The IGF1 gene is a strong genetic determinant of body size and serum level of IGFI across mammals (16–18). In dogs, the IGF1 promotor has been shown to be the most potent determi- nant of size (17), and IGFI levels in dogs are associated with

One of the two nonsynonymous mutations we identified in the IGF1R, Ala-37–Thr, is located at the edge of one of the putative

n 79 n 161 Category Nucleotide change Protein change Het Hom Het Nonsynonymous 244G>A Ala-37–Thr 1 0 0 0 1355G(cid:3)A Arg-407–His 1 0 0 0 Synonymous 948C(cid:3)A Gly-271–Gly 7 0 16 0 1545G>A Thr-470–Thr 1 0 0 0 1959C(cid:3)T Asn-608–Asn 0 0 1 0 1995G>T Arg-620–Arg 0 0 1 0 2343C(cid:3)T Thr-736–Thr 3 0 2 0 2745C>T Asn-870–Asn 1 0 3 0 3174G(cid:3)A Glu-013–Glu 48 20 67 29 4083C(cid:3)T Tyr-1316–Tyr 5 0 7 0 4103C>T Arg-1323–Arg 1 0 0 0 Intronic IVS2(cid:5)20C(cid:3)T 42 8 65 12 IVS8–20T(cid:3)C 49 13 67 34 IVS13(cid:1)17G>A 1 0 2 0 IVS13(cid:5)21A(cid:3)C 47 3 62 41 IVS13(cid:5)29(cid:1)30delGT 26 1 38 3 IVS15(cid:1)49insG 1 0 0 0 IVS15(cid:1)52A>G 0 0 1 0 IVS17(cid:2)5C>T 1 0 3 0 IVS21–34G(cid:3)A 46 15 59 42

Het, number of heterozygote carriers of variant; Hom, number of homozygote carriers of variant. unknown, novel variants are indicated in bold.

attenuation of IGFI-induced AKT phosphorylation in the lym- phocyte of the Arg-407–His carrier suggest that this mutation also inhibits IGFIR protein activity. Mutations in the close vicinity of this residue in IR are known to contribute to insulin-resistant diabetes (29, 30). Moreover, close to Arg-407 is the DAF-2 mutation Ala-580–Thr (e1365) that leads to dauer formation at 25°C (31). Finally, it is tempting to speculate that the altered IGF signaling induced by the synonymous mutation, waii>/ 1545G A (Thr-470–Thr), may be caused by the reduction of IGFIR levels, as demonstrated in the lymphocytes from the

ligand binding hot spots of the IGFIR extracellular domain in the L1 region (23). Our modeling using the crystal structure of the L1-CR1-L2 fragments of the IGFIR structure (24) shows that Ala-37 is positioned on a loop in the first L1 domain near residues that, when mutated, reduce the binding affinity of IGFI for IGFIR by 2- to 10-fold (Fig. 3, residues in pale pink) or aM 20-fold (Fig. 3, Phe-90 in red). A recently identified compound heterozygote missense mutation (22), Arg-108–Gln and Lys- 115–Asn, from a patient with intrauterine and postnatal growth retardation with features of IGF resistance is also located in this region of L1 (Fig. 3, residues in hot pink). These results suggest that the Ala-37–Thr 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 34–44 of the IR that are shown to be important for insulin binding (25). Align- ment 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 important function for insulin/IR binding (27). Interestingly, IGFIR resi- dues around Ala-37 are highly conserved among distant verte- brate species, indicating that sequence variants most probably affect function.

Ala37Thr Ala37Thr Arg407His Arg407His  

Another mutation found in our screen, Arg-407–His, is lo- cated in the region of the L2 domain where nonsynonymous mutations display defective proreceptor processing and trans- port to the plasma membrane without reduction in ligand affinity (28). Our finding of a reduction in IGFIR levels and concomitant

Table 2. Height and IGFI levels among centenarian carriers of either the Ala-37–Thr or the Arg-407–His mutation in the IGF1R gene

Fig.3. ThepositionofmutatedaminoacidsintheIGFIR.Thelocationsofthe mutated amino acids are indicated in the crystallographic structure encom- passing the first three domains of the IGFIR (L1-CR-L2 fragment) (24). Color- codedresiduesarethosewhenmutatedreducethebindingaffinityofIGFIfor IGFIR by 2- to 10-fold (pale pink) or (cid:3)20-fold (red) (23) or a compound heterozygote missense mutations (hot pink) from a patient with intrauterine and postnatal growth retardation (22). Arrows indicate the locations of the mutated amino acids identified in centenarians.

Carriers, Noncarriers, Variable n   6 n   163 P Height, cm 162   2.8 165   0.8 0.41 IGFI, ng/ml 165  21 121   6 0.04

sample, obtain a medical history including maximal height and weight, were obtained by using a standardized questionnaire. In such visits, offspring (and participating spouses) underwent similar evaluations as scribed (13, 14, 33). All blood samples were rapidly processed at the Clinical Research Center at the Albert Einstein College of Medicine for eration of transformed lymphocytes as a source of DNA and the policy of the Committee on Clinical Investigations of the Albert College of Medicine.

centenarian carrier, possibly resulting from the allele-specific differences in mRNA splicing, processing, or translational con- trol and regulation (32).