2.3. SNP genotyping

Whole blood samples were obtained from 347 centenarians (NECS: 266, LGP: 81), 260 NECS centenarian offspring, and 238 controls (NECS: 53 spousal controls and 106 septuagenarian offspring controls, LGP: 79 spousal controls).Of note,althoughserawerenotavailablefromallNECSsubjectsfor Hsp70assays,wholebloodwasavailableforgeneticstudies.DNAwasisolated from each subject’s peripheral blood leukocytes using the Qiagen DNA purification system (Qiagen, Valencia, CA). Real time PCR based SNP genotyping assays were purchased from Applied Biosystems Assays-on- DemandTMand performedin accordancewiththemanufacturer’sinstructions. Reaction products were analyzed on an ABI Prism 7900 Sequence detection system. HSP70-A1A and HSP70-A1B are encoding genes members of the hsp70 family located within the cluster of human leukocyte antigen class III genes on chromosome 6p21 in the neighborhood of the tumor necrosis factor

2.1. Study population

The criteria for eligibility, methods of recruitment, and study outcomes of the New England Centenarian Study (NECS) have been published elsewhere (Perls et al., 1999; Terry et al., 2003). Briefly, the NECS is an United States nationwide study of centenarians, their offspring, and controls. The controls include (a) spouses of the centenarian offspring and (b) offspring of individuals

Table 1

Characteristics of centenarians, centenarian offspring, and spousal controls enrolled in the NECS and in the LGP, expressed as frequency (percentage of available data) or as mean (standard deviation)

Mean   S.D. or n (%) LGP centenarians NECS centenarian offspring NECS controls LGP controls (n = 87) (n = 93) (n = 43) (n = 83) Mean age, years (SD) 99.9  + 7 2.6*** 72.0  7.2 72.9  7.9 64.4 1+ ¢ 6.3*** Females 64 (73.6) 62 (66.7)* 19 (44.2) 55 (68.8)** Current smoker 1 (1.6) 1 (2.6) 3 (3.6) 6(14.3) Hypertension 40 (47.6)** 24 (28.2)* 18 (47.4) 41 (51.9) History of myocardial infarction 9 (14.3)** 2 (2.4) 4 (10.5) 4 (5.4)

Statistical tests were carried-out to compare (1) LGP centenarians and NECS centenarian offspring (significance shown in the LGP centenarians column), (2) NECS centenarian offspring and NECS controls (significance shown in the NECS centenarian offspring column), and (3) LGP controls and NECS controls (significance shown in the LGP Controls column). Statistical significance is denoted as *p < 0.05, **p < 0.01, and ***p < 0.005.

(TNF) locus. These genes share the characteristic of having only one linkage disequilibrium (LD) block. Thus, we genotyped one tagSNP per gene: rs1043618 (hcv 11917510) in HSP70-A1A and rs6457452 (hcv3052604) in HSP70-A1B.

the controls (Table 1). The youngest ‘‘centenarian’’ is 95-years- old, while the oldest is a female, age 108. The oldest male is 106-years-old. The age range for the NECS centenarian offspring is 54–88 years, for the NECS controls is 57–89, and for the LGP controls is 39–74. Despite being of similar age ( p = 0.497), a higher proportion of NECS centenarian offspring are disease-free compared to the NECS controls. After examining age, gender, race, income, alcohol, cardiovascular disease, and a variety of other age-related diseases in a logistic regression model, low serum Hsp70 level was the only covariate associated with being a centenarian or a centenarian offspring.

2.4. Statistical analyses

Univariate and multivariate statistics comparing LGP centenarians, NECS centenarian offspring, NECS controls and LGP controls were computed using Stata 9.1 statistical software (StataCorp, 2005). Covariates included age, gender, current smoking status, presence of hypertension, and history of myocardial infarction. Fisher’s exact test was used for cells with values less than 5. We compared Hsp70 serum levels among (a) LGP centenarians versus NECS centenarian offspring, (b) LGP centenarians versus LGP controls,and (c) NECS centenarian offspring versus NECS controls using multiple linear regression adjusting for statistically significant covariates. Hardy-Weinberg equilibrium was determined among controls with the x2-test. Genotypic counts and allele frequencies were compared between cases and controls using multi- ple logistic regression. For each SNP, odds ratios (OR) and 95% confidence intervals (CI) were calculated.

The mean and median serum Hsp70 levels (ng/mL) in each group are shown in Table 2, both unstratified and stratified by sex. Fig. 1 graphically depicts the distribution of these measurements. Except for the LGP centenarians, females have slightly higher mean serum Hsp70 levels than males. Remarkably, centenarians and centenarian offspring have unadjusted mean serum Hsp70 levels close to seven-fold lower than those of the NECS controls and approximately three-fold lower than those of the LGP controls. After the mean Hsp70 levels were adjusted for significant covariates, the paired comparison revealed that Hsp70 is significantly lower in NECS offspring than in NECS controls (sex-adjusted p < 0.001) and

3. Results

3.1. Demographics and HSP analyses

As expected, the centenarians are older, predominantly female, and generally sicker than the centenarian offspring and

Serum Hsp70 levels in subject populations (ng/mL)

Males Females Both Sex-adjusted p-value NECS centenarian offspring (n = 93) Mean (S.D.) 0.95 (1.2) 1.10 (1.5) 1.05 (1.4) 0.99 (0.4) <0.001 Median (IQR) 0.61 (0.4) 0.55 (0.4) 0.56 (0.4) NECS controls (n = 43) Mean (S.D.) 6.58 (5.0) 7.36 (6.5) 6.93 (5.7) 6.95 (0.5) Median (IQR) 6.87 (8.0) 6.35 (10.4) 6.79 (9.2) Males Females Both Age-adjusted p-value LGP centenarians (n = 87) Mean (S.D.) 1.69 (2.4) 0.93 (1.1) 1.13 (1.6) 0.62 (0.8) 0.0577 Median (IQR) 0.51 (2.1) 0.49 (1.0) 0.5 (1.0) LGP controls (n = 83) Mean (S.D.) 2.81 (3.2) 3.17 (3.3) 3.07 (3.2) 3.59 (0.8) Median (IQR) 1.59 (3.8) 1.48 (4.9) 1.59 (4.2)

In (mean serum HSP70 levels) in ng/mL-13LGP CentenariansLGP Controls     NECS OffspringNECS Controls   a 4-2