Screening of the IGF1and IGF1RGenes for Mutations Associated with

Longevity. 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 9 were previously unknown, novel vari- ants that were not reported in the various SNP databases (Table 1). Five SNPs of the nine novel variants were in a coding region. i>/ Interestingly, two nonsynonymous mutations, i.e., 244G A ae> (Ala-37–Thr) and 1355G A (Arg-407–His), and two novel wei> synonymous mutations, i.e., 1545G A (Thr-470–Thr) and ny thasS>T 4103C T (Arg-1323–Arg), were found in centenarians with short stature and elevated IGFI that were not seen in controls. The overrepresentation of the two nonsynonymous mutations

Functional Studies of the Nonsynonymous Mutations Associated with

Longevity. To assess the functional impact of the mutations identified in centenarians, we analyzed immortalized lympho- cytes from centenarians by using an IGFIR ELISA. Immortal- AME3>A ized lymphocytes from the two nonsynonymous, 244G A (Ala- Uaei>/ 37–Thr) and 1355G A (Arg-407–His), and one synonymous, a iei>/ 1545G A (Thr-470–Thr), mutation carriers of female cente- narians showed a significant reduction in IGFIR levels compared with lymphocytes from 10 female centenarians without the wy te<< | mutations (P 0.03) (Fig. 1C). Moreover, when treated with IGFI, lymphocytes from the centenarian mutation carriers dem- onstrated reduced phosphorylation of AKT compared with the

A IGF1-1 IGF1-4 B 1355 G>A Arg 407 His 4 15.1 CGC>CAC 12 14 5 8.1 6 16 13 2.2 19 18 17 7 11 9 10 20 8.2 IGF1-2 2.1 15.2 IGF1-3 21.2 3 500 bp Size 100 bp siserohportcelE leG tneidarG gnirutaneD

Fig. 2. 2D gene scanning of human IGF1 and IGF1R genes. The entire coding regions and exon–intron junctions of the IGF1 and IGF1R genes were amplified by two-step PCR. Thirty short PCR fragments were distributed in 2D gels according to their size and melting temperature. (A) A 2D gene scanning pattern from acentenariansubjectwiththefragmentidentificationnumberandahetero-duplexbandinexon8.1andexon6oftheIGF1Rgeneisshown.(B)Thenovelgenetic variation in exon 6 was identified as 1355G(cid:3)A (Arg-407–His) by nucleotide sequencing.

lifespan (19). Severe short stature and extremely high IGFI levels were observed in a family with a mutation in the IGF1 gene, leading to an altered protein sequence associated with a de- creased affinity to the IGFIR (20). Interestingly, in our Ash- kenazi Jewish population, the coding region of the IGF1 gene was found to be remarkably free of any genetic variation, suggesting the possibility of selective pressure being exerted on this gene.

centenarian noncarriers (Fig. 1 D and E), suggesting that these mutations lead to decreases in IGF signaling.

Discussion

Despite the well established role of the GH/IGF axis in modu- lation 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 centenarians with the IGF1R mutations 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). The centenarian mutation carriers were on average 2.5 cm shorter than nonmutation carriers, but this difference was not statistically significant (Table 2), suggesting that the impact of these mutations on height is limited, probably because of compensation via increased IGFI levels and/or other mecha- nisms. In contrast, female offspring exhibited significantly higher serum IGFI and shorter stature, possibly because of variations in other genes involved in the IGF axis that may be more common than the IGF1R mutations described here. Comprehensive anal- ysis 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 enrich- ment of the variations in the coding region, especially the nonsyn- onymous mutations in centenarians compared with the control population, suggesting a contribution of multiple rare alleles lead- ing to a major phenotypic effect on human longevity in the general population (21). So far, only a few nonsynonymous IGF1R muta- tionshavebeenreportedfrompatientswithseverephenotypessuch 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’E´tude du Polymorphisme Humain cohort indicated that the Arg-407–His variation (ncbi.nlm.nih.gov/SNP/ sammiep_S pic   snp ss.cgi?subsnp id 46533818) was indeed very rare, heterozy- gocity being detected in only one individual from a total of 354 individuals. Together with our data, these results clearly demon- strate the rarity of nonsynonymous mutations among the normal population and, more importantly, its enrichment among long-lived humans.