Table 3 Association analysis of SHC1

SNP ID Centenarian Control Allele frequencies P Odds ((cid:1)2) ratio Major Hetero Minor Allele Major Hetero Minor Allele Centenarian Control homo homo count homo homo count SNP1 224 6 0 460 174 6 0 360 0.013 0.017 0.67 1.28 SNP2 223 7 0 460 174 5 1 360 0.015 0.019 0.64 1.28 SNP3 223 7 0 460 174 5 1 360 0.015 0.019 0.64 1.28 SNP4 212 18 0 460 160 18 2 360 0.039 0.061 0.15 1.60 SNP5 212 18 0 460 168 12 0 360 0.039 0.033 0.66 0.85 SNP6 210 20 0 460 165 15 0 360 0.043 0.042 0.90 0.96 SNP7 214 16 0 460 167 13 0 360 0.035 0.036 0.92 1.04 SNP8 229 1 0 460 180 0 0 360 0.002 0.000 0.38 0.00 SNP9 226 4 0 460 178 2 0 360 0.009 0.006 0.60 0.64 SNP10 224 6 0 460 175 5 0 360 0.013 0.014 0.92 1.07 SNP11 225 5 0 460 179 1 0 360 0.011 0.003 0.18 0.25 SNP12 210 20 0 460 164 16 0 360 0.043 0.044 0.95 1.02

Table 4 Haplotype analysis of SHC1 (1 major allele, 2 minor allele)

Haplotype ID SNP ID Haplotype frequencies 1 2 3 4 5 6 7 8 9 10 11 12 Control Control 1 1 1 1 1 1 1 1 1 1 1 1 1 0.795 0.780 2 1 1 1 1 1 2 1 1 1 1 1 2 0.044 0.041 3 1 1 1 1 2 1 1 1 1 1 1 1 0.037 0.031 4 1 1 1 2 1 1 1 1 1 1 1 1 0.036 0.053 5 1 1 1 1 1 1 2 1 1 1 1 1 0.026 0.034 Others             0.062 0.061  SNP12, showed high LD, indicating major SNP subgroups (Fig. 2, bottom). other SNP pairs were less than 0.1. Haplotypes were constructed on genotype data from 12 SNPs observed Haplotype frequencies were estimated maximization algorithm, with One major haplotype accounts for haplotypes, and other minor haplotypes the total. There was no significant frequency of haplotypes between Data access

Haplotype ID SNP ID Haplotype frequencies Chi-sqare Odds test P ((cid:1)2) ratio 1 2 3 4 5 6 7 8 9 10 11 12 Control Control 1 1 1 1 1 1 1 1 1 1 1 1 1 0.795 0.780 0.60 0.91 2 1 1 1 1 1 2 1 1 1 1 1 2 0.044 0.041 0.83 0.93 3 1 1 1 1 2 1 1 1 1 1 1 1 0.037 0.031 0.62 0.82 4 1 1 1 2 1 1 1 1 1 1 1 1 0.036 0.053 0.24 1.49 5 1 1 1 1 1 1 2 1 1 1 1 1 0.026 0.034 0.48 1.34 Others             0.062 0.061  

1 1 1 1 1 2 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 2 1 1           123 4 5 6 7 8 9 1011 12 SNP; 

SNP12, showed high LD, indicating the presence of two major SNP subgroups (Fig. 2, bottom). The r2 values for other SNP pairs were less than 0.1.

Haplotypes were constructed on the basis of the genotype data from 12 SNPs observed in SHC1 (Table 4). Haplotype frequencies were estimated by the expectation- maximization algorithm, with phase-unknown samples. One major haplotype accounts for about 80% of all haplotypes, and other minor haplotypes less than 22% of the total. There was no significant difference in the frequency of haplotypes between centenarians and controls.

Data access

The detailed data on this manuscript including sequences of oligonucleotide used for sequencing are available at http://hgp.gsc.riken.go.jp.

Fig. 2 Pairwise LD in SHC1, evaluated by r2 estimations. The LD between all pairs of SNPs (SNP and SNP , where i and j refer to the i j SNP IDs shown in Table 2) was evaluated by the LD measure r2 among 66 marker pairs studied in 410 Japanese participants, including centenarians and younger controls. SNP pairs in high LD (r2 (cid:1)0.9) are shown as blackened boxes. All other SNP pairs were in low LD (r2 <0.1) and are shown as white boxes. Two subgroups of SNPs in high LD with each other are shown below

Discussion

central role in controlling the oxidative stress response and aging, we investigated the possible association of SHC1 genetic variability with longevity in the Japanese popula- tion. We first investigated molecular variants in the 10-kb genomic region that contains the entire SHC1 gene (Fig. 1, Table 2). Among 12 SNPs identified in this study three were nonsynonymous sites. Although the frequency of these nonsynonymous SNPs was very small, their presence is likely to affect protein function. In particular, the A205V substitution occurs within a conserved phosphotyrosine- binding domain. The frequency of the other observed SNPs is also low, which reduces the statistical power to detect population differences at this locus. Ventura et al. [17] showed that p66shc expression in primary, immortal, and transformed human cells is regulated by the degree of methylation of its promoter. Accordingly, we sequenced 1 kb of the SHC1 promoter region; however, no variation was found among centenarians or controls.

somes analyzed, despite a reported frequency of 4% in the white population [15]. Homozygotes for SNP2, SNP3, and SNP4 were identified only among the younger controls. The other polymorphisms showed no significant difference in distribution between centenarians and con- trols. For each polymorphism the major allele showed a high frequency (>95%; Table 3). The genotype frequen- cies within our sample fitted Hardy-Weinberg equilibrium expectations with remarkable fidelity (data not shown).

Pairwise LD and haplotype analysis for SHC1

Estimation of the strength of pairwise LD allows detec- tion of risk variants by analyzing associated markers in LD. The r2 value is used in LD analyses because the power to detect a risk variant indirectly in n samples is equivalent to the power to detect it directly in nr2 samples [16]. The strength of LD for each pair of SNPs was measured using the r2 value for a total of 410 (230 centenarians and 180 controls) sets of genotypes. Figure 2 shows pairwise r2 values (1–0.9, blackened boxes) for assessment of patterns of significant disequilibrium values. Only two SNP pairs, SNP2/SNP3, and SNP6/

Association analyses were then performed using allelic and haplotype frequencies of observed SNPs in centenar- ians and control samples (Tables 3, 4). Although not statistically significant, we found some notable differ- ences in SHC1 between centenarians and younger controls. SNP8, with a reported frequency of 4% in the white population [15] was observed only in centenarians.

novel transforming protein (SHC) with an SH