(cid:2)2 test was performed to compare the difference in TSHR genotype frequencies. Genotype frequencies of all SNPs were found to be in Hardy-Weinberg equilibrium. Because two SNPs (in the TSHR) were involved in an independent new assay, the Bonferroni correction for multiple comparisons was used; thus << the threshold for statistical significance was P 0.025. Given that this is a relatively homogeneous population, tests for pop- ulation stratification were not performed. The haplotype asso- ciation test between cases (Ashkenazi Jewish centenarians) and controls was performed using Haploview 4 software (http:// www.broad.institut.org/haploview/haploview). Wilcoxon sta- tistics were calculated to test homogeneity between the groups.

To determine whether medications taken by partici- pants may affect TSH concentration, we applied a linear regression model where serum TSH levels served as a de- pendent variable, and various medications taken by our study subjects served as independent variables; no signif- icant effect of any medication was detected. Furthermore, stepwise regression of a mixed model, where probability to enter was 0.25 and probability to leave was 0.05, dem- onstrated that none of the medications had any significant effect on serum TSH. Drug categories and percentage of subjects using the drugs are listed in Table 2, as well as the percentage of subjects taking one or more drugs.

Heritability analysis

The phenotypic variance represents a combination of genetic (additive) variance, environmental variance, and the covariance between these two factors. However, we were only interested in the additive effect (narrow sense), which was measured as the covariance(i.e.theslope)oftherelationshipbetweentheaverage performance of the offspring over the average performance of their parents multiplied by 2 because there was only one parent involved.

The differences in age distributions between males vs. females had no effect on the outcome regarding TSH lev- els, even when plotted logarithmically. In fact, when lo- gistic regression was applied, adjusting for age, the P value was 0.91, indicating total similarity (Fig. 2).

Results

Demographic data

The distribution of our population by age and gender is depicted in Table 1. The median age of offspring (69 yr)

TABLE 1. Age, TSH, and FT4 in female and male controls, offspring, and probands (Ashkenazi Jewish centenarians) Controls Offspring Probands n (females/males) 163 (79/84) 366 (185/181) 232 (166/66) Age (yr) All 70 (53–80) 69 (59–79) 97 (95–105) Females 67 (52–80) 68 (59–79) 97 (95–103) Males 74 (59–80) 69 (59–79) 97 (95–103) TSH (mIU/liter) All 1.55 (0.63–3.93) 1.68 (0.65–4.79)a 1.97 (0.42–7.15)a Females 1.60 (0.60–4.7) 1.72 (0.51–6.3)a 2.00 (0.53–7.34)a Males 1.50 (0.55–4.50) 1.68 (0.65–5.9)a 1.93 (0.61–6.9)a FT4 (ng/dl) All 1.00 (0.69–1.7) 1.03 (0.67–2.0) 1.02 (0.62–2.02) Females 0.99 (0.74–1.5) 1.04 (0.66–1.9)a 1.04 (0.65–2.02)a Males 1.00 (0.57–1.7) 1.02 (0.67–2.0) 0.95 (0.48–2.06)a

Data are expressed as median (97.5% a P > < ( 0.05 vs. controls.

Control Offspring Proband 30 25 20 15 10 5 0 0.2 0.5 0.7 0.9 1.2 1.6 2.2 2.9 3.9 5.1 6.9 10.8 Serum TSH concentration (mIU/L) noitalupop ni egatnecreP

8 7 6 5 4 3 2 1 0 50 60 70 80 90 100 110 Age (Years) )L/Ulm( HST mureS

FIG. 2. Logarithmic plot of age distribution in males and females. Dashed line represents females, and continuous line represents

FIG. 1. TSH distribution in thyroid disease-free Ashkenazi Jewish centenarians, their offspring, and controls. The TSH frequency distribution curves were prepared using log-transformed values of TSH concentrations. Bin values were calculated by using the antilog function on the equal interval concentrations. Note: intervals on horizontal axis are not equal; the TSH data point is the upper TSH concentration in bin.

There was a logarithmically inverse correlation be- tween serum TSH concentrations and serum FT4 concen-      trations in all groups (r 0.13; P 0.01; Fig. 3A), but     not in the control (r 0.1; P 0.22; Fig. 3B).

Genetic analyses

Serum FT4 distribution

Because both Ashkenazi Jewish centenarians (6) and their offspring had significantly higher serum TSH concentrations compared with controls, we determined whether TSH concentrations in these groups might be inherited. Heritability analysis demonstrated a moder-

Serum FT4 concentrations were similar in offspring and controls with a median of 1.03 ng/dl (97.5% CI, 0.67–2.00) in the offspring and 1.00 ng/dl (97.5% CI, ~ eee 0.69–1.7) in the control group (P not significant; Table 2).

There was no gender-specific difference between serum FT4 concentrations in males in all groups. Female off- spring had higher serum FT4 levels than female controls [1.04 ng/dl (97.5% CI, 0.66–1.9) vs. 0.99 ng/dl (97.5%   CI, 0.74–1.5); P 0.02].

p=0.01, r=-0.13ASerumTSH (mIU/L)vwenaana  1Serum FT4(ng/dl)Log (TSH) = 0.6950695 - 0.2736748*FT4 (ng/dl)P=0.22, r=-0.1B 7.|        5-aSerum TSH (mIU/L) 04 06 O08 114 13°15 17Serum FT4 (ng/dl)

TABLE 2. Percentage of subjects taking drugs from various categories and percentage of subjects using medication(s)

% of total Drug categories Blood pressure 35.43 Heart disease 21.40 Diabetes mellitus 5.56 Dyslipidemia 27.30 Depression 13.44 Sleep medications 10.21 Aspirin 20.83 Anticoagulants 0.75 No. of drugs taken by same person 0 32.69 1 26.06 2 20.58 3 12.95 4 6.06 5 1.41 6 0.17 7 0.08

FIG. 3. The correlation between serum TSH and FT4 concentrations in all groups (A) and in controls (B).

Serum TSH in offspring (mIU/L)vO Ba aN @ ©1 2  3 4 5 6Serum TSH in proband (mIU/L)r=0.32p=0.007

quencies for these SNPs were not significantly different between offspring and centenarians.