Figure 1. A, Region in the apoC-III promoter containing a functional insulin response element. Dotted line represents functional insulin- element (C3IRE) from positions -490 to -449 (11,12). Solid line indicates the position of the sequence similar to the rat phospho- carboxykinase (PEPCK) insulin/phorbol ester response element (9,10), mapped to positions -463 to -454. Asterisks show two proxi- polymorphic sites located inside the C3IRE. T/C polymorphism at position -455 is located inside the rat PEPCK insulin/phorbol ester re- element-like region. Sequence of the entire area can be found in the GenBank: access number X13367. B, Amplification of the mismatch chain reaction (PCR) product with the formation of the MboI recognition site. Genomic DNA (i) is used for PCR, which generates -457G rather than -457C nucleotide via site-directed mutagenesis (ii). PCR product was digested with MboI restriction endonuclease. Thymine the -455 position (T allele) forms the authentic recognition site for MboI (fl GATC) (iii); in the case of cytosine at position -455 (C allele), the restriction site is absent (iv). C, An example of ethidium bromide-stained polyacrylamide gel from which allele determinations were made. 3, 4, and 5 are samples after digestion with MboI (lane 3, heterozygous -455T/C; lane 4, homozygous -455C; lane 5, homozygous -455T); 2, PCR product; lanes 1 and 6, the marker pBR322/AluI. bp 5 base pair.

for each group). Interestingly, the observed distribution of alleles in young (6–17 years) St. Petersburg citizens closely matched those in the group of Italian schoolchildren (11–13 years; 0.596/0.404) (7) and young (9–17 years) Canadian Indians (0.556/0.444) (19), suggesting that the initial allele distribution is similar in different populations (Table 1).

0.619, respectively, suggesting that a gradual loss of the T-allele proportion, with a relative accumulation of the , , C-allele proportion occurs with age (p .05 and p .02, , 1 respectively, and p .002 for pooled groups 2 3 vs group 4). The reliability of subject group size was confirmed with a two-proportions power analysis. A greater incidence of the C allele occurs around the age of 80 to 89 years and con- tinues with further aging (Figure 2). These results indicate that the variant (-455C) allele of the apoC-III T-455C poly- morphism is associated with longevity, and the factors in- volved relate to the elderly age.

The frequencies of T and C alleles in the total pool of 137 elderly individuals were 0.453 and 0.547, respectively (Ta- ble 2), indicating that, in an elderly Russian population, al- lele C prevails over T when compared with the distribution , of alleles at a young age (p .005). In group 1, allele C was found at a frequency of 0.465, which is quite close to its ini- tial presence in population (0.450). However, in groups 2 and 3, allele C was found with frequencies of 0.558 and

To investigate the possible pathways of the -455C al- lele’s relative accumulation, the statistical analysis of the allele distribution was performed on groups of patients

Table 2. ApoC-III -455 Polymorphism T/C Allele Distribution

Genotype (n) Age Range Total Mean Age C-Allele (y) (n) (y) TT TC CC Frequency x2 p Values Group 1 70–79 43 74.4 9 28 6 0.465 2.85 NS Group 2 80–89 52 84.4 6 34 12 0.558 6.68 ,.05 Group 3 90–106 42 94.5 3 26 13 0.619 9.10 ,.02 Total groups 11213 70–106 137 84.4 18 88 31 0.547 10.73 ,.005 Group 4 6–17 110 11.0 33 55 22 0.450 — —

Notes: NS not significant. p values were tested using test (value shown) for groups 1–3 vs group 4 (control).

with AH and IHD. No statistically significant difference could be demonstrated between the group of AH patients and healthy controls of the same age. Although not statisti- cally significant, a higher C-allele proportion was observed among AH patients in group 2 over their healthy counter- parts compared with the increase of the C-allele proportion among healthy subjects over AH patients in group 3 (Table 3). Analysis of the distribution of the T and C alleles in groups of subjects with or without IHD also failed to asso- ciate the variant allele with disease. In contrast to the allele distribution in AH, the C-allele proportion was increased in subjects without IHD in group 2 and in IHD patients in group 3 (Table 4). The distribution of alleles also showed no statistically significant difference when comparing a group of healthy individuals with the groups of AH pa- 1 tients, IHD patients, and AH IHD patients of the same . age (p .05 for each group, data not shown). A two-pro- portions power analysis comparing the groups of individu- als with or without AH or IHD in the same age groups demonstrated that the size of each group must be 2000 to 4800 subjects to make indicated differences in -455C-allele proportions statistically significant.

This finding does not support earlier reports of the variant promoter being involved in the development of hypertri- glyceridemia (19,21) and familial dysbetalipoproteinemia (21). Recently, it has been shown that at least in some sub- jects, the -455C allele can lead to a significant extension of the apoC-III abundance in vivo (27). The proposed mecha- nism involves partial inhibition of IRE response to insulin in subjects carrying the -455C allele of the apoC-III pro- moter (12), resulting in an increase of the plasma choles- terol and triglyceride levels and leading to atherosclerosis and further development of IHD. In contrast, a protective role of the haplotype containing the C allele of the -455 po- sition, unlike the T allele, has been reported with respect to the development of hypertriglyceridemia (18). Taken to- gether, it is unclear if any of the alleles is principally in- volved in the development of IHD or reflects solely ethnic or population differences.