centenarian carrier, possibly resulting from the allele-specific differences in mRNA splicing, processing, or translational con- trol 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 in a low frequency in human centenarians, more exhaustive studies will clarify whether longevity assuring genetic factors and related phenotypes exist for other compo- nents 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 stud- ies 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 variationsinthefullcodingsequenceandintron/exonboundariesoftheIGF1 and IGF1R genes by 2D gene scanning (35). To increase specificity, PCR ampli- fication was designed in two steps. First, two multiplex long-distance PCR coamplified 12 large fragments encompassing coding and splicing junction regions of IGF1 and IGF1R. These products served as template for four multi- plex short PCRs to amplify all target regions in 30 fragments (5 fragments for IGF1and25fragmentsforIGF1R).Primersarelistedinsupportinginformation (SI) Table 3. The mixture of amplicons after heteroduplexing was then sepa- ratedonthebasisofsizeandmeltingtemperatureina2Dgel.DNAfragments containing heterozygous sequence variation were then reamplified from the genomic DNA and sequenced to identify position and nature of sequence variation.

Materials and Methods

PopulationandSampleCollection.Inacasecontrolstudy,AshkenaziJewswere recruited as described (13, 14, 33). This population derived from an undeter- minedsmallnumber(estimatedtobeseveralthousands)offounders.External factors, including ecclesiastical edicts prohibiting all social contact with Jews, the Crusades, the establishment of the Pale of Settlement, numerous Po- groms,andethnicbigotryresultedinthesocialisolationandinbreedingofthe Ashkenazi Jews (34). Three hundred and eighty four probands with excep- tionallongevity[286femalesand98males,averageage97.7(0.2)years(mean SE), age range 95–108 years; 20% over the age of 100] were recruited to participate in the study. The participants’ ages were defined by birth certifi- cates 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 114femalesand174males[meanage67.8(0.46)years(meanSE),range49–88 years]. We further established a unique cohort of Ashkenazi Jewish descent withoutafamilyhistoryofunusuallongevity(approximateage70years)that servedascontrols[n(cid:1)312,meanage79.5(0.4)years(meanSE),57%female]. A research nurse visited the probands in the morning to draw a venous blood

Assays for IGFI, IGFIR, and Phospho-AKT. IGFI was measured in uniformly handled serum aliquots by using an ELISA Kit from DSL according to the manufacturer’s recommendations. IGFIR was measured in immortalized lym- phocyte cell extracts with an ELISA Immunoassay Kit from BioSource Interna- tionalaccordingtothemanufacturer’srecommendations.Lymphocyteswere isolated and immortalized as described (36). To assess the ability of IGFI to activatesignaltransductionviatheIGFIR,lymphocytesfromfemalecentenar- ians harboring mutations and those without were incubated with or without IGFI(100ng/ml)for25minafterwhichtotalcellproteinwasextractedand150 (cid:1)g of cell lysates was subjected to SDS/PAGE, immunoblotting with total and phospho-AKT antibodies from Cell Signaling, and densitometry using scan- ning autoradiography. Results are reported as means   SD and as box and whiskers plots. Statistical significance was determined by using unpaired t tests.

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