of other Slavic (mainly Ukrainian) and Jewish names, which represents a typical ethnic constitution in St. Petersburg.

atorily associated with ischemic heart disease (IHD) (21,23, 25). This increases a potential role of other contributing polymorphisms. Multiple haplotypes, including the poly- morphic sites of the cluster, have been reported to influence the plasma levels of lipids and triglycerides (6,12,22). How- ever, all studies that included IRE T-455C polymorphism analysis, had subjects with different forms of hyperlipi- demia and hypertriglyceridemia (7,18–21). No studies have analyzed this polymorphism in an aging population.

Whole venous blood was collected in ethylenediamine- tetraacetic acid, sample tubes, and genomic DNA was ex- tracted from peripheral blood leukocytes by a phenol-chlo- roform method (26) with minor modifications.

Two oligonucleotide primers were designed for amplifi- cation of the DNA region containing the T-455C polymor- 9 phism: reverse primer 5 -ATC TCA GCC TTT CAC ACT 9 9 GGA ATT T-3 (-351– -327) and forward primer 5 -GTC 9 TTC TGT GCC TTT ACT CCA AAG A-3 (-480– -456). 9 The forward primer contained, as the 3 -penultimate nucle- otide, a G rather than the native C residue. Thus, elongation fi of this primer led to site-directed mutagenesis (C G in the coding strand at position -457) and formation of a MboI site fi for the -455T (CATC GATC) allele, but not for the -455C fi (CACC GACC) allele (Figure 1).

The main purpose of our study was to determine whether the apoC-III T-455C polymorphism is important for sur- vival and which allele is associated with longevity. To study this question, we examined the distribution of the T-455C apoC-III polymorphism in a population-based sample of 137 elderly individuals (70–106 years old) with subgroups se- lected by age. A group of 110 healthy schoolchildren served as a control population to determine the initial distribution of the alleles.

m Amplification of 1 g of genomic DNA by polymerase chain reaction (PCR) was performed using 25 pmol of for- ward primer and 50 pmol of reverse primer with 0.8 unit of Taq polymerase (Institute of Genetics, Moscow, Russia) in m a total reaction volume of 30 l (20 mmol/l Tris HCl, pH 8.8; 10 mmol/l (NH ) SO ; 2.3 mmol/l MgCl ; and 50 4 2 4 2 m mol/l each deoxynucleoside triphosphate). Cycling condi- 8 tions were as follows: the initial cycle of 1 minute at 95 C, 1 8 8 minute at 52 C, 1 minute at 72 C; 29 cycles for 1 minute at 938 C, 1 minute at 568 C, 1 minute at 728 C; and a final exten- sion at 728 C for 5 minutes. A hot start was used to avoid nonspecific amplification. Nine m l of the PCR reaction products were diluted to 10 m l by the recommended restric- tion buffer containing 0.5 unit of MboI (MBI, Fermentas, Lithuania) and digested at 378 C overnight. Restriction prod- ucts were resolved on 10% polyacrylamide gels. Possible patterns were 154 bp (C in -455 position) and 131 bp 1 23 bp fragments (T in the -455 position; Figure 1C). P values were determined using the x2 test and two-tailed Fisher ex- act test to compare frequencies in different groups.

The T-455C apoC-III polymorphism, located within the functional IRE, demonstrates a greater frequency of the C-allele proportion with aging. However, we failed to find any statistically significant differences in the distribution of T and C alleles between age-dependent groups of patients with IHD and arterial hypertension (AH) and healthy con- trol groups of the same age.

M ATERIALS AND M ETHODS

A group of 147 elderly individuals (70–106 years old; mean age, 84.6 years) was selected from patients of the City Geriatric Center and the St. Petersburg Institute of Bioregu- lation and Gerontology in St. Petersburg, Russia. In this group, ten pairs of elderly siblings were present (20 subjects from 10 different families; mean age, 91.3 years); only the oldest individual from each family was analyzed. Together with individuals without elderly siblings, the study com- prised 137 subjects with a mean age of 84.4 years. Subjects were divided into several age-differentiated subgroups: group 1 (43 subjects, 70–79 years old; mean age, 74.4 years), group 2 (52 subjects, 80–89 years old; mean age, 84.4 years), and group 3 (42 subjects, 90–106 years old; mean age, 94.5 years). Information about AH and IHD was obtained based on long-standing clinical observations. The control group included 110 schoolchildren aged 6 to 17 years old (group 4; mean age, 11.0 years) from three sec- ondary schools in St. Petersburg. The ethnic composition in both the elderly and control groups was controlled via the analysis of last names and was found to be identical, with a major preponderance of Russian names and a minor presence

R D ESULTS AND ISCUSSION

We analyzed DNA samples from the following groups of St. Petersburg citizens: group 1 (43 subjects, 70–79 years old), group 2 (52 subjects, 80–89 years old), group 3 (42 subjects, 90–106 years old), and group 4 (110 subjects, 6–17 years old). The frequencies of T and C alleles in the control group (group 4) were 0.550 and 0.450, respectively. This distribution is comparable with 0.700/0.300 in a healthy adult white American population (18), and 0.690/0.310 (21) . or 0.655/0.345 (20) in healthy Dutch populations (p .05

AT cAGCCAGIGATGCCCGGTCT TCTGTGCCTITACTCCAAACATCCCCCAGCCCAA.. ~449B c#@ ... CCAAACATCCCC... Genomic DNACc*Gi) k .. .CCAAAGATCCCC... 1 PCR Product-480 -327“455Mbol |«iy .. -CCAAALGATCCCC...23 131Digested-455 PCR Products. ...CCAAAGACCCCC...ag —S—_154<— 154 bp+131 bp