Polymorphism of the cytokine genes in hospitalized patients with Puumala hantavirus infection

Satu Mäkelä1,, Mikko Hurme1, Ilpo Ala-Houhala1, Jukka Mustonen1, Anna-Maija Koivisto2, Jukka Partanen3, Olli Vapalahti4, Antti Vaheri4 and Amos Pasternack1

1 Medical School, University of Tampere and Tampere University Hospital, 2 Tampere School of Public Health, University of Tampere, 3 Finnish Red Cross Blood Transfusion Service, Helsinki, and 4 Haartman Institute, Department of Virology, University of Helsinki, Finland



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Nephropathia epidemica (NE) is a mild type of haemorrhagic fever with renal syndrome caused by Puumala (PUU) hantavirus. The clinical course of NE varies from asymptomatic to fatal. The aim of this study was to establish whether polymorphisms in the cytokine genes are associated with susceptibility to and outcome of NE.

Methods. The genotypes of the genes of tumour necrosis factor alpha (TNF{alpha}), interleukin-1{alpha} (IL-1{alpha}), IL-1ß and IL-1 receptor antagonist (IL-1RA) were analysed by polymerase chain reaction in 87 subjects, all hospital-treated for serologically confirmed acute NE. The control group comprised 400 healthy blood donors. Nineteen out of these 400 (5%) controls were PUU virus-seropositive.

Results. IL-1RA allele 2 and IL-1ß (base exchange polymorphism at position -511) allele 2 were strongly associated with each other in both groups. NE patients were more often IL-1RA-2 negative/IL-1ß-2 negative than PUU-seronegative blood donors (38 vs 27%, odds ratio 1.65, 95% confidence interval 1.0–2.7). However, there were no differences in the clinical severity of NE between the IL-1RA-2 negative/IL-1ß-2 negative and the other patients. The other allele frequencies studied evinced no statistically significant differences between the groups. Thirty-three out of 87 (38%) patients and 121 out of 381 (32%) seronegative controls were carriers of the high-producer genotype TNF2 allele. Several parameters showed the clinical course of NE to be more severe in TNF2 carriers than in non-carriers.

Conclusions. These data suggest that non-carriage of the IL-1RA allele 2 and IL-1ß (-511) allele 2 may contribute to susceptibility to NE. Furthermore, TNF{alpha} polymorphism seems to be associated with the outcome of NE.

Keywords: cytokine; gene polymorphism; hantavirus; haemorrhagic fever with renal syndrome; nephropathia epidemica; Puumala virus



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Nephropathia epidemica (NE) is a mild type of haemorrhagic fever with renal syndrome (HFRS) caused by Puumala (PUU) hantavirus. NE is endemic in Scandinavia, European Russia and the Balkans and also in many parts of Western Europe [1]. Approximately 1000 serological diagnoses are made in Finland annually, but as judged from the high seroprevalence (5%) in the population, most infections are subclinical and remain without serodiagnosis [2].

NE is clinically characterized by high fever, headache and back and abdominal pains [3]. Hypotension up to clinical shock is present in less than 10% of hospital-treated patients. Renal involvement results in transient massive proteinuria, haematuria and impairment of renal function followed by polyuria and spontaneous recovery. Transient haemodialysis is needed in a minority. Common laboratory findings are anaemia, leukocytosis, thrombocytopenia and a moderate elevation of C-reactive protein (CRP) levels [3]. There is considerable variability in the clinical severity of NE. It has previously been demonstrated that individuals with the HLA B8 DRB1*0301 haplotype suffer from a severe form of the disease [4], suggesting that host genetic factors influence the clinical picture.

The pathogenesis of NE is incompletely understood. An increase in capillary permeability is characteristic of various types of hantavirus infections, but the mechanism of vascular leakage remains to be elucidated. PUU virus causes no cytopathic effects in cultured cells but shows wide cell susceptibility in vitro. Immunological factors including cytokines may play an important role in the pathogenesis of NE [1]. Tumour necrosis factor alpha (TNF{alpha}) is known to induce vascular permeability, and to increase the expression of endothelial adhesion molecules [5]. Intravenous injection of TNF{alpha} induces a number of signs and symptoms similar to those seen in NE [5], and high plasma levels of TNFa have indeed been detected during the acute phase of NE [6]. Furthermore, increased expression of intercellular and vascular cell adhesion molecules has been described in the interstitial and tubular space of NE kidneys, respectively [1].

The key cytokines participating in the regulation of inflammatory response are TNF, interleukin-1 (IL-1), IL-6, IL-10 and IL-1 receptor antagonist (IL-1RA). Functionally these cytokines can be divided into proinflammatory (IL-1, IL-6 and TNF) and anti-inflammatory (IL-1RA and IL-10) molecules. Genetic factors have a substantial influence on the production of these cytokines, and changes (polymorphisms) in the cytokine genes may determine the amount of cytokine produced in the inflammatory reaction.

Several polymorphisms have been identified inside the promoter region of the TNF{alpha} gene [7]. Among these, there is a biallelic polymorphism at position -308, involving the substitution of guanine by adenosine in the uncommon allele TNF2 [8]. This latter allele has been found to correlate with enhanced TNF production [9]. A large number of studies have examined the importance of TNF genetics in susceptibility to autoimmune diseases, and some studies have also linked TNF2 polymorphism to the outcome of infections [7]. Recently, a study on TNF polymorphism in NE revealed that TNF allele 2-positive hospitalized NE patients suffered from a more severe NE than TNF allele 2-negative patients [10].

The IL-1 gene family on chromosome 2q13 codes for three proteins: IL-1{alpha}, IL-1ß and IL-1RA. All three genes are polymorphic. In the IL-1ß gene, there are at least two biallelic base-exchange polymorphisms: at position -511 [11] and at position +3953 [12], and in the IL-1{alpha} gene there is a base-exchange polymorphism at position -889 [13]. In intron 2 of the IL-1RA gene, there are variable numbers of an 86-bp repeat sequence; the most common allele 1 contains four repeats and allele 2 contains two repeats [14]. The frequency of IL-1RA allele 2 is increased in several diseases of inflammatory or autoimmune nature [15]. Healthy IL-1RA allele 2 carriers have been shown to have higher IL-1RA plasma levels than non-carriers [16]. However, the enhancing effect of IL-1RA allele 2 on IL-1RA plasma levels has required the presence of IL-1ß (-511) allele 2 or the absence of IL-1ß (+3953) allele 2 [16]. Furthermore, IL-1ß (-511) allele 2 has been found to be significantly associated with the presence of IL-1RA allele 2 [16]. It would, thus, seem plausible that the alleles determining a high agonist production and a high antagonist production are generally associated, which is obviously important in maintaining homeostasis.

In the present study we analysed allele frequencies and genotypes of the genes of the IL-1 complex and TNF{alpha} in a group of hospitalized NE patients and in a control group of healthy blood donors to address the possibility of an association with susceptibility to disease. In addition, we sought to establish whether the clinical course of NE is influenced by the polymorphisms of these cytokine genes.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Subjects
The study was carried out in Tampere University Hospital and in the University of Tampere Medical School, Finland. Blood samples were obtained from 87 patients, all of whom had been treated for serologically confirmed acute PUU virus infection at Tampere University Hospital. There were 61 males and 26 females aged from 15 to 77 (median 43) years. DNA specimens were gathered in two groups. Samples from 41 randomly selected patients who had been treated for NE in our hospital during the years 1982–1993 were obtained retrospectively. Another group consisted of 46 consecutive patients with NE during the years 1994–1998. Previously, Kanerva and co-workers have studied TNF gene polymorphism in 59 NE patients [10]. These patients were included in the present material.

The control group comprised 400 healthy blood donors. Blood samples were obtained from the Finnish Red Cross Blood Transfusion Center, Tampere, Finland. The donors were adults (18–60 years of age) and had no sign of infection during a 2-week period before the blood donation. Immunoglobulin G antibodies to PUU virus were examined by immunofluorescence test as described in a recent paper by Brummer-Korvenkontio and co-workers [2]. Nineteen out of these 400 (5%) controls were PUU virus-seropositive.

Analysis of gene polymorphisms
Genomic DNA was isolated from the blood samples by the salting-out method [17]. Polymerase chain reaction (PCR)-based genotyping of TNF{alpha} (base exchange polymorphism at position -308), IL-1RA (variable number of tandem repeats in exon 2), IL-1ß (base exchange polymorphisms at positions -511 and +3953) and IL-1{alpha} (base exchange polymorphism at position -889) was performed as previously described [8,1114].

Statistical analysis
Allelic frequencies (number of copies of a specific allele divided by the total number of alleles in the group) and carriage rates for infrequent 2-alleles (number of individuals with at least one copy of allele 2 divided by the total number of individuals within the group) were calculated in NE patients, and seronegative and seropositive controls. {chi}2-test was used to analyse, if there were differences in the carriage rates between NE patients and seronegative controls, or between the groups in general. Odds ratios (OR) were calculated after the {chi}2-test when appropriate, and 95% confidence intervals (95%CI) were determined. Tests for Hardy–Weinberg equilibrium were also made.

To describe the severity of NE, medians and ranges are given for skew-distributed continuous variables and percentages are used for categorical variables. The patients were grouped into carriers (including both homozygotes and heterozygotes) and non-carriers of specific alleles. Differences in clinical severity of NE between carriers and non-carriers were tested using Mann–Whitney U-test for numerical data and {chi}2-test or Fisher's exact test for categorical data. OR were calculated after the {chi}2-test where appropriate, and 95% CI were also determined. All testing was two-sided and statistically significant P-values are given. All tests were made with the SPSS (version 7.0) statistical software package.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Among the 400 healthy blood donors, 19 (5%) were PUU virus-seropositive. Thus, the prevalence of PUU virus antibodies in the control group was the same as previously reported for the Finnish population [2].

The IL-1RA and IL-1ß (-511) genotypes and corresponding allele frequencies in hospitalized NE patients and PUU virus-seronegative and -seropositive healthy blood donors are shown in Table 1Go.


View this table:
[in this window]
[in a new window]
 
Table 1. IL-1RA (variable number of tandem repeats in intron 2) and IL-1ß (-511) genotypes, corresponding allele frequencies and carriage rates of infrequent allele 2 in hospitalized NE patients, PUU virus-seronegative and seropositive healthy blood donors

 
There were no statistically significant differences in the carriage rates between patients and seronegative controls. However, when comparing the carriage rates of IL-1ß (-511) allele 2 between all three groups, a statistically significant difference was found (P=0.033). The carriage rate of IL-1ß (-511) allele 2 was lower in the NE patients compared with the seropositive blood donors (53 vs 84%). There were also fewer IL-1ß (-511) 2-carriers among the seronegative than among the seropositive blood donors (62 vs 84%). The difference in carriage rates of IL-1RA allele 2 between all three groups was not statistically significant (P=0.244), although there was a trend towards a lower carriage rate of IL-1RA allele 2 in the NE patients compared with the PUU virus-seronegative and seropositive controls (Table 1Go). The clinical severity of NE did not differ either between IL-1ß 2-carriers and non-carriers or between IL-1RA 2-carriers and non-carriers (data not shown). Furthermore, no statistically significant differences were observed between the groups in the allele distributions of the cytokine genes IL-1{alpha} (-889) and IL-1ß (+3953) (data not shown).

The IL-1RA allele 2 was strongly associated with IL-1ß (-511) allele 2 both in NE patients and in PUU virus-seronegative controls (P<0.001 in both groups). However, there were differences between the groups in the allele associations. In NE patients, the IL-1RA allele 2-carriers (designated IL-1RA-2 positive) were often carriers of the IL-1ß (-511) allele 2 and vice versa, and the IL-1RA-2 negative and IL-1ß-2 negative were associated. In the PUU virus-seronegative control group the IL-1RA-2 positive/IL-1ß-2 positive association was also obvious, but the IL-1RA-2 negative population contained almost equal numbers (95 and 104, respectively) of IL-1ß (-511) allele 2-carriers and non-carriers. Thus, NE patients were more often IL-1RA-2 negative/IL-1ß-2 negative than the PUU virus-seronegative controls (38 vs 27%, OR 1.65, 95%CI 1.0–2.7) (Table 2Go). Only two out of 19 (11%) PUU virus-seropositives were IL-1RA-2 negative/IL-1ß-2 negative. When non-carriers of IL-1RA-2/IL-1ß-2 against the remaining alleles were compared between all three groups, a statistically significant difference was found (P=0.029), suggesting a difference in the allele associations between the three groups. There were no significant differences in the clinical severity of NE between the IL-1RA-2 negative/IL-1ß-2 negative and the other patients (data not shown).


View this table:
[in this window]
[in a new window]
 
Table 2. Distribution of non-carriers and carriers of IL-1RA allele 2/IL-1ß (-511) allele 2 in hospitalized NE patients, PUU virus-seronegative and seropositive healthy blood donors

 
The TNF{alpha} (-308) genotypes and corresponding allele frequencies in hospitalized NE patients and PUU virus-seronegative and -seropositive healthy blood donors are shown in Table 3Go. The carriage rates revealed no statistically significant differences between the groups. However, there was a tendency towards an increased number of the high producer TNF{alpha} allele 2-carriers in the NE patients compared with both PUU virus-seronegative and seropositive blood donors (Table 3Go). Several clinical and laboratory parameters showed a more severe clinical course of NE in TNF2 carriers than in non-carriers (Table 4Go). Acute renal failure considered severe (the highest serum creatinine concentration measured during hospital care >500 µmol/l) was seen in 13 out of 33 (39%) TNF2 carriers and in 10 out of 54 (19%) TNF2 non-carriers (P=0.032, OR 2.9, 95%CI 1.1–7.6). Seven patients out of the total 87 (8%) were in clinical shock on admission and four of them required dialysis treatment during hospital care. Altogether 14 (16%) patients were dialysed. Six out of 33 (18%) TNF2 carriers suffered from shock compared with one out of 54 (2%) non-carriers (P=0.011, OR 11.8, 95%CI 1.3–102.9). Correspondingly, nine out of 33 (27%) TNF2 carriers required dialysis treatment compared with five out of 54 (9%) non-carriers (P=0.027, OR 3.7, 95%CI 1.1–12.2). In particular, both patients homozygous for TNF2 allele suffered from severe NE: both were dialysed because of marked renal insufficiency (maximal serum creatinine 929 and 987 µmol/l). No differences were observed between TNF2 carriers and non-carriers in the highest values of CRP, daily urine protein excretion and the amount of haematuria, or in the lowest values for blood thrombocyte count, or in the highest or lowest values for systolic and diastolic blood pressure observed during hospital care (data not shown). All patients recovered.


View this table:
[in this window]
[in a new window]
 
Table 3. TNF{alpha} (–308) genotypes, corresponding allele frequencies and carriage rate of high-producer allele 2 in hospitalized NE patients, PUU virus-seronegative and seropositive healthy blood donors

 

View this table:
[in this window]
[in a new window]
 
Table 4. Clinical findings in TNF2 carriers (genotypes 1.2 and 2.2) and non-carriers (genotype 1.1) in hospitalized NE patients. Medians (ranges) are given for continuous and percentages for categorical variables

 
The distributions of TNF{alpha} and IL-1 genotypes in patients and controls were in Hardy–Weinberg equilibrium, except the genotypes of IL-1{alpha} in the group of seronegative blood donors (P=0.005). However, this polymorphism did not reveal any disease susceptibility or influence to the outcome of NE.



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
This study found no statistically significant differences in the allele frequencies and genotypes of the genes of the IL-1 complex and TNF{alpha} between hospitalized NE patients and a control group of healthy blood donors (Tables 1Go and 3Go). However, there was a tendency towards an increased frequency of allele TNF2 and a decreased frequency of IL-1ß (-511) allele 2 in patients compared with controls. Furthermore, the frequency of non-carriers of IL-1RA allele 2/IL-1ß (-511) allele 2, was increased in the hospitalized NE patients compared with PUU virus-seronegative blood donors (Table 2Go). The present study also showed that the clinical course of NE was more severe in high-producer TNF2 allele carriers than in non-carriers (Table 4Go).

The prevalence of PUU virus antibodies in the control group of blood donors was the same as previously reported in the Finnish population [2]. Unfortunately, the voluntary nature of blood donating made it impossible to establish retrospectively whether these seropositive controls had previously been hospitalized because of an acute PUU virus infection. It is likely, however, that most of them had had a mild or asymptomatic disease, as comparison of diagnosis incidences and seroprevalences in fertile-aged women shows that in Finland on average only 13% of all PUU hantavirus infections are serodiagnosed [2]. In the present study, cytokine genotype distribution in this group of seropositive controls (with a probable mild disease) differed markedly from the genotype distribution of hospital-treated NE patients (obviously with a more severe form of disease). There were more IL-1ß (-511) 2-carriers and less non-carriers of IL-1RA allele 2/IL-1ß (-511) allele 2 among the seropositive controls than among patients. They also showed a tendency towards a higher carriage rate of IL-1RA allele 2 and a lower carriage rate of TNF{alpha} allele 2 compared with patients (Tables 1GoGo3Go).

Recently, Cox and co-workers have identified a common, eight-locus haplotype of the IL-1 gene cluster [18]. Their data indicate a linkage-disequilibrium across the region of chromosome 2q13. In the present study, a strong association emerged between IL-1RA allele 2 and IL-1ß (-511) allele 2. Functionally, the significance of this association is yet not entirely clear, but there are data suggesting that both of these alleles are required to maintain high IL-1RA plasma levels [16]. We found that the frequency of non-carriers of IL-1RA allele 2/IL-1ß (-511) allele 2 was increased in the hospitalized NE patients compared with the group of healthy, PUU virus-seronegative blood donors (Table 2Go). Hence, the present data suggest that IL-1RA allele 2/IL-1ß (-511) allele 2-polymorphism may contribute to susceptibility to NE. Associations between IL-1 complex genes have recently been analysed also in another viral disease, namely in Epstein–Barr virus infection. A significantly higher number of non-carriers of IL-1RA allele 2/IL-1ß (-511) allele 2 have been found among Epstein–Barr virus-seronegative than seropositive blood donors [19].

Our data suggest that TNF{alpha} polymorphism is unlikely to be of significance in susceptibility to NE, although there was a slight tendency towards an increased frequency of allele TNF2 in NE patients compared with controls. Previously, high-producer TNF alleles have been associated with susceptibility to autoimmune diseases, in which TNF is of pathophysiological importance [7]. Recently, Mira and colleagues demonstrated that the TNF2 allele occurs with an increased frequency in patients with septic shock compared with a group of blood donors [20]. In addition, mortality due to septic shock, was shown to be increased in patients with this allele, and every patient who was TNF2 homozygous had a fatal outcome [20].

Previously, the TNF gene polymorphism of 59 NE patients has been analysed by Kanerva and co-workers [10]. They showed that the clinical course of NE was more severe in TNF2 carriers than in non-carriers [10]. The same result was also predictably found in the present material that included the 59 patients of the earlier study [10]. Especially, both TNF2 homozygous patients evinced marked renal failure requiring transient dialysis therapy. Cytokines, including TNF{alpha}, may play an important role in the pathogenesis of NE [1]. It is, therefore, possible that high-producer allele TNF2 contributes to the outcome of NE. Individuals with the HLA B8 DRB1*0301 haplotype have previously been shown to suffer from a severe form of NE [4], and the TNF2 allele is known to be in strong linkage disequilibrium with this HLA haplotype in a northern European population [7]. It is, thus, possible that TNF2 allele is not an independent risk factor for severe NE but a passive component in the extended HLA haplotype. This issue cannot be evaluated from the present data, since the HLA haplotypes of the patients were not studied.

In conclusion, the results here show that non-carriage of IL-1RA allele 2 and IL-1ß (-511) allele 2 may contribute to susceptibility to NE. The data also support the hypothesis that the alleles determining high agonist and high antagonist production are generally associated. Furthermore, TNF{alpha} polymorphism seems to regulate the clinical course of NE. It would be interesting to carry out genetic studies on the more severe forms of hantavirus diseases and also on PUU virus infections with a subclinical or asymptomatic course.



   Acknowledgments
 
The study was financially supported by the Medical Research Fund of Tampere University Hospital and the Finnish Kidney Foundation. Part of the results were presented in abstract form at the ‘Nordic Conference on Renal Diseases’ in Tampere, Finland, May 1999. The skilful technical assistance of Mirja Ikonen is greatly appreciated.



   Notes
 
Correspondence and offprint requests to: Satu Mäkelä, MD, Medical School, FIN-33014 University of Tampere, Finland. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Kanerva M, Mustonen J, Vaheri A. Pathogenesis of Puumala and other hantavirus infections. Rev Med Virol1998; 8: 67–86[ISI][Medline]
  2. Brummer-Korvenkontio M, Vapalahti O, Henttonen H, Koskela P, Kuusisto P, Vaheri A. Epidemiological study of nephropathia epidemica in Finland 1989–96. Scand J Infect Dis1999; 31: 427–435[ISI][Medline]
  3. Mustonen J, Brummer-Korvenkontio M, Hedman K, Pasternack A, Pietilä K, Vaheri A. Nephropathia epidemica in Finland: a retrospective study of 126 cases. Scand J Infect Dis1994; 26: 7–13[ISI][Medline]
  4. Mustonen J, Partanen J, Kanerva M et al. Genetic susceptibility to severe course of nephropathia epidemica caused by Puumala hantavirus. Kidney Int1996; 49: 217–221[ISI][Medline]
  5. Tracey KJ, Cerami A. Tumor necrosis factor: a pleiotropic cytokine and therapeutic target. Annu Rev Med1994; 45: 491–503[ISI][Medline]
  6. Linderholm M, Ahlm C, Settergren B, Waage A, Tärnvik A. Elevated plasma levels of tumor necrosis factor (TNF)-alpha, soluble TNF receptors, interleukin (IL)-6 and IL-10 in patients with hemorrhagic fever with renal syndrome. J Infect Dis1996; 173: 38–43[ISI][Medline]
  7. Hajeer AH, Hutchinson IV. TNF-alpha gene polymorphism: clinical and biological implications. Microsc Res Tech2000; 50: 216–228[ISI][Medline]
  8. Wilson AG, di Giovine FS, Blakemore AI, Duff GW. Single base polymorphism in the human tumour necrosis factor alpha (TNF alpha) gene detectable by NcoI restriction of PCR product. Hum Mol Genet1992; 1: 353[Medline]
  9. Wilson AG, Symons JA, McDowell TL, McDevitt HO, Duff GW. Effects of a polymorphism in the human tumor necrosis factor alpha promoter on transcriptional activation. Proc Natl Acad Sci USA1997; 94: 3195–3199[Abstract/Free Full Text]
  10. Kanerva M, Vaheri A, Mustonen J, Partanen J. High-producer allele of tumour necrosis factor-alpha is part of the susceptibility MHC haplotype in severe Puumala virus-induced nephropathia epidemica. Scand J Infect Dis1998; 30: 532–534[ISI][Medline]
  11. di Giovine FS, Takhsh E, Blakemore AI, Duff GW. Single base polymorphism at -511 in the human interleukin-1 beta gene (IL1 beta). Hum Mol Genet1992; 1: 450
  12. Pociot F, Molvig J, Wogensen L, Worsaae H, Nerup J. A TaqI polymorphism in the human interleukin-1 beta (IL-1 beta) gene correlates with IL-1 beta secretion in vitro. Eur J Clin Invest1992; 22: 396–402[ISI][Medline]
  13. McDowell TL, Symons JA, Ploski R, Forre O, Duff GW. A genetic association between juvenile rheumatoid arthritis and a novel interleukin-1 alpha polymorphism. Arthritis Rheum1995; 38: 221–228[ISI][Medline]
  14. Tarlow JK, Blakemore AI, Lennard A et al. Polymorphism in human IL-1 receptor antagonist gene intron 2 is caused by variable numbers of an 86-bp tandem repeat. Hum Genet1993; 91: 403–404[ISI][Medline]
  15. Hurme M, Lahdenpohja N, Santtila S. Gene polymorphisms of interleukins 1 and 10 in infectious and autoimmune diseases. Ann Med1998; 30: 469–473[ISI][Medline]
  16. Hurme M, Santtila S. IL-1 receptor antagonist (IL-1Ra) plasma levels are co-ordinately regulated by both IL-1Ra and IL-1beta genes. Eur J Immunol1998; 28: 2598–2602[ISI][Medline]
  17. Miller SC, Ito H, Blau HM, Torti FM. Tumor necrosis factor inhibits human myogenesis in vitro. Mol Cell Biol1988; 8: 2295–2301[ISI][Medline]
  18. Cox A, Camp NJ, Nicklin MJ, di Giovine FS, Duff GW. An analysis of linkage disequilibrium in the interleukin-1 gene cluster, using a novel grouping method for multiallelic markers. Am J Hum Genet1998; 62: 1180–1188[ISI][Medline]
  19. Hurme M, Helminen M. Polymorphism of the IL-1 gene complex in Epstein-Barr virus seronegative and seropositive adult blood donors. Scand J Immunol1998; 48: 219–222[ISI][Medline]
  20. Mira JP, Cariou A, Grall F et al. Association of TNF2, a TNF-alpha promoter polymorphism, with septic shock susceptibility and mortality: a multicenter study. J Am Med Assoc1999; 282: 561–583[Abstract/Free Full Text]
Received for publication: 12. 4.00
Revision received 30. 1.01.