The IGF1 gene is a strong genetic determinant of body size and serum level of IGFI across mammals (16-18). In dogs, the IGF1 promoter has been shown to be the mostIn this region of L1 (Figure 3, residues in hot pink), these results suggest that the Ala-37Thr change may affect ligand binding but to a lesser extent than the mutations that are within the IGF binding pocket. This region also coincides with residues 3444 of the IR that are shown to be important for insulin binding (25). Alignment of IGFIR with the IR shows that Ala-37 is just one residue away from IR Phe-39, a residue important for insulin ligand specificity (26). Ala-37 is also adjacent to a highly conserved stretch of residues beginning with Glu-44, which has an important function for insulin/IR binding (27). Interestingly, IGFIR residues around Ala-37 are highly conserved among distant vertebrate species, indicating that sequence variants most probably affect function. Another mutation found in our screen, Arg-407His, is located in the region of the L2 domain where nonsynonymous mutations display defective proreceptor processing and transport to the plasma membrane without a reduction in ligand affinity (28). Our finding of a reduction in IGFIR levels and concomitant centenarian carrier, possibly resulting from allele-specific differences in mRNA splicing, processing, or translational control and regulation (32). This article provides strong evidence that the IGF1R can be a genetic determinant of human longevity. The role of GH/IGF pathway in human longevity is controversial because of the recognized evolutionary tradeoffs in the modulation of lifespan. Our findings suggest that IGF-related longevity genes do exist in human populations. Although these data suggest that such alterations occur at low frequency in human centenarians, more exhaustive studies will clarify whether longevity assuring genetic factors and related phenotypes exist for other components of the GH/IGF pathway. Indeed, our results suggest that centenarians may harbor individually rare but collectively more common genetic variations in genes encoding components of the GH/IGF signaling pathway. Additional comprehensive studies in the centenarians and their offspring may reveal other molecules within the GH/IGF pathway that are operative in human longevity. 

Mutation Screening of IGF1 and IGF1R. We screened genomic DNA isolated from blood of centenarians and controls to discover all possible genetic variations in the full coding sequence and intron/exon boundaries of the IGF1 and IGF1R genes by 2D gene scanning (35). To increase specificity, PCR amplification was designed in two steps. First, two multiplex long-distance PCR co-amplified 12 large fragments encompassing coding and splicing junction regions of IGF1 and IGF1R. These products served as a template for four multiplex short PCRs to amplify all target regions in 30 fragments (5 fragments for IGF1 and 25 fragments for IGF1R). Primers are listed in supporting information (SI) Table 3. The mixture of amplicons after heteroduplexing was then separated on the basis of size and melting temperature in a 2D gel. DNA fragments containing heterozygous sequence variation were then re-amplified from the genomic DNA and sequenced to identify the position and nature of sequence variation.

Population and Sample Collection. In a case-control study, Ashkenazi Jews were recruited as described (13, 14, 33). This population derived from an undetermined small number (estimated to be several thousands) of founders. External factors, including ecclesiastical edicts prohibiting all social contact with Jews, the Crusades, the establishment of the Pale of Settlement, numerous pogroms, and ethnic bigotry resulted in social isolation and inbreeding of the Ashkenazi Jews (34). Three hundred eighty-four probands with exceptional longevity [286 females and 98 males, average age 97.7 (0.2) years (mean SE), age range 95108 years; 20% over the age of 100] were recruited to participate in the study. The participants' ages were defined by birth certificates or dates of birth as stated on passports. Probands were required to be living independently at 95 years of age as a reflection of good health, although at the time of recruitment, they could be at any institution or level of dependency. In addition, probands had to have a first-degree offspring who was willing to participate in the study. The offspring group consisted of 114 females and 174 males [mean age 67.8 (0.46) years (mean SE), range 4988 years]. We further established a unique cohort of Ashkenazi Jewish descent without a family history of unusual longevity (approximate age 70 years) that served as controls [n=312, mean age 79.5 (0.4) years (mean SE), 57% female]. A research nurse visited the probands in the morning to draw a venous blood sample,I'm sorry, but I don't see any text enclosed in ``` ``` for me to proofread and improve. Could you please provide me with the text to work on?