Introduction

Diabetes is a chronic metabolic disease that affects 9% of adults all around the world 1. It is one of the major causes of morbidity and mortality among adults. In Malaysia, as per National Health and Morbidity Survey report 2015, prevalence of diabetes among adults of age 18 years and above is 17.5% 2. While World Health Organization (WHO), in the ‘Global Status Report on Noncommunicable Diseases 2014’, reported that the prevalence of diabetes among Malaysian adults is 11.1%, which is 1.2 times higher than the global prevalence1.

From the genetic point of view, the causes of diabetes can be divided into monogenic and polygenic disease. Usually, the monogenic defects cause type 1 diabetes mellitus (T1DM) by affecting the production of insulin from beta cells of pancreas, and it contributes to 1-5% cases of all diabetes 3. On the other hand, type 2 diabetes mellitus (T2DM) that contributes 90-95% of all diabetes, is a chronic multifactorial disease of adulthood 3,4. T2DM results from a complex interactions between multiple genetic and environmental factors, where pre-existing susceptible genes are being triggered by nongenetic environmental factors, such as consumption of junk food, decreased opportunity and motivation for physical activity, and awareness among the genetically susceptible individuals 5,6.

About a decade ago, the apolipoprotein E () gene was suggested as a susceptible gene that could induce pathogenesis of T2DM 7. Apolipoprotein E (apoE) is a small glycoprotein 8 with the molecular weight of 34 kDa 9, and is mainly secreted from the liver 10. The primary role of circulating apoE is to facilitate the interaction of chylomicron remnants and intermediate density lipoproteins (IDL) with lipoprotein receptors, namely the low-density lipoprotein (LDL) receptors (LDLr), and the chylomicron remnants or apoE receptors10,11. It also promotes the efficient uptake of these lipoproteins from the circulation and takes part in cellular cholesterol efflux and reverse cholesterol transportation 12. Concentration of plasma apoE and its binding capability is regulated by the gene, which is located in the long arm of chromosome 19, more precisely at 19q13.2 13. gene possesses three alleles, namely e2, e3 and e4, which are responsible for the production of corresponding E2, E3 and E4 plasma isoproteins 7. The gene is polymorphic in nature with having six genotypes, three of which are homozygous (e2/e2, e3/e3 and e4/e4) and three are heterozygous (e2/e3, e2/e4 and e3/e4) 14. Variations in the plasma apoE are due to cysteine–arginine interchanges in the chemical structure. ApoE3 contains a cysteine residue at 112 and an arginine residue at 158, while apoE2 contains cysteine residues at the both positions, and apoE4 contains arginine residue at the both positions 11,15. Corresponding single nucleotide polymorphisms at the genetic level is involved in the modification of amino acid sequences at the protein level 16.

Several studies on the role of gene in developing pathological conditions of diabetes have been conducted and the pathogenesis was further linked to lipid and lipoprotein metabolic abnormalities17,18,19,20,21. However, there are scarcity of similar studies on Malaysian populations. Hence, in this study we aimed to evaluate the relationship between gene polymorphism and T2DM among Malaysian subjects.

Methods

Apolipoprotein E gene polymorphism analysis

Genomic DNA was extracted from 200 ml of human whole blood using Invisorb Spin Blood Mini Kit (Stratec Molecular Gmbh, Germany) following the manufacturer’s protocol. Three common alleles of the gene were amplified by polymerase chain reaction (PCR) using TopTaq Master Mix Kit (QIAGEN, Germany). The final reaction volume was 25 ml for each sample, which consisted of 1.25 units TopTaq DNA polymerase, 1x PCR buffer solution, 3 mM MgCl and 200 mM of each dNTP and 5 ml of the genomic DNA. Concentration of each primer (forward: 5’-TAA GCT TGG CAC GGC TGT CCA AGG A-3’ and reverse: 5’-ACA GAA TTG GCC CCG GCC TGG TAC AC-3’; Invitrogen, USA) in the final reaction volume was 0.5 mM. Molecular grade glycerol (10% in each final reaction volume) was used due to high melting temperature of the selected primers. The PCR operation conditions included initial denaturation phase at 95C for 5 minutes, followed by 30 cycles of denaturation at 95C for 30 seconds, annealing at 60C for 20 seconds, extension phase at 72C for 20 seconds, and the final elongation at 72C for 10 minutes. Amplification was confirmed by 1% agarose gel electrophoresis stained with ethidium bromide. After confirmation of amplification, 10 ml of each amplified DNA was then digested at 37C for an hour by restriction enzyme (Invitrogen, USA). The digested products were then visualized in 4% agarose gel stained with ethidium bromide. A single band of about 244 base pairs (bp) in 1% agarose gel was considered confirmatory to the amplification (Figure 1). Seven bands at different levels were identified in 4% agarose gel after the digestion, the first four bands were used to identify different genotypes based on their presence/absence at the identical bp levels (Figure 1). The presence of the first two bands (at 94 and 81 bp) was used to identify the e2/e2 genotype, first two and fourth bands (at 94, 81 and 66 bp) represented e2/e3 genotype, all four bands (at 94, 81, 66 and 58 bp) represented the e2/e4 genotype, first and fourth bands (at 94 and 58 bp levels) represented the e3/e3 genotype, first and last two bands (at 94, 66 and 58 bp levels) represented the e3/e4 genotype, and last two bands (at 66 and 58 bp levels) represented the e4/e4 genotypes.

Results

Apolipoprotein E gene polymorphism and fasting glycemic status

Means of the FBG (±SD) in relation to different alleles between the study groups are shown in Figure 2. In both the study groups, e2 allele holds the highest mean FBG values followed by e4 and then e3 alleles. Mean differences were found to be statistically significant between alleles in diabetes group (p = 0.011), but not in the control group (p = 0.978). Within the diabetes group, both the e2 and e4 alleles had significantly higher mean FBG compared to e3 allele, p = 0.026 and 0.036, respectively.

Discussion

In this study, the distribution of gene polymorphism did not show any significant difference between cases and control groups. However, Hardy-Weinberg equilibrium test showed a significant difference in the allelic distribution between the Malay and Chinese race of non-diabetic subjects. Distribution of e2 and e4 alleles in Malay control subjects compared to Chinese controls was four and two times higher, respectively. Like Malay subjects, comparatively higher frequency of e4 allele was also observed in Indonesian populations, and certain parts of African, European and Oceania countries 22,23,24,25,26,27. This finding is also consistent with two studies conducted in Singapore, where Malay aboriginal subjects were recruited 28,29. On the other hand, the distribution of alleles within Chinese subjects follows the worldwide distribution 30, including the part of China, Japan, Taiwan and Korea13,21,31. Certain European countries also demonstrated similar distribution26,32. According to Corbo and Scacchi (1999), e3 allele is the most frequent among populations that have a long established agricultural economy, and e4 allele remains higher in populations where an economy of foraging still exists and food supply is, or was in the recent past, scarce and sporadically available30.

There was no significant association between alleles and T2DM in this study. It is noteworthy to mention that the percentages of the e2 and e4 alleles were slightly higher among the subjects with T2DM. Meanwhile, among the subjects with T2DM, mean FBG concentration was found significantly higher in subjects with e2 and e4 alleles compared to subjects with e3 allele. Similarly, the highest mean FBG was observed within e2 allelic control subjects, followed by e4 and the e3 allelic control subjects. Although plasma apoE protein does not have any direct effects on carbohydrate metabolism, its role in the development of obesity that eventually leads to the development of metabolic syndrome and insulin resistance had already been explored 33. Sima . reported a weak correlation between e4 allele with metabolic syndrome34. Whereas, Helkala . reported that e2-allele-carrying subjects had relatively higher fasting and 2-h blood glucose than the subject carrying other phenotypes35. Duman . also reported similar observation in a Turkish study population with T2DM 36.

Numerous studies have been conducted around the world regarding the relationship between the gene polymorphism and diseases that are caused by or lead to dyslipidemia37,38,39,40,41. It was initiated by exploring the role of the e2 allele (more specifically the e2/e2 genotype) in the pathogenesis of type III hyperlipidemia that could leads to premature coronary artery disease37. Subsequent studies discovered the role of e4 allele on the pathogenesis of Alzheimer’s diseases, atherosclerosis and cerebrovascular diseases as well38,39,40,41. A number of studies also tried to find out a relationship between gene polymorphism with T2DM and its complications. However, most of the studies were inconclusive. Only Liu and Errera suggested e2 allele as a risk factor for the development of T2DM in the Chinese and Brazilian populations17,18. The e2 allele was also reported to be associated with T2DM mediated nephropathy19,20,21. Increased risk of retinopathy in patients with T2DM was linked to the higher e4 allele frequency42. Most recently, Anthopoulos conducted a meta-analysis, where they suggested the e2 allele as a moderate risk factor for the T2DM43.

Almost all studies were conducted to prove a causal relationship between gene polymorphism and the risk of T2DM faced sample scarcity. However, original research and meta-analysis that reported a causal relationship between them recruited subjects with T2DM along with its complications, such as diabetic nephropathy, diabetic retinopathy, and/or diabetes with IHD, stroke.19,20,21,42,43. Similar to other previous studies, small sample size might be a cause behind the failure to find any association between gene polymorphism and T2DM in this study. The subjects included in the study were free from any other diabetes mediated complications. But this study has generated a hypothesis on the causal relationship between gene polymorphism and FBG concentration among the subjects with or without the T2DM.

Conclusions

Though this study represents preliminary data, it showed that subjects carrying e2 and e4 APOE alleles have poor glycemic control. Frequencies of these two alleles were also slightly higher among the subjects with T2DM. The e3 allelic subjects were found relatively safe in terms of the glycemic control and the occurrence of the T2DM. Further study with a larger sample size could help to improve the outcome of this research along with a proper conclusive causal relationship.

Abbreviations

ANOVA: Analysis of variance

APOE: Apolipoprotein E gene

apoE: Apolipoprotein E glycoprotein

bp: Base pair

EDTA: Ethylenediaminetetraacetic acid

FBG: Fasting blood glucose

IDL: Intermediate density lipoprotein

LDL: Low-density lipoprotein

LDLr: Low-density lipoprotein receptors

PASW: Predictive Analytics Software

PCR: Polymerase chain reaction

SD: Standard deviation

T1DM: Type 1 diabetes mellitus

T2DM: Type 2 diabetes mellitus

WHO: World Health Organization

Competing Interests

Authors declare no conflict of interests.

Authors' Contributions

KMHR and TBAR conceptualized and designed the work. KMHR carried out the experiment. KMHR and MSH conducted formal analysis. KMHR, MSH, NH, TBAR and HA prepared the original draft. MSH and NH reviewed and edited the drafts. All authors approved the manuscript.

Funding

This study was financially supported by the IIUM University Research Grant, EDW B 11-139-0478.

Acknowledgments

We extremely liked to acknowledge the supports from the Non-Communicable Disease Research Unite (NCD_RU), Kulliyyah of Medicine, International Islamic University Malaysia and the intellectual supports from the coordinator of the NCD_RU, Dr. Seikh Farid Uddin Akter.