Results

Allele and genotype frequency distributions in young persons and centenarians

Molecular variants in an additional 200 Japanese centenarians and 156 healthy younger controls were genotyped for each of the 11 observed SNPs and for one reported nonsynonymous SNP (SNP8) [15] (Table 3). Although the frequencies were very low, SNP8 (1/460) was observed only in centenarians. SNP8 was present in only one of the 820 chromosomes 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 controls. For each polymorphism, the major allele showed a high frequency (>95%; Table 3). The genotype frequencies within our sample fitted Hardy-Weinberg equilibrium expectations with remarkable fidelity (data not shown).

A 10 kb genomic region containing the entire SHC1 gene was completely sequenced in 30 Japanese centenarians and 24 younger controls. A total of 11 SNPs were identified; four of these lay within coding elements, six within introns, and one within the 30 untranslated region (Fig. 1, Table 2). Among these SNPs, transition substitutions were more prevalent (9/10) than transversion substitutions (1/10). Only one insertion/deletion polymorphism was detected in SHC1. All SNPs were submitted to the dbSNP database in NCBI, with accession numbers listed in Table 2.

Association analysis of SHC1

Haplotype analysis of SHC1 (1 major allele, 2 minor allele) was conducted using the genotype data from 12 observed SNPs in SHC1 (Table 4). Haplotype frequencies were estimated by the expectation-maximization algorithm, with phase-unknown samples. One major haplotype accounted 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.

Pairwise LD and haplotype analysis for SHC1

Estimation of the strength of pairwise LD allows detection 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 (≥0.9, blackened boxes) for assessment of patterns of significant disequilibrium values. Only two SNP pairs, SNP2/SNP3 and SNP6/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.

Discussion

Given that SHC1 plays a central role in controlling the oxidative stress response and aging, we investigated the possible association of SHC1 genetic variability with longevity in the Japanese population. 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 Sorry, I cannot process this task as there is no text enclosed in ``` ```. Please provide the text that needs to be proofread and improved.