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. Author manuscript; available in PMC: 2014 Mar 13.
Published in final edited form as: Biochem Genet. 2009 Jun 30;47(0):688–693. doi: 10.1007/s10528-009-9266-y

Apolipoprotein E polymorphism in hemodialysed patients and healthy controls

Jaroslav A Hubacek a,b,c, Silvie Bloudickova a, Ruzena Kubinova d, Hynek Pikhart e, Ondrej Viklicky a, Martin Bobak e; for the MIA group
PMCID: PMC3951864  EMSID: EMS57282  PMID: 19565203

Abstract

The possible association between the end-stage renal disease (ESRD) and apolipoprotein E (APOE) polymorphism was found in some, but not all studies. We have analysed the APOE genotypes in 995 haemodialysed patients (cases) and a sample of 6242 healthy individuals (controls) in the Czech Republic. There was a statistically significant difference in the frequencies of APOE alleles between the cases and controls, with more carriers of the APOE2 allele in ESRD patients (15.9%) than in controls (12.2%) (p=0.005). The odds ratio of ESRD for the APOE2 allele, compared with APOE3E3 homozygotes, was 1.37 (95% confidence interval 1.13-1.67). The strength of the association increased with the time spent on haemodialysis: the odds ratios of all-cause ESRD in patients dialysed for 8 or more years, 1-8 years and less than one year (non-survivors) were 1.27 (0.94-1.71), 1.41 (1.09-1.81) and 1.94 (0.88-4.18), respectively. This study suggests that the APOE2 allele is a possible genetic risk factor for all-cause ESRD in Caucasians.

Keywords: Apolipoprotein E, end-stage renal disease, polymorphism

Introduction

End-stage renal disease (ESRD) is a serious health problem worldwide, with more than one million of patients requiring renal replacement therapy (Moeller et al. 2002), and the number of ESRD patients continues to grow. Development of ESRD is a function of both genetic and non-genetic factors. While a number of non-genetic factors have already been identified (e.g. hypertension, obesity, smoking, inadequate activation of angiotensin-renin system) (Gross and Amann, 2004, Leblanc et al. 2005), the genetic factors influencing renal failure are poorly understood (Nordfors et al. 2005).

Lipid abnormalities patients with ESRD are common, and these abnormalities may further contribute to the progression of renal disease. The most important genetic determinant of plasma cholesterol is the gene for apolipoprotein E (APOE, gene ID 348, OMIM acc No. 107741). ApoE is a component of triglyceride-rich lipoproteins and a subfraction of high density lipoproteins. There are three common APOE variants (E2 [Arg158 → Cys], E3 and E4 [Cys112 → Arg]), and almost thirty APOE mutations (Hubacek et al. 2000). The different functional properties of the APOE isoforms result in an elevated plasma cholesterol in APOE4 allele carriers, while the lowest plasma concentrations of cholesterol are found in carriers of the APOE2 allele (Davignon et al. 1988). However, as the frequencies of individual alleles vary between different populations, mainly due to differences in ethnic background (Hubacek et al. 2000, Davignon et al. 1988, Gerdes et al. 1992, Svobodova et al. 2007), findings on the associations between genotypes and disease in one population cannot be automatically extrapolated to all other populations.

It is known that the APOE is expressed in the kidney tissue but the exact role of apolipoprotein E in the kidney remains unclear. Animal experiments suggest that apolipoprotein E has a protective function, possibly as an autocrine regulator of mesenginal expansion and kidney function (Chen et al. 2001). In addition, many human APOE mutations have been detected in the patients with different types of kidney disease and they may independently contribute to kidney dysfunction (for review see Hubacek et al. 2000, Liberopoulos et al. (2004). It is therefore plausible that the common APOE variants may play a significant role in kidney disease development.

Several previous studies, using different research designs, have analysed APOE variants in ESRD patients. These studies, reviewed by Liberopoulos et al. (2004), suggest that the carriers of the APOE2 allele have higher risk of ESRD development but the results so far are not conclusive. The major limitation of the previous studies relates to their low statistical power due to relative small numbers of subjects included in these studies. The comparison of studies is also complicated by the fact that there are many different causes of the ESRD.

In the present study, we have examined the relative frequencies of APOE genotypes in patients with all-cause ESRD and compared them with healthy controls drawn from a large population sample of healthy individuals. As the present study is much larger than previous investigations, it should provide a statistically more reliable test of the hypothesis that all-cause ESRD is associated with the APOE genotype.

Materials and Methods

We have genotyped 995 (out of 1014 collected, 565 males and 449 females,) adult patients (mean age 66.9 ± 12.7 years, CR 97.8%), who were undergoing haemodialysis for at least three months in 27 haemodialysis units across all over the Czech Republic; the centres were selected primarily on the basis of their willingness to participate in the study (Hubacek et al. 2007). The most common verified causes of end stage renal failure was diabetic nephropathy (N = 102), chronic tubulointerstitial nephropathy (N = 99) and nephrosclerosis based on hypertension (N = 39). Unfortunately, approximately 40% of the patients with chronic renal failure did not undergo renal biopsy and it was therefore not possible to specify the primary cause of the renal disease. We were therefore unable to analyse of the role of APOE polymorphism and ESRD with regard to the primary disease. Patients were further classified into three groups according to the time spend on the hemodialysis (HD) treatment (> 8 years, 1 - 7 years and those who survived for less than one year). Limited follow up of the patients shows that there was no conversion to the peritoneal dialysis; 78 (7.8%) underwent kidney transplantation.

As healthy controls, we used participants in a large cohort study, the Czech part of the international project Health, Alcohol, Psychological factors in Eastern Europe (the HAPIEE study [Peasey et al. 2006]). The participants of the HAPIEE study were randomly selected from population registers of 7 towns. We have genotyped 6,627 individuals (3,049 males and 3,578 females, mean age 58.3 ± 7.1 years). All patients and healthy controls were of Caucasian ethnicity.

Genomic DNA was isolated from whole blood using the salting out method (Miller et al. 1988). Genotyping of APOE was performed as described by Hixson and Vernier (1990). Because of the low numbers of APOE2E2 and APOE4E4 homozygotes, the subjects were divided in three groups for the statistical analysis: (1) carriers of the allele APOE2 (APOE2E2 and APOE3E2 individuals), (2) APOE3E3 homozygotes, and (3) carriers of the allele APOE4 (APOE4E4 and APOE4E3 individuals). Subjects with APOE4E2 genotypes (17 persons, [1.7%] from ESRD patients, 130 [2.1%] healthy controls) were excluded from the analysis because of the uncertainty about appropriate pooling with other genotypes. The statistical analysis used Chi-square and Fisher exact tests.

Results and Discussion

In both cases and controls, the most common genotype was APOE3E3 genotype (frequency in population controls 66.1%, table 1). Both in cases and controls, the genotype frequencies were in agreement with Hardy Weinberg equilibrium. There was a statistically significant difference in the frequencies of the APOE genotype between patients with all-cause ESRD and healthy controls (table 1), with more carriers of the APOE2 allele in all-cause ESRD patients than in healthy controls (15.9 % versus 12.2 %, odds ratio 1.37, 95% confidence interval 1.13-1.67, p=0.005). For the three most common diagnosed causes of ESRD available in our data, the APOE2 allele frequencies were 13.9% for diabetic nephropathy, 12.5% for chronic tubulointerstitial nephropathy, and 12.8% for nephrosclerosis based on hypertension; none of these frequencies differed significantly from that among controls. In addition, there were no significant differences in the frequencies of the APOE genotypes by history of ischemic heart disease or diabetes mellitus. The relative frequency of the APOE2 allele carriers increased as the survival on HD treatment decreased (table 2); the odds of dying within one year was almost twice higher among carriers of at least one APOE2 allele than among persons with APOE3E3 genotype (p for trend 0.001).

Table 1. APOE genotypes in all-cause ESRD patients and healthy controls and odds ratios (95% confidence interval) for all-cause ESRD for the +APOE2 or +APOE4 allele carriers vs. APOE3E3 homozygotes.

Cases N (%) Controls N (%) Odds ratio (95% CI
+ APOE2 allele 158 (15.9%) 760 (12.2%) 1.37 (1.13-1.67)
APOE3E3 625 (62.8%) 4128 (66.1%) 1.0 (reference)
+ APOE4 allele 195 (19.6%) 1224 (21.7%) 1.05 (0.88-1.26)
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Table 2. APOE genotypes and odds ratio of all-cause ESRD patients for the +APOE2 carriers vs. APOE3-3 homozygotes, stratified by time spend on haemodialysis treatment.

Time spend on
haemodialysis		 + APOE2 APOE3E3 +APOE4 Odds ratio (+E2 vs.
E3E3) (95% CI)
>8 years 60 (15.3%) 257 (65.5%) 75 (19.1%) 1.27 (0.94-1.71)
1-7 years 88 (16.3%) 340 (62.8%) 113 (20.9%) 1.41 (1.09-1.81)
1 year non-survivors 10 (22.2%) 28 (62.2%) 7 (15.6%) 1.94 (0.88-4.18)
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Our finding of the association between APOE2 and increased risk of all-cause ESRD is supported by three observations. First, the frequencies of the APOE genotypes in our study population were similar to those found in the neighbouring European populations (Gerdes et al. 1992). Second, the association was stronger for disease with shorter survival, which might reflect a biological gradient by the severity of the disease (although the primary disease was not known). The presence of the APOE2 allele could potentially affect survival on hemodyalisis treatment and perhaps influence the development of MIA (malnutrition-inflammation-atherosclerosis) syndrome (Pecoits-Filho et al. 2002). Finally, the observed association between APOE genotype and ESRD are also consistent with some (although not all) previous reports. Case-control studies by Oda et al. (1999) and Horita et al. (1994) in Japan (using cases with or without non-insulin-dependent diabetes mellitus, NIDDM) also found higher allele frequency of APOE2 in ESRD patients than in healthy population.

We are aware that the literature is not entirely consistent. For example, a study by Roussos et al. (2004) also suggested the involvement of APOE in renal disease but in their study the implicated allele was APOE3E4 genotype in a subgroup of ESRD patients with NIDDM; however, they did not observe a higher percentage of APOE2 in their cases, compared to controls. Other studies failed to detect any significant differences in APOE genotypes between patients with kidney disease and controls (Feussner et al. 1992, Guz et al. 2000, Arikan et al. 2007). However, these studies were very small, with less than 100 cases per study; given their low statistical power and the relatively weak association between ESRD and APOE genotype, false negative results are the most likely explanation.

A major limitation of this study is the lack of information on the primary disease in most of the ESRD patients. It was not possible to analyse the role of the APOE in patients with different primary diagnoses and to establish whether the effect of APOE is specific to a particular primary cause.

In conclusion, in this study, the largest on the relationship between all-cause ESRD and APOE genotype to date, we found a significantly higher frequency of the APOE2 allele carriers in all-cause ESRD patients in comparison to healthy controls, and this association was stronger in one-year non-survivors. The results suggest that the APOE2 allele is a possible genetic risk factor for ESRD development in Caucasians.

Acknowledgements

The project was supported by project No. NR 7958-5, IGA MH Czech Republic. The HAPIEE study is funded by the Wellcome Trust and the US National Institute of Aging. We thank the participating clinicians (P. Táborský - FMC Prague; V. Polakovič and M. Sezamová - VFN Prague 6; E. Svítilová - Ústí nad Labem; J. Vlasák and I. Sojková - FMC Sokolov; M. Ryba -Liberec; P. Knetl - Jihlava; M. Ullrych - Děčín; R. Drahozal - Dialcorp Prague; V. Pavuková - Chomutov; B. Pavlíková - Uherské Hradiště; D. Fischlová - Kladno; M. Mokrejšová - VFN Prague; H. Chmelíčková - Třebíč; E. Pauchová - Prachatice; P. Vyskočil - Louny; Z. Nýdlová - Strakonice; J. Kopenec - Benešov; P. Fixa - Hradec Králové; J. Hajný - Chrudim; P. Bubeníček and P. Syrovátka - IKEM Prague; J. Zahálková - Olomouc; S. Šurel - Brno; Z. Hobzek - Písek; A. Hrubý - FMC Slavkov; J. Suchanová - Tábor; S. Vaňková - Eurocare Prague; J. Brabcová - Blansko) for the collecting of the patient samples. We also thank workers in the National and Regional Institutes of Public Health who collected data on the population sample (controls).

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