS, Silvestri T, Vannier E, Duff GW, Miossec P. Genes Immun 2: 222-228, 2001.

[25] Di Giovine FS, Takhsh E, Blakemore Al, Duff GW. Hum MoI Genet 1: 450, 1992. [26] Pociot F, Molvig J, Wogensen L, Worsaae H, Nerup J. Eur J Clin Invest 22: 396-402, 1992.

[27] Tarlow JK, Blakemore AI, Lennard A, Solari R, Hughes HN, Steinkasserer A, Duff GW. Hum Genet 91: 403-404, 1993.

[28] Dennis RA, Trappe TA, Simpson P, Carroll C, Huang BE, Nagarajan R, Bearden E, Gurley C, Duff GW, Evans WJ, Kornman K, Peterson CA. J Physiol 560: 617-626, 2004.

[29] Duff GW. Am J Clin Nutr 83: 431S-435S, 2006.

[30] Pillay V, Gaillard MC, Halkas A, Song E, Dewar JB. Cytokine 12: 819-821, 2000. [31] Tishkoff SA, Williams SM. Nat Rev Genet 3: 611-621, 2002.

[32] Cauci S, Di Santolo M, Casabellata G, Ryckman K, Williams SM, Guaschino S. MoI Hum Reprod 13: 243-250, 2007. [33] Bioque G, Crusius JB, Koutroubakis I, Bouma G, Kostense PJ, Meuwissen SG, Pena AS. Clin Exp Immunol 102: 379-383, 1995.

[34] Liu K, Muse SV. Bioinformatics 21: 2128-2129, 2005. [35] Dudbridge F. Hum Hered 66: 87-98, 2008. [36] Suzuki K, Peake J, Nosaka K, Okutsu M, Abbiss CR, Surriano R, Bishop D, Quod MJ, Lee H, Martin DT, Laursen PB. Bur J Appl Physiol 98: 525-534, 2006.

[37] Melzer D, Perry JR, Hernandez D, Corsi AM, Stevens K, Rafferty I, Lauretani F, Murray A, Gibbs JR, Paolisso G, Rafiq S, Simon-Sanchez J, Lango H, Scholz S, Weedon MN, Arepalli S, Rice N, Washecka N, Hurst A, Britton A, Henley W, van de Leemput J, Li R, Newman AB, Tranah G, Harris T, Panicker V, Dayan C, Bennett A, McCarthy MI, Ruokonen A, Jarvelin MR, Guralnik J, Bandinelli S, Frayling TM, Singleton A, Ferrucci L. PLoS Genet 4: el000072, 2008.

[38] Rafiq S, Stevens K, Hurst AJ, Murray A, Henley W, Weedon MN, Bandinelli S, Corsi AM, Guralnik JM, Ferrucci L, Melzer D, Frayling TM. Genes Immun 8: 344-351, 2007. [39] Reiner AP, Wurfel MM, Lange LA, Carlson CS, Nord AS, Carty CL, Rieder MJ, Desmarais C, Jenny NS, Iribarren C, Walston JD, Williams OD, Nickerson DA, Jarvik GP. Arterioscler Thromb Vase Biol 28: 1407-1412, 2008. [40] Blakemore AI, Cox A, Gonzalez AM, Maskil JK, Hughes ME, Wilson RM, Ward JD, Duff GW. Hum Genet 97: 369-374, 1996. [41] Crusius JB, Pena AS, Van Oosten BW, Bioque G, Garcia A, Dijkstra CD, Polman CH. Lancet 346: 979, 1995. [42] El-Omar EM, Carrington M, Chow WH, McCoIl KE, Bream JH, Young HA, Herrera J, Lissowska J, Yuan CC, Rothman N, Lanyon G, Martin M, Fraumeni JF, Jr., Rabkin CS. Nature 404: 398- 402, 2000.

[43] Gremlich S, Fratta S, Rebellato E, Uras R, Reymondin D, Damnon F, Germond M, Gerber S. Hum Reprod 23: 1200-1206, 2008. [44] Langdahl BL, Lokke E, Carstens M, Stenkjaer LL, Eriksen EF. J Bone Miner Res 15: 402-414, 2000.

[45] Van der Paardt M, Crusius JB, Garcia-Gonzalez MA, Baudoin P, Kostense PJ, Alizadeh BZ, Dijkmans BA, Pena AS, van der Horst-Bruinsma IE. Rheumatology (Oxford) 41: 1419- 1423, 2002.

[46] Witkin SS, Gerber S, Ledger WJ. Clin Infect Dis 34: 204-209, 2002.

Claims

1. Method for the determination of the athletic predisposition in the Caucasian population comprising the following steps: a) genotyping of a subject for the presence of at least one polymorphic site located in the IL-IRN gene (Gene Bank, ID: 3557) encoding for the receptor antagonist of interleukin IL-I (IL-lra) , alone or in association with at least one polymorphic site located in the cytokine IL-lβ gene, JL-IB (Gene Bank, ID: 3553); b) predicting the athletic phenotype on the basis of the presence of the one or more polymorphic sites.

2. Method according to claim 1, wherein said genotyping is carried out by allele-specif ic amplification, DNA sequencing, allele-specific hybridisation, and/or RT- PCR analysis.

3. Method according to anyone of the claims 1-2, wherein said at least one polymorphic site of IL-IRN gene consists of pentaallelic polymorphic site at the level of intron 2 containing a variable number of 86 tandem repeats (VNTR) and said at least one polymorphic site of IL-IB gene consists of the transition of C/T bases at position -511 of promoter or at position +3954 of exon 5 of IL-IB.

4. Method according to anyone of the claims 1-3, wherein the pentaallelic polymorphism amplification at the level of intron 2 containing a variable number of 86 tandem repeat (VNTR) is carried out by the following amplification primer pair i) S'-CTCAGCAACACTCCTAT- 3' (SEQ ID No:l) and 5' -TCCTGGTCTGCAGGTAA-BMSEQ ID No: 2) .

5. Method according to anyone of the claims 1-4, wherein the amplification of the DNA region comprising the IL-IB promoter is carried out by the following amplification primer pair ii) S'-TGGCATTGATCTGGTTCATC-S' (SEQ ID No:3) and 5' -GTTTAGGAATCTTCCCACTT-B' (SEQ ID No : 4 ) followed by the assessment of the polymorphism consisting in the base transition C/T at position -511 of IL-IB promoter by restriction enzymes or sequencing.

6. Method according to anyone of the claims 1-5, wherein the amplification of DNA region comprising the IL-IB exon 5 is carried out by the following amplification primer pair iii) δ'-GTTGTCATCAGACTTTGACC- 3' (SEQ ID No: 5) and δ'-TTCAGTTCATATGGACCAGA-S' (SEQ ID No: 6) followed by the assessment of the polymorphism consisting in the base transition C/T at position +3954 of IL-IB exon 5.

7. Method according to anyone of the claims 1-6, wherein said primers are labelled with a fluorophore, biotin, radioisotope.

8. Method according to anyone of the claims 1-7, wherein the presence of allele 2 at the level of IL-IRN gene intron 2 containing VNTR is positively associated to the athletic phenotype .

9. Method according to anyone of the claims 1-8, wherein the presence of genotype 1/2 IL-IRN VNTR is associated to a high athletic predisposition.

10. Method according to anyone of the claims 1-8, wherein the presence of genotype l/l IL-IRN VNTR is negatively associated to the athletic phenotype .

11. Method according to anyone of the claims 1-9, wherein the presence of the three sites haplotype (- 51I)C- (+3954) T- (VNTR) 2 is associated to a high athletic predisposition.

12. Method according to anyone of the claims 1-9, wherein the presence of the two site haplotype (+3954)T- (VNTR) 2 or three sites haplotype (-511) T- (+3954) T- (VNTR) 2 is associated to a medium-high athletic predisposition.

13. Method according to anyone of the claims 1-9, wherein the presence of the two sites haplotype (- 51I)C- (VNTR) 2 or ( -511) T- (VNTR) 2 is associated to a medium athletic predisposition.

14. Method according to anyone of the claims 1-9, wherein the presence of the two sites haplotype (+3954) C- (VNTR) 2 is associated to a moderate athletic predisposition.

15. Method according to anyone of the preceding claims, wherein the presence of a polymorphism in linkage disequilibrium with allele 2 of VNTR polymorphism at the level of intron 2 of IL-IRN gene is positively associated to the athletic phenotype.

16. Kit for the determination of athletic predisposition in the Caucasian population comprising at least a primer pair for the amplification of the pentaallelic polymorphism at the level of intron 2 containing a variable number of 86 tandem repeat ( VNTR ) .

17. Kit for the determination of athletic predisposition in the Caucasian population comprising the following components: a) primer pair for the amplification of the pentaallelic polymorphism at the level of intron 2 containing a variable number of 86 tandem repeat

CVNTR) ; b) primer pair for the amplification of the polymorphism consisting in the base transition C/T at position -511 of the promoter of IL-IB; c) primer pair for the amplification of the polymorphism consisting in the base transition C/T at position +3954 of exon 5 of JL-IB.

18. Kit according to anyone of the claims 16-17 wherein said primer pair a) consists in:

- 5' -CTCAGCAACACTCCTAT-B' (SEQ ID Nθ:l) and

- 5' -TCCTGGTCTGCAGGTAA-S' (SEQ ID No: 2) ; said primer pair b) consists in: - 5' -TGGCATTGATCTGGTTCATC-S' (SEQ ID No: 3) and

- 5' -GTTTAGGAATCTTCCCACTT-B' (SEQ ID NO :4) ; said primer pair c) consists in:

- 5' -GTTGTCATCAGACTTTGACC-S' (SEQ ID No: 5) and

- 5' -TTCAGTTCATATGGACCAGA-S' (SEQ ID No: 6) .

19. Immunologic kit for the determination of athletic predisposition in the Caucasian population comprising at least one monoclonal or polyclonal antibody specific for the protein IL-lra.

20. Use of monoclonal or polyclonal antibodies specific for the IL- Ira protein for the quantitative analysis of said protein levels for the detection of athletic predisposition in the Caucasian population.

21. Use of the genotype 1/2 IL-IRN as a marker for the detection of high level athletic predisposition.

22. Use of the three sites haplotype ( -511) C- (+3954 ) T- (VNTR) 2 as a marker for the detection of high level athletic predisposition.