Polymorphisms of the tumour necrosis factor {alpha} gene at position -308 and TNFd microsatellite in primary IgA nephropathy

Serhan Tuglular, Patricia Berthoux and François Berthoux

Nephrology, Dialysis and Renal Transplantation Department, University North Hospital, Saint Etienne, France



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Appendix
 References
 
Background. The development of glomerular inflammation in immunoglobulin A nephropathy (IgAN) has been associated with various cytokines, including tumour necrosis factor {alpha} (TNF{alpha}). A biallelic polymorphism in the promoter region of the TNF{alpha} gene (TNFA), at position -308, has been described (TNFA-1 and TNFA-2) and is associated with increased TNF{alpha} production for the TNFA-2 allele. Another microsatellite polymorphism has been described for TNFd, which is functional and associated with increased production of TNF{alpha} for the d3 allele.

Methods. We have studied these two polymorphisms in 242 Caucasian patients with biopsy-proven IgAN (169 male, 73 female), who were followed from 1990 to 1999, and in 210 appropriate local Caucasian controls (133 male, 77 female) for comparison of genotypes and allelic distribution.

Results. The respective frequencies of A1/A1, A1/A2 and A2/A2 TNFA genotypes were 76.4, 22.3 and 1.3% in IgAN vs 78.1, 19.5 and 2.4% in controls (P=NS). For TNFd, the frequencies of the respective genotypes d3/d3, d3/non-d3 and non-d3/non-d3 were significantly different ({chi}2=12.30, P=0.002, Pc=0.013) with an increased frequency of the low-producer genotype non-d3/non-d3 in IgAN patients (24 vs 12%). The combination of TNFA and TNFd polymorphisms demonstrated that compared with controls, patients with non-A2 and non-d3 alleles (low producers) were more common (18 vs 9%; P=0.006). In the genotype/clinical phenotype correlations, we could not demonstrate significant differences between the different subgroups of patients. However, high-producer TNF{alpha} patients (A2 and d3 alleles) had more chronic renal failure than others (36.6 vs 22.9%) at last follow-up and their survival without chronic renal failure (Kaplan–Meier) was lower. Nevertheless, TNF{alpha} polymorphisms were not an independent risk factor for the progression of the disease.

Conclusions. TNFA and TNFd polymorphisms seem to influence the occurrence or initiation of the disease, but do not play a significant role, if any, in the progression of IgA nephritis.

Keywords: gene polymorphism; genotype/phenotype correlation; IgA nephropathy; promoter region (-308); tumour necrosis factor {alpha}; TNFd microsatellites



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Appendix
 References
 
Immunoglobulin A nephropathy (IgAN) is recognized as the most common form of primary glomerulonephritis worldwide and in many countries accounts for 10% of patients reaching end-stage renal disease (ESRD). About 50–60% of the patients progress to chronic renal failure (CRF) over 20–30 years with 20–35% reaching ESRD [1]. The exact mechanism of mesangial IgA1 deposition still remains partially unknown. Once deposited, however, an inflammatory process is triggered resulting in monocyte/macrophage infiltration of the glomeruli and in mesangial proliferation with increase in matrix components.

Various immune mediators have been demonstrated to play a role in this inflammatory process. Tumour necrosis factor {alpha} (TNF{alpha}) [2] is one of the most important cytokines, with various immunological functions. It is produced early in the inflammatory process, generating a cascade of other mediators and upregulation of some adhesion molecules and other cytokines, such as interferon gamma (IFN{gamma}), interleukin-6 (IL-6), IL-8, IL-10 and TNF{alpha} itself. TNF{alpha} is predominantly produced by monocytes/macrophages and in turn is a strong activator of phagocytic cells. Monocytes/macrophages are the dominant infiltrating cells in the kidney of IgAN patients and the degree of hypercellularity is correlated with the degree of irreversible glomerular injury [3]. Together with infiltrating monocytes and macrophages, mesangial cells also are capable of producing TNF{alpha} and increased expression of TNF{alpha} by mesangial cells has been reported in IgAN [4,5]. Increased production of TNF{alpha} by peripheral blood mononuclear cells from patients with IgAN has been demonstrated previously [6,7]. Finally, plasma levels and urinary excretion of TNF{alpha} were shown to be elevated in patients with IgAN [8]. Taken together, these data led us to investigate whether a genetically controlled cytokine, such as TNF{alpha}, could play a significant role in patients with IgAN.

The TNF{alpha} gene (TNFA) is located on the short arm of chromosome 6, in the class III region, within the major histocompatibility complex (MHC) in a position defined as 250 kb centromeric to HLA-B locus and about 850 kb telomeric to HLA-DR locus [9]. A polymorphism in the promoter region of the TNF{alpha} gene has been described at nucleotide position -308 and consists of a single mutation, with the presence of guanine in the common allele 1 (TNFA-1) as opposed to adenine in the relatively rare allele 2 (TNFA-2). TNFA-2, a stronger activator of transcription than TNFA-1, is associated with the ‘high producer TNF{alpha} phenotype [10]. The TNFA-2 allele has also been linked with the extended haplotype HLA-A1/B8/DR3/DQ2, which is reported to be associated with autoimmunity [9].

Another microsatellite polymorphic region has been described [11], located 8 kb downstream to the TNFA gene, and consists of two microsatellites designated TNFd and TNFe. Further study [12] showed that TNFd was highly informative and functional. TNFd polymorphism consisting of dinucleotide repeats (GA) and the d3 allele was associated with greater production of TNF{alpha} cytokine. Finally, both TNF-A2 and TNF-d3 alleles were associated with overproduction of TNF{alpha}.

The aims of this study were: (1) to determine the allelic frequency and the genotype distribution for these two TNF{alpha} polymorphisms, -308 TNFA and TNFd, in patients with IgAN compared to appropriate controls; and (2) to study the genotype/phenotype correlation in terms of clinical and pathological data at diagnosis and at last follow-up.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Appendix
 References
 
We studied 242 (169 male, 73 female; sex ratio: male/female=2.32) consecutive Caucasian patients with biopsy-proven IgAN diagnosed during the years 1990–1999 (incident cases). None of these patients had any evidence of secondary causes, such as cirrhosis, lupus nephritis or Henoch-Schönlein purpura. At the time of diagnosis (first renal biopsy, RB1), the respective age of these patients was 41.3 (14.8) for males and 39.5 (14.3) years for females; the mean and median proteinurias were 0.94 (1.28) and 0.50 g/24 h and the mean and median serum creatinines were 128.1 (±122.6) and 92 µmol/l. The mean and median durations of follow-up of these patients from onset of the disease were 120.2 and 98.1 months, respectively. All these patients were adequately followed and treated for arterial hypertension and heavy proteinuria and most of them were receiving angiotensin I converting enzyme inhibitor (ACEI) or angiotensin II receptors antagonists (AIIA). The patients with severe pathological scores had received high dose steroids whenever indicated. Therefore, this does not correspond to the natural history of such a cohort.

The control group was matched for gender to reflect the male predominance of IgAN and consisted of 210 local Caucasian healthy adults (133 male, 63.3%; 77 female) and will represent the general population. Each patient and control gave informed consent to this genetic study.

Clinical data
We collected the following parameters for these patients at the date of their first renal biopsy: the amount of proteinuria (g/24 h), the presence of arterial hypertension, plasma creatinine (µmol/l) and the presence of CRF, defined according to the K/DOQI Clinical Practice Guidelines for Chronic Kidney Disease as a glomerular filtration rate <60 ml/minx1.73 m2 and calculated by the Cockroft–Gault equation.

Pathological data at RB1
The scoring was performed by our renal pathologist as a routine procedure and without knowledge of subjects' clinical data. We used the global optical score (GOS) ranging from 0 to 20, which is the sum of glomerular (0–6), vascular (0–5), interstitial (0–5) and tubular (0–4) indices [13]. The mean GOS for the cohort was 7.09 (3.17).

Clinical progression
The primary endpoint for clinical progression was the occurrence of CRF. In a previous study [13], the independent risk factors present at diagnosis (RB1) and predictive of ultimate CRF were 24 h proteinuria, occurrence of arterial hypertension and GOS. They were chosen as secondary endpoints together with end-stage renal failure (ESRF). Survival curves without CRF were also computed.

Genotyping of -308 TNF{alpha} polymorphism
Genomic DNA was obtained from peripheral blood mononuclear cells with a classical phenol chloroform extraction method. A polymerase chain reaction coupled with restriction fragment length polymorphism (PCR–RFLP) was used for amplification and accurate genotyping. The sequence of the primers chosen for the amplification of a 289 bp DNA fragment, including the polymorphic -308 nucleotide, were the following: 5'-AGGCAATAGGTTTTGAGGGCCAT-3' (sense) and 5'-CAGCGGAAAACTTCCTTGGT-3' (antisense). The PCR technique was performed on 250 ng of extracted DNA: DNA amplification with 1.25 U of Taq DNA polymerase (Gibco BRL) in 20 mM Tris–HCl containing 50 mM KCl, 1.5 mM MgCl2, 200 µmol/l dNTP and 0.5 µM of each primer. After an initial denaturing time of 5 min at 90°C, PCR reactions were run for 35 cycles including 30 s at 94°C, 30 s at 60°C and 1 min at 72°C.

The PCR product was digested by 5 U of the enzyme NcoI at 37°C for 3 h and then subjected to electrophoresis in 2% agarose gel (Metaphor agarose; FMC BioProducts) stained with ethidium bromide. The TNFA-1 allele gave two bands at 244 and 20 bp and the TNFA-2 allele gave only one band at 264 bp. Since the very light band of 20 bp migrates too fast to be detected on the gel, the TNFA-1 allele was in practice characterized by only one band at 244 bp. Finally, the genotypes were defined as 1/1 (homozygous) in case of a unique band at 244 bp, as 2/2 (homozygous) in case of a unique band at 264 bp and as 1/2 (heterozygous) in case of two bands—one at 244 bp and one at 264 bp.

Genotyping of TNFd microsatellite polymorphism
The polymorphism is based on the number of GA repeats. It was detected by PCR followed by polyacrylamide gel electrophoresis (PAGE) to separate the different DNA fragments of 126, 130, 132, 134 and 138 bp. The primers used were: TNFd12 sense, 5'-CATAGTGGGACTCTGTCTCCAAAG-3'; TNFd11 antisense, 5'-AGATCCTTCCCTGTGAGTTCTGCT-3'. For the PCR, we used 250 ng of extracted DNA, amplified with 1.25 U of Taq polymerase (Gibco BRL) in 20 mM Tris–HCl containing 50 mM KCl, 1.5 mM MgCl2, 200 µM of each DNTP and 0.4 µM of each primer. After an initial denaturation at 94°C for 2 min, PCR reactions were run for 30 cycles including 15 s at 94°C, 15 s at 55°C, 30 s at 72°C and one cycle with 2 min at 72°C and 1 min at 30°C.

The PCR products obtained were submitted to PAGE (12% acrylamide/bisacrylamide 19:1) and the bands obtained permitted the characterization of the different alleles d1, d2, d3, d4, d5 and d7. No d6 was found.

For both polymorphisms, we have verified that the genotype distributions observed were in accordance with the Hardy–Weinberg equilibrium.

Statistical analysis
Comparisons between patients and controls. The different allelic frequencies and genotype distributions were compared using the {chi}2 contingency table for qualitative variables. When applicable, the P-value obtained was corrected (Pc) for the number of alleles (six for TNFd).

The power of this study for allelic comparison was at least 90% based on the total number of alleles (individuals x2), a risk {alpha}=0.05, a risk ß=0.10 and permitted detection of a 10% difference.

Clinicopathological parameters among patients with different genotypes. The qualitative variables, such as hypertension, CRF and ESRF, were compared using the {chi}2 contingency table. The quantitative variables, such as proteinuria, GOS and serum creatinine, were compared using either the unpaired t-test or non-parametric test (Mann–Whitney U-test) when applicable. Survival without significant CRF was analysed according to the Kaplan–Meier method, where the event is CRF as already defined; time zero is the date of onset/discovery of the disease and not the date of renal biopsy; and the final date is either the date of the event or the date of the last follow-up (censored patients). The curves obtained were compared according to the log-rank test. For significant results, a multivariate Cox regression analysis was performed with the following covariates at RB1: 24 h proteinuria, GOS, hypertension and sex.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Appendix
 References
 
TNF genotype (-308 position) distribution and allelic frequencies
The genotype (A1A1, A1A2, A2A2) distribution and the allelic (A1, A2) frequencies in the group of patients vs controls are given in Tables 1Go and 2Go. There was no statistical difference according to the contingency table test, even after split for gender.


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Table 1.  TNFA (polymorphism at position -308) genotype distribution

 

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Table 2.  TNFA (polymorphism at position -308) allelic frequencies

 

TNFd microsatellite polymorphism
The results are given in Tables 3Go and 4Go. The most frequent allele is d3. With respect to genotypes, individuals were classified as d3 homozygous (d3/d3), d3 heterozygous (d3/non-d3) and non-d3/non-d3. The distribution is significantly different between IgAN patients and controls with more non-d3 individuals (24.4 vs 11.9%) and fewer d3 heterozygous individuals (49.6 vs 61.4%) in the IgAN group.


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Table 3.  TNFd (microsatellite polymorphism) genotype distribution

 

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Table 4.  TNFd (microsatellite polymorphism) allelic frequencies

 

Combined TNFA and TNFd genotypes and alleles
The A2 allele and d3 allele are known to be associated with hyperproduction of TNF{alpha} cytokine. We have combined these genotypes as follows: individuals with A2 and d3 alleles were considered as ‘high producers’, individuals with A2 or d3 allele were considered as ‘intermediate producers' and individuals with neither A2 nor d3 alleles were considered as ‘low producers’. The results are given in Table 5Go and, compared with controls, show a greater number of non-A2/non-d3 individuals (low producers) in the IgAN group (17.8 vs 8.6%; P=0.006).


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Table 5.  Combined TNFA and TNFd genotypes according to the presence or absence in individuals of A2 and/or d3 alleles

 

Validity of the data
The number of observed genotypes was not significantly different by the {chi}2 test from the theoretical numbers calculated from the Hardy–Weinberg equilibrium using the allelic frequencies (see Appendix).

Genotype/phenotype correlations: clinicopathological data
TNFA genotypes. We have compared patients with the A1A1 genotype to non-A1A1 (A1A2+A2A2), i.e. patients with the A2 allele. The results are given in Table 6Go. There was a trend towards more severe disease in patients with the A2 allele (more proteinuria, more CRF and more ESRF), but there was no significant difference. New cases reaching CRF were 5/42 (11.9%) in patients with the A2 allele vs 9/153 (5.9%) in patients with the A1A1 genotype (P=NS).


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Table 6.  Correlations between TNFA genotypes (A1A1 vs non-A1A1, i.e. with A2 allele) and clinicopathological data in IgAN patients

 
TNd3 genotypes. We compared patients with d3/d3, d3/non-d3 and non-d3/non-d3 genotypes. The results are given in Table 7Go. There was no significant difference. The d3/d3 subgroup had a tendency to have more severe disease (more proteinuria, more CRF and more ESRF). In this subgroup, the number of new patients reaching CRF was six out of the 50 at risk (12.0%) compared with 4/48 (8.3%) for the non-d3/non-d3 subgroup (P=NS).


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Table 7.  Correlations between TNFd genotypes (d3/d3, d3/non-d3, non-d3/non-d3) and clinicopathological data in IgAN patients

 
Combined TNFA and TNFd3 genotypes. The patients were classified into three subgroups: A2 and d3, A2 or d3 and non-A2/non-d3. The results are given in Table 8Go and show no significant difference. However, the A2 and d3 subgroup was associated with more severe disease [more CRF, more ESRF and more new CRF patients (10.3 vs 5.7% for non-A2/non-d3; P=NS)].


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Table 8.  Correlations between combined TNF genotypes (A2 and d3, A2 or d3, non-A2/non-d3) and clinicopathological data in IgAN patients

 
Survival without CRF according to the different genotypes. Patient survivals without CRF (as defined above) are given in Table 9Go for the entire cohort and for the different genotype combinations. High-producer patients (A2 and d3 alleles) have a significantly lower survival compared with all others combined (intermediate+low producers) (log-rank test: P=0.04). However, in a Cox regression model including already known risk factors, the high-producer phenotype was not an independent risk factor for progression.


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Table 9.  Patient survival without CRF (Kaplan–Meier)

 



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Appendix
 References
 
In this report, we did not find a significant difference between IgAN patients and appropriate controls for both TNFA genotypes and allelic distribution. However, we found a significant difference for the TNFd genotype non-d3/non-d3, which was two times more frequent in patients with IgAN than in appropriate controls (P=0.002, Pc=0.013). In addition, the TNF{alpha} ‘low producer’ phenotype (non-A2 and non-d3) was associated with the occurrence or initiation of IgA nephritis (P=0.006). The significance of this finding is unclear to us. We could imagine a natural selection eliminating high-producer individuals who presented highly severe disease and died from ESRF and complications. We have found in the literature only three studies dealing with this subject. In one from Taiwan, with 111 IgAN vs 100 controls [14] restricted to the -308 TNFA polymorphism, the allele frequencies of TNFA-1 and TNFA-2 were 94.1 and 5.9%, respectively, in the patients and were not different from controls. There was no correlation with clinical severity. The only association was between TNFA-2 and gross haematuria (which is not a risk factor). The second study from Korea [15] concerned 76 IgAN vs 100 controls: the genotype A2/A2 was more prevalent in the progressor group, but note that in our study we have found only 3 patients with this genotype. In the third study from Finland [16], the TNF study also was limited to the -308 polymorphism. In a cohort of 167 patients with IgAN they found a reduced allelic frequency of TNFA-2: 9.0 vs 16.2% in 400 local controls. The TNFA-2 allele appeared as protective for the disease; however, progression of the disease did not differ significantly with TNF genotypes.

One of the aims of genetic studies is to provide knowledge about the prognosis of a given disease. IgAN shows large inter-individual variations in the rate of progression to CRF. Since the decline in renal function occurs rather slowly but most often regularly, it is important to identify the possible predictors of poor prognosis. It has been demonstrated that TNFA A2/A2 individuals on one hand and TNF d3/d3 individuals on the other produced higher amounts of TNF{alpha} than TNF A1/A1 and TNF non-d3/non-d3, respectively [10]. Normal individuals with the TNFA-2 allele in heterozygous combination (genotype A1/A2) also demonstrated increased TNF{alpha} levels [17]. The -308 TNFA polymorphism has been associated with increased risk and severity of infectious diseases [18] and infections after renal transplantation [19]. The TNFd polymorphism has been associated with more graft-vs-host disease and early mortality in bone marrow graft [20].

In our study, we could not demonstrate a statistically significant difference between the different genotypes and the clinicopathological data. However, patients with the TNF{alpha} high-producer phenotype (A2 and d3) usually have more severe disease; the prevalence of CRF was 36.6% in this subgroup compared with 22.9% [P=NS (0.08)] in the others and their survival was significantly lower (P=0.04) only in monovariate analysis. A possible pitfall of our study is that serum or urine TNF{alpha} levels were not measured in these patients and therefore could not be correlated with the genotypes. It would be equally interesting to investigate mesangial TNF{alpha} expression of the different genotypes.

There are other recently identified functional polymorphisms of the TNFA gene that are reported to influence TNF{alpha} production: a -863 C to A substitution in the promoter region of the TNFA gene associated with reduced circulating levels of TNF{alpha} [21] and a -238 G to A substitution in the promoter region of the TNFA gene associated with increased TNF levels [22]. It should also be mentioned that polymorphism of the TNFß gene (TNFB) may modulate the production of both TNFß (lymphotoxin {alpha}) and TNF{alpha} cytokines [22]. Further investigation of TNFB gene polymorphism might be needed to get a complete picture of the role of TNF{alpha} in IgAN.

The demonstration of the exact role of TNF{alpha} in the evolution of the inflammatory process in glomerulonephritis may also have therapeutic implications. Administration of TNF{alpha} increased the severity of injury in experimental models of nephritis, while specific inhibition reduced the injury [23]. However, the potential of inhibiting TNF{alpha} for the control of glomerular injury in human IgAN needs to be further investigated. Undoubtedly, the glomerular injury encountered in IgAN is influenced by a network of different cytokines and finding a monolithic explanation is unlikely.

In this study, we were dealing with sporadic cases of IgAN and not with rare familial cases of IgAN in whom a recent study [24] by whole-genome scanning demonstrated a gene, called IGAN1, located at 6q 22–23. This gene lies 80 cM distal to the MHC loci and is at a great distance for linkage disequilibrium.

In conclusion, we found in a large cohort of patients a significant association between the occurrence of IgA nephritis and the TNF{alpha} low-producer phenotype (non-A2/non-d3). However, we could not demonstrate significant and independent influences of the various TNF{alpha} genotypes on the progression of the disease.

Conflict of interest statement. None declared.



   Appendix
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Appendix
 References
 
For example for TNFA (Tables 1Go and 2Go) in the patients: A1 allele frequency=0.876; A2 allele frequency=0.124; A1A1 genotype=(0.876)2x242=186; A2A2 genotype=(0.124)2x242=4; A1A2 genotype=2 (0.876x0.124)x242=53. The genotype distribution observed was 185, 3 and 54, respectively, which is very close to the theoretical numbers ({chi}2=0.153, P=NS).

For example for TNFd (Tables 3Go and 4Go): d3 allele frequency=0.508; non-d3 allele frequency=0.492; d3d3 genotype=(0.508)2x242=62; non-d3/non-d3 genotype=(0.492)2x242=59; d3/non-d3 genotype=2(0.508x0.492)x242=121. The genotype frequency observed was 63, 59 and 120, respectively, which is very close to the theoretical numbers ({chi}2=0.012, P=NS).



   Notes
 
Correspondence and offprint requests to: Prof. François Berthoux, Nephrology, Dialysis and Renal Transplantation Department, University North Hospital, F-42055 Saint-Etienne Cédex 2, France. Email: francois.berthoux{at}chu-st-etienne.fr or francois.berthoux{at}wanadoo.fr Back

Present address: S. Tuglular, Department of Internal Medicine, Division of Nephrology, Tophanelioglu Cad 13/15, 81130 Altunizade, Istanbul, Turkey. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Appendix
 References
 

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Received for publication: 20. 6.02
Accepted in revised form: 23.10.02