Analysis of apolipoprotein E genetic polymorphism in a large ethnic Hakka population in southern China

Abstract There is currently no data about the genetic variations of APOE in Hakka population in China. The aim of this study was to analyze the allelic and genotypic frequencies of APOE gene polymorphisms in a large ethnic Hakka population in southern China. The APOE genes of 6,907 subjects were genotyped by the gene chip platform. The allele and genotype frequencies were analyzed. Results showed that the ∊3 allele had the greatest frequency (0.804) followed by ∊2 (0.102), and ∊4 (0.094), while genotype ∊3/∊3 accounted for 65.43% followed by ∊2/∊3 (15.85%), ∊3/∊4 (14.13%), ∊2/∊4 (3.01%), ∊4/∊4 (0.84%), and ∊2/∊2 (0.74%) in all subjects. The frequencies of the ∊4 allele in Chinese populations were lower than Mongolian and Javanese, while the frequencies of the ∊2 allele were higher and ∊4 allele lower than Japanese, Koreans, and Iranian compared with the geographically neighboring countries. The frequencies of ∊2 and ∊4 alleles in Hakka population were similar to the Vietnamese, Chinese-Shanghai, Chinese-Kunming Han and Chinese-Northeast, and French. The frequency of ∊2 in Hakka population was higher than Chinese-Dehong Dai and Chinese-Jinangsu Han. The low frequency of the APOE ∊4 allele may suggest a low genetic risk of Hakka population for cardiovascular disease, Alzheimer’s disease, and other diseases.


Introduction
Apolipoprotein E (ApoE) is a multifunctional protein that plays an important role in lipoprotein metabolism, and is involved in the metabolism of very low density lipoproteins (VLDL) and chylomicrons (Blum, 2016). There are three major isoforms of human ApoE including E2 (OMIM 107741.0001), E3 (OMIM 107741.0015), and E4 (OMIM 107741.0016), as identified by isoelectric focusing. The gene coding for ApoE is APOE (OMIM 107741), which is located on chromosome 19 in band 19q13.32 (Mahley, 1988;Siest et al., 1995). The polymorphisms in the fourth exon of APOE gene determine three common alleles (e2, e3 and e4) coding for three major isoforms of ApoE (Martin et al., 2000;Kantarci et al., 2004;Kumar et al., 2017).

DNA extraction
Blood samples were stored in 2-mL vacuum tubes containing ethylenediaminetetraacetic acid (EDTA) from each participant. Genomic DNA was extracted from the samples using QIAamp DNA Blood Mini Kit (Qiagen, Germany) according to the manufacturer's instructions. DNA concentration and purity were quantified using Nanodrop 2000 TM Spectrophotometer (ThermoFisher Scientific, Waltham, MA), and only good quality DNA (A260/280 ratio > 1.7) was stored at -80°C up to the day of analysis.

Polymerase chain reaction and genotyping
The single nucleotide polymorphisms of APOE gene rs429358 and rs7412 were genotyped using a commercially available kit (Sinochips Bioscience Co., Ltd, Zhuhai, Guangdong, China). PCR assays was performed according to the following protocol: 50°C for 2 min, pre-denaturation at 95°C for 15 min, followed by 45 cycles at 94°C for 30 s and 65°C for 45 s. The amplified products were revealed using an APOE Gene typing Detection kit (gene chip assay) (Sinochips Bioscience Co., Ltd, Zhuhai, China).
SPSS statistical software version 19.0 was used for data analysis. The data are reported as the means ± SD. Chi-square and Fisher's exact tests were used to compare the allele and genotype frequencies. Descriptive analysis was used to compare allele frequencies between the Hakka population and published data of other ethnic groups. A value of p < 0.05 was considered as statistically significant.

Discussion
ApoE is one of the important apolipoproteins in plasma, which is mainly synthesized, secreted, and metabolized in the liver (Schneider et al., 1981;Rall Jr et al., 1982b). It is involved in the transport, storage, and metabolism of lipids, and has the effects of repairing tissues, inhibiting platelet aggregation, and regulating immunity (van den Elzen et al., 2005). Studies have found that APOE gene polymorphisms are closely associated with coronary heart disease, hyperlipidemia, cerebral infarction, Alzheimer's disease, multiple sclerosis, chronic hepatitis, and other diseases (Ghiselli et al., 1981;Corder et al., 1993;Faivre et al., 2005;Price et al., 2006;Rovin et al., 2007;Kathiresan et al., 2008). ApoE4 is associated with decreased longevity, increased plasma total and LDL cholesterol, and increased prevalence of cardiovascular disease and Alzheimer's disease. Different populations have different frequencies of genetic polymorphisms of APOE (Gerdes et al., 1996).
We compared the allele frequencies estimated here for APOE e2, e3, and e4 allele with respect to previously published reports in other ethnic populations ( Table 2). Comparison of our results with the geographically neighboring countries showed that the frequencies of e4 allele in Chinese populations were lower than in Javanese (Svobodova et al., 2007a,b) populations, while the frequencies of the e2 allele were higher and of the e4 allele lower than in Japanese (Hallman et al., 1991;Gerdes et al., 1992) and Koreans (Hong et al., 1997). In addition, the analysis showed that the frequencies of e2 and e4 allele in Hakka population were similar to the Vietnamese (Nghiem et al., 2004), Chinese-Shanghai (Yang et al., 2003), Chinese-Kunming Han (Tang et al., 2005), Chinese-Northeast (Zhou et al., 2005), and French (Boerwinkle et al., 1986;Gueguen et al., 1989;Bailleul et al., 1993).
Comparing our results with other Chinese populations, the frequencies of the e2 and e4 alleles in the Hakka population were highly similar to the Chinese-Shanghai, Chinese-Kunming Han, and Chinese-Northeast, while the frequency of e2 in the Hakka population was higher than Chinese-Dehong Dai (Tang et al., 2005) and Chinese-Jiangsu Han (Liang et al., 2009) (Figure 2). This suggests that the risk of some diseases in the Hakka population of Southern China may be different from those of other populations. Since e4 polymorphism is associated with increased risk of cardiovascular disease, Alzheimer's disease, and other diseases, our findings suggest a low genetic risk in the Hakka population for these diseases.
In some reports, the subjects were relatively few and the results did not represent the actual gene frequencies of that region and population. Here, the Apolipoprotein E genetic polymorphism was analyzed in a large ethnic Hakka population in southern China, and is the first performed on a large sample of the population of this area. Our sample size is one of the largest of all studies, and thus should more accurately assess the APOE gene allele and genotype frequencies of the Hakka population in southern China. Our next step is to increase the sample size of the study. A number of investigations have demonstrated that carriers of e4 allele are characterized by a lower life expectancy (Hyman et al., 1996;Gerdes et al., 2015). Thus, we are going to investigate the APOE gene polymorphisms in people living in Jiaoling, which is considered the hometown of longevity in China. 744 Apolipoprotein E polymorphism in southern China  Smit et al., 1988;Knjiff et al., 1993 Finnish 2245 0.044 0.748 0.208 Lehtimäki et al., 1990;Salo et al., 1993;Hallman et al., 1991 Germans 1211 Valveny et al., 2010;Gerdes et al., 1992;Lucotte et al., 1997;Muros and Rodríguez-Ferrer, 1996 French 1228 0.108 0.771 0.121 Bailleul et al., 1993;Gueguen et al., 1989;Boerwinkle et al., 1986 Belgians 189 0.069 0.762 0.169 Engelborghs et al., 2003 UK 734 0.089 0.767 0.144 Corbo et al., 1995;Lucotte et al., 1997 Greeks 551 0.054 0.878 0.068 Marios et al., 1995;Sklavounou et al., 2010 Danish 466

Conclusions
The frequencies of the e4 allele in Chinese populations were lower than in Mongolians and Javanese, while the frequencies of the e2 allele were higher and of the e4 allele lower than in Japanese and Koreans, which are geographically neighboring countries. The frequencies of the e2 and e4 alleles in the Hakka population were similar to the Vietnamese, Chinese-Shanghai, Chinese-Kunming Han and Chinese-Northeast, and French, while the frequency of e2 in the Hakka population was higher than Chinese-Dehong Dai and Chinese-Jinangsu Han. Our findings suggest a low genetic risk in the Hakka population for some diseases. the Center for Precision Medicine, Meizhou People's Hospital (Huangtang Hospital), Meizhou Hospital Affiliated to Sun Yat-sen University for their helpful comments on the manuscript. This study was financially supported by National Major Scientific and Technological Special Project for "Significant New Drugs Development" during the Thirteenth Five