arm of chromosome 1 (1p13.3- p12) (25). The TSHR gene is located on the short arm of chromosome 14 (14q31-q32.1) and spans approximately 60 kb with 10 exons (26). We selected SNPs in the TSH-B and TSHR genes based on their known or potential functional effects and/or their previous association with thyroid function (18, 19, 27). We genotyped two SNPs in the TSH-B gene promoter/enhancer region (rs179247 and rs12101255) and five SNPs in the TSHR gene (rs12050077, rs10149689, rs10995190, rs2239130, and rs2231280) using a custom-designed Illumina GoldenGate assay (Illumina Inc., San Diego, CA). The genotyping success rate was greater than 96% for all SNPs. The minor allele frequencies of these SNPs were greater than 0.05 in our study population. All SNPs were in Hardy-Weinberg equilibrium in each group separately and in the pooled groups.  Statistical analyses  Data are presented as means S.E.M., unless otherwise noted. Comparison of continuous variables between two groups was performed using an unpaired t test or Mann-Whitney rank sum test. Comparison of categorical variables between two groups was performed using a chi-square test or Fisher exact test. Correlations between two continuous variables were assessed using Pearson correlation coefficient or Spearman rank correlation coefficient. Heritability estimates were calculated using the SOLAR program (28). Logistic regression analysis was used to examine the association between SNPs and longevity status after adjusting for age and sex. Haplotype frequency was estimated using the EH program (29). Linkage disequilibrium coefficient r2 was calculated using Haploview software (30). A P value of less than 0.05 was considered statistically significant. All analyses were performed using SAS software (version 9.1; SAS Institute Inc., Cary, NC), unless otherwise noted.Arm of chromosome 1. We selected four SNPs (rs10494170, rs12046203, rs12740035, and rs7523360) of TSH-B gene and genotyped them in both Ashkenazi Jewish centenarians and controls. The allelic frequencies of these SNPs between the Ashkenazi Jewish centenarians and controls did not show significant differences. Due to the association between TSH receptor (TSHR) and TSH expression, we further examined SNPs in this gene. SNPs (rs12050077 and rs10149689) were genotyped using the PSQ HS 96A Pyrosequencer, according to the manufacturer's recommendations (Pyrosequencing, Uppsala, Sweden; www.pyrosequencing.com). Briefly, a PCR product was generated from a primer pair that included one primer covalently coupled to biotin, the biotinylated template was bound to streptavidin-coated Sepharose HP beads, and this mixture was then annealed to a sequencing primer. Stepwise elongation of a sequencing primer strand upon sequential addition of a specified sequence of deoxynucleotide triphosphates and the degradation of nucleotides by apyrase were carried out simultaneously. As the sequencing reaction progressed, the DNA strand was extended, and the sequence was determined from the measured signal output of light upon nucleotide incorporation. The resulting peaks in the pyrogram were analyzed using Pyrosequencing software. Primer sequences are available from the authors upon request. Error rates based on blind replicates were estimated to be 0.5%. Stated age was verified by checking birth certificates or U.S. passports. Jewish religion was self-reported (mother and father Jewish), and subjects were considered Ashkenazi Jews if their four grandparents were originally Ashkenazi Jews. Information on medical history, demographic characteristics, and clinical data were obtained uniformly using a structured questionnaire. All subjects underwent a physical examination and were asked to provide a blood sample. Informed written consent was obtained in accordance with the policy of the Committee on Clinical Investigation of the Albert Einstein College of Medicine. Exclusion criteria included acute or debilitating medical conditions.

Serum TSH Distribution Statistical Analysis Serum TSH concentrations in the offspring were significantly higher compared with controls [median, 97.5% CI: 1.68 (0.65-4.79) and 1.55 (0.63-3.93) mIU/liter, respectively; P < 0.02; Table 1). Moreover, serum TSH distribution was also shifted to higher TSH concentrations in offspring compared with controls, including the TSH concentrations at peak frequency (Fig. 1), suggesting a population shift phenomenon. TSH distribution in Ashkenazi Jewish centenarians, recently published (4) and shown only for comparative purposes, was further shifted to higher TSH concentrations (Fig. 1) compared with offspring, suggesting a genetic predisposition for higher TSH.

All analyses were conducted using Statistical Analysis Software (SAS Institute, Cary NC), version 9.12. Because serum TSH and FT4 were not normally distributed, nonparametric tests including the Kruskal-Wallis one-way ANOVA by ranks and Mann-Whitney rank sum test were used for group comparisons. A chi-square 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 population stratification were not performed. The haplotype association test between cases (Ashkenazi Jewish centenarians) and controls was performed using Haploview 4 software (http://www.broad.institut.org/haploview/haploview). Wilcoxon statistics were calculated to test homogeneity between the groups.