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.

LinkagedisequilibriumbetweenthetwoSNPswashigh (r2   0.95), suggesting interaction between them. The fre- quency of GA haplotype was significantly greater among both Ashkenazi Jewish centenarians and their offspring eS compared with controls (0.57 and 0.53 vs. 0.46; P  0.0001 and P 0.01, respectively).

with adjustment for gender, we found that the median TSH in those who had rs10149689 (G allele) and rs12050077 (A allele) was significantly higher than those who had neither of the alleles [median (97.5% CI), 1.42 (0.56 to 3.33) vs. 1.68 (0.63 to 6.06) mIU/liter in controls, P   0.02; and 1.49 (0.46 to 3.74) vs. 1.70 (0.68 to 5.38) mIU/liter, P   0.049 in offspring]. Similarly, the median TSH level in the combined groups (control and offspring) associated with GA haplotype was significantly higher compared with the individuals without the GA [median (97.5% CI), 1.78 (0.59 to 7.01) vs.1.54 (0.50 to 5.29) mIU/liter; P   0.04].

FIG. 4. Hereditability analysis of serum TSH in Ashkenazi Jewish centenarians (probands) and their offspring.

ate degree of heritability (h2  0.32; P  0.007) for serum TSH in Ashkenazi Jewish centenarians and their offspring (Fig. 4).

To examine further the role of genetic factors, performed a case-control genetic analysis of TSH and TSHR variants (4 and 2, respectively). SNPs were a part of a larger group of SNPs in independent dataset comprised of individuals from same genetic origin. Although the SNPs in the TSH-B this dataset did not show significant changes cases and controls, the SNPs in the TSHR did significant differences. Thus, we chose to examine ther those SNPs using a different technology and an independent dataset. The allelic frequency for SNPs in the TSHR gene was significantly different paring Ashkenazi Jewish centenarians and [rs10149689 G allele frequency, 0.57 vs. 0.48; P   odds ratio (OR), 1.56] and (rs12050077 A allele   quency, 0.57 vs. 0.46; P 0.0001; OR 1.68). allelic frequency also differed between the offspring controls (rs10149689 G allele frequency, 0.53 vs.   P 0.08; OR 1.22; and rs12050077 A allele  0.53 vs. 0.46; P 0.01; OR 1.32; Fig. 5). Allelic

Carriers of the favorable allele in the combined groups (offspring and control) have higher TSH levels [median (97.5% CI), 1.70 (0.59 to 5.34) vs.1.45 (0.50–3.99) mIU/ liter; P   0.005], suggesting that the high prevalence of the TSHR SNPs is associated with higher serum TSH levels. However, when the centenarians’ data were entered into the equation, the differences within the genotype (carriers/ noncarriers of the favorable allele) are less significant [me- dian (97.5% CI), 1.89 (0.61–5.07) vs. 1.60 (0.49–4.50) mIU/liter; P   0.04], suggesting that additional factors are involved. Finally, adjusting for age at recruitment dimin- ished the differences, suggesting that age is a crucial com- ponent of elevated TSH.

Regulatory elements prediction

Using enhancer (http://www.cs.helsinki.fi/u/kpalin/EEL) andpromoterpredictionsoftware(25),welocatedourSNPs between two enhancer regions and in close proximity to the promoterarea.Becausethiswasanassociationstudyandthe SNPs were located outside the gene, we have predicted that they are in linkage disequilibrium with a regulatory genetic variant that is located either in the enhancer or in the pro- moter. Thus, these results demonstrate a possible effect on gene expression that leads to an increase TSH levels.

o60s     2E o4532z 03 @Probandz DOffspring202 WContro!22=011510149689 (G) 1812050077 (A)*p=0.01 vs, Controls**p=0.001 vs. Controls

Further studies are needed, however, to clarify this

Discussion

Our data show that offspring of the centenarian group have raised serum TSH concentrations compared with

FIG. 5. GA haplotype prevalence in Ashkenazi Jewish centenarians (probands), their offspring, and controls.

extension, suggesting that genetic determinants of creased thyroid function may modulate life span.

tenarian group who had elevated serum TSH levels also had higher TSH concentrations, suggesting a heritable trait that we documented by heritability analysis. After searching for genetic markers that could be associated with, and possibly responsible for, the raised serum TSH in centenarians and their offspring, we demonstrated for the first time that two SNPs (rs10149689 and rs12050077 of the TSHR), located between enhancer regions and close to the promoter region, were significantly associated with raised TSH concentrations in the Ashkenazi Jewish cen- tenarians and their offspring compared with controls, and showed strong linkage disequilibrium. Our new results raise the possibility that these genetic changes exert an effect on TSHR gene expression that leads to increased TSH and may be, at least in part, transmitted from indi- viduals with exceptional longevity to their offspring.

Thyroid hormone has profound impact on metabolic rate and body temperature by various mechanisms, in- cluding modulating mitochondrial activity, membrane composition and ion permeability, and substrate cycling (29). The relationship between longevity and hypothy- roidism is considered to be due to lower levels of reactive oxygen species production and oxidative stress in re- sponse to changes in both obligate and facultative ther- mogenesis (30).