Genetic Variation of Apolipoprotein E ( ApoE ) in Surabaya , Palu and Alor Populations of Indonesia

Apolipoprotein E (ApoE) has been considered to play an important role in cardiovascular disorders. Several studies reported that genetic variation in ApoE locus influence plasma lipoprotein level. The objectives of this study was to compare the frequency of ApoE genotypes and alleles in some populations of Indonesia. One hundred and ninety five voluntarily unrelated apparently healthy individuals were recruited from Surabaya, Palu and Alor representing the western, middle and eastern populations of Indonesia, respectively. Blood samples were collected from each subject for DNA extraction. The common allelic variants of ApoE were screened using polymerase chain reaction (PCR) and restriction fragment length polymorphism. Three alleles i.e. ε2, ε3 and ε4 were identified and five genotypes i.e. ApoE ε2/ε2, ApoE ε2/ε3, ApoE ε3/ε3, ApoE ε2/ε4, ApoE ε3/ ε4 were found in three populations studied, while ApoE ε4/ε4 was absent in Surabaya, representing the western populations of Indonesia. The frequency of ε2, ε3 and ε4 alleles in the western population were 0.208, 0.701 and 0.092 respectively; in the middle population were 0.242, 0.618 and 0.140 respectively and in the eastern population of Indonesia were 0.267, 0.466 and 0.267 respectively. The highest frequency of ε2 and ε4 allele was found in the eastern population of Indonesia. The distribution of ε2 allele were not significantly different among all Indonesian populations, but significantly different were found in ε3 and ε4 allele in the eastern population compared to those in the western and middle populations of Indonesian. It can be concluded that the frequency of three ApoE alleles in the western and middle populations of Indonesia was not significantly different however, significantly different was observed in the frequency of ApoE ε3 and ε4 alleles from the eastern compared to those in the western and middle populations of Indonesia.


Introduction
Apolipoprotein E (ApoE), a 34-kDa glycoprotein, plays an important role in the transport and metabolism of plasma cholesterol and triglycerides through its ability to interact with cell surface of low density lipoprotein (LDL) receptor and LDL receptor-related protein (LRP).ApoE exists in three isoforms (ApoE2, E3, and E4) that differ in their cysteine/arginine content at two polymorphic sites (Corella et al., 2002).Three common ApoE alleles have been identified: ApoE2, ApoE3, and ApoE4 (Elousa et al., 2004).The encoded 299-residue plasma protein ApoE is a surface component of primarily triglyceride-rich lipoproteins, such as very low-density lipoproteins (VLDLs), chylomicron remnant, and high density lipoproteins (HDLs).ApoE is the main ligand for clearance of VLDLs and chylomicron remnant, and as such affects circulating concentrations of lipoproteins and plasma levels of cholesterol and triglycerides (Mahley & Ji, 1999).Genetic variation in the ApoE locus significantly affects plasma lipoprotein concentrations.The allele of ApoE is associated with increased risk of two major causes of death in low-mortality populations: ischemic heart disease and Alzheimer's disease.The presence of the ε4 allele is associated with increased LDL cholesterol, whereas the presence of the ε2 allele is associated with decreased LDL cholesterol and may protect against mortality due to coronary heart disease and Alzheimer's (Douglas, 2002;Jurcovicova et al., 2006, Minihane et al, 2007).Meta-analyses suggest that ApoE4 carrier may have a 40-50% increased CVD risk, however the associations reported in individual studies are highly heterogeneous and environmental factors influence genotype-phenotype associations (Minihane et al., 2007).Both ApoE2 and ApoE4 are also associated with increases in plasma triglycerides although the ApoE2 allele is associated with a lower CVD risk in women in the Framingham Study (Elousa et al., 2004).The frequencies of these three ApoE alleles are highly variable in different populations (Singh et al., 2006).Globally, ε3 allele is the most frequently found, constituting 60-90% of the allelic variation, ε2 allele constitutes 0-20% and ε4i allele 10-20% of allelic variation (Elousa et al., 2004;Gerdes et al., 1996).
Indonesia is a country in Southeast Asia and Oceania, comprising 17,508 islands.Across its many islands, Indonesia consists of a variety of distinct ethnic, linguistic, and religious groups.Most Indonesians are descendent from Austronesian-speaking peoples whose languages can be traced to Proto Austronesian, which is likely originated from Taiwan.The other major grouping is Melanesians, inhabiting the eastern part of Indonesia (Dawson & Gillow, 1994).There are around 300 distinct native ethnicities in Indonesia, and 742 different languages and dialects (Merdekawaty, 2006).The objectives of this work is to study ApoE genotypes and allele polymorphism in Surabaya, Palu and Alor populations, representing the western, middle and eastern populations of Indonesia.

Materials and Methods
All individuals were recruited as part of a comprehensive study on the "Interaction of biochemical genetic traits and other markers in malaria endemic areas in Indonesia".All subjects were voluntarily unrelated apparently healthy individuals and all of their grandparents were born and belong to a particular ethnic in the study area.Among 195 subjects involved in the study, 82, 68 and 45 individuals were from Surabaya, Palu and Alor representing the western, middle and eastern populations of Indonesia respectively.Written informed consent was obtained from all individuals, and ethical clearance for the project was provided by Human Research and Ethics Committee of the Faculty of Medicine Universitas Gadjah Mada, Yogyakarta.Approximately 10 mL venous blood was drawn from ante cubital vein of each individuals employing EDTA anticoagulated vacutainer (Terumo).The blood was stored in 4 o C and brought to the laboratory in Yogyakarta prior to laboratory examination.

DNA analysis
Genomic DNA was isolated from blood leukocytes using a standard phenolchloroform extraction procedure.Polymerase c h a i n r e a c t i o n ( P C R ) a m p l i f i c a t i o n was undertaken using forward primer: 5'-TCCAAGGAGCTGCAGGCGGCGA-3' and reverse primer: 5'-GCCCGGCCTG GTACACTGCCA-3' to produce a 218-bp product.The mixture was a mix Promega 12.5 uL, forward and reverse primers 1 μL, water 9.5 μL, and DNA 1 μL was subjected in PCR reaction.PCR conditions used in amplification were 94°C for 1 min, followed by 40 cycles of 94°C for 30 sec, 60°C for 30 sec, and 70°C for 90 sec for extension and a final extension cycle of 72°C for 10 min.

Hastuti et al.
Amplified DNA 8 μL was digested with 2.5 U Afl III and 5 U Hae II for 24 h at 37 o C. The result was analyzed on 4% agarose gel and visualized by ethidium bromide staining.Digested product were 145-bp, 168-bp and 195 bp wich were specific for ApoE3, E2 and E4 (Zivelin et al., 1997).The genotype of ApoE in populations was compared employing Chi square analysis, and significant difference was agreed when p<0,05.

Results
Despite abundant reports on the genetic variation of ApoE gene locus, the distribution of ApoE alleles particularly ε2, ε3 and ε4 are scarcely available in Indonesian populations, if any.In this report variation of ApoE gene locus were found in three Indonesian populations studied (Figure 1 and Figure 2).The frequency of ApoE genotypes and alleles were shown in Table 1 and Figure 2.  Having considered that the population of Surabaya, Palu and Alor represented the western, middle and eastern populations in Indonesia (figure 3), the frequency of ApoE ε2, ε3 and ε4 alleles were not significantly different between the western and middle populations of Indonesia.Similarly, the frequency of ApoE ε2 allele for all three populations were not significantly different (p>0.05).It is interesting that the frequency of ApoE ε2 and ε4 allele increased from the western to the eastern population, whereas the frequency of ApoE ε3 allele was high in the western and declined to the eastern population of Indonesia.Significant difference in ApoE ε3 and ApoE ε4 alleles were found between the western and eastern populations of Indonesia as well as between the middle and the eastern populations of Indonesia (P<0,05).In contrast there was no significant difference for ApoE ε2 allele frequency between the western and the middle populations of Indonesia.

Discussion
The frequency of ApoE ε2, ε3, ε4 alleles in this study were 0.207 -0.267, 0.466 -0.701 and 0.092 -0.267 respectively.It can be observed from previous reports that the frequency of ApoE allele varied from population to population all of the world (Al-Bustan et al., 2005;Belkovets et al., 2001, Mastana et al., 1998;Rodrigues et al, 2005;Siest et al., 1995, Thelma et al., 2001).This present result finds high frequency of ApoE ε2 allele in all three Indonesian populations studied compared to its frequency found in other Asians (Belkovets et al., 2001).Variation of ApoE ε4 allele frequency ranges from 5% in Taiwan & Sardinia to 40% in the Pygmies, while variation of ApoE ε3 allele frequency ranges from 49% in Papua New Guinea to 90% in Sardinia (Siest et al., 1995).In this study, the frequency of ApoE ε4 allele in the eastern population of Indonesia was the highest compared to the other two populations.It may be due to their relative isolation leading to endogamous mating which results in the increase frequency of rare variant.Compared with the ApoE ε4 frequency in Papua New Guinea population (49%) it was shown that its frequency in the eastern population of Indonesia was lower.However, it was still the highest compared with the other two populations.In other word, the frequency of ApoE ε4 allele is lower even absent in the rest of populations of Indonesia.
It has been acknowledged that populations of the western part of Indonesia were strongly influenced by Mongoloid gene pool, while those in the eastern part of Indonesia were strongly influenced by Melanesian gene pool (Sofro, 1982).If this the case, higher frequency of ApoE ε4 allele is peculiar for Melanesian populations.The influence of these two gene pools in Indonesian populations has been also confirmed by the distribution of various genetic markers and genetic relationship analysis (Sofro, 1982).
Distribution of ApoE ε3/ε3 genotype and the frequency of ApoE ε3 allele in Mongolian population were reported to be higher and significantly different with Western population and Han nations in China.In addition, ApoE ε4 allele was somewhat high in Oceania (0.221 ± 0.149) than in Africa (0.209 ± 0.090), while populations in India and other Asians showed higher ApoE ε3 allele frequency than in European (Gerdes et al., 1996).
In European, the frequency of ApoE ε4 allele increase from the South to the North along with gradien frequency of cardiovascular disease.The ApoE ε4 allele also involves not only in response to diet but also to other genetic factors (Couderc & Bailleul, 1998).North European (Finland, Germany) tends to have high frequency of ApoE ε4 (14-19%) than south European (France, Italy) (7-12%).Nigeria, Japan dan Finland papulations have relative low frequency of ApoE ε2 (3-4%).Mexican-American and American-Indian also have low ApoE ε2 allele frequency (2-4%).In the tribes from South American-Indian, ApoE ε2 allele was not commonly found (Eichner et al., 2002).ApoE ε2 allele frequency in this study was 0.207 -0.267.The study by Gajra et al.(1994) found ε2 allele frequency in Javanese population was 0.06.ApoE ε2 allele found high in South of Africa (0.19) (Masemola et al., 2007) also in Xinjiang Uygur and Han population in China (0.193) (Yang et al.,2004).The summary of the distribution of various ApoE allele frequency among major human population as depicted in Table 2 provides worldwide polymorphism of this gene locus.
A comprehensive population genetic study three decades ago revealed that a clinal pattern was observed in the distribution of genetic markers such as Glyoxylase (GLO), Glutamate-piruvic transaminase (GPT), Phosphogluconate dehydrogenase (PGD ) , and Haptoglobin (Hp) across the Indonesian archipelago.There was a trend toward increasing GPT 1 , PGD C and and Hp 1 frequencies and decreasing GLO 1 gene frequency towards the east.For systems with more specific alleles such as Phosphoglucomutase (PGM) and Transferrin (Tf), the distribution of PGM 2 and Tf D1 showed a western limit as far as Java and the distribution of TfD Chi and HbE showed an eastern limit as far as Halmahera in the north and Timor in the south (Sofro, 1982).In a more recent study, Indonesian populations can be genetically grouped into three clusters i.e. cluster I consisted of the populations with strong influence from the Mongoloid gene pool, whose mostly living in the western part of the country, cluster II consisted of populations who shared both Mongoloid and the Austro-Melanesian gene pool, whose living in the middle part of the country and cluster III signified by Austro-Melanesian gene pool mostly living in the eastern part of the Indonesian archipelago (Lanni 2002).ApoE gene polymorphism from these three respective populations also provide more or less similar pattern.No difference was observed in the distribution of ApoE ε2 allele in all populations.The frequency of ApoE ε2, ε3, and ε4 alleles were not different either between the western and the middle population, but the frequency of ApoE ε3 and ε4 alleles was different in the eastern populations compared to the western and middle populations.
The present study confirms genetic variability of ApoE genotypes and alleles across world populations.The subjects examined were all healthy individuals however, the impact of this genetic variability is important in relation to diseases.Speculation may be made and is important to clarify the association of ApoE gene with cardiovascular and other disorders in various populations (Abboud et al., 2008;Jasinska-Myga et al., 2007) Studies investigating the relationship of ApoE polymorphism to vascular disease risk in different ethnic groups are also important and may provide new insights into underlying mechanisms contributing to the etiology of vascular disease (Erdembileg & Lars, 2008).This result support that the western and middle Three alleles and five genotypes were distributed in all populations studied with the exception of ApoE ε4/ε4 which was not found in Surabaya.The frequency of three ApoE alleles in the western and middle populations of Indonesia was not significantly different however, significant difference was observed in the frequency of ApoE ε3 and ε4 alleles from the eastern compared to those in the western and middle populations of Indonesia.

Figure 2 .
Figure 2. Distribution of ApoE alleles in Surabaya, Palu and Alor representing the western, middle and eastern populations of Indonesia ( )

Table 2 .
Distribution of various APOE alleles (frequency range) among major human populations Indonesian has different gene pool with their eastern counterpart, so the characteristics of disease or management of patients might be somewhat different.This study can be extended to other populations in Indonesia with unique gene pool suffering from any disorders possibly influenced by ApoE genotype.It is concluded that genetic variation of ApoE gene was found in three populations i.e.Surabaya, Palu and Alor, each representing populations with strong influence of Mongoloid gene pool, populations sharing Mongoloid and Austro-Melanesian gene pool and populations with strong influence of Austro-Melanesian gene pool respectively.