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Biological Research

versão impressa ISSN 0716-9760

Biol. Res. v.34 n.3-4 Santiago  2001

http://dx.doi.org/10.4067/S0716-97602001000300011 

Genetic polymorphism at position -308 in the promoter
region of the tumor necrosis factor (TNF): Implications
of its allelic distribution on susceptibility or resistance to
diseases in the Chilean population

JIMENA CUENCA1, CLAUDIO A. PÉREZ1, ADAM J. AGUIRRE1, IRENE SCHIATTINO2 and J. CARLOS AGUILLÓN 1

1Disciplinary Immunology Program, Institute of Biomedical Sciences (ICBM),
Faculty of Medicine, University of Chile, Santiago de Chile
2School of Public Health, Faculty of Medicine, University of Chile. Santiago de Chile

 

Corresponding author: Dr. Juan Carlos Aguillón. Disciplinary Immunology Program, ICBM, Faculty of Medicine University of Chile. Independencia 1027, Santiago. Casilla 13898, Correo 21. Telephone: (562) 678-6724. Fax: (562) 735-3346. E-mail: jaguillo@machi.med.uchile.cl

Received: September 7, 2001. In Revised Form: September 24, 2001. Accepted: September 26, 2001

 

ABSTRACT

Several single-nucleotide polymorphisms have been identified in the human TNF gene promoter. The polymorphism at position _308 (TNF-308), which involves substituting G for A and designing the TNF2 allele, leads to a higher rate of TNF gene transcription than the wild-type TNF1 allele in in vitro expression studies. It has also been linked to increased susceptibility to a variety of illnesses. Using PCR-RFLP analysis we detected significant differences in the TNF-308 genotypes of Chilean and other populations. We conclude that there is a gradient in the distribution of the TNF2 allele according to ethnicity; we have also hypothesized that populations bearing a higher proportion of the TNF2 allele may have an increased predisposition toward or incidence of several chronic metabolic, degenerative, inflammatory and autoimmune diseases.(Biol Res 2001; 34 3-4: 237-241)

 

Cytokines are expressed during the effector stages of the innate and acquired immunity by a large variety of cells and tissues. Although cytokines act at very low concentrations (pg/ml), their effect is closely related to their circulating levels. Thus, deregulation of the gene expression that increases the cytokine production may alter the homeostasis of the organism, resulting in organ-specific or even systemic failures. Cytokine unbalance is responsible for the pathogenesis of diverse infections and inflammatory diseases, and the Tumor Necrosis Factor (TNF), among other cytokines, has been described to play a central role in these processes (Beutler and Grau, 1993).

TNF belongs to a family of proteins that includes lymphotoxin a (LTa) and lymphotoxin ß (LTß). Although T-cells can produce TNF, activated monocytes and macrophages are the major source of TNF, which is synthesized as a 20 kDa pro-protein and cleaved by TNF, converting the enzyme to a 17 kDa monomer. Under physiological conditions, TNF circulates as a stable cone-shaped homotrimer that mediates its effects by binding to two receptor molecules, TNFRI (p55) and TNFRII (p75). TNFRI is thought to be the dominant pathway and has been implicated in the majority of known TNF effects, including induction on macrophage activity, up-regulation of adhesion molecules, and nuclear factor kB activation (Smith et al., 1994).

By the mid 1980s, the TNF protein had been purified and its gene cloned, sequenced, and mapped to the Major Hystocompatibility Complex (MHC) Class III region on the short arm of chromosome six. The TNF gene is tandemly arranged with that for LTa and LTß within the TNF locus, a 7 kb region 250 kb centromeric to the HLA B locus, 400 kb telomeric to the C2/BF locus and 1000 kb from the MHC Class II DR genes (Dunham et al., 1987).

TNF is associated with a broad spectrum of biological effects, and is the first factor involved in the cytokine cascade that promotes inflammation and essential for activating macrophages in host defense against invading microbes during infection. Thus, TNF can mediate both beneficial and deleterious effects depending on the nature of the disease process. TNF is now recognized to be involved in stimulating cytokine production, enhancing the expression of adhesion molecules and neutrophil activation. It is also a co-stimulator for T-cell activation and antibody production by B cells (Vassalli, 1992).

Although the circulating TNF levels are highly variable (Aguillón et al., 2001), the up-regulation of TNF gene expression has been involved in the pathogenesis of a large variety of illnesses with inflammatory and autoimmune components, some of which are associated with the MHC, including systemic lupus erythematosus (Jacob et al., 1990), rheumatoid arthritis (Breunan et al., 1992), inflammatory bowel diseases (Bouma et al., 1996), and ankylosing spondylitis (Rudwaleit et al., 2001). Another group includes acute and chronic infectious processes, such as septic shock syndrome (Tracey and Cerami, 1993), cerebral malaria (McGuire et al.,1994), and acquired immunodeficiency syndrome (Hober et al., 1989).

TNF production may be regulated at the transcriptional, post-transcriptional, and translational levels. It has been suggested that variability in the promoter and coding regions of the TNF gene may modulate the magnitude of the secretory response of this cytokine (Bouma et al.,1996). Polymorphisms in the TNF gene may affect transcriptional regulation, because levels of secretion of TNF by human monocytes and peripheral blood mononuclear cells in vitro are associated with extended HLA haplotypes and microsatellite alleles linked to TNF in the MHC Class III region (Bouma et al., 1996). Several single-nucleotide polymorphisms have been identified in the human TNF gene promoter. The most important has been identified at position _308 (TNF-308), involving the substitution of guanine (G) for adenine (A) (Wilson et al., 1992). This polymorphism, which designed the TNF2 allele, leads to a higher rate of TNF gene transcription than the wild-type TNF1 allele in in vitro expression studies, and it has been linked to an increased susceptibility to a variety of illnesses (Abraham and Kroeger, 1999).

The purpose of this work is to study the allelic distribution for the polymorphism TNF-308 in the Chilean population with respect to other ethnic origins, and our hypothesis is that this genetic variation may influence resistance or susceptibility to specific diseases. To this aim, we examined the distribution of the TNF-308 genotypes on healthy individuals from the Metropolitan Area of Santiago de Chile (166 individuals, 131 women and 35 men with a mean age of 36 years and a range of 19-71) by polimerase-chain reaction (PCR)-restriction fragment length polymorphism (Wilson et al., 1992). Briefly, a 107 bp region of the TNF gene was amplified using the following primers: 5'-AGG CAA TAG GTT TTG AGG GCC AT-3' and 5'-TCC TCC CTG CTC CCG GAT TTC CG-3'. PCR was performed by 30 cycles at 94ºC, 60ºC, 72ºC for 30s, 35s and 1 min, respectively. Finally, an extension cycle at 72ºC for 10 min was done. Nco I digestion of the amplified product and subsequent electrophoresis revealed the two alleles of TNF-308: TNF1 (G/G genotype -fragments of 87 bp and 20 bp-), TNF2 (G/A genotype -fragments of 107 bp, 87 pb and 20 bp- A/A genotype -fragment of 107 bp-). For the statistical analysis we used the One-sample Test for Binomial Proportions.

As shown in Table I, we observed a significant difference in the TNF-308 genotype frequencies of the Chilean population when they were compared to Caucasian TNF-308 genotypes (American, Australian, Danish, English, German and, Swedish). No differences were observed with the Chinese TNF-308 genotypes. When the comparison was made with French, Spanish and West African TNF-308 genotypes only the A/A genotype showed a significant difference. The comparison with the Japanese TNF-308 genotype provided a significant difference only for the G/G and G/A genotypes. From the data summarized in Table I, we can conclude that there is a gradient in the distribution of the TNF2 allele according to the ethnicity. Thus, Caucasians and Japanese have the higher and lower percentage of TNF2 allele, respectively. The percentage of TNF2 alleles observed in French, Spanish, and West African populations is very similar, although lower than Northern European Caucasians. The Japanese, Chilean, and Chinese populations showed the lowest percentage rates of the TNF2 allele. Although the presence of G/G and G/A genotypes in the French, Spanish and West African population are similar to the Chilean population, the difference resides in the A/A genotype. Surprisingly, the distribution of the TNF2 allele in the Chilean population is more closely related to the Chinese than the Spanish population.

As mentioned above, the presence of the TNF2 allele is associated with increased susceptibility to a variety of illnesses, such as inflammatory bowel disease (Bouma et al., 1996), rheumatoid arthritis (Danis et al., 1995), susceptibility and mortality by septic shock (Mira et al., 1999), systemic lupus erythematosus (Rood et al., 2000), Alzheimer's disease (Collins et al., 2000), and ankylosing spondylitis (Rudwaleit et al., 2001) among others. Interestingly, all these associations described so far in Caucasian populations are consistent with the differences in the genotype frequencies observed in the population where the polymorphism TNF-308 has been studied (Table I). We hypothesize that populations bearing a higher proportion of the TNF2 allele may have an increased predisposition toward or incidence of several chronic metabolic, degenerative, inflammatory and, autoimmune diseases.

Table I
Frequency of polymorphism -308 of the TNF promoter in different populations.


POPULATION

  GENOTYPE FREQUENCIES (%) SOURCE
STUDIED
G/G
p-v
G/A
p-v
A/A
p-v
 

Chilean
American
Australian
Chinese
Danish
English
French
German
Japanese
Spanish
Swedish
West African

83.1
68.2*
58.0*
83.2
65.8*
59.0*
81.6
67.0*
97.0*
82.4
56.5*
81.6

-
0.00
0.00
0.99
0.00
0.00
0.69
0.00
0.00
0.82
0.00
0.61

16.3
29.1*
39.0*
15.7
30.5*
37.0*
17.2
28.0*
3.0*
15.7
40.5*
17.0

-
0.00
0.00
0.75
0.00
0.00
0.91
0.00
0.00
0.77
0.00
0.94

0.6
2.7*
3.0*
1.1
3.7*
3.0*
1.2*
5.0*
0.0
1.9*
3.0*
1.4*

-
0.00
0.00
0.48
0.00
0.00
0.00
0.00
0.89
0.00
0.00
0.00
Cuenca et al., 2001
Wingerchuk et al., 1997
Milner et al., 1999
Lee et al., 2000
Rasmussen et al., 2000
Keatings et al., 2000
Mira et al., 1999
Höhler et al., 1998
Ishii et al., 2000
Vinasco et al., 1997
Rosmond et al., 2001
Conway et al., 1997

* Statistically significant (p-value ­ 0.05)

Data available in the literature show a definitive positive association to the TNF2 allele in at least two models of disease. The first example is rheumatoid arthritis, where pathology has been positively associated with the presence of the TNF2 allele in the Caucasian population (Danis et al., 1995), while no association has been demonstrated in the Spanish population (Vinasco et al., 1997) or in our own studies with the Chilean population (Cuenca et al., 2000). In the second case, obesity has been associated to the TNF2 allele in the German Caucasian population (Brand et al., 2001), while no association was detected in a study performed in the Japanese population (Ishii et al., 2000). Altogether these results show that according to the similarity between the Japanese and Chilean TNF2 allele frequencies, obesity in the Chilean population may not be associated to the TNF2 allele. Moreover, we could postulate that due to the lower TNF2 frequency in the Chilean population, it may be less exposed to TNF2 allele-associated diseases. Obviously, this last hypothesis remains to be tested experimentally.

Among the known genetic polymophisms that appear to affect presence, severity, and risk or progress of a disease, the TNF2 allele stands out. The TNF2 allele represents a stable mutation of part of the TNF promoter gene that increases TNF protein expression by altering the level of TNF gene transcription. However, an important unresolved question is why the TNF2 allele has persisted at such a high population frequency despite conferring what appears to be a marked survival disadvantage. As with other detrimental gene mutations that persist in the human population, an unrecognized selective advantage to carriers of the TNF2 allele may exist. For example, the presence of the TNF2 allele may increase the resistance of the host to local infection (by increasing local production of TNF at the infection site) even while increasing the risk for chronic, inflammatory, or autoimmune diseases.

In conclusion, this work suggests that a single base pair alteration in the promoter region of the TNF gene, contained in the cluster of HLA Class III genes on 1 or 2 copies of chromosome 6, may result in a variable increased risk to a variety of diseases, depending on ethnicity,.

ACKNOWLEDGEMENTS

This work was supported by FONDECYT-CHILE 1990936. We thank Dr. Rosario Billetta for editorial suggestions and Ms. Juana Orellana and Ms. Ruth Mora for their technical assistance.

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