Departments of Internal Medicine, 1Section Nephrology, 2Genetics, Biology and Biochemistry, 3Clinical Pathology, Analysis Laboratory Service of Transplant Immunology and 4Epidemiology Unit of Cancer of the University of Torino, S.Giovanni-Molinette Hospital, Torino, Italy
Correspondence and offprint requests to: Piero Stratta, MD, Department of Internal Medicine, Section Nephrology, S.Giovanni Molinette Hospital, Corso Bramante 88, I-10126, Torino, Italy. Email strattanefro{at}hotmail.com
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Abstract |
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Methods. The effect of combination of variant alleles of four genes encoding for three components of the RAS [angiotensin converting enzyme insertion/deletion (ACE I/D), angiotensin II receptor 1 (AT1R 1166A/C), angiotensinogen (AGT M235T)] and for NOS (ecNOS4b/a) on the development and progression of membranous GN (MGN) were evaluated in a longitudinal study comparing 117 patients with serum creatinine (s-Cr) <1.5 mg/dl at renal biopsy and follow-up 5 years (KaplanMeier and Cox multivariate analysis). The control group consisted of DNA from 171 organ donors.
Results. We found no relationship between single or combined variations of the four gene polymorphisms and development of MGN. Among single gene variations, there were no independent genetic risk factors for the progression of renal disease, after adjustment for age, sex, hypertension, proteinuria, s-Cr, chronicity and activity index. However, double variation coincidences such as the combination of the allele a of ecNOS4b/a and both the allele D of ACE I/D (2 =4.80, P = 0.028; HR = 1.97, 95% CI 0.983.96) and the allele T of AGT (M235T) (
2 = 5.09, P = 0.024; HR = 2.84, 95% CI 1.395.82) exerted an additional effect that was higher than that of the single gene variations.
Conclusion. This study is the first to demonstrate a role for an interaction between simultaneous variations of genes encoding for NOS and components of RAS in the progression of MGN. Interactions between various polymorphisms may explain conflicting results obtained in previous studies that examined single gene variations, since the effect of a single locus variation may be influenced by the simultaneous presence of other variant alleles in polygenic diseases such as primary GN. However, the small sample sizes and possible multiple interactions limited the interpretation of the current findings, which may represent true biological interaction or simply statistical interactions or spurious results due to the small sample sizes.
Keywords: angiotensin converting enzyme I/D polymorphism; endothelial nitric oxide synthase polymorphism; angiotensin II type 1 receptor polymorphism; angiotensinogen 235 polymorphism; membranous glomerulonephritis; reninangiotensin system
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Introduction |
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For angiotensin converting enzyme (ACE), there is an insertion/deletion (I/D) polymorphism in intron 16 that correlates with the inter-individual ACE level variability in plasma and tissues. The DD genotype correlates with the highest plasma enzyme concentrations and with the clinical course of cardiovascular diseases [4,5].
The AII receptor 1 (AT1R), which mediates most of the cardiovascular effects of AII, is expressed by vascular smooth muscle cells. The adenine/cytosine (A/C) polymorphism at base 1166 of the AT1R gene is associated with cardiovascular phenotypes, and the CC genotype underlies a condition associated with increased cardiovascular risk [6].
Angiotensinogen (AGT) secretion in hepathocytes is regulated by AII. The molecular variant encoding threonine instead of methionine at position 235 (T allele) is associated with higher plasma AGT levels and contributes to the hypertensive phenotype in Caucasian and Japanese populations [7].
For NOS, different polymorphisms of the endothelial constitutive NOS (ecNOS) gene have been described, including an allelic variant in intron 4 (ecNOS4b/a) that is associated with lower levels of plasma NO metabolites and with the smoking-related risk for coronary artery disease [8].
Taken together, these findings demonstrate that the variant alleles of these four polymorphisms may operate as risk factors for a number of cardiovascular events. Importantly, these single variant alleles have also been shown to be also associated with faster progression of various renal diseases [9,10]. In the case of primary GN, several authors including ourselves [11] have suggested that genetic variability in certain components of RAS and NOS may modify the progression of GN with prominent IgA deposits (IgA-GN) [12], whereas others have failed to find a positive association [13]. One group found no association between genetic polymorphisms of the RAS system and focal segmental glomerulosclerosis (FSGS) in children [14].
If two or more polymorphisms of candidate genes contribute even slightly to a genetic background for cardiovascular disease, their contribution may become additive or multiplicative during coincidence of double or more than double combinations of variant alleles.
For example, an additive interaction between ACE I/D and AT1R 1166 A/C or AGT M235T has been demonstrated in patients with coronary disease and cardiac hypertrophy, and a synergistic effect of DD and CC or DD and TT genotypes significantly increased the odds ratios in this patient population [15]. However, other results occur in patients bearing multiple candidate unfavourable genotypes, as was demonstrated for risk of disease in patients simultaneously carrying the variant D allele of ACE I/D and the non-variant M allele of AGT M235T [16].
Previous studies evaluating combined polymorphisms have reported no effects of coincidence of variant alleles of ACE I/D and ecNOS4b/a in the progression of IgA-GN, membranous GN (MGN) and FSGS [17], but an additive effect of the DD and MM genotypes of ACE I/D and AGT M235T polymorphisms in IgA-GN [18,19].
MGN is the most common cause of nephrotic syndrome in adults, and eventually progresses to ESRF in 1030% of patients at 10-year follow-up. Moreover, many studies suggest that genetic factors play an important role in the development of MGN.
The purpose of the present study was to evaluate the relationships between combinations of variant alleles of genes encoding for three components of RAS (ACE, AT1R and AGT) as well as for ecNOS and the development and progression of primary MGN.
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Subjects and methods |
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Study groups included 117 patients with primary MGN. Only patients with biopsy-proven diagnosis of MGN and serum creatinine (s-Cr) <1.5 mg/dl at biopsy were enrolled. We excluded cases with diagnosis of underlying diseases (autoimmune disorders, malignancy, chronic infections) and patients with follow-up <5 years or with incomplete records.
In all patients, we investigated age, sex, follow-up (in months), s-Cr concentration, urinary protein excretion (grams per 24 h) and blood pressure at the time of biopsy. Hypertension was defined as systolic blood pressure 140 mmHg and diastolic blood pressure
90 mmHg. Renal pathological lesions were scored in order to calculate Chronicity Index (from 0 to 4 for glomerular sclerosis and interstitial fibrosis) and Activity Index (from 0 to 4 for mesangial proliferation and interstitial infiltration).
We also registered the number of patients that received ACE-inhibitors (ACE-I), corticosteroids and immunosuppressants at any time during follow-up.
The control group consisted of organ donors from the local bank (n = 171: 87 males and 84 females) that gave informed consent to furnish local genotype frequencies.
Study design
Study I. The effect of single and combined polymorphisms in the development of primary MGN was evaluated by comparing frequency and odd ratios of different genotypes among patients and controls.
Study II. The effect of single and combined polymorphisms in the progression of MGN, taking into account other putative risk factors, such as age, sex, s-Cr, proteinuria, hypertension and renal pathology, was studied by evaluating kidney survival in patients with s-Cr 1.5 mg/dl at biopsy and follow-up
5 years. Genotype frequencies were compared in subgroups reaching (progressors) and not reaching (non-progressors) the primary end-point. The primary end-point was designated as the time at which s-Cr doubled or when patients began regular dialysis treatment (RDT).
Extraction of genomic DNA and genotype determinations
Genomic DNA was extracted from peripheral blood lymphocytes according to a standard salting-out method.
ACE. The 287 bp insertion/deletion (I/D) polymorphism in intron 16 of the ACE gene was determined as described previously [20]. PCR products of 190 bp for the D allele and 490 bp for the I allele were resolved on a 2% agarose/0.5x TBE gel by ethidium bromide staining.
AII receptor 1. The A/C transversion at base 1166 in the 3' untranslated region of the AT1R gene was determined as described previously [20]. A 349 bp PCR product was digested with DdeI for 2 h at 37°C and resolved on a 2% agarose/0.5x TBE gel by ethidium bromide staining. After restriction digest, the 1166C allele was cut into 139 and 210 bp fragments.
AGT. The C/T transition in exon 2 of the AGT gene that causes a methionine/threonine substitution at codon 235 (M235T) was determined by PCR amplification of the region using a modified primer (forward 5'-AGGCTGTGACAGGATGGAAGACTGGCTGCTCGTTGA-3', reverse 5'-CTGCCCATCTCCAAGGCCTGACTG-3') introducing a HindII restriction site in the presence of the C allele. The 163 bp PCR product was digested with HindII at 37°C for 3 h and resolved on a 2.5% agarose/0.5x TBE gel by ethidium bromide staining. After restriction digest, the T allele (235T) was cut into 128 and 35 bp fragments.
NOS. The 27 bp variable number of tandem repeats polymorphism in intron 4 of the ecNOS gene was determined according to a method described previously [8]. The five repeat ecNOS4b allele (b) was detected as a 420 bp fragment on a 2% agarose/0.5x TBE gel, while the four repeat ecNOS4a allele (a) is detected as a 393 bp fragment.
Statistical analyses
Clinical and demographic parameters were reported as means±SD or actual numbers and percentages. Student's tests and 2 test were used when appropriate. The time course from the renal biopsy to the end-point (initiation of RDT or doubling s-Cr) was analysed by the KaplanMeier method and the Cox proportional hazard model (Hazard Ratio =HR), taking into account the effect of several covariates.
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Results |
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Study I. Development of GN
Single gene study. Both patients and controls were in HardyWeinberg equilibrium. There were no significant differences in genotype frequencies for each gene between cases and controls, and this was supported by odds ratios from univariate and multivariate analysis adjusted for sex and other polymorphisms. A separate analysis was performed for the three genotypes of each polymorphism that examined individuals with at least one variant allele (Table 2).
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In individuals with two variant alleles (at least one for each polymorphism), there were no significant differences in the odds ratios for combinations of couples of genotypes in patients vs controls (Table 3).
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At baseline, patients were comparable for age, proteinuria, hypertension and scores for Chronicity and for Activity Index (data not shown). The evaluation of the time interval from the renal biopsy to the doubling of s-Cr concentrations or beginning of RDT, performed by KaplanMeier analysis, showed a faster progression in patients bearing DD and ID genotypes of the ACE I/D polymorphism vs ACE II, but this difference did not reach statistical significance (2 =3.30; P = 0.069). Although the Cox proportional multivariate analysis confirmed an increased risk for patients bearing the D allele, this increase did not attain statistical significance (Table 4, A). Analysis of clinical and histological covariates revealed that only hypertension (HR = 2.28; 95% CI 1.184.41) was a significant risk factor, whereas proteinuria (HR = 1.62; 95% CI 0.465.65), ACE-I therapy (HR = 1.58; 95% CI 0.753.34), Chronicity Index (HR = 1.24; 95% CI 0.861.79) and Activity Index (HR = 1.07; 95% CI 0.741.53) were not significant factors. Evaluation of Chronicity Index indicated that HR represented a slightly increased risk, probably because our patients were chosen only if s-Cr concentration was <1.5 mg/dl. Therefore, pathological signs of chronicity were present only in a minority of cases and the Chronicity Index was low (96% patients showed a Chronicity Index <2).
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Discussion |
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Although several studies have evaluated genetic backgrounds that predispose to the progression of primary GN, there has been little work examining the combined effects of different polymorphisms.
To our knowledge, our study is the first to evaluate the effect of simultaneous coincidence of variant alleles of four polymorphisms on the development and progression of MGN. However, small sample sizes and low statistical power (most of the associations reported in Table 4 for combined genetic factors had a statistical power <50% for estimation of statistical significance with alpha = 0.05) limited the interpretation of our study. Thus, although the present findings point to a biological interaction, our results may simply represent a statistical interaction.
In this current study, we failed to detect associations between these polymorphisms and the development of MGN, and there were no effects of single gene variation on the progression of renal disease. However, additive effects may be operative, such as the coincidental presence of variant alleles of genes for the RAS in combination with NOS. This association might act synergistically to induce an increased risk for progression of GN, a risk that is higher than when the single allele is present. An additive role disturbed local NOS modulation is an intriguing possibility since a disrupted balance between vasodilator and vasoconstrictor agents at the level of renal microcirculation may act as crucial event in worsening renal damage. Thus, in addition to studying the NOS polymorphism, we analysed many other NOS genes that have been shown to be involved in the progression of renal disease [3]. Therefore, the findings from the present study strongly suggest that a better understanding of inter-individual differences in the course of chronic renal damage in renal diseases must not be restricted to studying candidate genes of the RAS system but must also include examination of NOS gene polymorphisms.
The possibility that combinations of various genetic polymorphisms act as operative risk factors for the progression of primary GN may explain conflicting findings in previous studies. In fact, the actual phenotype of a single genotype might be modulated by the simultaneous presence of variations at other loci. It is conceivable that disparities in results from previous studies examining single genes may have stemmed from variable prevalences of other genetic risk factors in different populations. For example, the risk factor due to the D allele of the ACE I/D polymorphism may be enhanced by the coincidence of other variant alleles, and this was supported by our results. In contrast, risk factors linked to certain alleles may be underestimated in the absence of other synergistic variations, as was shown for the T allele of AGT and the a allele of NOS in our study.
The present results remain to be confirmed in larger samples from comparable patient populations, especially since it is not possible to exclude the presence of other unmeasured polymorphisms of genes having effects on renal diseases that could not be detected through linkage disequilibrium with the polymorphisms studied. The relationships between these genetic risk factors and therapeutic responsiveness are of great potential importance and warrant further investigations to examine the role of NOS. Finally, we wish to re-emphasize that the observed statistical interaction between genes does not necessarily point to biological significance.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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