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Apolipoprotein E genetic polymorphism influence the susceptibility to nephropathy in type 2 diabetes patients
Journal Pre-proof Apolipoprotein E genetic polymorphism influence susceptibility to nephropathy in type 2 diabetes patients
the
Kuralay K. Atageldiyeva, Rita Nemr, Akram Echtay, Eddie Racoubian, Sameh Sarray, Wassim Y. Almawi PII:
S0378-1119(19)30670-5
DOI:
https://doi.org/10.1016/j.gene.2019.144011
Reference:
GENE 144011
To appear in:
Gene
Received date:
4 June 2019
Revised date:
12 July 2019
Accepted date:
24 July 2019
Please cite this article as: K.K. Atageldiyeva, R. Nemr, A. Echtay, et al., Apolipoprotein E genetic polymorphism influence the susceptibility to nephropathy in type 2 diabetes patients, Gene (2019), https://doi.org/10.1016/j.gene.2019.144011
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© 2019 Published by Elsevier.
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Apolipoprotein E genetic polymorphism influence the susceptibility to nephropathy in type 2 diabetes patients
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Kuralay K. Atageldiyeva 1, Rita Nemr 2, Akram Echtay 3, Eddie Racoubian 4, Sameh Sarray 5,6,
of Medicine, Nazarbayev University, Astana, Kazakhstan, 2 Department of Internal
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1 School
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Wassim Y. Almawi 1,6
Medicine, LAU Medical Center - Rizk Hospital, Beirut, 3 Department of Adult Endocrinology and
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Diabetes, Rafic Hariri University Hospital, Beirut, 4 St. Marc Medical Center, Beirut, Lebanon, 5
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College of Medicine and Medical Sciences, Arabian Gulf University, Manama, Bahrain, 6 Faculty of
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Sciences, El-Manar University, Tunis, Tunisia.
Running Title:
Apolipoprotein E variants in Diabetic Nephropathy
Corresponding Author: Wassim Y. Almawi, Ph.D. Department of Biomedical Sciences, School of Medicine, Nazarbayev University, Nur-Sultan City (Astana), Kazakhstan. Tel. +7 (7172) 90 6340; E-mail. [email protected]
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ABSTRACT Background. An association between Apolipoprotein E (Apo E) alleles and genotypes and diabetic nephropathy (DN) was suggested, but with inconsistent results. We tested the relationship between serum lipids, Apo E alleles and genotypes with type 2 diabetes (T2DM), and DN pathogenesis.
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Methods. Study subjects comprised 1,389 normoglycemic controls, and 1,422 T2DM patients, of
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whom 825 were normoalbuminuric (DWN), and 597 presented with nephropathy (DN).
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Results. Significantly lower Apo 2, and higher Apo 4 allele frequencies was seen among T2DM
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patients than controls. Significantly higher frequency of 3/4, and lower frequencies of 3/3,
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2/3, and 4/4 carriers was seen among T2DM cases. Apo 2-carrying individuals were more frequently found in controls than in patients, while significantly higher frequency of 4-carrying
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genotypes was seen in T2DM cases. Significantly higher 2, and lower 3 allele frequencies were
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noted for DN group compared to DWN group. Significantly higher frequency of 2-containing
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2/3 and 2/4, and lower frequencies of 3/3 carriers was seen among DN cases. Apo 3/3 was associated with higher total cholesterol, LDL-cholesterol, and triglyceride levels in DN patients, and significantly higher triglyceride levels were seen in 2/3-carrying DN patients. Logistic regression analysis confirmed the association of Apo 3-containing 3/3, 2/3, and 3/4, and Apo 2-containing 2/4 with DN, after controlling for key covariates. Conclusion. The results of this case-control study provide evidence that the 2 and 3 alleles of APOE modify lipid profile, and constitute independent risk factors of DN in type 2 diabetes. The molecular mechanisms underlying this risk is discussed. Key Words:
Apolipoprotein E; diabetic nephropathy; genotyping; type 2 diabetes mellitus.
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Introduction Diabetic nephropathy (DN) is a major microvascular complication of diabetes, and the most common cause of end stage renal disease [1,2]. It is estimated that one third of type 2 diabetes (T2DM) patients will develop evidence of DN, and that a substantial number of them would require dialysis. This is likely to increase further with the global rise in the prevalence of T2DM
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and obesity, coupled with increased longevity. DN pathogenesis is multifactorial with a
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heterogeneous pathological manifestation. DN is characterized by clinical albuminuria, and is
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aggravated by hyperglycemia, hypertension, smoking and dyslipidemia [3,4]. Previous studies
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confirmed genetic predisposition to DN, highlighted by familial clustering of elevated urine
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albumin:creatinine ratio (ACR) cases among T2DM subjects [5,6], and by the association of genetic variants with the presence and severity of DN [7,8].
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Several candidate genes were described to be associated with DN, including those associated
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with inflammation [9,10], coagulation [11,12], and metabolism [13,14]. The latter include
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apolipoprotein E (Apo E), a 299 amino acid, 34 kDa glycosylated plasma protein, which plays a key role in lipid and lipoprotein metabolism [15,16]. The gene encoding Apo E is located on chromosome 19 (19q13.2), and two specific polymorphisms rs741 and rs429358 identify three major alleles: 2, 𝜀3, and 𝜀4, and corresponding coding proteins E2 (Arg158 Cys), E3 (parent isoform), and E4 (Arg112Cys), and corresponding three homozygous (E3/E3, E2/E2, E4/E4) and three heterozygous (E2/E3, E2/E4, and E3/E4) phenotypes [17,18]. Functionally, Apo alleles and genotypes influence lipid profile. This was evidenced by the association of Apo 2 alleles with lower blood cholesterol and low-density lipoprotein cholesterol (LDL-C) levels [19,20], stemming from reduced Apo-E2 binding to LDL receptor (LDLR) and LDL-
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related protein (LRP), and consequently removal of decomposed products of triglyceride (TG)-rich lipoproteins [21,22]. On the other hand, 4 alleles are associated with higher blood cholesterol and low-density lipoprotein cholesterol (LDL-C) levels [19,20], due to reduced LDLR affinity and increased LRP affinity, which in turn accelerate the metabolism of triglyceride-rich lipoproteins, and hence elevate LDL serum levels [21,23]. The altered lipid profile induced by Apo variants
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precipitates atherogenic events [17,22], with higher circulating cholesterol sub-fractions playing a
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significant etiologic role in the development of overt albuminuria [23,24].
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A direct relationship between dyslipidemia [high total cholesterol and LDL-C, and low high-
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density lipoprotein cholesterol (HDL-C)] and DN was documented [3,4,13], and reduction in LDL-
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cholesterol and triglyceride (TG) levels were associated with reduced risk for progression from moderate to severe albuminuria [24]. The contribution of Apo alleles as regulators of lipid
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metabolism to DN pathogenesis was previously reported, highlighted by the association of the ε2
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allele with increased risk of DN [25], macroalbuminuria [23] and renal failure [26]. The
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heterogeneity in ethnic background in the reported studies, and Apo gene-gene and geneenvironment interaction warrants careful scrutiny in assigning a role for Apo E isoforms in DN pathogenesis. In this study we investigated the relationship between Apo alleles and genotypes and DN, particularly the association between Apo E isoforms and lipid profile (total cholesterol, triglycerides, LDL-C, and HDL-C) in T2DM patients with (DN) and without (DWN) nephropathy.
SUBJECTS AND METHODS Study Subjects. From 2012 to 2014, a total of 1,422 consecutive unrelated adult Lebanese T2DM patients of Arab origin, were recruited from the outpatient diabetes clinics of LAU Medical
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Center-Rizk Hospital, Rafic Hariri University Hospital, Saint George Hospital University Medical Center, and St. Marc Medical Center in Beirut, Lebanon. The study was carried out in accordance with the Helsinki Declaration of 1975 guidelines, and was approved by the University of institutional review board, and informed consent was obtained from all subjects. The diagnosis T2DM was based on clinical and laboratory features, and none of the patients ever
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had ketoacidosis. T2DM treatment included diet (16.5%) and/or oral anti-diabetic drugs (65.6%),
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and/or insulin (17.9%); all subjects who required insulin were treated with oral drugs for at least
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two years. DM medications were given as mono- (23.1%), double- (65.2%), or triple- (11.7%)
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agents. Patients were subdivided into those with DN (n = 597), and those without nephropathy
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(DWN; n = 825), according to their 24-h urinary albumin excretion rate (AER). DN was defined as AER of > 30 mg over 24-h period determined as the mean of 3 consecutive daily measurements,
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and/or high plasma creatinine levels (> 176 µmol/L), without coexisting renal diseases (15), while
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normoalbuminuria was defined as an AER persistently <30 mg/24-h.
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Obesity was defined as body mass index [BMI; calculated as per weight divided by height squared (kg/m2)] of 30 or higher. Blood pressure (BP) was measured twice with participants in the sitting position following a 5 min rest. Hypertension was defined as blood pressure >140/90 mm Hg on two separate occasions, and/or the use of antihypertensive therapy (ACE inhibitors, angiotensin receptor blockers, beta blockers, diuretics, and calcium antagonists), which were administered as mono- (27.8%), double- (13.1%), triple- (4.3%), or quadruple- (1.1%) drug regimens. Control group included 1,389 healthy euglycemic Lebanese subjects of both genders, with no known personal or family history of diabetes, and were matched to controls with regards to gender distribution and geographical origin. Demographic details recorded included age, gender,
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ethnic origin, BMI, age at onset and duration of diabetes, first-degree family history of diabetes, history of chronic diabetes complications and other systemic illness, and treatment of diabetes, including date of initiation and/or discontinuation of oral agents or insulin.
Biochemical Analysis. Venous blood samples were collected after an overnight fast for measuring glucose in fluoride oxalate tube by hexokinase method (Cobas Integra 800; Roche, Mannheim,
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Germany). Total hemoglobin and HbA1c levels were measured after hemolyzing EDTA anti-
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coagulated blood by colorimetric and immunoturbidimetric methods, respectively, on COBAS
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Integra 800; the Hb to HbA1c yielded percent HbA1c levels. Serum lipids (total cholesterol, HDL and LDL, and triglycerides) were measured by enzymatic colorimetric method (Integra 800;
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Roche), creatinine assayed by Jaffe reaction method (Integra 800). Additional testing for liver-
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function tests, renal-function tests, serum electrolytes, was done on Dade Boehring instrument.
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Apo E Genotyping. Total genomic DNA was isolated from leukocyte-rich interphase layer of EDTA anti-coagulated blood by the phenol-chloroform method, dissolved in nuclease-free water, and stored at 4°C pending assay. Apo genotyping was done by PCR-RFLP analysis using the following primers: (forward) 5’-TCC AAG GAG CTG CAG GCG GCG CA-3’, and (reverse) 5’-ACA GAA TTC GCC CCG GCC TGG TAC ACT GCC A-3, and Cfo I restriction enzyme (New England Biolabs, Ipswich, MA), as detailed elsewhere [27]. Digested PCR products were electrophoresed on 5% NuSieve agarose gel (Karlan, Rockland, ME). Controls were in Hardy–Weinberg equilibrium.
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Statistical analysis. Statistical analysis was performed on SPSS v. 25.0 software (IBM, Armonk, NY). Data were expressed as mean SD (continuous variables), or as percentages of total (categorical variables). Pearson 2 or Fisher’s exact test were used to assess inter–group significance, and Student’s t-test was used to determine differences in means. Allele frequencies were calculated by the gene-counting method, and depending on the data type, (two-tailed)
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Pearson chi-square test or Fisher exact test was used. Correction for multiple comparison was
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made using the Bonferroni method, as per: Pc (corrected) = [1 – (1 – P)n]; n being the number of
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comparisons made. Logistic regression was used to estimate odds ratios (OR) and 95% confidence
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intervals (CI). The regression model utilized included the Apo E genotype groups, lipid profile, HbA1c, age of T2DM onset and duration of T2DM, gender, hypertension, smoking, and other
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Results
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major risk factors for DN; statistical significance set at P <0.05.
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Characteristics of Study Subjects. The characteristics of DN & DWN patients and control subjects are shown in Table 1. The three groups were matched for gender (P = 0.596) and age (P = 0.785), with T2DM patients having a higher BMI (P = 0.008), and prevalence of obesity (P = 0.005) together with higher total cholesterol, LDL-cholesterol, and triglyceride levels, and lower HDLcholesterol levels. Among T2DM patients, the prevalence of hypertension (P <0.001), HbA1c (P 0.03), urea (P = 0.972), total cholesterol (P = 0.001), HDL cholesterol (P = 0.032), urea (P =0.009) and creatinine (P = 0.014) were significantly different between both T2DM patient groups.
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Distribution of Apo E Alleles and Genotypes. Results presented in Table 2 demonstrated significantly lower Apo 2 (P <0.001) and significantly higher Apo 4 (P <0.001) allele frequencies among T2DM patients compared to non-diabetic control subjects; Apo 3 allele frequency being comparable between both study groups (P = 0.12). Significantly higher frequency of 3/4 (P <0.001), and significantly lower frequencies of 3/3 (P = 0.003), 2/3 (P <0.001), and
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homozygous 4/4 (P = 0.005) genotype carriers was seen among T2DM cases, thus assigning
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positive and negative nature to these genotypes, respectively (Table 2). Individuals with 2 allele
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(2-containing) were more frequently found in controls than in T2DM patients (P <0.001; 2 =
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39.73), while significantly higher frequency of 4-carrying genotypes was seen in T2DM cases
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than in control subjects (P <0.001; 2 = 93.37) (Table 2). These differences persisted after applying the Bonferroni correction for multiple comparisons.
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Next, we examined the distribution of Apo alleles and genotypes among T2DM patients with
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nephropathy, T2DM patients with no evidence of nephropathy serving as controls. Results from
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Table 3 demonstrated significantly higher Apo 2 (P <0.001), but significantly lower Apo 3 (P = 0.006) allele frequencies among patients with nephropathy (DN group) compared to control patient group (DWN). Interestingly, Apo 4 allele frequency was comparable between both patient groups (P = 0.322). Significantly higher frequency of the 2-containing genotypes 2/3 (P <0.001) and 2/4 (P <0.001), and significantly lower frequencies of 3/3 (P <0.001) genotype carriers was seen among DN cases, thus assigning DN-susceptible and DN-protective nature to these genotypes, respectively. These differences persisted after applying the Bonferroni correction for multiple comparisons. Individuals with 2-containing allele (E2 group) were more frequently found in DN patients (P <0.001), while significantly higher frequency of 3-carrying
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genotypes (E3 group) was seen in DWN control group than in DN patient group (P = 0.023). However, the association of E3 group with DN became non-significant after correcting for multiple comparisons.
Effect on Lipid Profile. Total cholesterol (P <0.001), LDL-C (P = 0.019), HDL-C (P = 0.032), and
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triglyceride (P = 0.003) serum concentrations were significantly different between T2DM cases
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and control subjects (Table 1). We addressed the functional consequence of carriage of specific
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Apo genotype on serum lipid profile in DN and DWN patients. Homozygous 3/3 was
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associated with significant increases in total cholesterol (P <0.001), LDL-cholesterol (P = 0.032),
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and triglyceride (P = 0.046) levels in DN patients compared with DWN patients (Table 4). On the other hand, significantly higher triglyceride levels were seen in 2/3-carrying DN patients.
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Furthermore, high levels of total cholesterol were significantly more common in Apo E2 (P =
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0.012) and Apo E3 (P = 0.024) carriers, while elevation in serum triglycerides was noted in Apo E3
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carriers (P = 0.020) (Table 4). No significant differences were noted between Apo E4-containing and no Apo E4 phenotype carriers among controls (Table 4).
DN risk associated with Apo E genotypes. Predictors of DN were assessed by performing logistic regression analysis, with the dependent variable being DN, and the independent potentially confounding variables being lipid profile, HbA1c, age of T2DM onset and duration of T2DM, gender, hypertension, and Apo genotypes. Apo 3-containing 3/3 (P = 0.002) 2/3 (P <0.001), and Apo 2-containing 2/4 (P <0.001) were the selected variable associated with DN (Table 5). These differences persisted after applying the Bonferroni correction for multiple
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comparisons. Adjusting for lipid profile, HbA1c, age of T2DM onset and duration of T2DM, gender, hypertension, and smoking, Apo 3-containing 3/3 (P = 0.009), 2/3 (P = 0.001), 3/4 (P = 0.013), and Apo 2-containing 2/4 (P = 0.016) were the selected variable associated with DN (Table 5). However, the association of 3/4 (Pc = 0.063) and 2/4 (Pc = 0.077) with DN was lost after correcting for multiple comparisons. Furthermore, carriage of 2 allele (E2 group; P =
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0.003) or 4 allele (E4 group; P = 0.021) were associated with increased risk of DN. Unlike the E2
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group (Pc = 0.009), the association of E4 group with DN was lost upon correcting for multiple
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comparisons (Pc = 0.062).
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Discussion
We report on the differential association between the Apo alleles and genotypes and the risk
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of DN in Lebanese T2DM patients. Our tested hypothesis is that Apo E isoform facilitates the
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pathogenesis and progression of DN, in part by precipitating dyslipidemia [25,26], and also by
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promoting pro-inflammatory changes in the kidney independent of its effect on lipid metabolism [28,29]. This case-control study was the first to investigate the association of Apo gene polymorphism with DN among an Arab (Lebanese) population. We provide evidence that Apo 2 is an independent risk factor of DN, and that E2-containing phenotypes (2/3 + 2/4) distinguish DN patients. This association did not weaken when adjusted for key covariates, in particular HbA1c, age of T2DM onset and duration. Originally, DN patients were planned to be subgrouped into those with microalbuminuria and those with macroalbuminura. Due to the comparable frequencies of APOE alleles and genotypes in both subgroups (data not shown), this was simplified by combining microalbuminuria and macroalbuminuria patients into one group.
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Differential distribution of ε2 and ε4 alleles, and ε2-containing and ε4-containing genotypes was seen between unselected T2DM patients and euglycemic control subjects. This was in agreement with a Turkish [30] and Egyptian [31], which also demonstrated differential distribution of ApoE genotpyes among T2DM patients and healthy subjects. However, our results were in apparent disagreement with an earlier French [32] study, and a Turkish study involving a very small sample size (46 DN patients, 56 T2DM patients without nephropathy, and
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35 healthy individuals) [33], both of which reported on lack of association of ApoE alleles and
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genotypes with T2DM. In contrast to our findings and those of others, they showed that the Apo
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ε2 allele and ε2-carrying genotypes were negatively associated with DN [32,33].
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Heterogeneity in Apo allele frequencies were described for several ethnicities, with a north-to-south gradient in the distribution of the ε4 allele reported in Europe, ranging from 0.15
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in Finland [34] to 0.07 in Italy [35]. A south-to-north gradient in Apo 4 distribution was also
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described for Africans, highlighted by prevalence rates of 0.37 in South Africa [36], 0.205 in
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Nigeria [37], 0.102 in Morocco [38], and 0.095 in Tunisia [39]. The variation in the distribution of the Apo E alleles is driven by geographical distances, along with genetic drift, as was suggested [40]. The prevalence of the ε4 allele reported here for control subjects (0.202) was higher than the rate reported for Lebanese elsewhere [41,42]. These apparent differences with the studies of El Shamieh [41], and Mahfouz [42] are attributed to differences in sample size, and also to the selection of control subjects from all Lebanese regions, given that the distribution of Apo E genotypes is geographically determined [27,40]. The association of the 2 allele with increased risk of DN are consistent with earlier results on Caucasian [43] and non-Caucasian [15,16]. Araki et al. reported that carriage of the 2 allele
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was associated with 3.1-fold increased risk of DN type 1 diabetes [43]. Our results were reminiscent of a recent Chinese study involving 429 DN and 416 DWN patients [15]. Similar to our findings, they reported on the positive association of 2 allele and 2/3 genotype, and negative association (DN protective) of 4 allele and 3/4 genotype with DN. The notable differences between the two studies is the negative association of homozygous 4/4, and positive association of homozygous 2/2 in Chinese [15]. It is noteworthy that the magnitude of the
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association was weak for both 4/4 (0.93% vs. 3.13%), and 2/2 (1.63% vs. 0.48%), thus
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warranting careful assessment of this association.
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APOE 4 allele was not associated with reduced risk of DN among Lebanese T2DM patients. Our results were in agreement with an earlier Chinese study which reported on the lack
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of association of ApoE 4 allele with renal disease [44]. Our findings were in apparent
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disagreement with another Chinese (Taiwan) study, which reported that apo 4 carriers were at
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greater risk of progression of DN than non-apo 4 carriers [45], and with Chinese [15], Korean
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[46], and Thai [47] studies, which suggested a protective role of APOE 4 for DN in the studied (East Asian) populations. These apparent discrepancies are reconciled by differences in study sample size [46,47], clinical presentation [15,45,46], and ethnic background, since most studies on the topic were done on predominantly East Asian populations [15, 45-47]. Apo 3, more so than Apo 4, was associated with significant increases in total cholesterol and triglyceride levels in DN patients. This was in contrast to earlier studies documenting association of Apo 4 with elevated plasma cholesterol [48, 49]. While the reasons behind these apparently contradictory findings are not understood, and beyond the scope of the study, it is tempting to speculate that they may be the result of altered plasma lipoprotein distributions in
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advanced nephropathy, as was suggested [50]. Furthermore, Apo E2 appears to constitute a DN risk factor. Compared to other Apo E isoforms, ApoE2 has the lowest binding ability to apoE receptor, resulting in impaired liver uptake and clearance of chylomicrons (CM) or VLDL remnants [50, 51]. This delay in CM or VLDL clearance facilitates the development of type III hyperlipidemia, and subsequently renal vascular atherosclerosis [49, 51]. Apo E2 may exacerbate kidney damage through a non-lipid mediated pathway, involving accelerated damage of renal mesangial cells [52,
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53]. This in turn promotes the development of chronic kidney disease, and likely ESRD [53].
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In conclusion, we demonstrated an association between ApoE alleles and genotypes with
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altered risk of DN in Lebanese T2DM patients. Our study has strengths. It was sufficiently powered, that DN and control T2DM patients were ethnically matched, thus minimizing the
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inherent problems of differences in genetic background, and that potential covariates were
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controlled for. Our study has also a number of limitations. Most T2DM patients in DN and control
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group were treated with statins, which may affect lipid profile results. Future studies aimed at
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evaluating only non-medicated patients or to control by medication use will be of significance towards linking specific APOE genotype with altered lipid profile. Another limitation is in the study design (retrospective case-control study), which prompts the speculation of cause-effect relationship, and that it was limited to Lebanese Arabs, thus necessitating parallel studies on related and distant ethnic groups. Follow up studies are needed to confirm the contribution of ApoE alleles and genotypes with T2DM and its complications.
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Funding Sources This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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Table 1 Clinical Characteristic of Patients and Controls Controls (1,389) 607:782
DN (n = 597)
P1
375: 450
281: 316
0.596
57.5 ± 10.8
60.1 ± 11.0
60.1 ± 10.5
0.985
23.4 ± 3.4
27.5 ± 4.2
27.9 ± 4.4
0.118
Waist-hip ratio 3
0.91 ± 0.08
0.92 ± 0.09
0.94 ± 0.09
0.049
Obesity (>30 kg/m2) 2
162 (11.7)
315 (38.2)
177 (29.6)
NA
12.1 ± 6.3
13.1 ± 6.3
0.005 0.010
NA
47.9 ± 10.8
46.7 ± 10.3
0.074
319 (53.5)
Age at study (years)3
Duration of diabetes
3
Age of onset (years) 3
310 (22.3)
Hypertension 2
121.5 ± 14.1
DBP (mmHg) 3
78.0 ± 10.4
141.43 ± 28.6
140.3 ± 26.0
81.0 ± 13.6
81.8 ± 11.7
0.717 0.373
e-
SBP (mmHg) 3
< 0.001 0.540
332 (40.2)
pr
Mean BMI
(kg/m2) 3
oo
Gender (M/F) 2
f
DWN (n = 825)
Characteristic
5.3 ± 0.7 5.1 ± 1.1
12.5 ± 5.6 9.2 ± 3.6
12.8 ± 4.8 9.9 ± 3.9
4.9 ± 1.5
5.1 ± 1.3
5.4 ± 1.3
<0.001
Triglycerides (mmol/L) 3
1.5 ± 0.8
1.7 ± 1.3
2.0 ± 1.3
0.003
HDL (mmol/L) 3
1.5 ± 0.9
1.1 ± 0.3
1.0 ± 0.3
0.032
3.0 ± 1.6
4.1 ± 1.3
4.1 ± 1.4
0.579 < 0.001 < 0.001
Glucose
(mmol/L) 3 (mmol/L) 3
al
Total cholesterol
Pr
HbA1c (%) 3
rn
LDL (mmol/L) 3
5.9 ± 2.5
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Urea (mmol/L) 3
6.4 ± 2.4
8.8 ± 5.9
0.020
51.9 (37.7 – 57.3 (51.9 – 109.9 (74.2 – 74.0) 72.5) 198.4) Creatinine clearance 94.2 (78.3 – 102.1 (91.2 – 83.9 (57.3 – (ml/min/1.73 m2) 125.3) 134.6) 127.3) 1. Pearson chi-square (categorical variables); Student t-test (parametric) and Mann-Whitney U-test (nonCreatinine (mmol/L) 4
parametric) for continuous variables. 2.
Number of subjects (percent total)
3.
Mean ± SD
4.
Median (range)
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Table 2
T2DM (1,422)
P
Pc 2
2
OR (95% CI)
2
550 (19.8) 1
403 (14.2)
<0.001
<0.001
31.62
0.67 (0.58 – 0.77)
3
1,668 (60.0)
1,650 (58.0)
0.12
0.32
2.39
0.91 (0.83 – 1.02)
4
560 (20.2)
791 (27.8)
<0.001
<0.001
45.1
1.53 (1.35 – 1.73)
3/3
574 (41.3)
509 (35.8)
0.003
0.015
9.07
1.00 (Reference)
2/3
366 (26.3)
198 (13.9)
<0.001
<0.001
67.64
0.45 (0.37 – 0.55)
4/4
111 (8.0)
76 (5.3)
0.005
0.025
7.93
0.65 (0.48 – 0.88)
3/4
154 (11.1)
434 (30.5)
<0.001
<0.001
160.41
3.52 (2.88 – 4.31)
2/4
184 (13.2)
205 (14.4)
0.37
0.90
0.81
1.10 (0.89 – 1.37)
E2
550 (39.6)
403 (28.3)
< 0.001
< 0.001
39.73
0.60 (0.51 – 0.71)
E3
1,094 (78.8)
1,141 (80.2)
0.33
0.86
0.94
1.09 (0.91 – 1.32)
E4
449 (32.3)
715 (50.2)
<0.001
<0.001
93.37
2.12 (1.82 – 2.47)
e-
Pr
al
rn
Carrier 3
Jo u
Genotype
oo
f
Allele
Controls (1,389)
pr
Apolipoprotein E Polymorphism in T2DM cases and Euglycemic controls
1. Number (percent total)
2. Pc = corrected P per Bonferroni method. 3. E2 = 2/3 + 2/4 ; E3 = 3/3 + 2/3 + 3/4 ; E4 = 4/4 + 3/4 + 2/4.
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Table 3 Apolipoprotein E Polymorphism in T2DM patients with or without nephropathy DN (597)
P
Pc 2
OR (95% CI)
2
186 (11.3) 1
217 (18.2)
< 0.001
< 0.001
1.75 (1.42 – 2.16)
3
957 (58.0)
630 (52.8)
0.006
0.018
0.81 (0.70 – 0.94)
4
507 (30.7)
346 (29.0)
0.322
0.688
0.92 (0.78 – 1.08)
3/3
335 (40.6)
174 (29.1)
2/3
88 (10.7)
110 (18.4)
4/4
44 (5.3)
33 (5.5)
3/4
261 (31.6)
2/4
1.00 (Reference)
< 0.001
< 0.001
2.40 (1.58 – 3.64)
0.236
0.740
1.45 (0.79 – 2.66)
174 (29.1)
0.136
0.519
0.78 (0.92 – 1.79)
97 (11.8)
106 (17.9)
Pr
< 0.001
< 0.001
2.11 (1.40 – 3.19)
E2
185 (22.4)
216 (36.4)
< 0.001
< 0.001
1.96 (1.47 – 2.62)
E3
684 (82.9)
458 (76.6)
0.023
0.110
0.68 (0.49 – 0.94)
E4
402 (48.7)
313 (52.5)
0.284
0.812
1.16 (0.90 – 1.51)
al
e-
pr
<0.001
Jo u
Carrier 2
<0.001
rn
Genotype
oo
f
Allele
DWN (825)
DN, diabetic nephropathy ; DWN, diabetes without nephropathy 1.
Number (percent total)
2.
Pc = corrected P per Bonferroni method.
3.
E2 = 2/3 + 2/4 ; E3 = 3/3 + 2/3 + 3/4 ; E4 = 4/4 + 3/4 + 2/4.
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TABLE 4 Effect of Apolipoprotein E genotypes on serum lipid profile Total Cholesterol
HDL-cholesterol
LDL-Cholesterol
DWN
DN
P
DWN
DN
P
DWN
DN
P
DWN
DN
P
5.1 ±
5.4
<0.001
1.8 ±
1.9
0.07
1.1 ±
1.2
0.36
3.2 ±
3.3
0.74
1.2
±
1.0
±
0.4
±
1.3
±
5.5
1.3
±
<0.001
1.6 ±
1.9
0.9
±
1.4 5.5
1.4
± 1.5
4.7 ±
5.5
1.3
±
1.9 ±
1.9
1.4
±
<0.001
1.9
0.9
±
1.4 E2/E3
5.2 ±
5.6
1.2
± 1.3
0.087
1.0
0.4
±
0.300
1.1 ±
1.0
0.3
±
1.4
1.6 ±
Jo u
E3/E3
0.31
1.1 ±
0.3
al
5.4 ±
rn
E4
1.5
0.020
pr
4.7 ±
0.4
Pr
E3
1.3
e-
1.3
oo
f
E2
Triglycerides
0.046
2.2
0.7
± 1.4
5.0 ±
4.2
0.3
±
1.0 ±
1.0
0.2
±
0.22
4.0 ±
3.9
1.5
±
1.1 ±
1.2
0.4
± 0.4
0.76
1.5 0.904
5.0 ±
4.2
0.4
±
0.3 0.021
0.81
1.1
0.3
1.5
1.7 ±
0.88
1.1
0.032
1.1 0.354
3.5 ±
3.5
0.4
± 1.3
0.535
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1.4
±
0.187
2.0 ±
1.7
1.2
±
1.2 E3/E4
5.5 ±
5.5
1.5
±
5.0 ±
5.1
1.3
±
1.2 ±
1.1
0.4
±
1.1 0.746
1.9 ±
1.9
1.4
±
1.5 E2/E4
0.329
1.9 ±
1.6
1.1
±
0.667
1.0 ±
1.0
0.3
±
2.5
0.8
±
0.191
1.1 ±
1.2
0.4
±
1.0
0.4
0.774
4.2 ±
4.2
1.6
±
Pr
al rn
0.972
1.3 0.433
3.0 ±
3.4
1.1
± 1.3
E2 = E2/E3 + E2/E4; E3 = E3/E3 + E2/E3 + E3/E4; E4 = E4/E4 + E3/E4 + E2/E4
Jo u
0.242
1.2
0.3
e-
1.3
3.1 ±
0.3
1.4 0.708
0.572
f
4.9
oo
5.5 ±
pr
E4/E4
27
0.258
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TABLE 5 Estimates for the OR of ApoE Polymorphism in DN vs. DWN patients Multivariate
Pc 1
OR (95% CI)
P
Pc 1
aOR 2 (95% CI)
3/3
0.002
0.010
1.00 (Reference)
0.009
0.044
1.00 (Reference)
2/3
< 0.001
< 0.001
2.40 (1.58 – 3.64)
0.001
0.005
2.48 (1.42 – 4.31)
4/4
0.236
0.740
1.45 (0.79 – 2.66)
0.192
0.656
1.83 (0.74 – 4.51)
3/4
0.136
0.519
1.29 (0.92 – 1.79)
0.013
0.063
1.70 (1.12 – 2.59)
2/4
< 0.001
< 0.001
2.11 (1.40 – 3.19)
0.016
0.077
2.18 (1.15 – 4.13)
E2 3
< 0.001
< 0.001
2.09 (1.47 – 2.98)
0.003
0.009
2.11 (1.29 – 3.45)
E3 3
0.534
0.899
1.16 (0.73 – 1.84)
0.305
0.644
1.41 (0.73 – 2.69)
E4 3
0.203
0.494
1.23 (0.89 – 1.70)
0.021
0.062
1.62 (1.08 – 2.44)
oo
pr
e-
rn
al
Allele/Genotype
f
P
Pr
Univariate
Pc = corrected P per Bonferroni method.
2.
aOR = adjusted OR; covariates that were controlled for were lipid profile, HbA1c, age of
Jo u
1.
T2DM onset and duration of T2DM, gender, hypertension, and smoking, 3.
E2 = E2/E3 + E2/E4; E3 = E3/E3 + E2/E3 + E3/E4; E4 = E4/E4 + E3/E4 + E2/E4
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Albumin excretion rate
Apo E
Apolipoprotein E
BMI
Body mass index
CI
Confidence interval
DN
Diabetic nephropathy
DWN
Diabetes without nephropathy
OR
odds ratios
T2DM
Type 2 diabetes
Jo u
rn
al
Pr
e-
pr
oo
AER
f
Abbreviations List
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HIGHLIGHTS Apolipoprotein E alleles and genotypes were linked with diabetic nephropathy.
Lower Apo 2, and higher Apo 4 allele frequencies was seen among diabetic patients
Higher Apo 2 and lower Apo 3 allele frequencies were seen in diabetic nephropathy
Apo 3-containing 3/3, 2/3, and 3/4, and 2-containing 2/4 linked with DN
Jo u
rn
al
Pr
e-
pr
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f
the
Kuralay K. Atageldiyeva, Rita Nemr, Akram Echtay, Eddie Racoubian, Sameh Sarray, Wassim Y. Almawi PII:
S0378-1119(19)30670-5
DOI:
https://doi.org/10.1016/j.gene.2019.144011
Reference:
GENE 144011
To appear in:
Gene
Received date:
4 June 2019
Revised date:
12 July 2019
Accepted date:
24 July 2019
Please cite this article as: K.K. Atageldiyeva, R. Nemr, A. Echtay, et al., Apolipoprotein E genetic polymorphism influence the susceptibility to nephropathy in type 2 diabetes patients, Gene (2019), https://doi.org/10.1016/j.gene.2019.144011
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© 2019 Published by Elsevier.
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Apolipoprotein E genetic polymorphism influence the susceptibility to nephropathy in type 2 diabetes patients
oo
f
Kuralay K. Atageldiyeva 1, Rita Nemr 2, Akram Echtay 3, Eddie Racoubian 4, Sameh Sarray 5,6,
of Medicine, Nazarbayev University, Astana, Kazakhstan, 2 Department of Internal
Pr
1 School
e-
pr
Wassim Y. Almawi 1,6
Medicine, LAU Medical Center - Rizk Hospital, Beirut, 3 Department of Adult Endocrinology and
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Diabetes, Rafic Hariri University Hospital, Beirut, 4 St. Marc Medical Center, Beirut, Lebanon, 5
rn
College of Medicine and Medical Sciences, Arabian Gulf University, Manama, Bahrain, 6 Faculty of
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Sciences, El-Manar University, Tunis, Tunisia.
Running Title:
Apolipoprotein E variants in Diabetic Nephropathy
Corresponding Author: Wassim Y. Almawi, Ph.D. Department of Biomedical Sciences, School of Medicine, Nazarbayev University, Nur-Sultan City (Astana), Kazakhstan. Tel. +7 (7172) 90 6340; E-mail. [email protected]
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ABSTRACT Background. An association between Apolipoprotein E (Apo E) alleles and genotypes and diabetic nephropathy (DN) was suggested, but with inconsistent results. We tested the relationship between serum lipids, Apo E alleles and genotypes with type 2 diabetes (T2DM), and DN pathogenesis.
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Methods. Study subjects comprised 1,389 normoglycemic controls, and 1,422 T2DM patients, of
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whom 825 were normoalbuminuric (DWN), and 597 presented with nephropathy (DN).
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Results. Significantly lower Apo 2, and higher Apo 4 allele frequencies was seen among T2DM
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patients than controls. Significantly higher frequency of 3/4, and lower frequencies of 3/3,
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2/3, and 4/4 carriers was seen among T2DM cases. Apo 2-carrying individuals were more frequently found in controls than in patients, while significantly higher frequency of 4-carrying
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genotypes was seen in T2DM cases. Significantly higher 2, and lower 3 allele frequencies were
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noted for DN group compared to DWN group. Significantly higher frequency of 2-containing
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2/3 and 2/4, and lower frequencies of 3/3 carriers was seen among DN cases. Apo 3/3 was associated with higher total cholesterol, LDL-cholesterol, and triglyceride levels in DN patients, and significantly higher triglyceride levels were seen in 2/3-carrying DN patients. Logistic regression analysis confirmed the association of Apo 3-containing 3/3, 2/3, and 3/4, and Apo 2-containing 2/4 with DN, after controlling for key covariates. Conclusion. The results of this case-control study provide evidence that the 2 and 3 alleles of APOE modify lipid profile, and constitute independent risk factors of DN in type 2 diabetes. The molecular mechanisms underlying this risk is discussed. Key Words:
Apolipoprotein E; diabetic nephropathy; genotyping; type 2 diabetes mellitus.
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Introduction Diabetic nephropathy (DN) is a major microvascular complication of diabetes, and the most common cause of end stage renal disease [1,2]. It is estimated that one third of type 2 diabetes (T2DM) patients will develop evidence of DN, and that a substantial number of them would require dialysis. This is likely to increase further with the global rise in the prevalence of T2DM
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and obesity, coupled with increased longevity. DN pathogenesis is multifactorial with a
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heterogeneous pathological manifestation. DN is characterized by clinical albuminuria, and is
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aggravated by hyperglycemia, hypertension, smoking and dyslipidemia [3,4]. Previous studies
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confirmed genetic predisposition to DN, highlighted by familial clustering of elevated urine
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albumin:creatinine ratio (ACR) cases among T2DM subjects [5,6], and by the association of genetic variants with the presence and severity of DN [7,8].
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Several candidate genes were described to be associated with DN, including those associated
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with inflammation [9,10], coagulation [11,12], and metabolism [13,14]. The latter include
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apolipoprotein E (Apo E), a 299 amino acid, 34 kDa glycosylated plasma protein, which plays a key role in lipid and lipoprotein metabolism [15,16]. The gene encoding Apo E is located on chromosome 19 (19q13.2), and two specific polymorphisms rs741 and rs429358 identify three major alleles: 2, 𝜀3, and 𝜀4, and corresponding coding proteins E2 (Arg158 Cys), E3 (parent isoform), and E4 (Arg112Cys), and corresponding three homozygous (E3/E3, E2/E2, E4/E4) and three heterozygous (E2/E3, E2/E4, and E3/E4) phenotypes [17,18]. Functionally, Apo alleles and genotypes influence lipid profile. This was evidenced by the association of Apo 2 alleles with lower blood cholesterol and low-density lipoprotein cholesterol (LDL-C) levels [19,20], stemming from reduced Apo-E2 binding to LDL receptor (LDLR) and LDL-
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related protein (LRP), and consequently removal of decomposed products of triglyceride (TG)-rich lipoproteins [21,22]. On the other hand, 4 alleles are associated with higher blood cholesterol and low-density lipoprotein cholesterol (LDL-C) levels [19,20], due to reduced LDLR affinity and increased LRP affinity, which in turn accelerate the metabolism of triglyceride-rich lipoproteins, and hence elevate LDL serum levels [21,23]. The altered lipid profile induced by Apo variants
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precipitates atherogenic events [17,22], with higher circulating cholesterol sub-fractions playing a
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significant etiologic role in the development of overt albuminuria [23,24].
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A direct relationship between dyslipidemia [high total cholesterol and LDL-C, and low high-
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density lipoprotein cholesterol (HDL-C)] and DN was documented [3,4,13], and reduction in LDL-
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cholesterol and triglyceride (TG) levels were associated with reduced risk for progression from moderate to severe albuminuria [24]. The contribution of Apo alleles as regulators of lipid
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metabolism to DN pathogenesis was previously reported, highlighted by the association of the ε2
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allele with increased risk of DN [25], macroalbuminuria [23] and renal failure [26]. The
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heterogeneity in ethnic background in the reported studies, and Apo gene-gene and geneenvironment interaction warrants careful scrutiny in assigning a role for Apo E isoforms in DN pathogenesis. In this study we investigated the relationship between Apo alleles and genotypes and DN, particularly the association between Apo E isoforms and lipid profile (total cholesterol, triglycerides, LDL-C, and HDL-C) in T2DM patients with (DN) and without (DWN) nephropathy.
SUBJECTS AND METHODS Study Subjects. From 2012 to 2014, a total of 1,422 consecutive unrelated adult Lebanese T2DM patients of Arab origin, were recruited from the outpatient diabetes clinics of LAU Medical
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Center-Rizk Hospital, Rafic Hariri University Hospital, Saint George Hospital University Medical Center, and St. Marc Medical Center in Beirut, Lebanon. The study was carried out in accordance with the Helsinki Declaration of 1975 guidelines, and was approved by the University of institutional review board, and informed consent was obtained from all subjects. The diagnosis T2DM was based on clinical and laboratory features, and none of the patients ever
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had ketoacidosis. T2DM treatment included diet (16.5%) and/or oral anti-diabetic drugs (65.6%),
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and/or insulin (17.9%); all subjects who required insulin were treated with oral drugs for at least
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two years. DM medications were given as mono- (23.1%), double- (65.2%), or triple- (11.7%)
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agents. Patients were subdivided into those with DN (n = 597), and those without nephropathy
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(DWN; n = 825), according to their 24-h urinary albumin excretion rate (AER). DN was defined as AER of > 30 mg over 24-h period determined as the mean of 3 consecutive daily measurements,
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and/or high plasma creatinine levels (> 176 µmol/L), without coexisting renal diseases (15), while
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normoalbuminuria was defined as an AER persistently <30 mg/24-h.
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Obesity was defined as body mass index [BMI; calculated as per weight divided by height squared (kg/m2)] of 30 or higher. Blood pressure (BP) was measured twice with participants in the sitting position following a 5 min rest. Hypertension was defined as blood pressure >140/90 mm Hg on two separate occasions, and/or the use of antihypertensive therapy (ACE inhibitors, angiotensin receptor blockers, beta blockers, diuretics, and calcium antagonists), which were administered as mono- (27.8%), double- (13.1%), triple- (4.3%), or quadruple- (1.1%) drug regimens. Control group included 1,389 healthy euglycemic Lebanese subjects of both genders, with no known personal or family history of diabetes, and were matched to controls with regards to gender distribution and geographical origin. Demographic details recorded included age, gender,
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ethnic origin, BMI, age at onset and duration of diabetes, first-degree family history of diabetes, history of chronic diabetes complications and other systemic illness, and treatment of diabetes, including date of initiation and/or discontinuation of oral agents or insulin.
Biochemical Analysis. Venous blood samples were collected after an overnight fast for measuring glucose in fluoride oxalate tube by hexokinase method (Cobas Integra 800; Roche, Mannheim,
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Germany). Total hemoglobin and HbA1c levels were measured after hemolyzing EDTA anti-
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coagulated blood by colorimetric and immunoturbidimetric methods, respectively, on COBAS
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Integra 800; the Hb to HbA1c yielded percent HbA1c levels. Serum lipids (total cholesterol, HDL and LDL, and triglycerides) were measured by enzymatic colorimetric method (Integra 800;
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Roche), creatinine assayed by Jaffe reaction method (Integra 800). Additional testing for liver-
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function tests, renal-function tests, serum electrolytes, was done on Dade Boehring instrument.
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Apo E Genotyping. Total genomic DNA was isolated from leukocyte-rich interphase layer of EDTA anti-coagulated blood by the phenol-chloroform method, dissolved in nuclease-free water, and stored at 4°C pending assay. Apo genotyping was done by PCR-RFLP analysis using the following primers: (forward) 5’-TCC AAG GAG CTG CAG GCG GCG CA-3’, and (reverse) 5’-ACA GAA TTC GCC CCG GCC TGG TAC ACT GCC A-3, and Cfo I restriction enzyme (New England Biolabs, Ipswich, MA), as detailed elsewhere [27]. Digested PCR products were electrophoresed on 5% NuSieve agarose gel (Karlan, Rockland, ME). Controls were in Hardy–Weinberg equilibrium.
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Statistical analysis. Statistical analysis was performed on SPSS v. 25.0 software (IBM, Armonk, NY). Data were expressed as mean SD (continuous variables), or as percentages of total (categorical variables). Pearson 2 or Fisher’s exact test were used to assess inter–group significance, and Student’s t-test was used to determine differences in means. Allele frequencies were calculated by the gene-counting method, and depending on the data type, (two-tailed)
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Pearson chi-square test or Fisher exact test was used. Correction for multiple comparison was
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made using the Bonferroni method, as per: Pc (corrected) = [1 – (1 – P)n]; n being the number of
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comparisons made. Logistic regression was used to estimate odds ratios (OR) and 95% confidence
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intervals (CI). The regression model utilized included the Apo E genotype groups, lipid profile, HbA1c, age of T2DM onset and duration of T2DM, gender, hypertension, smoking, and other
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Results
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major risk factors for DN; statistical significance set at P <0.05.
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Characteristics of Study Subjects. The characteristics of DN & DWN patients and control subjects are shown in Table 1. The three groups were matched for gender (P = 0.596) and age (P = 0.785), with T2DM patients having a higher BMI (P = 0.008), and prevalence of obesity (P = 0.005) together with higher total cholesterol, LDL-cholesterol, and triglyceride levels, and lower HDLcholesterol levels. Among T2DM patients, the prevalence of hypertension (P <0.001), HbA1c (P 0.03), urea (P = 0.972), total cholesterol (P = 0.001), HDL cholesterol (P = 0.032), urea (P =0.009) and creatinine (P = 0.014) were significantly different between both T2DM patient groups.
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Distribution of Apo E Alleles and Genotypes. Results presented in Table 2 demonstrated significantly lower Apo 2 (P <0.001) and significantly higher Apo 4 (P <0.001) allele frequencies among T2DM patients compared to non-diabetic control subjects; Apo 3 allele frequency being comparable between both study groups (P = 0.12). Significantly higher frequency of 3/4 (P <0.001), and significantly lower frequencies of 3/3 (P = 0.003), 2/3 (P <0.001), and
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homozygous 4/4 (P = 0.005) genotype carriers was seen among T2DM cases, thus assigning
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positive and negative nature to these genotypes, respectively (Table 2). Individuals with 2 allele
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(2-containing) were more frequently found in controls than in T2DM patients (P <0.001; 2 =
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39.73), while significantly higher frequency of 4-carrying genotypes was seen in T2DM cases
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than in control subjects (P <0.001; 2 = 93.37) (Table 2). These differences persisted after applying the Bonferroni correction for multiple comparisons.
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Next, we examined the distribution of Apo alleles and genotypes among T2DM patients with
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nephropathy, T2DM patients with no evidence of nephropathy serving as controls. Results from
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Table 3 demonstrated significantly higher Apo 2 (P <0.001), but significantly lower Apo 3 (P = 0.006) allele frequencies among patients with nephropathy (DN group) compared to control patient group (DWN). Interestingly, Apo 4 allele frequency was comparable between both patient groups (P = 0.322). Significantly higher frequency of the 2-containing genotypes 2/3 (P <0.001) and 2/4 (P <0.001), and significantly lower frequencies of 3/3 (P <0.001) genotype carriers was seen among DN cases, thus assigning DN-susceptible and DN-protective nature to these genotypes, respectively. These differences persisted after applying the Bonferroni correction for multiple comparisons. Individuals with 2-containing allele (E2 group) were more frequently found in DN patients (P <0.001), while significantly higher frequency of 3-carrying
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genotypes (E3 group) was seen in DWN control group than in DN patient group (P = 0.023). However, the association of E3 group with DN became non-significant after correcting for multiple comparisons.
Effect on Lipid Profile. Total cholesterol (P <0.001), LDL-C (P = 0.019), HDL-C (P = 0.032), and
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triglyceride (P = 0.003) serum concentrations were significantly different between T2DM cases
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and control subjects (Table 1). We addressed the functional consequence of carriage of specific
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Apo genotype on serum lipid profile in DN and DWN patients. Homozygous 3/3 was
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associated with significant increases in total cholesterol (P <0.001), LDL-cholesterol (P = 0.032),
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and triglyceride (P = 0.046) levels in DN patients compared with DWN patients (Table 4). On the other hand, significantly higher triglyceride levels were seen in 2/3-carrying DN patients.
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Furthermore, high levels of total cholesterol were significantly more common in Apo E2 (P =
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0.012) and Apo E3 (P = 0.024) carriers, while elevation in serum triglycerides was noted in Apo E3
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carriers (P = 0.020) (Table 4). No significant differences were noted between Apo E4-containing and no Apo E4 phenotype carriers among controls (Table 4).
DN risk associated with Apo E genotypes. Predictors of DN were assessed by performing logistic regression analysis, with the dependent variable being DN, and the independent potentially confounding variables being lipid profile, HbA1c, age of T2DM onset and duration of T2DM, gender, hypertension, and Apo genotypes. Apo 3-containing 3/3 (P = 0.002) 2/3 (P <0.001), and Apo 2-containing 2/4 (P <0.001) were the selected variable associated with DN (Table 5). These differences persisted after applying the Bonferroni correction for multiple
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comparisons. Adjusting for lipid profile, HbA1c, age of T2DM onset and duration of T2DM, gender, hypertension, and smoking, Apo 3-containing 3/3 (P = 0.009), 2/3 (P = 0.001), 3/4 (P = 0.013), and Apo 2-containing 2/4 (P = 0.016) were the selected variable associated with DN (Table 5). However, the association of 3/4 (Pc = 0.063) and 2/4 (Pc = 0.077) with DN was lost after correcting for multiple comparisons. Furthermore, carriage of 2 allele (E2 group; P =
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0.003) or 4 allele (E4 group; P = 0.021) were associated with increased risk of DN. Unlike the E2
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group (Pc = 0.009), the association of E4 group with DN was lost upon correcting for multiple
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comparisons (Pc = 0.062).
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Discussion
We report on the differential association between the Apo alleles and genotypes and the risk
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of DN in Lebanese T2DM patients. Our tested hypothesis is that Apo E isoform facilitates the
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pathogenesis and progression of DN, in part by precipitating dyslipidemia [25,26], and also by
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promoting pro-inflammatory changes in the kidney independent of its effect on lipid metabolism [28,29]. This case-control study was the first to investigate the association of Apo gene polymorphism with DN among an Arab (Lebanese) population. We provide evidence that Apo 2 is an independent risk factor of DN, and that E2-containing phenotypes (2/3 + 2/4) distinguish DN patients. This association did not weaken when adjusted for key covariates, in particular HbA1c, age of T2DM onset and duration. Originally, DN patients were planned to be subgrouped into those with microalbuminuria and those with macroalbuminura. Due to the comparable frequencies of APOE alleles and genotypes in both subgroups (data not shown), this was simplified by combining microalbuminuria and macroalbuminuria patients into one group.
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Differential distribution of ε2 and ε4 alleles, and ε2-containing and ε4-containing genotypes was seen between unselected T2DM patients and euglycemic control subjects. This was in agreement with a Turkish [30] and Egyptian [31], which also demonstrated differential distribution of ApoE genotpyes among T2DM patients and healthy subjects. However, our results were in apparent disagreement with an earlier French [32] study, and a Turkish study involving a very small sample size (46 DN patients, 56 T2DM patients without nephropathy, and
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35 healthy individuals) [33], both of which reported on lack of association of ApoE alleles and
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genotypes with T2DM. In contrast to our findings and those of others, they showed that the Apo
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ε2 allele and ε2-carrying genotypes were negatively associated with DN [32,33].
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Heterogeneity in Apo allele frequencies were described for several ethnicities, with a north-to-south gradient in the distribution of the ε4 allele reported in Europe, ranging from 0.15
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in Finland [34] to 0.07 in Italy [35]. A south-to-north gradient in Apo 4 distribution was also
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described for Africans, highlighted by prevalence rates of 0.37 in South Africa [36], 0.205 in
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Nigeria [37], 0.102 in Morocco [38], and 0.095 in Tunisia [39]. The variation in the distribution of the Apo E alleles is driven by geographical distances, along with genetic drift, as was suggested [40]. The prevalence of the ε4 allele reported here for control subjects (0.202) was higher than the rate reported for Lebanese elsewhere [41,42]. These apparent differences with the studies of El Shamieh [41], and Mahfouz [42] are attributed to differences in sample size, and also to the selection of control subjects from all Lebanese regions, given that the distribution of Apo E genotypes is geographically determined [27,40]. The association of the 2 allele with increased risk of DN are consistent with earlier results on Caucasian [43] and non-Caucasian [15,16]. Araki et al. reported that carriage of the 2 allele
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was associated with 3.1-fold increased risk of DN type 1 diabetes [43]. Our results were reminiscent of a recent Chinese study involving 429 DN and 416 DWN patients [15]. Similar to our findings, they reported on the positive association of 2 allele and 2/3 genotype, and negative association (DN protective) of 4 allele and 3/4 genotype with DN. The notable differences between the two studies is the negative association of homozygous 4/4, and positive association of homozygous 2/2 in Chinese [15]. It is noteworthy that the magnitude of the
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association was weak for both 4/4 (0.93% vs. 3.13%), and 2/2 (1.63% vs. 0.48%), thus
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warranting careful assessment of this association.
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APOE 4 allele was not associated with reduced risk of DN among Lebanese T2DM patients. Our results were in agreement with an earlier Chinese study which reported on the lack
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of association of ApoE 4 allele with renal disease [44]. Our findings were in apparent
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disagreement with another Chinese (Taiwan) study, which reported that apo 4 carriers were at
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greater risk of progression of DN than non-apo 4 carriers [45], and with Chinese [15], Korean
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[46], and Thai [47] studies, which suggested a protective role of APOE 4 for DN in the studied (East Asian) populations. These apparent discrepancies are reconciled by differences in study sample size [46,47], clinical presentation [15,45,46], and ethnic background, since most studies on the topic were done on predominantly East Asian populations [15, 45-47]. Apo 3, more so than Apo 4, was associated with significant increases in total cholesterol and triglyceride levels in DN patients. This was in contrast to earlier studies documenting association of Apo 4 with elevated plasma cholesterol [48, 49]. While the reasons behind these apparently contradictory findings are not understood, and beyond the scope of the study, it is tempting to speculate that they may be the result of altered plasma lipoprotein distributions in
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advanced nephropathy, as was suggested [50]. Furthermore, Apo E2 appears to constitute a DN risk factor. Compared to other Apo E isoforms, ApoE2 has the lowest binding ability to apoE receptor, resulting in impaired liver uptake and clearance of chylomicrons (CM) or VLDL remnants [50, 51]. This delay in CM or VLDL clearance facilitates the development of type III hyperlipidemia, and subsequently renal vascular atherosclerosis [49, 51]. Apo E2 may exacerbate kidney damage through a non-lipid mediated pathway, involving accelerated damage of renal mesangial cells [52,
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53]. This in turn promotes the development of chronic kidney disease, and likely ESRD [53].
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In conclusion, we demonstrated an association between ApoE alleles and genotypes with
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altered risk of DN in Lebanese T2DM patients. Our study has strengths. It was sufficiently powered, that DN and control T2DM patients were ethnically matched, thus minimizing the
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inherent problems of differences in genetic background, and that potential covariates were
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controlled for. Our study has also a number of limitations. Most T2DM patients in DN and control
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group were treated with statins, which may affect lipid profile results. Future studies aimed at
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evaluating only non-medicated patients or to control by medication use will be of significance towards linking specific APOE genotype with altered lipid profile. Another limitation is in the study design (retrospective case-control study), which prompts the speculation of cause-effect relationship, and that it was limited to Lebanese Arabs, thus necessitating parallel studies on related and distant ethnic groups. Follow up studies are needed to confirm the contribution of ApoE alleles and genotypes with T2DM and its complications.
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Funding Sources This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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Table 1 Clinical Characteristic of Patients and Controls Controls (1,389) 607:782
DN (n = 597)
P1
375: 450
281: 316
0.596
57.5 ± 10.8
60.1 ± 11.0
60.1 ± 10.5
0.985
23.4 ± 3.4
27.5 ± 4.2
27.9 ± 4.4
0.118
Waist-hip ratio 3
0.91 ± 0.08
0.92 ± 0.09
0.94 ± 0.09
0.049
Obesity (>30 kg/m2) 2
162 (11.7)
315 (38.2)
177 (29.6)
NA
12.1 ± 6.3
13.1 ± 6.3
0.005 0.010
NA
47.9 ± 10.8
46.7 ± 10.3
0.074
319 (53.5)
Age at study (years)3
Duration of diabetes
3
Age of onset (years) 3
310 (22.3)
Hypertension 2
121.5 ± 14.1
DBP (mmHg) 3
78.0 ± 10.4
141.43 ± 28.6
140.3 ± 26.0
81.0 ± 13.6
81.8 ± 11.7
0.717 0.373
e-
SBP (mmHg) 3
< 0.001 0.540
332 (40.2)
pr
Mean BMI
(kg/m2) 3
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Gender (M/F) 2
f
DWN (n = 825)
Characteristic
5.3 ± 0.7 5.1 ± 1.1
12.5 ± 5.6 9.2 ± 3.6
12.8 ± 4.8 9.9 ± 3.9
4.9 ± 1.5
5.1 ± 1.3
5.4 ± 1.3
<0.001
Triglycerides (mmol/L) 3
1.5 ± 0.8
1.7 ± 1.3
2.0 ± 1.3
0.003
HDL (mmol/L) 3
1.5 ± 0.9
1.1 ± 0.3
1.0 ± 0.3
0.032
3.0 ± 1.6
4.1 ± 1.3
4.1 ± 1.4
0.579 < 0.001 < 0.001
Glucose
(mmol/L) 3 (mmol/L) 3
al
Total cholesterol
Pr
HbA1c (%) 3
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LDL (mmol/L) 3
5.9 ± 2.5
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Urea (mmol/L) 3
6.4 ± 2.4
8.8 ± 5.9
0.020
51.9 (37.7 – 57.3 (51.9 – 109.9 (74.2 – 74.0) 72.5) 198.4) Creatinine clearance 94.2 (78.3 – 102.1 (91.2 – 83.9 (57.3 – (ml/min/1.73 m2) 125.3) 134.6) 127.3) 1. Pearson chi-square (categorical variables); Student t-test (parametric) and Mann-Whitney U-test (nonCreatinine (mmol/L) 4
parametric) for continuous variables. 2.
Number of subjects (percent total)
3.
Mean ± SD
4.
Median (range)
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Table 2
T2DM (1,422)
P
Pc 2
2
OR (95% CI)
2
550 (19.8) 1
403 (14.2)
<0.001
<0.001
31.62
0.67 (0.58 – 0.77)
3
1,668 (60.0)
1,650 (58.0)
0.12
0.32
2.39
0.91 (0.83 – 1.02)
4
560 (20.2)
791 (27.8)
<0.001
<0.001
45.1
1.53 (1.35 – 1.73)
3/3
574 (41.3)
509 (35.8)
0.003
0.015
9.07
1.00 (Reference)
2/3
366 (26.3)
198 (13.9)
<0.001
<0.001
67.64
0.45 (0.37 – 0.55)
4/4
111 (8.0)
76 (5.3)
0.005
0.025
7.93
0.65 (0.48 – 0.88)
3/4
154 (11.1)
434 (30.5)
<0.001
<0.001
160.41
3.52 (2.88 – 4.31)
2/4
184 (13.2)
205 (14.4)
0.37
0.90
0.81
1.10 (0.89 – 1.37)
E2
550 (39.6)
403 (28.3)
< 0.001
< 0.001
39.73
0.60 (0.51 – 0.71)
E3
1,094 (78.8)
1,141 (80.2)
0.33
0.86
0.94
1.09 (0.91 – 1.32)
E4
449 (32.3)
715 (50.2)
<0.001
<0.001
93.37
2.12 (1.82 – 2.47)
e-
Pr
al
rn
Carrier 3
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Genotype
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f
Allele
Controls (1,389)
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Apolipoprotein E Polymorphism in T2DM cases and Euglycemic controls
1. Number (percent total)
2. Pc = corrected P per Bonferroni method. 3. E2 = 2/3 + 2/4 ; E3 = 3/3 + 2/3 + 3/4 ; E4 = 4/4 + 3/4 + 2/4.
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Table 3 Apolipoprotein E Polymorphism in T2DM patients with or without nephropathy DN (597)
P
Pc 2
OR (95% CI)
2
186 (11.3) 1
217 (18.2)
< 0.001
< 0.001
1.75 (1.42 – 2.16)
3
957 (58.0)
630 (52.8)
0.006
0.018
0.81 (0.70 – 0.94)
4
507 (30.7)
346 (29.0)
0.322
0.688
0.92 (0.78 – 1.08)
3/3
335 (40.6)
174 (29.1)
2/3
88 (10.7)
110 (18.4)
4/4
44 (5.3)
33 (5.5)
3/4
261 (31.6)
2/4
1.00 (Reference)
< 0.001
< 0.001
2.40 (1.58 – 3.64)
0.236
0.740
1.45 (0.79 – 2.66)
174 (29.1)
0.136
0.519
0.78 (0.92 – 1.79)
97 (11.8)
106 (17.9)
Pr
< 0.001
< 0.001
2.11 (1.40 – 3.19)
E2
185 (22.4)
216 (36.4)
< 0.001
< 0.001
1.96 (1.47 – 2.62)
E3
684 (82.9)
458 (76.6)
0.023
0.110
0.68 (0.49 – 0.94)
E4
402 (48.7)
313 (52.5)
0.284
0.812
1.16 (0.90 – 1.51)
al
e-
pr
<0.001
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Carrier 2
<0.001
rn
Genotype
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f
Allele
DWN (825)
DN, diabetic nephropathy ; DWN, diabetes without nephropathy 1.
Number (percent total)
2.
Pc = corrected P per Bonferroni method.
3.
E2 = 2/3 + 2/4 ; E3 = 3/3 + 2/3 + 3/4 ; E4 = 4/4 + 3/4 + 2/4.
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TABLE 4 Effect of Apolipoprotein E genotypes on serum lipid profile Total Cholesterol
HDL-cholesterol
LDL-Cholesterol
DWN
DN
P
DWN
DN
P
DWN
DN
P
DWN
DN
P
5.1 ±
5.4
<0.001
1.8 ±
1.9
0.07
1.1 ±
1.2
0.36
3.2 ±
3.3
0.74
1.2
±
1.0
±
0.4
±
1.3
±
5.5
1.3
±
<0.001
1.6 ±
1.9
0.9
±
1.4 5.5
1.4
± 1.5
4.7 ±
5.5
1.3
±
1.9 ±
1.9
1.4
±
<0.001
1.9
0.9
±
1.4 E2/E3
5.2 ±
5.6
1.2
± 1.3
0.087
1.0
0.4
±
0.300
1.1 ±
1.0
0.3
±
1.4
1.6 ±
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E3/E3
0.31
1.1 ±
0.3
al
5.4 ±
rn
E4
1.5
0.020
pr
4.7 ±
0.4
Pr
E3
1.3
e-
1.3
oo
f
E2
Triglycerides
0.046
2.2
0.7
± 1.4
5.0 ±
4.2
0.3
±
1.0 ±
1.0
0.2
±
0.22
4.0 ±
3.9
1.5
±
1.1 ±
1.2
0.4
± 0.4
0.76
1.5 0.904
5.0 ±
4.2
0.4
±
0.3 0.021
0.81
1.1
0.3
1.5
1.7 ±
0.88
1.1
0.032
1.1 0.354
3.5 ±
3.5
0.4
± 1.3
0.535
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1.4
±
0.187
2.0 ±
1.7
1.2
±
1.2 E3/E4
5.5 ±
5.5
1.5
±
5.0 ±
5.1
1.3
±
1.2 ±
1.1
0.4
±
1.1 0.746
1.9 ±
1.9
1.4
±
1.5 E2/E4
0.329
1.9 ±
1.6
1.1
±
0.667
1.0 ±
1.0
0.3
±
2.5
0.8
±
0.191
1.1 ±
1.2
0.4
±
1.0
0.4
0.774
4.2 ±
4.2
1.6
±
Pr
al rn
0.972
1.3 0.433
3.0 ±
3.4
1.1
± 1.3
E2 = E2/E3 + E2/E4; E3 = E3/E3 + E2/E3 + E3/E4; E4 = E4/E4 + E3/E4 + E2/E4
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0.242
1.2
0.3
e-
1.3
3.1 ±
0.3
1.4 0.708
0.572
f
4.9
oo
5.5 ±
pr
E4/E4
27
0.258
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TABLE 5 Estimates for the OR of ApoE Polymorphism in DN vs. DWN patients Multivariate
Pc 1
OR (95% CI)
P
Pc 1
aOR 2 (95% CI)
3/3
0.002
0.010
1.00 (Reference)
0.009
0.044
1.00 (Reference)
2/3
< 0.001
< 0.001
2.40 (1.58 – 3.64)
0.001
0.005
2.48 (1.42 – 4.31)
4/4
0.236
0.740
1.45 (0.79 – 2.66)
0.192
0.656
1.83 (0.74 – 4.51)
3/4
0.136
0.519
1.29 (0.92 – 1.79)
0.013
0.063
1.70 (1.12 – 2.59)
2/4
< 0.001
< 0.001
2.11 (1.40 – 3.19)
0.016
0.077
2.18 (1.15 – 4.13)
E2 3
< 0.001
< 0.001
2.09 (1.47 – 2.98)
0.003
0.009
2.11 (1.29 – 3.45)
E3 3
0.534
0.899
1.16 (0.73 – 1.84)
0.305
0.644
1.41 (0.73 – 2.69)
E4 3
0.203
0.494
1.23 (0.89 – 1.70)
0.021
0.062
1.62 (1.08 – 2.44)
oo
pr
e-
rn
al
Allele/Genotype
f
P
Pr
Univariate
Pc = corrected P per Bonferroni method.
2.
aOR = adjusted OR; covariates that were controlled for were lipid profile, HbA1c, age of
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1.
T2DM onset and duration of T2DM, gender, hypertension, and smoking, 3.
E2 = E2/E3 + E2/E4; E3 = E3/E3 + E2/E3 + E3/E4; E4 = E4/E4 + E3/E4 + E2/E4
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Albumin excretion rate
Apo E
Apolipoprotein E
BMI
Body mass index
CI
Confidence interval
DN
Diabetic nephropathy
DWN
Diabetes without nephropathy
OR
odds ratios
T2DM
Type 2 diabetes
Jo u
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al
Pr
e-
pr
oo
AER
f
Abbreviations List
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HIGHLIGHTS Apolipoprotein E alleles and genotypes were linked with diabetic nephropathy.
Lower Apo 2, and higher Apo 4 allele frequencies was seen among diabetic patients
Higher Apo 2 and lower Apo 3 allele frequencies were seen in diabetic nephropathy
Apo 3-containing 3/3, 2/3, and 3/4, and 2-containing 2/4 linked with DN
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al
Pr
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pr
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f