The study was approved by the Clinical Research Ethics Committee of the Chinese University of Hong Kong. Informed







4000 men and women aged 65 and over living in the community were invited to attend a health check carried out in the School of Public Health of the Chinese University of Hong Kong, by placing recruitment notices in community centers for the elderly and housing estates. Several talks were also given at these centers explaining the purpose, procedures and investigations to be carried out. Subjects were volunteers, and the aim was to recruit a stratified sample so that approximately one third were in each of these age groups: 65-69, 70-74, 75+. Subjects who were unable to walk without assistance, or had bilateral hip replacement, were excluded. The study sample differed from the general Hong Kong population of the same age group, in that using general population data from the government census for comparison, they were more educated and was more likely to be married. The study was approved by the Clinical Research Ethics Committee of the Chinese University of Hong Kong. Informed written consent was obtained. The study commenced in 2001, and the subjects continued to be followed up. The results after five years of follow-up are reported in this study. During this period, 614 subjects did not return for follow up while 233 subjects died (Fig 1). There was no difference in frequencies of the three ACE gene polymorphisms between participants who returned

At baseline, a 10ml venous specimen of blood was collected from 1841 men and 1957 women and placed into EDTA tubes. Blood samples were stored at -70C until DNA extraction (within one year after collection). Genomic DNA was

Genotyping of Alu insertion/deletion (I/D) polymorphism in the ACE gene was performed by polymerase chain reaction with forward and reverse primers (5'- AGAGAGACTCAAGCACGCCC-3' and 5'- ACCCAAGTGCCAGTGATGTT-3'). Reactions were set up in a volume of 25 ml containing 0.2 mM of each primer, 1X PCR buffer, 2 mM MgCl2, 0.2mM of each dNTP, 0.6 U Taq polymerase (Fermentas Life Sciences). About 50 ng of DNA was used in each sample. The initial denaturation was performed at 95oC for 5 min, and it was followed by 35 cycles of 94oC for 30 sec, 64oC for 30 sec and 72oC for 30 sec, and a final extension at 72oC for 10 min. This method yielded amplification products of 439 bp for the D allele and 727 bp for the I allele. The products were visualized in 2% agarose gels



Chi-square test was used to test for statistical difference between categorical variables, and students’ t-test and analysis variance for continuous variables, between the three genotypes groups. Analysis of co-variance was used to adjust for confounding variables, adjusting for multiple hypothesis testing. Incidence of disease was calculated as the number without disease at baseline developing the disease at follow-up. Cox regression analysis was used to examine mortality rates between the three genotype groups. Proportional hazard model

At baseline, a questionnaire was administered to participants, which included demographics, history of chronic disease, use of ACE inhibitors, smoking habit, physical activity level, and dietary intake including alcohol. Physical activity level was assessed using the Physical Activity Scale of the Elderly (PASE) (22). Dietary intake was assessed using a food frequency questionnaire and administered by an interviewer (23). Body weight was measured with subjects wearing a light gown, by the Physical Balance Beam Scale (Healthometer, Bridgeview, Illinois, USA). Height was measured by the Holtain Harpenden stadiometer (Holtain Ltd, Crosswell, UK). Supine blood pressure was measured in the right arm and both ankles using a standard mercury sphygmomanometer and a hand held Doppler with an 8-MHz Doppler probe. (Pocket Doppler Model 841-A, Parks Medical Electronics Inc, Aloha, Or, USA). The average reading of two measurements at each site was used. The ABI was calculated by dividing the blood pressure at the posterior tibial site by the brachial artery site for each side, and the lowest of the two values was used. An ABI value of <0.9 has been used as a cut off value to indicate the presence of peripheral vascular disease (24), and also as an indicator of atherosclerosis (25). The procedure was carried out for 1999 men and 1999 women and the baseline results are reported elsewhere (26). During the fifth year of follow-up, participants returned and a medical history was obtained and ABI measurement repeated. The Death Registry was searched



ACE gene polymorphism results were available in 1841 men and 1957 women from whom blood samples for DNA extraction had been obtained. For men, the frequencies of I/I, I/D and D/D genotype were 815 / 1841 (44.3%), 842 / 1841 (45.7%), and 184 / 1841 (9.9%) respectively. Genotype distributions in both sexes followed Hardy Weinberg equilibrium. For women, the frequencies were 46%, 43% and 10.9% respectively. The percentage of subjects with ABI <0.9 did not differ significantly between the three polymorphism

Mean ABI, percentage of subjects with ABI <0.9, and potential confounders in the relationship between genetic factors and ABI are listed by genotype groups in Table 1a & b. In women only, participants with the D/D genotype had the greatest reduction in mean ABI value. This group also had the highest mean age. After adjusting for all potential confounding factors, the D/D group had the greatest progression of atherosclerosis (using reduction in ABI as an indicator), followed by I/I, and I/D group (Table 2). No significant