The R219K polymorphism of the ABCA1 gene was recently identified by Clee et al. [16]. The PCR primer was as below: 5′- GTA TTT TTG CAA GGC TAC CAG TTA CAT TTG ACA A-3′ and 5′-GAT TGG CTT CAG GAT GTC CAT GTT GGA A-3′. PCR was conducted with denaturation at 95°C for 5 min, anneal- ing at 60°C for 1 min and primer extension at 72°C for 1.5 min for a total of 30 cycles. After digestion with EcoNI, the resulting frag- ment is 177-bp for the R allele and 107and 100 bp fragments for the K allele.

DNA mutation and polymorphism

cell by phenol extraction. CETP mutation in intron 14 (Int14A) was determined by polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) method as described by Inazu et al. [4]. Briefly, genomic DNA was amplified by PCR in Program TEMP control system PC-800 (ASTEC, Fukuoka) using designed primer as below: 5′-AGC ATC TGC CTT GTG GGT-3′ and 5′AAG CTC TGT CAG CCT CGG CAC CCA GTT TCC CCT CCA GCC CAC ACA TA-3′. The product was digested with 5 U NdeI (Gibco BRL, Gainthersburg, Md., USA) at 37°C with 10 µl reaction mixture. The digested sample was electrophoresed on 3.0% Nusive 3:1 agarose gel, stained with ethidium bromide, and visualized with an UV transilluminator. If the G→A mutated allele contained two fragments of 138 and 47 bp were observed af- ter NdeI digestion.

Statistical analyses

Values were expressed as mean ±SD. Statistical analysis was car- ried out using StatView version 4.5 (Abacus Concepts, Berkley, Calif., USA). Because distribution of triglyceride and lipopro- tein(a) were markedly skewed, these variables were logarithmically transformed for further statistical analyses. Comparisons between centenarians and controls were calculated by Student’s unpaired t test. Analysis of variance (ANOVA) was performed to compare the three groups. The χ2 test was performed to detect differences in al- lele frequencies. Multiple regression analysis was calculated with HDL-C levels as dependent, and genetic markers and modifiable factors as independent variables to determine the quantitative ef- fects of covariates. Because the nonfasting blood sample was uti- lized triglyceride was not included for multiple regression analysis. Differences were considered significant at P<0.05.

CETP mutation in exon 15 (442D:G) was determined by PCR- RFLP as described by Takahashi et al. [5]. The primer was: 5′- CTC AAG CTT TGG GGC AGA AGG GAA T-3′ and 5′-CAG GAA TTC TGT CTG GGC CTT CTC TC-3′, generating a 392-bp fragment. After digestion by Taq1 the wild-type allele shows 240 and 70 bp fragments while the muted allele shows additional 76 bp.

Results

CETP Taq1B polymorphism was determined as described by Fumeron et al. [27] with minor modification. PCR was conducted using originally designed primers with denaturation at 95°C for 5 min, annealing at 60°C for 1 min and primer extension at 72°C for 1.5 min for a total of 30 cycles. The PCR product was digested with 5 U Taq1 at 65°C for 30 min, then electrophoresis on 2% agarose gel for Taq1B genotyping.

Candidate gene polymorphisms in the subjects

The distribution of genotype and allele frequencies for RCT gene polymorphisms in centenarians and controls is presented in Table 2 and Fig. 1. In both groups, the poly- morphisms of PvuII and HindIII of the LPL gene were in strong linkage disequilibrium by using maximum likeli- χ hood estimation ( 2=80.2, P<0.001 for centenarians, χ 2=64.1, P<0.001 for controls). Among seven polymor- phisms in four genes no significant gender differences were observed in genotype distribution and allele fre- quencies either in centenarians or in controls (data are not shown).

PvuII and HindIII polymorphisms of the LPL gene were deter- mined as described by Anderson et al. [28]. PvuII restriction site in intron 6 was amplified by the following primer: 5′-ATG GCA CCC ATG TGT AAG GTC-3′ and 5′-GTG AAC TTC TGA TAA CAA TCT C-3′, and the primer for HindIII polymorphisms in in- tron 8 was: 5′-TGA AGC TCA AAT GGA AGA GT-3′ and 5′- TCA AAG CAA ATG ACT AAA-3′. PCR products were digested overnight at 37°C with PvuII or HindIII. After digestion with PvuII the presence of restriction site resulted in 330- and 110-bp fragments, represented as P(+). The HindIII restriction site pro- vides a 600-bp fragment, indicated as H(+).

Table

Genotype distributions of genes involved in reverse cholesterol transport between centenarians and controls; com- parison of genotype distribu- tions between centenarians and controls were calculated by χ

test

Canditade gene n Genotype (%) P CEPT intron 14 Homozygote Heterozygote Wild type 0.429 Centenarians 256 0 (0.0) 2 (0.8) 254 (99.2) Controls 190 0 (0.0) 3 (1.5) 187 (98.5) CEPT exon 15 Homozygote Heterozygote Wild type 0.689 Centenarians 256 0 (0.0) 16 (6.3) 240 (93.7) Controls 190 0 (0.0) 13 (6.8) 167 (93.2) Total CEPT mutation Homozygote Heterozygote Wild type 0.606 Centenarians 256 0 (0.0) 18 (7.0) 238 (93.0) Controls 190 0 (0.0) 16 (8.4) 174 (91.6) CEPT Taq1B B1B1 B1B2 B2B2 0.286 Centenarians 256 73 (28.5) 132 (51.6) 51 (19.9) Controls 190 65 (34.2) 84 (44.2) 41 (21.5) LPL PvuII P (−/−) P (−/+) P(+/+) 0.869 Centenarians 256 20 (7.8) 101 (39.4) 135 (52.7) Controls 190 14 (7.3) 71 (37.4) 105 (55.6) LPL HindIII H (−/−) H (−/+) H(+/+) 0.562 Centenarians 256 14 (5.5) 75 (29.3) 167 (65.2) Controls 190 11 (5.8) 60 (31.6) 119 (62.6) HLC-514T CC CT TT 0.201 Centenarians 256 63 (24.6) 132 (51.6) 61 (23.8) Controls 190 60 (31.6) 94 (49.5) 36 (18.9) ABCA1 R219K RR RK KK 0.887 Centenarians 256 63 (25.2) 132 (52.8) 55 (22.0) Controls 190 48 (25.3) 96 (50.7) 45 (23.8)