E and longevity among Han Chinese population

Zhang Jian-Gang a,d, Ma Yong-Xing b, Wang Chuan-Fu b, Lu Pei-Fang b, Zhen Song-Bai b, Gu Nui-Fan c, Feng Guo-Yin c,

a Section of Human Genome Project, Shanghai Research Center of Life Sciences, Chinese Academy of Sciences, 320Yue Yang Road, Shanghai 200031, People’s Republic of China b Department of Aging and Antiaging, Shanghai Institute of Gerontology and Geriatric, Huadong Hospital, 221West Yan An Road, Shanghai 200040, People’s Republic of China c Shanghai Mental Center, 600South Wan Ping Road, Shanghai 200030, People’s Republic of d Shanghai Brain Research Institute, Chinese Academy of Sciences, 320Yue Yang Road,

Received 25 February 1998; received in revised form 15 May 1998; accepted 21 May

Abstract

The apolipoprotein E gene (APOE), which locates on chromosome 19, has three common APOE alleles encoding APOE*2, APOE*3, APOE*4. It has been considered to be closely associated with human longevity among Western population. However, the allele frequency varies among racial and ethnic groups. We studied the relationship between APOE and longevity among 1562 Han Chinese (246 female and 1316 male) whose ages ranged from 20–108 years old in Shanghai metropolitan area and Lian Yungang, Jiangsu province. % APOE was amplified by one-stage PCR using the forward primer: 5 -GGC ACG GCT GTC % % % CAA GGA GCT-3 and reverse primer: 5 -GAT GGC GCT GAG GCC GCG CT-3 . The PCR product was digested directly with 5 U of CfoI and separated by a 20% polyacrylamide (acrylamide:bisacrylamide(cid:30)29:1) nondenaturing gel. The allele frequency of APOE*4in the

* Corresponding author. Tel.: (cid:27)86 21 64748700; fax: (cid:27)86 21 64333084; e-mail: du@iris.shlc.ac.cn

0047-6374:98:$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0047-6374(98)00067-0

] very old age group ( 85 years) was significantly different from that in the youth (20–39 years), middle (40–59 years) and old (60–84 years) age group (2.5% vs 8.4, 7.9 or 7.6%; B P 0.05), respectively. © 1998 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Apolipoprotein E; Longevity; Han Chinese; PCR; RFLP

1. Introduction

Many factors relating to longevity have been studied in the past and it has been argued that genes play a key role in the aging process. At present, two main developments in support of the hypothesis (Jazwinski, 1996) can be found. The first one was from efforts to test evolutionary theories of aging and the second one arose from studies of genes modulating life span. Smith and Walford (1977) and Walford and Bergmann (1979) found the evidence that the main histocompatibility genes H-2 in mice had played an important role in life-span by inducing an appropriate immune response or by influencing DNA repair capacity. Although there is evidence in support of the contention that a genetic component contributes to the determination of human longevity, the only association reported so far is the HLA locus, which was reported by three independent investigations (Proust et al., 1982; Takata et al., 1987; Yong-Xing et al., 1997). In order to search for genetic influences on human longevity in Han Chinese, we studied a candidate gene, Apolipoprotein E (apoE) which had been well characterized in the Western population.

2.1. Subjects

Blood samples were collected from 1562 health people (246 female and 1316 male, Table 1) with ages ranging from 20 to 108 years old. A total of 1506 unrelated samples were gathered from Shanghai area and 56 people were selected : from Lian Yungang city, Jiangsu province, 600 km away from Shanghai. The selected healthy subjects must meet the following criteria: good physical and mental health confirmed by clinical, radiological, biological examinations and a brief cognitive test (Ming-Yuan et al., 1990).

Regarding their ages, all subjects were divided into the following subgroups: youth (20–39 years old), middle (40–59 years old), old (60–84 years old) and very ] old ( 85 years old) age group, respectively.

The acid citrate dextrose (ACD) was used as anticoagulant (Sambrook et al.,

PCR buffer contains: 50 mmol:l KCl, 10 mmol:l Tris–HCl (pH 8.3), 2.5 mmol:l MgCl , 0.1 mg:ml gelatin, 0.45% NP40, 0.45% Tween 20, which was 2 m autoclaved and stored at (cid:28)20°C. Just before use, 0.6 l of 10 mg:ml Proteinase m K (in H O) was added into 100 l of the buffer solution; Lysis buffer contains: 2 0.32 mol:l sucrose, 10 mmol:l Tris–HCl (pH 7.5), 5 mmol:l MgCl , 1% Triton 2 X-100; 10(cid:29) PCR reaction buffer contains: 0.67mol:l Tris–HCl (pH 8.8), 0.166 b mol:l (NH ) SO , 0.067 mol:l MgCl , 0.72% (v:v) -mercaptoethanol (Sigma). 4 2 4 2

2.3.1. Isolation of genomic DNA from peripheral blood samples

The protocol for isolation of genomic DNA from peripheral blood samples in this study was the same as that described by Russell (1989), except for some m minor modifications. Briefly, 100 l anticoagulent peripheral blood was mixed with 0.8 ml lysis buffer in a 1.5-ml Eppendorf microcentrifuge tube and cen- trifuged at 13000(cid:29)g for 0.5 min; the supernatant was removed and the pellet was resuspended using vortex mixer after adding another 0.8 ml of lysis buffer to centrifuge at 13000(cid:29)g for 0.5 min; After removing the supernatant, the pellet m was resuspended in 50 l of PCR buffer and incubated at 60°C for 1.5 h and at 95°C for another 15 min to inactivate the proteinase K. The solution was ready for PCR amplification directly.