2.2. Methods

Trained and qualified personnel conducted the households and home visits.Informationondemographicvariables,healthstatus,healthbehav- ior and physical activity were obtained byusinga standardized question- naire. Participants were asked to fast overnight. The peripheral blood, fasting serum and serum samples were separated for genome DNA extraction and laboratory testing of lipid and glucose parameters accord- ing to the previously described methods (Sun et al., 2006). The diagnosis for dyslipidemia is according to the National Cholesterol Education Program/Adult Treatment Panel III criteria (NCEP ATP III) (2002).

FPG: fasting plasma glucose; TC: total cholesterol; TG: triglycerides; HDL-c: high-density lipoprotein cholesterol; LDL-c: low-density lipoprotein cholesterol; BMI: body mass index; HR: heart rate; SBP: systolic blood pressure; DBP: diastolic blood pressure; All the data are listed as means ± standard deviations except number.

Table 2

Genotype and allele frequencies for CETP and APOE polymorphisms.

Gene Group n Genotype (%) P P HWE additive CETP Oldest-old VV IV II Overall 372 74 (19.9) 170 (45.7) 128 (34.4) 0.200 hTG 105 32 (30.5) 47 (44.8) 26 (24.8) 0.297 Control 267 42 (15.7) 123 (46.1) 102 (38.2) 0.628 0.002 hTG hTC 56 10 (17.9) 24 (42.9) 22 (39.3) 0.447 Control 316 64 (20.3) 146 (46.2) 106 (33.5) 0.292 0.701 hTC Mixed-dys 32 8 (25.0) 14 (43.8) 10 (31.3) 0.491 Control 243 40 (16.5) 113 (46.5) 90 (37.0) 0.653 0.475 Mixed Controls 340 70 (20.6) 170 (50.0) 100 (29.4) 0.885 0.347 APOE Oldest-old ε4-carrier Non-ε4-carrier Overall 372 52 (14.0) 320 (86.0) 0.247 hTG 105 20 (19.0) 85 (81.0) NA Control 267 32 (12.0) 235 (88.0) NA 0.077 hTG hTC 56 16 (28.6) 40 (71.4) NA Control 316 36 (11.4) 280 (88.6) NA 0.001 hTC Mixed-dys 32 12 (37.5) 20 (62.5) NA Control 243 28 (11.5) 215 (88.5) NA b0.001 Mixed Controls 340 80 (23.5) 260 (76.5) 0.072 0.001

hTG:hypertriglyceridemia;hTC:high-cholesterol;Mixed-dys:mixeddyslipidemia;AccordingtothecriteriaofNationalCholesterolEducationProgram/AdultTreatmentPanelIIIcriteria (NCEP ATP III).

hTG:hypertriglyceridemia;hTC:high-cholesterol;Mixed-dys:mixeddyslipidemia;AccordingtothecriteriaofNationalCholesterolEducationProgram/AdultTreatmentPanelIIIcriteria

frequent in oldest-old group (23.5% vs 14.0% in controls vs. oldest-old, respectively; P = 0.001). No contribution of CETP I405V for longevity wasfound(P= 0.347).Stratifiedanalysisintheoldest-oldgroupaccord- ing to the NCEP ATP III criteria suggested that APOE*4 also significantly associated with risk for hTC (P = 0.001) and mixed dyslipidemia (P b 0.001). In addition, we also found that CETP genotype distribut- ed differently between hTG and Control groups (P = 0.002) with hTG a higher proportion of VV genotype in the hTG subgroup (30.5% vs15.7% in hTG vs. controls, respectively).

As noted in Fig. 1, BMI, alcohol drinking, and TC increased the risk of hTG in our oldest-old subjects, and HDL-c significantly decreased the risk (P b 0.05). The odds of hTG for subjects with CETP*V allele was 1.88 times greater than subjects without the risk allele in the univariate polymorphism logistic regression model. After adjustment for APOE*4, age, BMI, alcohol drinking, TC, HDL-c, and LDL-c, the risk contribution was consistent (P b 0.05). When coupled with APOE genotypes contain- ingε4 at least one risk allele,theodds ofbeingaffected increasedto 4.36 times greater for subjects withtheCETP*V allelethan thosewithneither risk allele. In addition, the interaction item estimated by logistic regres- sion between CETP*V and APOE*4 significantly increased the risk of hTG (P = 0.002, OR = 3.25). There was no evidence for an increased risk for hTG due to the presence of APOE*4 itself (P = 0.079). In addition, we failed to repeat the findings that CETP conferred the risk of hTG and the interaction with APOE in our middle-aged controls (data not shown).

As shown in Table 3, according to whether individuals carry the risk allele (APOE*4 and CETP*V), Student t-test suggested higher TC (P = 0.004), TG (P = 0.044) and LDL-c (P = 0.007) in oldest-old females with APOE*4 compared to APOE*ε2, whichwasalso observed in controls (data not shown). In addition, TG was significantly increased in subjects with CETP*V compared to CETP*I (P = 0.003).

A cumulative effect of CETP*V allele was observed to increase the risk forhTG(P = 0.003,OR = 2.989,OR = 1.499).Accordingtothe fortrend VV VI genotypecombinationwithAPOE*4,theriskforhTGincreasedinsubjects with both CETP*V and APOE*4 compared to subjects with neither risk alleles (P = 0.001, OR = 19.091, OR = 1.909). After adjust- for trend VV VI ment for age and BMI (Model 2), association of CETP*V for hTG was con- sistent, whether CETP alone (P = 0.001, OR = 3.264, OR = for trend VV VI 1.335) or coupled with APOE*4 carriers (P = 0.042, OR = for trend VV 7.849, OR = 1.107) in oldest-old females. No contributions of APOE*4 VI for hTG was found with or without covariate adjustment (Table 4).

The exhaustive data mining MDR analysis was used to evaluate the impact of interactions between the genotypes of CETP and APOE poly- morphisms on hTG in oldest-old females. The 2-locus model that com- prised CETP and APOE emerged as the best disease predicting model with the highest level of statistical significance (MDR: TBA = 0.559, CVC = 10/10, P = 0.024; GMDR adjusted for age, BMI and alcohol drinking: TBA= 0.562, CVC = 10/10, P = 0.038).Overall, theparamet- ric and nonparametric results were consistent for the significant inter- actions between CETP and APOE.

Table 3

Comparisons of parameters between subjects with risk/reference alleles in oldest-old females.