Distribution of apoe polymorphism among primary school children in some northern provinces of Viet Nam

161 HNUE JOURNAL OF SCIENCE DOI: 10.18173/2354-1059.2017-67 Chemical and Biological Science 2017, Vol. 62, Issue 10, pp. 161-168 This paper is available online at DISTRIBUTION OF APOE POLYMORPHISM AMONG PRIMARY SCHOOL CHILDREN IN SOME NORTHERN PROVINCES OF VIETNAM Nguyen Thi Hong Hanh 1 , Duong Thi Anh Dao 1 and Tran Quang Binh 2 1 Faculty of Biology, Hanoi National University of Education 2 National Institute of Nutrition Abstract. APOE gene plays an important role in immunity and metabolism regulating, especially in lipid metabolism. Cross-sectional study was conducted on 300 children at the age of 6-11 in Hanoi, Thai Nguyen, and Hai Duong province to investigate the varying distribution of the APOE genotype by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR -RFLP) method. Popular alleles at rs429358 and rs7412 were T allele (91.4% in Hanoi, 94.3% in Thai Nguyen, and 89.6% in Hai Duong, P = 0.178) and C allele (90.8% in Hanoi, 93.6% in Thai Nguyen, and 94.0% in Hai Duong, P = 0.699), respectively. There was no statistically significant difference between Hanoi, Thai Nguyen, and Hai Duong in the distribution of ε2, ε3, ε4 allele frequency. The ε2, ε3, ε4 allele percentages in Hanoi were 9.2%, 82.2% and 8.6%; in Thai Nguyen were 6.4%, 87.9% and 5.7%; and in Hai Duong were 6.0%, 83.6% and 10.4%, respectively. It can be concluded that some provinces in northern of Vietnam is homogeneous in terms of APOE genotype distribution in 6-11 year-old children. Keywords: Apoliporotein E, 6-11 year-old children, northern provinces of Vietnam. 1. Introduction Apoprotein E gene (APOE), coding for apolipoprotein E protein (apo E) - a multifunctional protein, which is produced primarily in the liver and macrophages in peripheral tissues; in astrocytes and glial cells in the central nervous system. APOE gene plays an important role in immunity and metabolism regulating, especially in lipid metabolism [1]. APOE gene located on chromosome 19, in position 13.2 and in a cluster with APOC1 and APOC2 [2]. APOE gene is polymorphic, including 195 single nucleotide polymorphisms (SNP). The differences at SNP rs429358 and rs7412 in exon 4 form three alleles ε2, ε3, ε4, which encode for apo E2, apo E3 and apo E4 isoforms, respectively. The differences in the amino acid sequence alter the structure and function of apo E [2]. Received July 24, 2017. Revised November 28, 2017. Accepted December 5, 2017. Contact Nguyen Thi Hong Hanh, e-mail address: hanhnth@hnue.edu.vn Nguyen Thi Hong Hanh, Duong Thi Anh Dao and Tran Quang Binh 162 The isoforms of apo E display different roles in lipid metabolism, neurobiology, and neurodegenerative diseases [3]. The apo E isoforms differ in their ability to interact with specific cell-surface receptors (LDL receptor, the LDL receptor-related protein, the VLDL receptor, the apoE receptor-2, and gp330). The E4 isoform has a higher affinity than E2 and E3 isoforms [4]. E3 isoform is a natural form with the highest frequency in human population. Thus, the ε3 allele is considered neutral in the APOE genotype. Meanwhile, ε4 and ε2 alleles have been reported related to some diseases in human populations. The ε4 variant is known to be risk factors for Alzheimer's disease in different ethnic groups [5, 6]. Besides, ε4 allele carriers also have higher risk of dyslipidemia [7], cardiovascular disease, type II diabetes [8], and stroke due to ischemia [9] than ε3 homozygous. In contrast, the ε2 allele was considered as a protective factor of dyslipidemia [10]. Especially, in children, similar relationship between APOE genotype and dyslipidemia also has been reported repeatedly [11, 12]. However, according to our understanding, the study of APOE genotypes and alleles distribution in primary school children in Vietnam is still limited. Therefore, studies were conducted to determine the distribution of the APOE gene in 6-11 year-old children in Hanoi, Thai Nguyen, and Hai Duong help provide data for follow-up cohort studies on the relationship between APOE gene with some diseases. 2. Content 2.1. Subjects and methods * Study population Based on the survey results on the 7,750 children in Hanoi Capital, 1,116 children in Hai Duong province and 1,823 children in Thai Nguyen province, 300 children at the age of 6-11 were selected to the study, except children is suffering from acute or chronic diseases such as tuberculosis, HIV/AIDS... Of that total, subjects include 163 children in Hanoi (64 girls, 99 boys), 67 children in Hai Duong (26 girls, 41 boys), 70 children in Thai Nguyen (30 girls, 40 boys). All of them are Kinh, the major ethic in Vietnam. The research protocol was approved by The Institutional Review Board of the National Institute of Hygiene and Epidemiology, Vietnam (IRB-VN01059-25). Written consent to participate in the study was given by the parents of all subjects. * Blood collection All children were taken 2 ml peripheral blood in the morning after fasting for at least 08 hours by the medical staff of the Medlatec hospital, Thai Nguyen Medical University Hospital and Hai Duong Hospital. Blood was collected in EDTA (Ethylene Diamine Tetraacetic Acid) anticoagulant tubes. The blood was stored in the cold box moved Laboratory Molecular Genetics, Institute of Hygiene and Epidemiology within 3 hours. Blood samples were centrifuged for 10 min at 4°C to extract the plasma. The blood samples were stored at -80°C before DNA extraction. Distribution of APOE polymorphism among primary school children in some northern provinces of Vietnam 163 * DNA extraction method Peripheral blood samples were obtained from each participants and genomic DNA was extracted from peripheral blood leukocytes, using Wizard® Genomic DNA Purification Kit (Promega Corporation, USA). * Genotyping method Genotyping of the APOE was performed by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method according to Hixson and Vernier method [13] with some modification [14]. * Statistical analysis The statistical analyses were done with the statistical software package SPSS16.0 (SPSS Inc., Chicago, Illinois). Genotypic and allele frequency was determined for Hardy- Weinberg equilibrium by Fisher's exact test. Genotypic and allele frequency were compared by Chi-square test. P-values < 0.05 were considered as significant. 2.2. Results and Discussion 2.2.1. Genotype and allele frequencies of APOE rs429358 and rs7412 polymorphism Genotype of each SNP in all subjects were determined by PCR-RFLP method. The results were shown in Tables 1 and 2. Table 1. The distribution of APOE rs429358 genotypes and alleles Genotypes/ alleles Hanoi (n = 163) Thai Nguyen (n = 70) Hai Duong (n = 67) P Genotypes, n (%) T/T 136 (83.4%) 62 (88.6%) 53 (79.1%) 0.537 T/C 26 (16.0%) 8 (11.4%) 14 (20.9%) C/C 1 (0.6%) 0 (0%) 0 (0%) Alleles, n (%) T 298 (91.4%) 123 (94.3%) 120 (89.6%) 0.178 C 28 (8.6%) 8 (6.7%) 14 (10.4%) H-W equil. P 0.840 0.612 0.340 H-W equil.: test for Hardy-Weinberg equilibrium using Chi-square test. P-values obtained by Chi-square test. Table 1 shows that in all groups the genotypes observed are in agreement with those expected under Hardy-Weinberg equilibrium (P > 0.05). At the rs429358 polymorphism, the most common genotype in Hanoi, Thai Nguyen and Hai Duong population was T/T (83.4%, 88.6%, 79.1%, respectively). The minor allele was C allele (8.6%, 6.7% and 10.4%). The exact test for population differentiation indicates that there was no significant variation in genotype and allele distribution among Hanoi, Thai Nguyen and Hai Duong with P = 0.537. Nguyen Thi Hong Hanh, Duong Thi Anh Dao and Tran Quang Binh 164 Table 2 shows that, contrary to the APOE rs429358 polymorphism, in APOE rs7412 the minor allele was T allele (9.2%, 6.4% and 6.0% in Hanoi, Thai Nguyen and Hai Duong, respectively). The allele and genotype distribution in whole sample was under Hardy-Weinberg equilibrium. There was no difference in the genotype and allele distribution among Hanoi, Thai Nguyen and Hai Duong (P = 0.560). Table 2. The distribution of APOE rs7412 genotypes and alleles Genotypes/ alleles Hanoi (n = 163) Thai Nguyen (n = 70) Hai Duong (n = 67) P Genotypes, n (%) C/C 136 (83.4%) 61 (87.1%) 59 (88.1%) 0.569 T/C 24 (14.7%) 9 (12.9%) 8 (11.9%) T/T 3 (1.8%) 0 (0%) 0 (0%) Alleles, n (%) C 296 (90.8%) 131 (93.6%) 126 (94.0%) 0.699 T 27 (9.2%) 9 (6.4%) 8 (6.0%) H-W equil. P P = 0.129 P= 0.565 P = 0.603 H-W equil.: test for Hardy-Weinberg equilibrium using Chi-square test. P-values obtained by Chi-square test. Genotype and allele distribution of APOE rs429358 polymorphism in our study are slightly different from some other populations (Hapmap project). According to research results of Tejedor et al. (2014) in 4660 middle-aged Spanish men [15], the distribution of T/T, T/C and C/C genotypes were 81.5%, 17.6% and 0.9%, respectively. Research by Tejedor et al. [15] in the rs7412 polymorphism also showed that the genotype distribution was C/C (88.2%), T/T (0.5%). Figure 1. The genotype frequencies of APOE rs429358 in Vietnamese children population compared with some populations according HapMap database (CHB: Han Chinese in Beijing, China; JPT: Japanese in Tokyo, Japan; YRI: Yoruban in Inbadan, Nigeria; HnVN: Kinh in Hanoi, Vietnam; TVN: Kinh in Thai Nguyen, Vietnam; HVN: Kinh in Hai Duong, Vietnam) Distribution of APOE polymorphism among primary school children in some northern provinces of Vietnam 165 The genotype and allele distribution in both SNPs in whole sample as well as in each province were under Hardy-Weinberg equilibrium. It suggested that sample might represent for population. Therefore, we compared the distribution of APOE rs429358 and rs7412 polymorphism in this study with some other populations in the world according HapMap database shown in Figures 1 and 2. Figure 2. The genotype frequencies of APOE rs7412 in Vietnamese children population compared with some populations according HapMap database (ASW: African Ancestry in South-West USA; CHB: Han Chinese in Beijing, China; CHD: Chinese in Colorado, USA; GIH: Gujarati Indians in Texas, USA; JPT: Japanese in Tokyo, Japan; YRI: Yoruban in Inbadan, Nigeria; HnVN: Kinh in Hanoi, Vietnam; TVN: Kinh in Thai Nguyen, Vietnam; HVN: Kinh in Hai Duong, Vietnam) The figure shown that in SNP rs429358 (Figure 1), the proportion of allele C in this study was greater than other populations such as Japanese in Tokyo, Japan population (JPT) and Yoruban in Inbadan, Nigeria population (YRI). 100% of Han Chinese in Beijing population (CHB) was only allele T. In SNP rs7412 (Figure 2), the more common allele in all population was allele C. The allele distribution in Kinh in Hanoi, Vietnam (HnVN) population (T allele occupy 9.2%) was similar to African Ancestry in South-West USA (ASW) population and Han Chinese in Beijing, China (CHB) population. The allele distribution in Kinh in Thai Nguyen, Vietnam (TVN) population (T allele occupy 6.4%) and Kinh in Hai Duong, Vietnam (HVN) population (T allele occupy 6.0%) were similar to Chinese in Colorado, USA (CHD) population and Japanese in Tokyo, Japan (JPT) population. The proportion of T allele in this study was significantly different from Gujarati Indians in Texas, USA (GIH) population. 2.2.2. The distribution of APOE polymorphism according to ε isoform APOE is polymorphic, with three major alleles ε2, ε3 and ε4, which differ from each other by only one or two nucleotides at positions SNP rs7412 and rs429358. ε2 has T at rs7412 and T at rs429358. Meanwhile, ε3 has C at rs7412 and T at rs429358. ε4 has C at rs7412 and C at rs429358 [2]. The genotype and allele distribution of APOE polymorphism in both SNP according ε isoforms was presented in Table 3. Nguyen Thi Hong Hanh, Duong Thi Anh Dao and Tran Quang Binh 166 Table 3. The distribution of APOE polymorphism Genotypes/ alleles Hanoi (n = 163) Thai Nguyen (n = 70) Hai Duong (n = 67) P Genotypes, n (%) ε2/ε2 3 (1.8%) 0 (0%) 0 (0%) 0.641 ε2/ε3 22 (13.5%) 9 (12.9%) 6 (9%) ε2/ε4 2 (1.2%) 0 (0%) 2 (3%) ε3/ε3 111 (68.1%) 53 (75.7%) 47 (70.1%) ε3/ε4 24 (14.7%) 8 (11.4%) 12 (17.9%) ε4/ε4 1 (0.6%) 0 (0%) 0 (0%) Alleles, n (%) ε2 30 (9.2%) 9 (6.4%) 8 (6.0%) 0.725 ε3 268 (82.2%) 123 (87.9%) 112 (83.6%) ε4 28 (8.6%) 8 (5.7%) 14 (10.4%) P-values obtained by Chi-square test. The results showed that six different genotypes were found. The most common genotype was ε3/ε3 (70.4% in whole subjects), whereas the rare genotype was ε4/ε4 (0.3% in whole subjects). There was no difference in genotype and allele distribution among Hanoi, Thai Nguyen and Hai Duong (P = 0.641). Compared with other studies showed that ε3/ε3 genotype proportion was slightly difference among some population such as Vietnamese population (63.0%) [16], French population (69.3%) [17], and Brazilian population (61.0%) [18]. According to research results in nine native American populations living at subtropical latitudes of South America, the incidence of the most common allele (ε3) ranged from 78% to 98%, while the ε4 allele prevalence varied from 0% to 17%. The rare allele ε2 was found in five of the nine populations investigated [19]. The two main mechanisms commonly invoked to explain geographic gradients in gene frequencies are gene flow and natural selection [20]. Another complication in the interpretation of the spatial pattern observed for the APOE variants is the differences in lifestyle, environmental factors, and risk-factor profiles. The reconstruction of human demographic history is of direct biological and anthropological interest. Additionally, the history of effective population size has a profound effect on important quantities such as the extent of linkage disequilibrium and is therefore important for medical association studies. There have been many attempts for demographic inference from contemporary molecular data representing different molecular mutation systems [21]. In this study, we have not yet considered the influence of this polymorphism and some environmental factors on the disease in children. This is a potential limitation of our study because the genetic characteristics of ethnic, differences in nutritional factors, physical activities, and socio-economic factors have different impacts on the relationship of genes and disease in different ethnic groups. Distribution of APOE polymorphism among primary school children in some northern provinces of Vietnam 167 3. Conclusion The main message of our study is that some provinces in northern of Vietnam are homogeneous in terms of APOE genotype distribution. The ε2, ε3, ε4 allele percentage in Hanoi were 9.2%, 82.2% and 8.6%; in Thai Nguyen were 6.4%, 87.9% and 5.7%; and in Hai Duong were 6.0%, 83.6% and 10.4%, respectively. Popular alleles at rs429358 and rs7412 were T allele (91.4% in Hanoi, 94.3% in Thai Nguyen, and 89.6% in Hai Duong, P = 0.178) and C allele (90.8% in Hanoi, 93.6% in Thai Nguyen, and 94.0% in Hai Duong, P = 0.699), respectively. Acknowledgements. This study was supported by grant No. B2014-17-47 (Study on the association of TMEM18, APOE genes with obesity and dyslipidemia in primary school children) from the Ministry of Education and Training, Vietnam. REFERENCES [1] Liu C.C., Kanekiyo T., Xu H., Bu G., 2013. Apolipoprotein E and Alzheimer disease: risk, mechanisms and therapy. Nature Reviews. Neurology, Vol. 9, No. 2, pp. 106-118. 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