Nephrology, Dialysis and Renal Transplantation Department, University North Hospital, Saint Etienne, France
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Abstract |
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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 (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
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 (KaplanMeier) was lower. Nevertheless, TNF
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 ; TNFd microsatellites
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Introduction |
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Various immune mediators have been demonstrated to play a role in this inflammatory process. Tumour necrosis factor (TNF
) [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
), interleukin-6 (IL-6), IL-8, IL-10 and TNF
itself. TNF
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
and increased expression of TNF
by mesangial cells has been reported in IgAN [4,5]. Increased production of TNF
by peripheral blood mononuclear cells from patients with IgAN has been demonstrated previously [6,7]. Finally, plasma levels and urinary excretion of TNF
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
, could play a significant role in patients with IgAN.
The TNF 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
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
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 cytokine. Finally, both TNF-A2 and TNF-d3 alleles were associated with overproduction of TNF
.
The aims of this study were: (1) to determine the allelic frequency and the genotype distribution for these two TNF 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.
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Subjects and methods |
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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 CockroftGault 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 (06), vascular (05), interstitial (05) and tubular (04) 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 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 (PCRRFLP) 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 TrisHCl 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 bandsone 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 TrisHCl 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 HardyWeinberg equilibrium.
Statistical analysis
Comparisons between patients and controls. The different allelic frequencies and genotype distributions were compared using the 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 =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 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 (MannWhitney U-test) when applicable. Survival without significant CRF was analysed according to the KaplanMeier 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.
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Results |
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TNFd microsatellite polymorphism
The results are given in Tables 3 and 4
. 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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Combined TNFA and TNFd genotypes and alleles
The A2 allele and d3 allele are known to be associated with hyperproduction of TNF 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 5
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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Validity of the data
The number of observed genotypes was not significantly different by the 2 test from the theoretical numbers calculated from the HardyWeinberg 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 6. 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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Discussion |
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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 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
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 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
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
expression of the different genotypes.
There are other recently identified functional polymorphisms of the TNFA gene that are reported to influence TNF production: a -863 C to A substitution in the promoter region of the TNFA gene associated with reduced circulating levels of TNF
[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
) and TNF
cytokines [22]. Further investigation of TNFB gene polymorphism might be needed to get a complete picture of the role of TNF
in IgAN.
The demonstration of the exact role of TNF in the evolution of the inflammatory process in glomerulonephritis may also have therapeutic implications. Administration of TNF
increased the severity of injury in experimental models of nephritis, while specific inhibition reduced the injury [23]. However, the potential of inhibiting TNF
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 2223. 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 low-producer phenotype (non-A2/non-d3). However, we could not demonstrate significant and independent influences of the various TNF
genotypes on the progression of the disease.
Conflict of interest statement. None declared.
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Appendix |
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For example for TNFd (Tables 3 and 4
): 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 (
2=0.012, P=NS).
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Notes |
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Present address: S. Tuglular, Department of Internal Medicine, Division of Nephrology, Tophanelioglu Cad 13/15, 81130 Altunizade, Istanbul, Turkey.
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References |
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