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1 From the Division of Genomic Medicine, University of Sheffield School of Medicine and Biomedical Science, Sheffield, United Kingdom
2 Presented at the conference "Living Well to 100: Nutrition, Genetics, Inflammation," held in Boston, MA, November 15–16, 2004. 3 Reprints not available. Address correspondence to GW Duff, Division of Genomic Medicine, Medical School, University of Sheffield, Beech Hill Road, Sheffield, S10 2RX, United Kingdom. E-mail: g.w.duff{at}sheffield.ac.uk.
ABSTRACT
For many chronic diseases, the influence of genetics is subtle and complex and does not conform to simple Mendelian patterns of inheritance as is seen with single-gene disorders. Genetic variation can influence the propensity for the initiating event, the progression to a clinical disease state, and the trajectory of disease. One example of how genetic variations may affect complex diseases is provided by the interleukin 1 family of cytokines. This cytokine family plays a key role in mediating inflammation, which is a central component of many chronic diseases, including coronary artery disease and rheumatoid arthritis. Recent research has identified many sequence variations in the regulatory DNA of the genes coding for important members of the interleukin 1 family, and these variations are associated with differential effects on the inflammatory response. These in turn alter the risk of some diseases in which inflammation plays a role and also affect physiologic responses, such as the inflammatory response to exercise. As this new genetic knowledge is developed and extended, it may be possible to make health care interventions at an earlier stage, before clinical disease is established, rather than after tissues have been permanently damaged.
Key Words: Inflammation • interleukin 1 • cytokines • genetic variation • single-nucleotide polymorphism • haplotype • cardiovascular disease • rheumatoid arthritis • ankylosing spondylitis • inflammatory response to exercise
ROLE OF GENETIC VARIATION IN DISEASE SUSCEPTIBILITY AND PROGRESSION
Many common diseases run in families, but although they are inherited to some extent, their inheritance does not conform to simple Mendelian patterns. With Mendelian genetics, the interaction between genes and environment that creates the phenotype is dominated by the genetic component. With many of the "single-gene" diseases that show Mendelian patterns of inheritance, eg, sickle cell disease or cystic fibrosis, the diseases are often rare and the first signs are evident in childhood. In the case of common diseases, what is inherited is a higher risk of developing the disease rather than the certainty of disease.
In common multifactorial diseases, the interaction between genes and the environment is subtle and complex: susceptibility genes modulate the effect of environmental risk factors, making the initial pathologic event more or less likely (Figure 1). Whether this pathologic event triggers the development of a clinically detectable disease, and how fast the disease develops, is influenced by genetic modifiers that exaggerate or suppress disease progression.
FIGURE 1.. Paradigm to describe the interactions between genes and the environment in the production of the clinical phenotype for multifactorial diseases.
One example in which genes may act as both susceptibility factors and modifiers for some disease states can be found in the cytokine system. Cytokines are intercellular signal molecules that coordinate the host inflammatory response to injury. They include interleukins, tumor necrosis factors, interferons, and hematopoietic growth factors. The inflammatory response is usually tightly controlled, so the response is commensurate with the challenge, eradicating pathogens and repairing injured tissues yet limiting the cost to the host. But inflammation is also an important component in the pathogenesis of many chronic diseases; cytokines, for example, have been identified as key mediators in diseases such as rheumatoid arthritis (1-3), asthma (4, 5), psoriasis (6, 7), ulcerative colitis (8), periodontitis (9, 10), diabetes (11), coronary heart disease (12), postmenopausal osteoporosis (13, 14), and Alzheimer disease (15, 16).
It is possible to hypothesize that genetic variation affecting the activity of certain cytokine genes may produce individuals with a more exaggerated or prolonged inflammatory response. Such individuals may be more susceptible to inflammatory diseases, and evidence is growing that genetic variations in the inflammatory pathways do in fact influence the susceptibility and clinical severity of these diseases.
THE INTERLEUKIN 1 CYTOKINE FAMILY
Interleukin 1 (IL-1) plays an important role in the up- and down-regulation of the acute inflammatory response. It also plays a major role in a wide range of inflammatory and autoimmune diseases (17, 18). Although at least 10 members of the IL-1 family are now known (19, 20), 3 major components have been well studied: the proinflammatory agonists IL-1 and IL-1ß (encoded by the genes IL-1A and IL-1B, respectively) and the antiinflammatory protein IL-1 receptor antagonist (IL-1Ra, encoded by the gene IL-1RN) (19, 21). The genes of the IL-1 family have all been mapped to a 430-kb section of DNA on the long arm of human chromosome 2 (Figure 2; 22).
FIGURE 2.. Relative positioning of the genes for interleukin 1 (IL-1A), IL-1ß (IL-1B), and IL-1 receptor antagonist (IL-1RN) on the long arm of chromosome 2. Hatched areas denote exons. kb, kilobase.
The actions of IL-1 appear to be mediated by a relatively well-known biochemical pathway. IL-1 binds to the type 1 IL-1 receptor on the cell surface to form a complex. The cellular membrane IL-1 receptor-associated protein (IL-1RAcP) then binds to the complex, initiating intracellular signaling pathways, such as the B kinase pathway, or those involving various small GTPases (23). This results in the activation of transcription factors that in turn increases the expression of proinflammatory genes encoding chemokines, cytokines, acute-phase proteins, cell adhesion molecules, degradative metalloproteinases, and other enzymes (23). IL-1Ra, on the other hand, acts as a competitive inhibitor by binding to the IL-1 receptor without recruiting IL-1RAcP to the complex, therefore not inducing any intracellular signaling response (21).
INFLUENCE OF GENETIC VARIATION ON DISEASE: IL-1RN DELETION IN MICE
Some years ago, a study investigated whether knocking out the IL-1RN gene would reveal the relevant pathophysiologic effects of IL-1. That is, if the negative half of this finely balanced system were removed, what would be the consequences of the unopposed activities of the positive IL-1 and IL-1ß ligands (24)? The results of this study were, at the time, quite surprising. Most of the mice with an active IL1-RN gene survived for the 550 d of the study and were healthy throughout. Their IL-1RN knockout littermates with unopposed IL-1 biological activity, on the other hand, showed an excess mortality: 93% died or were culled on humane grounds by the end of the study, with a median age at death of 159 d. Between 200 and 400 d from birth, the odds ratio for mortality exceeded 10. It is important to note that the IL1-RN-deficienct mice were not subjected to any unusual environmental challenges and were reared the same as their littermates.
Autopsies showed that the IL-1RN knockout mice had major inflammatory lesions in the large arteries, particularly in the aorta and its primary and secondary branches at bifurcations and flexures. Some mice had ruptured aortic aneurisms, indications of internal hemorrhage, previous myocardial infarctions, and interstitial nephritis. Interestingly, the veins, capillaries, and the pulmonary circulation, the low-pressure parts of the vascular system, were not affected. In another study involving a mouse with a different genetic background, however, deleting the IL-1RN gene did not seem to affect the arteries (25, 26). Instead, the synovium of the joints was inflamed, resulting in a destructive arthritis similar to what is seen in human rheumatoid arthritis.
The results of these studies seem to indicate that removing one of the body's natural mechanisms to dampen the immune response to injury (by knocking out the IL-1RN gene) results in the appearance of immune-related chronic diseases, although the ways in which this was manifested differed between the 2 mouse strains. Also, it can be deduced that the IL-1 gene system plays a role in maintaining healthy arteries and joints. These are structures that are exposed to constant mechanical stress in life and are likely to require a continuous repair response. Disrupting the balance of this system seems to accelerate degenerative diseases, at least in mice, and perhaps in humans.
SINGLE-NUCLEOTIDE POLYMORPHISMS AND THE HAPLOTYPE PHENOMENON
The studies described above involved the functional ablation of an entire gene of the IL-1 family. However, much of the variation in genomes between individuals from the same species is due to much smaller changes—single base changes called single-nucleotide polymorphisms (SNPs).
There are an estimated 7–10 million SNPs in the 3 billion–nucleotide human genome (27). Fortunately, SNPs are often inherited together and travel as blocks from generation to generation, which is known as linkage disequilibrium. Linkage disequilibrium can be described as the inheritance of same-chromosome SNPs together at a frequency greater than chance. Groups of co-inherited SNPs on the same chromosome are called haplotypes, and the haplotype phenomenon means that the number of tests necessary to map a particular chromosomal region can be reduced by using marker SNPs rather than having to map the entire region. For the IL-1 gene cluster, for example, Cox et al (28) identified a common, 8-locus haplotype of the IL-1 gene cluster with strong linkage disequilibrium that allowed for a more rational design to genetic studies of IL-1 polymorphisms and their relation to disease.
In 2002 we took 25 individuals of diverse ethnicity, sequenced their DNA across the entire IL-1 region, and then used 48 equally spaced SNPs across the region to make a detailed gene map to determine linkage disequilibrium (29). Areas of high linkage disequilibrium were identified, interspersed with areas of low linkage disequilibrium. Therefore, distinct haplotypes within the IL-1 gene cluster can be detected and characterized, and the population frequencies of different haplotype patterns can be determined.
Some evidence suggests that IL-1 polymorphism may influence gene expression. Shirodaria et al (30) reported that IL-1 concentrations in the gingival crevicular fluid of patients with severe periodontal disease was almost 4-fold higher in carriers of IL-1A(–889) allele 2 than in those who were homozygous for allele 1. In healthy blood donors, plasma IL-1ß concentrations were significantly higher in those homozygous for the IL-1A(–889) allele 2 than in heterozygotes (P < 0.008) and those homozygous for allele 1 (P < 0.02) (31). The IL-1B(+3954) allele 2 has also been associated with increased IL-1 production (32).
INFLUENCE OF GENETIC VARIATION ON DISEASE: ASSOCIATION BETWEEN IL-1 GENE POLYMORPHISM AND CHRONIC DISEASES
The question that then arises is do SNPs, such as those that exist in the IL-1 gene cluster, show a clinically significant association with disease risk and progression? Rheumatoid arthritis (RA) is an example of a multifactorial inflammatory disease with some degree of familial clustering, and it is well established that genes of the major histocompatibility complex, closely linked with HLA-DRB1, account for >30% of the genetic component of the disease (33).
Cox et al (34) used transmission disequilibrium testing to study linkage between genes in the IL-1 gene cluster and RA. A total of 195 families from the Arthritis Research Campaign's National Repository in the United Kingdom were genotyped for 9 polymorphic markers and genes in the IL-1 gene cluster. Although no single allele showed a significant association with development of RA per se, there was evidence for linkage of several markers in the IL-1 gene cluster in a subset of patients with erosive RA (rapid loss of cartilage and bone) and who were heterozygous for the HLA-DRB1 allele (Figure 3). This study provided preliminary evidence suggesting that the IL-1 gene cluster is linked to erosive RA.
FIGURE 3.. Linkage association of interleukin 1 (IL-1) gene markers with erosive rheumatoid arthritis in families heterozygous for the HLA DRB1 allele 1. Data are from Cox et al (34). The horizontal axis represents the position along the chromosome of the tested markers. The vertical axis is a measure of association. The line for statistical significance is shown. It can be seen that a significant association with erosive rheumatoid arthritis occurred across the region containing IL-1A, IL-1B, and IL-1RN. kb, kilobase.
Recently, it was also shown through the use of transmission disequilibrium testing that the IL-1 gene cluster contains a major susceptibility locus for another kind of arthritis, ankylosing spondylitis (AS) (35). AS is a common, heritable inflammatory arthropathy. Although the gene HLA-B27 is almost essential for its inheritance, it is not sufficient to explain the familial pattern of occurrence of the disease (36). There was previously a suggestive linkage of AS to chromosome 2q12-13, which is the region containing the IL-1 gene cluster (37). To confirm that linkage, 227 white, British affected sibling-pair families and 317 parent-case trios with AS and 200 healthy blood donors were studied (35). There were 930 cases (66% male), who were on average 43.9 y old and had been affected for an average of 21.6 y. The study showed a statistically significant association between markers in the IL-1A, IL-1B, and IL-RN genes and AS, thus strengthening the link between the IL-1 region and AS (35).
Polymorphisms of the IL-1 gene cluster have also been associated with an increased risk of coronary artery disease, a condition now intimately associated with inflammation (for review, see reference 38) (12, 17, 39). Berger et al (P Berger, M Nunn, K Stephenson, J Sorrell, F di Giovine, G Duff, unpublished observations, 2000) examined 504 patients referred for angiography for chest pain on exertion. Patients were classified as having 1-, 2-, or 3-vessel disease if angiography showed >50% stenosis; patients with no significant disease or mild disease (<30% stenosis) were classified as controls. Odds ratios for cholesterol (1.74), lipoprotein(a) (2.55), and smoking (3.92) were as expected. Strikingly, patients who were homozygous for a common IL-1 gene polymorphism, IL-1B(–511) allele 2, had an odds ratio that was almost as high as that for smoking (3.88; Figure 4).
FIGURE 4.. Odds ratios for risk of coronary artery occlusion associated with cholesterol, lipoprotein(a) [Lp(a)], smoking, and IL-1B(+3954) allele 2 homozygosity in 504 patients examined for chest pain on exertion. IL-1, interleukin 1. Data from Berger et al (P Berger, M Nunn, K Stephenson, J Sorrell, F di Giovine, G Duff, unpublished observations, 2000).
Furthermore, when the patients were stratified according to total cholesterol (<200, 200–239, and >239 mg/dL) and IL-1 genotype, a distinct interaction was apparent: IL-1 genotype had no effect on the odds ratios for patients with total cholesterol concentrations <200 mg/dL, whereas for patients with total cholesterol concentrations >239 mg/dL, the odds ratio increased from 2.7 in patients who were homozygous for IL-1B(–511) allele 1 to 5.91 for patients who were homozygous for IL-1B(–511) allele 2 (P Berger, M Nunn, K Stephenson, J Sorrell, F di Giovine, G Duff, unpublished observations, 2000). A similar pattern was seen for LDL cholesterol. In a further analysis of this study, patients who were homozygous for allele 2 of the IL-1B(+3954) SNP had twice the median C-reactive protein concentrations as did patients who were homozygous for allele 1 (4.33 and 2.01 mg/L, respectively; P = 0.001).
A separate study, involving 1850 patients, tested for association between IL-1RN polymorphism and the risk of restenosis after coronary stent implantation (40). Carriers of IL-1RN(+2018) allele 2 had a 22% lower risk of restenosis 6 mo after implantation (odds ratio: 0.78; 95% CI: 0.63, 0.97). In addition, there was a significant interaction between the presence of allele 2 and age, with a progressively stronger protective effect in younger patients.
INFLUENCE OF GENETIC VARIATION ON REPAIR: ASSOCIATION BETWEEN IL-1 GENE POLYMORPHISM AND INFLAMMATORY RESPONSE TO EXERCISE
IL-1 genotype also appears to be related to the inflammatory muscle response after exercise (41). Inflammation appears to play an important role in the repair and regeneration of skeletal muscle damage that occurs with exercise.
Dennis et al (41) tested the hypothesis that the severity of the inflammatory response in muscle after an acute bout of resistance exercise is associated with SNPs previously shown to alter IL-1 activity. From 100 young men screened for their haplotype pattern in the IL-1 gene cluster, 24 were selected and performed standard resistance leg exercises. Muscle biopsies were performed before and 24 and 72 h after exercise for measurement of inflammatory marker messenger RNA levels (IL-1ß, IL-6, and tumor necrosis factor ) and CD68+ macrophage levels. Subjects homozygous for IL-1B(+3954) allele 1 or IL-1B(–3737) allele 2 had an 2-fold higher median induction of inflammatory markers, but no increase in macrophages, indicating an increased cytokine production per macrophage. The IL1-RN(+2018) SNP maximized the response specifically within these groups and was associated with increased macrophage recruitment. This study showed for the first time that IL-1 genotype is associated with the inflammation of skeletal muscle after acute resistance exercise that may potentially affect the adaptation to chronic resistance exercise (41).
CONCLUSION
In summary, the IL-1 gene region acts as a good example of how genetic variation may influence disease manifestation and progression. The 3 known major genes, IL-1A, IL-1B, and IL-1RN, are all polymorphic, and clear haplotypes can be identified within the gene cluster and across individual genes. These haplotypes show functional differences in terms of messenger RNA and IL-1 production and in relation to other downstream biomarkers of inflammation, such as C-reactive protein (PB Berger, M Nunn, K Stephenson, GW Duff, GG Kee, JP McConnell, unpublished observations, 2001). Furthermore, in several inflammatory diseases, genetic linkage and association has been detected with the IL-1 region, and IL-1 genes have been associated with the physiologic repair responses in healthy muscle after exercise.
Most of modern medical practice is concerned with the treatment of patients once clinical disease has emerged; much less opportunity or capacity exists to prevent disease development at the preclinical stage. With an improved understanding of the genetic basis of multifactorial diseases and the interactions between genes and the environment, it may become possible to predict susceptibility to diseases and to make positive interventions in susceptible individuals before irreparable damage occurs to tissues and organs. In this way, perhaps at an individual level, we can envisage a rational approach to disease prevention in the future.
ACKNOWLEDGMENTS
GD is a consultant to Interleukin Genetics Inc and owns shares in the company. Interleukin Genetics has patents issued and pending on the use of IL-1 and tumor necrosis factor genetics as risk factor tests for various diseases with inflammatory components.
REFERENCES