1 Diabétologie, Hôpital Bichat, Assistance Publique-Hôpitaux de Paris, Paris, France
2 Département de Génétique, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux Paris, Paris, France
3 EA 3516, Faculté Xavier-Bichat, Université Paris VII, Paris, France
4 INSERM U525, Hôpital Pitié-Salpêtrière, Paris, France
5 Médecine Interne, Hôpital Universitaire, Poitiers, France
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ABSTRACT |
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Diabetic nephropathy results from the interaction of genetic factors with chronic hyperglycemia. Atrial natriuretic peptide (ANP) may affect the course of diabetic nephropathy by inducing afferent arteriolar dilatation and efferent constriction within the glomerulus (13). In type 1 diabetic subjects, high glomerular filtration rate and ANP concentration correlated with each other (4), and ANP infusion increases glomerular filtration rate, filtration fraction, and albuminuria (5). ANP favors diabetic hyperfiltration (13). Thus, the ANP gene is a candidate gene for diabetic nephropathy, but investigations (6,7) on this topic have been controversial.
We report here a systematic analysis of the ANP gene in large case-control and follow-up studies of type 1 diabetic subjects (8,9). We characterized genetic variability at the ANP gene locus and then investigated the association between polymorphisms and diabetic nephropathy. We carried out both genotype- and haplotype-based association analyses using newly elaborated statistical methods (10).
The characteristics of the 489 participants in the cross-sectional Genetique de la Nephropathie Diabetique (GENEDIAB) study (8) are given in Table 1. Allele frequencies for the nonsynonymous polymorphisms are available in the online appendix (available at http://diabetes.diabetesjournals.org). All of these single nucleotide polymorphisms (SNPs) were in Hardy-Weinberg equilibrium. No significant association was found between the distribution of any of these SNPs and the stages of nephropathy (online appendix). The pairwise linkage disequilibrium matrix is also available in the online appendix. Eight haplotypes had a frequency >1% and accounted for >94% of all haplotypes. No association was found between ANP gene haplotypes and nephropathy stages (online appendix).
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A careful systematic search for contributive SNPs in the coding and noncoding regions of the ANP gene led us to genotype five SNPs in both the case-control and follow-up studies. The association of the 2238C allele with the progression of diabetic nephropathy was significant only in poorly controlled subjects in the SURGENE study, suggesting that the interaction might only be detectable under certain circumstances. Such a stress-the-genotype interaction is classical in complex traits and has been characterized for the ACE I/D polymorphism (11). However, because the cross-sectional study was negative, we cannot exclude a stratification effect, even though the interaction with glycemic control was defined to be tested a priori in our analysis (9).
One major advantage of haplotype analyses is a considerable reduction in the number of statistical tests required to investigate several polymorphisms; the number of haplotypes is usually much smaller than the number of tests involving all possible interactions between genotypes. Therefore, such analyses do not necessarily inflate the number of tests. Besides, it allows for differentiation of the true effect of a polymorphism from those due to its linkage disequilibrium with other "functional" variants. This was especially true here; the effect of the C708T polymorphism on renal disease progression observed in univariate analysis was in fact the consequence of its linkage disequilibrium with the T2238C polymorphism. However, even though we found that the observed effect of the T2238C polymorphism was consistent on the two haplotypes by which it was carried, replication in other populations and studies would be required because it cannot be excluded that the observed association is artificial and, for instance, due to multiple testing.
The GENEDIAB multicenter cross-sectional study (8) aimed to assess the association between genetic variations and the severity of diabetic nephropathy. Because all subjects had past or present proliferative diabetic retinopathy, they had expressed their risk of renal complications due to diabetes duration and control. In the SURGENE study (9), the same issue was investigated on a follow-up basis with Caucasian participants recruited at a single center in the Angers region. End-stage renal disease was the only exclusion criterion at baseline (12 patients), so survival bias is unlikely. The baseline characteristics of the participants and their rate of progression are very similar to those reported elsewhere (12).
The first work (6) investigating ANP gene polymorphisms and type 1 diabetic nephropathy found that the 2238C allele was associated with lower severity of the disease. This result was not reproduced elsewhere (7). Our case-control study was also inconclusive. However, ANP haplotypes carrying the 2238C allele accelerated the course of renal disease in SURGENE. The reasons for this discrepancy are unclear. The participants of the Italian and German studies were also Caucasian, and the reported genotype frequencies were similar (6,7). Survival bias and population stratification cannot be ruled out in case-control studies. We believe that differences in study design were probably a major cause of the differences in the results obtained.
The substitution of a T for a C at position 2238 eliminates the regular stop codon. A new stop codon arises six nucleotides further on, and translation results in a polypeptide with two additional arginines (13). Nannipieri et al. (6) found plasma ANP levels and albumin transcapillary escape rates to be lower in type 1 diabetic patients carrying the 708T and 2238C alleles. However, another study (14) found no association between ANP T2238C polymorphism and ANP levels in Japanese subjects. Finally, the functional effect of ANP T2238C polymorphism was evaluated three times after low-, normal, and high-salt diets in 105 Polish subjects. Following all three diets, plasma ANP levels were strongly associated with T2238C polymorphism and were lowest in TT subjects (15) (A. Ciechanowicz, personal communication). Thus, the functional significance of this polymorphism remains to be determined.
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RESEARCH DESIGN AND METHODS |
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The follow-up cohort, SURGENE, is a prospective single-center study of 310 type 1 diabetic patients (Angers, University Hospital, France) selected on diabetes duration >3 years, diagnosis before age 40 years, and no other chronic disease (whatever their retinopathy or nephropathy stages), except end-stage renal disease at baseline (9). The median follow-up duration was 6 years (range 210). For technical reasons, only 301 patients were analyzed (characteristics in Table 2). A renal event was defined as progression to a further stage of nephropathy, thereby defining the progressors against the nonprogressors during the study.
All participants gave written informed consent, and the studies were approved by the ethics committee of Angers University Hospital (Angers, France).
Diabetic nephropathy classification.
Diabetic nephropathy stages (8) were defined as follows: no nephropathy, normal urinary albumin (<30 mg/24 h, 20 µg/min, or 20 mg/l) and plasma creatinine <150 µmol/l without antihypertensive treatment in two or three of three consecutive assessments; incipient nephropathy, microalbuminuria (30300 mg/24 h, 20200 µg/min, or 20200 mg/l) without antihypertensive treatment and plasma creatinine <150 µmol/l; established nephropathy, past or present macroalbuminuria (>300 mg/24 h, 200 µg/min, or 200 mg/l) in patients on antihypertensive treatment or macroalbuminuria without antihypertensive treatment and plasma creatinine <150 µmol/l; and advanced nephropathy, past or present macroalbuminuria with or without antihypertensive treatment and plasma creatinine >150 µmol/l or renal replacement therapy.
Genotyping.
Several SNPs were already described (14) for the ANP gene. We carried out a systematic search for new SNPs by direct sequencing of the DNA of a series of 48 subjects. The promoter region was not sequenced because previous results (14) showed a single, uncommon polymorphism. We used direct sequencing to genotype the two cohorts. Three PCR products were analyzed for the case-control study and two for the follow-up study (see technical conditions in the online appendix). Thus, we genotyped known variations (nucleotide numbering according to genomic position, GenBank accession number K02043): G663A, C708T, G1837A, A1869G (these two SNPs for only the case-control study), T2238C, T2325C, T2332C, and T2455C, and we also identified a new SNP at position 2311 (G2311T) and a new insertion/deletion polymorphism at position 2497 (2497 I/D). The polymorphisms G1837A and A1869G were in strong linkage disequilibrium with G663A and T2238C, respectively. We therefore did not genotype them in SURGENE subjects.
Statistical analysis.
Statistical analysis was carried out with Statview software (SAS Institute, Cary, NC). Data are presented as means ± SD or medians (ranges) for skewed distributions. Groups were compared using parametric (ANOVA or Students t test) or nonparametric (if not normally distributed, Kruskal-Wallis or Mann-Whitney U tests) tests for continuous variables. For survival analysis (with progression to a higher stage of diabetic nephropathy, i.e., defining a renal event, as the outcome variable) time to first renal event curves were generated by Kaplan-Meier estimation and compared, using the log-rank test, both on all participants and separately in those having poor (HbA1c higher than the median, i.e., 8.5%) versus good (HbA1c at median or less) glycemic control (9). Coxs proportional hazards model was used to investigate the relationship between several candidate prognostic variables and the outcome variable in the genotype-based analyses.
Association between ANP gene haplotypes and nephropathy status (no nephropathy versus nephropathy [incipient, established, and advanced pooled]) was investigated by means of a maximum likelihood method that we recently proposed (10) for haplotype-based association analysis. This method allows us to simultaneously estimate haplotype frequencies and covariate-adjusted haplotypic odds ratios by comparison with a reference haplotype.
Here this method was extended to take into account survival data analysis by use of a parametric Weibull model to describe the association between haplotype and survival outcome, progression to a further stage of nephropathy. This allowed us to estimate the haplotype effects, expressed as HRRs (see the online appendix for details) (10).
By setting appropriate constraints on parameters, we were also able to use these models to test for homogeneity in certain haplotype effects. For example, we investigated whether the effects of the GCCTT and GTCGT haplotypes could be assumed to be equal by means of the likelihood-ratio criterion (10).
Finally, differences in haplotype frequency distributions according to the stages of nephropathy were investigated with Arlequin software (16).
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ACKNOWLEDGMENTS |
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We thank Philippe Coudol, Valérie Boccio, and Franck Péan for technical assistance and Vanessa Auger and Françoise Defrance for secretarial assistance.
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FOOTNOTES |
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Address correspondence and reprint requests to Ronan Roussel, Diabétologie, Hôpital Bichat, 46 rue Henri Huchard, 75877 Paris Cedex 18, France. E-mail: ronan.roussel{at}polytechnique.org
Received for publication July 17, 2003 and accepted in revised form January 27, 2004
ANP, atrial natriuretic peptide; SNP, single nucleotide polymorphism
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REFERENCES |
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