Journals | Policy | Permission

Journal of Clinical Medicine Research

Original Article

Volume 9, Number 8, August 2017, pages 695-700

Detection of Apolipoprotein E Gene Polymorphism and Blood Lipid Level in Hemodialysis Patients

Considerable evidence has indicated that hemodialysis patients are often accompanied by lipid metabolism disorders, cardiovascular complications in patients with one of the important factors [1-5]. However, the etiology and underlying mechanism are not entirely clear. It may be related to renal dysfunction, dialysis adequacy, the type of dialysis membrane and uremia basic diseases, genetic and other factors [6-9]. Apolipoprotein E (ApoE) is an important component of very low-density lipoprotein (VLDL), high-density lipoprotein (HDL) and chylomicrons, which may play an important role in lipid metabolism [10-12]. Recently, an interesting study demonstrated that triacylglycerol (TAG) hydrolysis mediated by lipoprotein lipase in VLDL is accompanied by the release of surface material containing phospholipids, free cholesterol and ApoE and ApoCs (CII and CIII). The released molecules are accepted by HDL, and new HDL-sized ApoE-containing particles are also generated. A decrease in the number of HDL particles or abnormalities in their structure is associated with unfavourable changes in the features of VLDL remnants [11].

Its genetic polymorphism is an important genetic factor affecting lipid metabolism. As a matter of fact, approximately 14-17% variation of plasma cholesterol concentration can be associated with genetic polymorphism [13-16].

In a study reported by Echeverria et al, they performed a cross-sectional study to investigate the impact of 13 polymorphisms of nine genes affecting lipid metabolism. Polymorphisms were identified in the selected genes for their role in the development of atherogenic dyslipidemia including triglycerides and HDL [17]. At present, more and more studies on the relationship between ApoE gene polymorphism and lipid metabolism are being conducted in patients with cardiovascular and cerebrovascular diseases. Dyslipidemia plays a major role determining the epidemic cardiovascular burden attributed to the metabolic syndrome. ApoE is involved on cholesterol and triglycerides metabolism regulation. If ApoE polymorphism influences lipid metabolism, it may bring on induced susceptibility to the development of metabolic syndrome and cardiovascular disease [18-20].

However, the effect of ApoE gene polymorphism on lipid metabolism in hemodialysis patients has not been reported in China. In this study, polymerase chain reaction-restriction fragment length (PCR-RFLP) was used to detect the polymorphism of ApoE gene in hemodialysis patients and explore the effect of ApoE gene polymorphism on lipid metabolism in hemodialysis patients.

A total of 112 patients with hemodialysis (58 males and 54 females) were included in this study, and the median age was 51 ± 11 years. Dialysis time was 2 - 21 years, an average of 6.1 ± 3.7 years. The primary disease was 86 cases of chronic glomerulonephritis, four cases of polycystic kidney disease, two cases of chronic pyelonephritis, two cases of chronic interstitial nephritis, two cases of systemic lupus erythematosus, and 16 cases of unknown causes. Hemodialysis was performed 12 h per week, by using arteriovenous fistula and acetate fiber membrane dialyzer. Anticoagulation with heparin sodium was applied in vitro, and blood flow was 200 - 250 mL/min. Bicarbonate dialysate was used, and dialysis fluid flow was approximately 500 mL/min. Patients did not take lipid-lowering drugs within 3 months, but those patients who had diabetes, liver dysfunction or were taking β-blockers were excluded.

A total of 372 healthy subjects (212 males and 160 females) were included, with a median age of 41 ± 8 years.

Total cholesterol (TC) and triglyceride (TG) were measured with enzymatic assay (British RANDOX Company). High-density lipoprotein cholesterol (HDLC) and low-density lipoprotein cholesterol (LDLC) were using direct method (Germany Bao Ling Man products). ApoA1, ApoB, ApoE, and Lp(a) were tested using immunoturbidimetry (provided by WAKO Co., Ltd, Japan).

Electrocardiogram, X-ray, blood tests, liver function, plasma protein, serum creatinine, urea nitrogen, and uric acid were determined before and after dialysis, and the KT/V value was calculated.

The diagnosis of high blood pressure, ischemic heart disease, and cardiac insufficiency was made with reference to the related literature [9]. Cardiac enlargement was diagnosed based on the dry weight increase of greater than 50% on the chest X-ray radiograph at three times of measurement; cerebrovascular disease diagnosis was confirmed according to clinical manifestations in combination with CT examination.

By use of multi-PCR-RFLP analysis, the detailed procedures were described as follows. Primer design: the upstream sequence was 5′-AACAACTGACCCCGGTGGGG-3′, the downstream sequence was 5′-ATAA AT ATAAAATATAAATAATGGCGCTGAGGCCGCGCTC-3′, the length of the amplified product was 312 bp, and the length of the product fragment was 91, 83, 72, 61, 48, and 35 bp, respectively.

Rapid extraction and purification method of genomic DNA derived from small amounts of blood were performed according to the instructions provided by the Huashun products.

The 10 × buffer 2.5 µL, 25 mM magnesium chloride 2 µL, dNTP 200 µM, Taq DNA polymerase 1 U, primer 1 pmmol/µL, template DNA 1 µg, water added to 25 µL. PCR procedure was 95 °C pre-denaturation for 4 min into the cycle, with each cycle at 94 °C degeneration for 40 s, 60 °C refolding for 1 min, 70 °C extension for 3 min, a total of 35 cycles, the last 70 °C heat for 7 min terminate the reaction. The amplified product 5 µL was identified by 1.2% agarose gel electrophoresis.

Successful product 20 µL was amplified by adding 10 × buffer 2 µL, calf serum 0.2 µL, endonuclease HhaI5u, 65 °C warm bath for 4 h, 3 µL of the digested product, 3 h of polyacrylamide gel electrophoresis (voltage 150 V), and silver nitrate staining was performed to record the results by taking images (Fig. 1).

Click for large image

Figure 1. Electrophoresis results of the enzyme and the cleavage products. 1: PCR product (312 bp); 2: genotype ε3/4 (91, 72, 61, and 48 bp); 3: marker; 4: ε2/4 (91, 83, 72, 61, and 48 bp) (91, 61, and 48 bp); 5: ε2/3 (91, 83, 61, and 48 bp); 6: ε4/4 (72, 61, and 48 bp); 7: ε3/3 (91, 61, and 48 bp); 8: marker; 9: ε2/2 (91, 83, and 61 bp).

The number of groups between the use of t-test or analysis of variance, composition than with the Chi-square test, correlation analysis using Spearman rank correlation analysis.

The main performance of TG and ApoB levels was significantly increased, and HDLC and other indicators were significantly reduced (Table 1). According to the criteria of normal values made by our hospital laboratory, 37 cases (33%) of the patients who received hemodialysis had higher TG levels than the normal upper limit (1.7 mmol/L), and 10 cases (9.4%) had higher levels of ApoB than normal upper limit (300 mg/dL), whereas HDLC levels were found lower than the normal lower limit (0.85 mM) in 11 cases (10.4%). TC levels of all patients were in the normal range or below normal.

Click to view

Table 1. Comparisons of Blood Lipid Levels Between the Control Group and the Hemodialysis Group, the Hemodialysis Patients With Hypertension and the Normal Blood Pressure Group, and the Cardiovascular Disease Group and the Non-Cardiovascular Disease Group

TG, HDLC, sex, age, height, body weight, body mass index, KT/V, serum albumin, ex vivo blood flow during hemodialysis and correlation with serum creatinine, urea nitrogen and uric acid levels before and after dialysis were recorded during the study. The results showed that TG was significantly correlated with serum albumin level and extracorporeal circulation blood flow during dialysis, and the correlation coefficients were -0.367** and -0.202*, respectively. HDLC was significantly associated with KT/V, gender (one for males and two for females), and the correlation coefficients were 0.302** and 0.364**, respectively (*P < 0.05 and **P < 0.01); however, the level of HDLC in male patients was significantly lower than that in females.

The incidence of hypertension was 73.6% (78/106) and cardiovascular disease was 25% (26/104), of which six cases had ischemic heart disease; heart failure was found in five cases; 21 cases were found to have cardiac enlargement, and cerebrovascular accident occurred in three cases. There was no significant difference in the level of blood lipid between hypertensive group and non-hypertensive group, and the level of TG in cardiovascular disease group was significantly higher than that in the group without cardiovascular disease (Table 1).

This study found that all six genotypes of ApoE, genotype ε3/3 and allele ε3 had the highest frequency, as shown in Table 2.

Click to view

Table 2. Distribution of ApoE Genotype and the Allele Frequency

In order to study the pure effects of alleles ε2 and ε4 on blood lipids, ε2/4 type was removed and divided into three groups according to different genotypes: ε2/2 + ε2/3, ε3/3, and Ε3/4 + ε4/4. The results showed that the levels of TC, TG and LDLC in ApoE genotype ε3/4 + ε4/4 were significantly higher in hemodialysis group (Table 3).

Click to view

Table 3. Comparison of Blood Lipid Levels in Different ApoE Genotypes in Hemodialysis Patients

In this study, TG and ApoB were significantly increased in patients with hemodialysis, while TC and HDLC were significantly decreased. TG levels were associated with serum albumin and decreased blood circulation during dialysis. The decrease of HDLC level was related to dialysis insufficiency and gender, and male patients were more significant. The mechanism of lipid metabolism disorder in hemodialysis patients is not yet clear. Elevated levels of TG and ApoB may be due to increased TG-rich lipoprotein production in chronic renal insufficiency, reduced degradation, decreased activity of liver TG and lipoprotein lipase [21, 22], and decreased serum albumin at the feedback caused by increased liver lipoprotein production. This study shows that hemodialysis extracorporal blood circulation can significantly affect the level of TG. The mechanism may be due to the increase of dialysis permeability when large volume of extracorporal blood circulation was performed, in which more lipoproteins and other large and medium molecular substances, lipoprotein lipase inhibitors and ApoC III clearance can be removed, meanwhile lipoprotein lipase activity was increased. Serum cholesterol levels were mainly due to reduction of lipoprotein synthesis in the liver and intestinal tract [22]. Decrease of HDLC levels is mainly related to inadequate dialysis and malnutrition. It has been reported that 50-70% of hemodialysis patients had increased plasma Lp(a) levels, higher than 0.3 g/L, inconsistent with the results of this article [8, 23], perhaps due to different conditions, different ways, different time to start doing hemodialysis and treatment course, and all the above factors can affect the metabolism of Lp(a) [22]; Lp(a) level was increased significantly in diabetic patients; however, it was found irrelevant to diabetes in this study. In addition, variability of Lp(a) levels is huge dependent on the size of samples. If the number of samples is quite small, it tends to have a greater impact on the observed results. It has been reported that the incidence of hypertension was as high as 50-90% in hemodialysis patients, whereas the incidence of cardiovascular disease was 30-50% [6, 8]. In our study, the incidence of hypertension in hemodialysis patients was 73.6%, and the incidence of cardiovascular disease was 25%. Interestingly, TG levels in serum were significantly associated with cardiovascular disease.

ApoE has three isomers (E2, E3, and E4), encoded by the three alleles ε2, ε3, and ε4 on chromosome 19, respectively. In vitro studies have shown that the binding capacity of E2 with its receptor is low, leading to the delay of degradation of the chylomicrons in blood and VLDL residue catabolism, the reduction of intermediate density lipoprotein to LDL conversion, the increase of intermediate density lipoprotein, and the decrease of LDL concentration. The net effect of E2 results in an increase of TG and a decrease of cholesterol, while the effect of E4 is just on the opposite [9]. But in fact, the mechanisms that ApoE involved in lipid metabolism and atherosclerosis may be very complex. Clinical and animal experimental studies have come to the inconsistent results. Dallongeville et al reported that the TG levels of alleles ε2, ε4 carriers were higher than those of E3 carriers [14]. Animal experiments show that ApoE-deficient animals can cause severe lipid metabolism disorders and atherosclerosis, which can be improved by transgenic treatment [24]. In this study, the levels of TC, TG and LDLC in the control group and hemodialysis group were increased in the order of ε2/2 + ε2/3, ε3/3, and ε3/4 + ε4/4, which was not consistent with some reported results. It is reported that the effect of ApoE genotype ε on TG level is quite different. The consensus is that the level of TG in the normal human and coronary heart disease patients is higher in the allele ε2 carriers than that in ε3 carriers. On the other hand, TG level of the allele ε4 carriers either higher or lower than that of the allele ε3 carriers was obtained in previous studies [7].

These results showed that ApoE genotype had different effects on the blood lipid level of different people. In this study, hemodialysis patients and normal people carrying allele ε4 were prone to hyperlipidemia, yet the mechanisms need to be investigated in future studies.

1. Taguchi K, Moriyama A, Kodama G, Nakayama Y, Fukami K. The coexistence of multiple myeloma-associated amyloid light-chain amyloidosis and fabry disease in a hemodialysis patient. Intern Med. 2017;56(7):841-846.
   doi pubmed
2. Silva CA, Barreto FC, Dos Reis MA, Moura Junior JA, Cruz CM. Targeted screening of fabry disease in male hemodialysis patients in Brazil highlights importance of family screening. Nephron. 2016;134(4):221-230.
   doi pubmed
3. Banach M, Aronow WS, Serban MC, Rysz J, Voroneanu L, Covic A. Lipids, blood pressure and kidney update 2015. Lipids Health Dis. 2015;14:167.
   doi pubmed
4. Taheri S, Baradaran A, Aliakbarian M, Mortazavi M. Level of inflammatory factors in chronic hemodialysis patients with and without cardiovascular disease. J Res Med Sci. 2017;22:47.
   doi pubmed
5. Elsayed ET, Nassra RA, Naga YS. Peroxisome proliferator-activated receptor-gamma-coactivator 1alpha (PGC-1alpha) gene expression in chronic kidney disease patients on hemodialysis: relation to hemodialysis-related cardiovascular morbidity and mortality. Int Urol Nephrol. 2017.
   doi pubmed
6. Stefoni S, Scolari MP, Cianciolo G, Mosconi G, De Sanctis LB, De Pascalis A, La Manna G, et al. Membranes, technologies and long-term results in chronic haemodialysis. Nephrol Dial Transplant. 2000;15(Suppl 2):12-15.
   doi pubmed
7. Roy GC, Sutradhar SR, Barua UK, Datta NC, Debnath CR, Hoque MM, Hossain AS, et al. Cardiovascular complications of chronic renal failure - an updated review. Mymensingh Med J. 2012;21(3):573-579.
   pubmed
8. Senti M, Romero R, Pedro-Botet J, Pelegri A, Nogues X, Rubies-Prat J. Lipoprotein abnormalities in hyperlipidemic and normolipidemic men on hemodialysis with chronic renal failure. Kidney Int. 1992;41(5):1394-1399.
   doi pubmed
9. Locatelli F, Bommer J, London GM, Martin-Malo A, Wanner C, Yaqoob M, Zoccali C. Cardiovascular disease determinants in chronic renal failure: clinical approach and treatment. Nephrol Dial Transplant. 2001;16(3):459-468.
   doi pubmed
10. Chmurzynska A, Malinowska AM, Twardowska-Rajewska J, Gawecki J. Elderly women: homocysteine reduction by short-term folic acid supplementation resulting in increased glucose concentrations and affecting lipid metabolism (C677T MTHFR polymorphism). Nutrition. 2013;29(6):841-844.
    doi pubmed
11. Cwiklinska A, Gliwinska A, Senderowska Z, Kortas-Stempak B, Kuchta A, Dabkowski K, Jankowski M. Impact of phosphatidylcholine liposomes on the compositional changes of VLDL during lipoprotein lipase (LPL)-mediated lipolysis. Chem Phys Lipids. 2016;195:63-70.
    doi pubmed
12. Rideout TC, Movsesian C, Tsai YT, Iqbal A, Raslawsky A, Patel MS. Maternal Phytosterol Supplementation during Pregnancy and Lactation Modulates Lipid and Lipoprotein Response in Offspring of apoE-Deficient Mice. J Nutr. 2015;145(8):1728-1734.
    doi pubmed
13. Walden CC, Hegele RA. Apolipoprotein E in hyperlipidemia. Ann Intern Med. 1994;120(12):1026-1036.
    doi
14. Dallongeville J, Lussier-Cacan S, Davignon J. Modulation of plasma triglyceride levels by apoE phenotype: a meta-analysis. J Lipid Res. 1992;33(4):447-454.
    pubmed
15. Konialis C, Spengos K, Iliopoulos P, Karapanou S, Gialafos E, Hagnefelt B, Vemmos K, et al. The APOE E4 Allele Confers increased risk of ischemic stroke among Greek carriers. Adv Clin Exp Med. 2016;25(3):471-478.
    doi pubmed
16. Lahoz C, Schaefer EJ, Cupples LA, Wilson PW, Levy D, Osgood D, Parpos S, et al. Apolipoprotein E genotype and cardiovascular disease in the Framingham Heart Study. Atherosclerosis. 2001;154(3):529-537.
    doi
17. Echeverria P, Guardiola M, Gonzalez M, Vallve JC, Bonjoch A, Puig J, Clotet B, et al. Association between polymorphisms in genes involved in lipid metabolism and immunological status in chronically HIV-infected patients. Antiviral Res. 2015;114:48-52.
    doi pubmed
18. Hanh NT, Nhung BT, Dao DT, Tuyet LT, Hop LT, Binh TQ, Thuc VT. Association of apolipoprotein E polymorphism with plasma lipid disorders, independent of obesity-related traits in Vietnamese children. Lipids Health Dis. 2016;15(1):176.
    doi pubmed
19. Teixeira AA, Marrocos MS, Quinto BM, Dalboni MA, Rodrigues CJ, Carmona Sde M, Kuniyoshi M, et al. Diversity of apolipoprotein E genetic polymorphism significance on cardiovascular risk is determined by the presence of metabolic syndrome among hypertensive patients. Lipids Health Dis. 2014;13:174.
    doi pubmed
20. Berkinbayev S, Rysuly M, Mussayev A, Blum K, Baitasova N, Mussagaliyeva A, Dzhunusbekova G, et al. Apolipoprotein Gene Polymorphisms (APOB, APOC111, APOE) in the development of coronary heart disease in ethnic groups of Kazakhstan. J Genet Syndr Gene Ther. 2014;5(2):216.
    pubmed
21. Olmer M, Renucci JE, Planells R, Bouchouareb D, Purgus R. Preliminary evidence for a role of apolipoprotein E alleles in identifying haemodialysis patients at high vascular risk. Nephrol Dial Transplant. 1997;12(4):691-693.
    doi pubmed
22. Klin M, Smogorzewski M, Ni Z, Zhang G, Massry SG. Abnormalities in hepatic lipase in chronic renal failure: role of excess parathyroid hormone. J Clin Invest. 1996;97(10):2167-2173.
    doi pubmed
23. Namba T, Masaki N, Matsuo Y, Sato A, Kimura T, Horii S, Yasuda R, et al. Arterial stiffness is significantly associated with left ventricular diastolic dysfunction in patients with cardiovascular disease. Int Heart J. 2016;57(6):729-735.
    doi pubmed
24. Shimano H, Yamada N, Katsuki M, Shimada M, Gotoda T, Harada K, Murase T, et al. Overexpression of apolipoprotein E in transgenic mice: marked reduction in plasma lipoproteins except high density lipoprotein and resistance against diet-induced hypercholesterolemia. Proc Natl Acad Sci U S A. 1992;89(5):1750-1754.
    doi pubmed

This article is distributed under the terms of the Creative Commons Attribution Non-Commercial 4.0 International License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Journal of Clinical Medicine Research is published by Elmer Press Inc.