possible compensatory response to reduced IGFIR signaling, and smaller stature in female offspring of centenarians, led us to explore the IGF1R gene in more detail.

We conducted sequence analysis of the IGF1 and IGF1R genes of female centenarians and controls. We found an overrepresentation of heterozygous mutations in the IGF1R gene among centenarians compared to controls. These mutations are associated with high serum IGFI levels and reduced activity of the IGFIR, as measured in transformed lymphocytes. Therefore, genetic alterations in the human IGF1R that result in altered IGF signaling pathway confer an increase in susceptibility to human longevity, suggesting a role of this pathway in modulation of human lifespan.

In conclusion, our study provides evidence for the role of the IGF signaling pathway in human longevity. We demonstrated a gender-specific increase in serum IGFI associated with smaller stature and identified partial loss-of-function mutations in the IGF1R gene that are overrepresented among centenarians compared to controls. These findings suggest that altered signaling in the insulin/IGFI pathway may influence human lifespan. However, further studies are needed to fully understand the complex role of this regulatory network for exceptional longevity in human populations.Small reductions in auxological growth are highly reminiscent of a report on female mutant mice haploinsufficient for the IGFIR, which display nearly identical features, including gender-specific lifespan extension (15). The overrepresentation of the two nonsynonymous mutations among centenarians compared with controls was statistically significant (P 0.02). Of the nine centenarian carriers, IGFI levels and height were available for six carriers. When we compared the mutation carriers (n 6, 66% female) vs. noncarriers (n 163, 67% female) among centenarians, the mutation carriers had significantly higher IGFI levels (165 tly4 21 vs. 121 6 ng/ml, P 0.04) than noncarriers after adjustment for gender and age (Table 2). Although the mutation carriers were slightly shorter (162 2.8 vs. 165 Se | 0.8 cm, P 0.41) than noncarriers, this difference was not statistically significant.

We comprehensively screened genomic DNA from a subset of female centenarians with heights below the mean for that population (n 79) and female controls (n 161) for all possible genetic variations throughout the coding exons and splice junctions of the IGF1 and IGF1R genes by using 2D gene scanning and DNA sequencing (Fig. 2). Remarkably, we did not find a single variation in the IGF1 gene, suggesting that the coding regions of this gene are highly conserved. In contrast, the IGF1R gene was highly polymorphic with a total of 20 sequence variants, among which nine were previously unknown novel variants that were not reported in the various SNP databases (Table 1). Five SNPs of the nine novel variants were in a coding region.

To assess the functional impact of the mutations identified in centenarians, we analyzed immortalized lymphocytes from centenarians by using an IGFIR ELISA. Immortalized lymphocytes from the two nonsynonymous, 244G A (Ala-37Thr) and 1355G A (Arg-407His), and one synonymous, 1545G A (Thr-470Thr), mutation carriers of female centenarians showed a significant reduction in IGFIR levels compared with lymphocytes from 10 female centenarians without the mutations (P 0.03) (Fig. 1C). Moreover, when treated with IGFI, lymphocytes from the centenarian mutation carriers demonstrated reduced phosphorylation of AKT compared with the centenarian noncarriers (Fig. 1 D and E), suggesting that these mutations lead to decreases in IGF signaling.

Despite the well-established role of the GH/IGF axis in modulation of lifespan in laboratory animals, its role in human longevity has been controversial. The identification of new nonsynonymous mutations in the IGF1R gene that result in reduced IGF signaling among centenarians suggests a similar role for this pathway in modulation of human lifespan as observed in model organisms. The centenarian mutation carriers display manifestations of elevated serum IGFI levels and a trend toward smaller height, reminiscent of the phenotypes observed in mice haploinsufficient for the IGF1R with extended lifespan (15). Comprehensive analysis of all genes within this axis will be required to firmly establish a role of the IGFI axis in modulation of human longevity.

In contrast, we identified a diverse set of genetic alterations in the IGF1R gene in this population. Of particular interest is the enrichment of the variations in the coding region, especially the nonsynonymous mutations in centenarians compared with the control population, suggesting a contribution of multiple rare alleles leading to a major phenotypic effect on human longevity in the general population (21). So far, only a few nonsynonymous IGF1R mutations have been reported from patients with severe phenotypes such as intrauterine growth retardation (20, 22). The prevalence of such nonsynonymous mutations in the normal population has been obscure because of the relatively low number of control individuals studied in previous reports (n 100). Resequencing of the IGF1R gene in the Centre d'Etude du Polymorphisme Humain cohort indicated that the Arg-407His variation (ncbi.nlm.nih.gov/SNP/snp\_ss.cgi?subsnp\_id=46533818) was indeed very rare, heterozygosity being detected in only one individual from a total of 354 individuals. Together with our data, these results clearly demonstrate the rarity of nonsynonymous mutations among the normal population and, more importantly, its enrichment among long-lived humans.