Lack of relationship in long-term type 1 diabetic patients between diabetic nephropathy and polymorphisms in apolipoprotein {varepsilon}, lipoprotein lipase and cholesteryl ester transfer protein

Samy Hadjadj1, Yves Gallois2, Gilles Simard2, Béatrice Bouhanick1, Philippe Passa3, André Grimaldi4, Pierre Drouin5, Jean Tichet6, Michel Marre,1, on behalf of the GENEDIAB (Genétique de la Nephropathie Diabétique) Study Group,7 and the D.E.S.I.R. (Données Epidémiologiques sur le Syndrome d'Insulino-Résistance) Study Group,7

1 Médecine B and 2 Biochimie B, University Hospital, Angers, 3 Saint-Louis Hospital, Paris, 4 La Pitié Hospital, Paris, 5 Jeanne D'Arc Hospital, Dommartin Les Toul and 6 Institut Régional de la Santé, La Riche, France 7 See appendix



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Background. Genetic susceptibility contributes to the risk of diabetic nephropathy. Lipid disorders may favour diabetic nephropathy. Thus polymorphisms in lipid metabolism are candidates for the genetic component of risk for diabetic nephropathy.

Methods. We searched for a contribution of the genetic polymorphisms of lipoprotein lipase (LPL), cholesteryl ester transfer protein (CETP) and apolipoprotein {varepsilon} (Apo E) to the development of diabetic nephropathy by studying 494 type 1 diabetic patients with proliferative retinopathy and various stages of diabetic nephropathy (GENEDIAB Study). The selection process ensured that all patients had expressed their risk of chronic complications due to uncontrolled diabetes. Thus the nephropathy stages were largely influenced by genetic background. The lipid profile included fasting plasma total cholesterol (TC), triglycerides (TG), apolipoprotein A1 (Apo A1) and B (Apo B), and lipoprotein (a) (Lp(a)). Genetic polymorphisms were determined by PCR-based detection of Apo {varepsilon} (e2/e3/e4), LPL (mutation Asn 291 Ser) and CETP (TaqIB B1/B2).

Results. One hundred and fifty-seven patients (32%) had no nephropathy, 104 (21%) incipient nephropathy, 126 (25%) established nephropathy and 107 (22%) advanced nephropathy. There was a significant relationship between the stages of diabetic nephropathy and TC (P=0.002), TG (P<0.0001), Apo B (P=0.0007) or Lp(a) (P=0.038), but not Apo A1. However the genetic polymorphism distributions of LPL, CETP and Apo {varepsilon} did not differ in terms of renal complications. The study power to reject the null hypothesis was 58% for the Apo {varepsilon} genotypes.

Conclusion. These results support no or only marginal effects of a genetic basis for lipid disturbances encountered in diabetic nephropathy.

Keywords: apolipoprotein E; cholesteryl ester transfer protein; diabetic nephropathy; genetics; lipoprotein lipase; type 1 diabetes



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An increased concentration of plasma lipids is a risk factor for nephropathy in type 1 (insulin-dependent) diabetes [1]. An adverse lipid profile can be secondary to glomerular disease, but recent follow-up studies suggest that it may cause nephropathy, as normoalbuminuric type 1 diabetic patients who developed diabetic nephropathy have been found to have higher initial triglycerides (TG) or LDL-cholesterol concentrations than those who did not [1]. This hypothesis is also supported by experimental studies showing that manipulations in lipid concentrations can alter glomerular disease [2].

Genetic polymorphisms of the enzymes and proteins involved in lipid metabolism (lipoprotein lipase (LPL), cholesteryl ester transfer protein (CETP) and apolipoprotein {varepsilon} (Apo E)) have been shown to affect plasma lipid concentrations [35]. These polymorphisms were related to coronary heart disease (CHD) in case-control or prospective studies [6,7]. Genetic susceptibility linking lipids to diabetic nephropathy is also supported by familial studies: the plasma TC and Apo B concentrations of healthy relatives of type 1 diabetic patients with microalbuminuria were reported to be higher than those of healthy relatives of normoalbuminuric patients [8]. The hypothesis of a possible association between polymorphism in Apo {varepsilon} and diabetic nephropathy has been examined in several studies, but results were discordant [911]. Similar studies have not been done with LPL or CETP polymorphisms.

We tested the hypothesis of a role for a genetic basis for risk of diabetic nephropathy through the alteration of the lipid profile in a large multicentre, cross-sectional study of type 1 diabetic patients selected according to proliferative retinopathy. This strategy was used to remove any uncertainty due to diabetes control and duration on the development of diabetic nephropathy [12].

The tested polymorphisms were those currently known to affect lipid metabolism and cardiovascular disease progression in the general population, i.e. LPL N291S [3], CETP TaqIB [4,6] and Apo {varepsilon} [5,7]. The plasma concentrations of total cholesterol (TC), triglycerides (TG), apolipoprotein A1 (Apo A1), apolipoprotein B (Apo B) and lipoprotein (a) (Lp(a)) were also measured in the fasting patients.



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Patients
The GENEDIAB Study is a cross-sectional, multicentre, prospective study designed to test the contribution of genetic factors to the development of renal complications in type 1 diabetes mellitus [12]. Briefly, 494 type 1 diabetic patients (mean age 44±12 years, 56% men, mean type 1 diabetes duration 29±10 years) were recruited on the basis of type 1 diabetes onset before 35 years and past or present proliferative diabetic retinopathy. Details of their characteristics are given elsewhere [12]. Control subjects were 359 sex- and age-matched healthy persons taking part in a prospective study in the French general population. The studies were approved by the Angers and Bicetre Hospital ethics committees (both in France) and all participants gave written informed consent.

Renal involvement was staged as described previously [12]: (i) no nephropathy: normal urinary albumin excretion rate (<30 mg/24 h, 20 µg/min or 20 mg/l) and plasma creatinine <150 µmol/l without hypertension or antihypertensive treatment; (ii) incipient nephropathy: microalbuminuria (30–300 mg/24 h, 20–200 µg/min or 20–200 mg/l) without antihypertensive treatment, and plasma creatinine <150 µmol/l; (iii) 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 (iv) advanced nephropathy: past or present macroalbuminuria with or without antihypertensive treatment and plasma creatinine <150 µmol/l, or renal replacement therapy.

CHD was documented by the patient's record, a structured questionnaire and by the presence of Q wave on ECG (Minnesota code criteria 1.1.1–1.3.6), analysed blindly by an ECG comittee. CHD was staged as absent, angina pectoris or myocardial infarction.

Biochemical determinations
The TC and TG, Apo A1, Apo B and Lp(a) were assayed in a subset of 170 fasting patients. TC and TG were determined using enzyme-colorimetric methods (Cholesterol CHOD PAP from Boehringer Mannheim, Mannheim, Germany and Unimake 7 from Roche Diagnostic Systems, Basel, Switzerland). Plasma Apo A1, Apo B and Lp(a) (assay sensitivity 0.026 g/l) were measured using a nephelometric method (OUED, OSAN and OQHL, respectively; Behring Diagnostics, Marburg, Germany).

LPL, CETP and APO {varepsilon} genotyping
Five hundred microgrammes of DNA were extracted from a 15 ml blood sample from each patient by the phenol chloroform method. The Apo {varepsilon}, LPL and CETP genotypes were determined after PCR. All genotypes were determined blindly by two technicians with any disagreement resulting in the genotyping being repeated until agreement was obtained. The genotype of Apo {varepsilon} could not be ascertained in three subjects, those for LPL in four subjects and for CETP in one subject.

Apo {varepsilon} alleles were identified by the Cys and Arg residues in position 112 and 158 using a previously described restriction isotyping method with two primers: Apo {varepsilon} F4 (5'-ACA-GAA-TTC-GCC-CCG-GCC-TGG-TAC-AC-3') and Apo {varepsilon} F6 (5'-TAA-GCT-TGG-CAC-GGC-TGT-CCA-AGC-A-3') [13]. Fragments resulting from enzymatic digestion were electrophoresed in 10% polyacrylamide gel then treated with ethidium bromide, and genotypes were scored by UV illumination. Apo e2 corresponds to Cys112 Cys158; Apo e3 to Cys112 Arg158 and Apo e4 to Arg112 Arg158. The Apo e2 allele carriers (Apo E2) were compared with Apo e3/e3 homozygotes (Apo E3) and Apo e4 allele carriers (Apo E4) to study the effect of Apo {varepsilon} genotype. Six Apo e2/e4 patients were excluded from the analysis, as they could not be classified as Apo E2 or Apo E4.

The LPL genotypes were determined by identifying the mutation Asn291 Ser, by mismatch PCR using two primers: LPL L1 (5'-CTG-CTT-CTT-TTG-GCT-CTG-ACT-GTA-3') and LPL L6 (5'-GCC-GAG-ATA-CAA-TCT-TGG-TG-3') and RsaI digestion [14]. PCR fragments were electrophoresed in 2% agarose gel stained with ethidium bromide, and genotypes were scored by UV illumination.

CETP gene polymorphism was identified by the presence (B1) or absence (B2) of a restriction site for TaqIB in intron 1 of the gene after PCR amplification with primers D (5'-CAC-TAG-CCC-AGA-GAG-AGG-AGT-GCC-3') and R (5'-CTG-AGC-CCA-GCC-GCA-CAC-TAA-C-3') [15,16]. Fragments resulting from enzymatic digestion were electrophoresed in 1.5% agarose gel stained with ethidium bromide, and genotypes were scored by UV illumination.

Statistical analysis
The Statview V® software (SAS Institute Inc. Cary, NC, USA) was used on an Apple MacIntosh computer. The study power as well as the number needed to screen were computed with Sample Power 1.0 (SPSS Inc. Chicago, IL, USA). Data are presented as mean±standard deviation (SD), or median (range) if variables were not normally distributed. Whenever variable distribution was skewed, data were log-transformed for analysis. Analysis of variance (ANOVA) was used to study lipid concentrations except Lp(a), according to various stages of nephropathy or lipid genetic polymorphisms. A non-parametric Kruskal–Wallis test was used to study the concentration of Lp(a) according to stage of nephropathy, as more than half of the patients yielded Lp(a) concentrations below assay sensitivity. {chi}2 or {chi}2 for trend tests were used to assess the distributions of genetic polymorphisms among different populations and according to stages of diabetic nephropathy. The Bonferroni correction for repeated statistical analyses was applied. The level of significance was set at 0.05.



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As previously described [12], 157 patients (32%) had no nephropathy, 104 (21%) incipient nephropathy, 126 (25%) established nephropathy and 107 (22%) advanced nephropathy. CHD was absent in 438 subjects, and angina pectoris and myocardial infarction were found in 19 and 37 patients, respectively. The degree of CHD was not related to the stage of diabetic nephropathy ({chi}2=8.73; P=0.19).

A significant effect of the stage of nephropathy was noticed on fasting TC (F=6.65; P=0.0022), TG (F=8.83; P<0.0001), Apo B (F=6.03; P=0.0007), Lp(a) (H=12.43; P=0.006) but not on Apo A1 (F=0.44; P=0.72) (Table 1Go). Patients were also analysed according to CHD: TC, Apo B and Lp(a) levels were higher in those patients with CHD (Table 1Go).


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Table 1. Fasting plasma lipid concentrations and stages of diabetic nephropathy and coronary heart disease in the fasting patients of the GENEDIAB study

 
The distribution of Apo {varepsilon} and CETP genotypes was not different in patients and controls ({chi}2=5.69, P=0.34, and {chi}=1.21, P=0.55, respectively) and was in Hardy–Weinberg equilibrium in both populations (Table 2Go). The LPL N291S mutation was less common in the GENEDIAB population than in the control group, but the difference was not statistically significant after Bonferroni correction ({chi}2=5.12; P=0.07) (Table 2Go).


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Table 2. Distribution of polymorphisms involved in lipid metabolism in the 494 patients ofthe GENEDIAB study and in 359 age- and sex-matched healthy subjects

 
The distribution of Apo {varepsilon} genotypes did not differ according to the stage of nephropathy ({chi}2 for trend=0.02; P=0.89) (Table 3Go). No association was found between Apo {varepsilon} polymorphism and renal failure (advanced nephropathy) ({chi}2=1.52; P=0.47). There was no significant association between the stage of renal involvement and LPL ({chi}2 for trend=2.40; P=0.12) or CETP genotypes ({chi}2 for trend=1.15; P=0.28). The allelic frequencies were also not different (Table 3Go). There was no relationship between CHD stages and Apo {varepsilon}, LPL and CETP polymorphisms (data not shown).


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Table 3. Lipid polymorphisms and stages of diabetic nephropathy in the GENEDIAB study

 
The genotype distributions were not different in the 170 fasting patients, compared with the whole cohort (data not shown). The lipid profile in the fasting patients was analysed according to the studied polymorphisms regardless of the stage of nephropathy. Plasma TC concentration was low in Apo E2 (5.40±0.89 mmol/l), intermediate in Apo E3 (5.66±0.73 mmol/l) and high in Apo E4 patients (5.94±0.92 mmol/l) (F=7.25; P<0.03). A similar trend was found for Apo B: 0.85±0.20 g/l in Apo E2, 0.98±0.25 g/l in Apo E3 and 1.03±0.30 g/l in Apo E4 (F=2.77; P=0.06). Fasting TG and Apo A1 concentrations were not related to the Apo {varepsilon} genotype (F=0.2, P=0.82, and F=2.04, P=0.13, respectively).

No significant effect of the LPL polymorphism was noticed on the lipid concentrations, but only four patients with the mutation were included in this analysis (data not shown).

Plasma TC, TG, Apo A1 and Apo B concentrations did not differ according to the CETP genotype (F=0.02, P=0.98; F=0.16, P=0.85; F=0.57, P=0.57; and F=0.02, P=0.98, respectively). However, in patients with no or incipient nephropathy (66 subjects), Apo A1 was low in B1B1 (1.41±0.25 g/l), intermediate in B1B2 (1.48±0.36 g/l) and high in B2B2 patients (1.71±0.33 g/l) (F=2.63; P=0.08).



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In this study, we found no significant association between diabetic nephropathy and genetic polymorphisms in Apo {varepsilon}, LPL and CETP. To our knowledge, the present study is the first to examine diabetic nephropathy, and LPL and CETP polymorphisms. Conversely, we found that plasma lipids were related to the severity of nephropathy and CHD (with the exception of Apo A1) in these patients.

Sample bias may be present in such a cross-sectional study, but it is unlikely to explain our results. First, the patient and control groups were in Hardy– Weinberg equilibrium for all the studied polymorphisms. Secondly, there was a relationship between Apo {varepsilon} polymorphism and total cholesterol in our patients, similar to that found in the general population [7]. Thirdly, the plasma concentrations of TC, TG, Apo B and Lp(a) were related to the stages of diabetic nephropathy, which confirms previous findings [17].

Plasma lipids have been recognized as predictors of diabetic nephropathy in longitudinal follow-up studies in type 1 diabetic patients [1,18]. However, a role for a genetically determined lipid profile on the diabetic nephropathy constitution is still under investigation. Genetic susceptibility to diabetic nephropathy was supported by familial studies and one study indicated that this genetic component is linked to familial susceptibility to cardiovascular diseases [19]. The plasma TC and Apo B concentrations of healthy relatives of type 1 diabetic patients with microalbuminuria were reported to be higher than those of healthy relatives of normoalbuminuric patients [8]. As Apo {varepsilon}, LPL and CETP activities can affect these plasma lipids through the studied polymorphisms [35], a possible association with diabetic nephropathy and CHD was worth being investigated.

We failed to show an association between diabetic nephropathy and Apo {varepsilon} polymorphism, although this polymorphism has a positive effect on TC concentration. This is in accordance with two other reports [9,11]. Recently, Chowdhury et al. reported that the Apo e2 allele was associated with nephropathy in type 1 diabetic patients [10]. However, plasma lipids were not reported [10]. According to this study [10], the number needed to screen to evidence a difference in Apo {varepsilon} genotype between those patients without and those with any stage of diabetic nephropathy was 293, with a study power of 90%. Our present study with 484 patients is, however, negative, and the statistical power derived from our data was 58.1% to reject the null hypothesis.

We found no association between LPL N291S polymorphism and diabetic nephropathy. However, this polymorphism was a plausible candidate, since it is associated with increases in TG and decreases in HDL-cholesterol [3]. These lipid alterations are predictors of progression to renal disease in diabetic and non-diabetic patients [1]. The present data must be interpreted cautiously since the LPL N291S mutation was infrequent, encountered in only eight out of 494 patients of the GENEDIAB study. Further prospective investigations are therefore required on the LPL N291S polymorphism in type 1 diabetic patients.

The polymorphism of the CETP gene was of interest since CETP activity has been found to be higher in type 1 diabetic patients with diabetic nephropathy than in those with normal urinary albumin excretion [20]. The fact that patients with the B1 allele had higher levels of CETP [4] provided the impetus to search for a role of the CETP gene polymorphism in diabetic nephropathy. We were not able to show any association between the stages of nephropathy and the CETP genotypes. A borderline effect of CETP B1/B2 polymorphism could be observed on Apo A1, as expected from studies in the general population [4]. Our data and previous reports [20] indicate no or a trivial role for CETP in the constitution of diabetic nephropathy or CHD in type 1 diabetic patients.

The association found between Lp(a) and diabetic nephropathy is consistent with previous reports in type 1 diabetic patients [17]. Plasma Lp(a) levels are genetically determined, but they can also be influenced by environmental factors. Thus the present findings can be interpreted as consequences, rather than causes of diabetic nephropathy.

In conclusion, we found no significant association between diabetic nephropathy in long-term type 1 diabetic patients, and polymorphisms involved in lipid metabolism. As this study is cross-sectional, these rather negative data may be due to lack of study power, although a large number of type 1 diabetic patients at high risk were recruited in this multicentre study. An alternative explanation may be that genetically based alterations in the lipid profile do not play a major role in the constitution of diabetic nephropathy.



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The GENEDIAB (Genétique de la Nephropathie Diabétique) Study Group composition was as follows.



   Investigators in clinical centers (number of recruited/selected patients)
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Philippe Passa, Abderamane Bouallouche in Hôpital Saint-Louis, Paris (82/72); Michel Rodier, Florence Galtier, Jacques Bringer in Nîmes-Montpellier (64/59); Michel Marre, Béatrice Bouhanick in Angers (coordinating center) (67/54); Caroline Sert, André Grimaldi in Hôpital La Pitié, Paris (57/51); Laurent Dusselier, Thérèse Crea, Pierre Drouin in Nancy (47/35); Zoubida Kahal, Dominique Simon in Créteil (31/29); Lucy Chaillous, Bernard Charbonnel in Nantes (27/27); Anne Muller, Richard Marechaud in Poitiers (25/25); Serge Halimi in Grenoble (27/25); Henry Sackmann, Jean-Pierre Tauber in Toulouse (23/22); Hervé Mayaudon, Bernard Bauduceau in Saint-Mandé (21/20); Nicolas Paquot, André Scheen in Liège (19/18); Patrick Miossec, Jean-Raymond Attali in Bondy (21/18); Isabelle Cerf, Guillaume Charpentier in Corbeil-Essonnes (13/12); Charles Thivolet in Lyon (11/11); Dominique Tielmans, Pierre-Jean Guillausseau in Hôpital Lariboisière, Paris (11/11); Jean-Yves Poirier, Hubert Allannic in Rennes (5/5); all centers are in France, except for Liège in Belgium.



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Philippe Passa (president), Jean-Raymond Attali, Bernard Bauduceau, André Grimaldi, Pierre-Jean Guillausseau, Michel Marre, Michel Rodier.



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François Alhenc-Gelas (INSERM U367, Paris), Bernard Bauduceau, Michel Rodier, Philippe Pezard (Angers).

The D.E.S.I.R. (Données Epidémiologiques sur le Syndrome d'Insulino-Résistance) Study Group is described in: Gallois Y, Vol S, Caces E, Balkau B and the DESIR study group: Distribution of fasting serum insulin, measured by enzymoimmunoassay, in an unselected population of 4032 individuals, references values, according to age and sex. Diabetes Metab 1996; 22: 427–431



   Acknowledgments
 
We thank the type 1 diabetic subjects who took part in this study. We also thank Franck Pean, Vincent Benoit, Gaëlle Jouet-Pastre and Gwenaëlle Brossard for technical assistance, and Laëtitia Martin, Isabelle Gouleau and Line Godiveau for secretarial assistance. The GENEDIAB Study was supported by grants from the Juvenile Diabetes Formation International (grant No. 194/55), by INSERM research network (grant No. 94/325), by CNAM-INSERM research (grant No. 4 AIC18), by a Bristol-Myers-Squibb–INSERM grant (grant No. 86–002), by a DRC-AP research grant (931007) and by the Association Diabète Risque Vasculaire (Angers, France). The English text was checked by Dr Owen Parkes.



   Notes
 
Correspondence and offprint requests to: Michel Marre, Endocrinologie, Diabétologie, Hôpital Bichat, 46 rue Henri Huchard, F-75018 Paris, France. Back



   References
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Received for publication: 9.11.99
Revision received 2. 8.00.