One of the most intriguing questions to arise from our current understanding of the science of aging is whether genes associated with longevity in various animal models have the same involvement in human aging processes. Centenarians, who are the best examples of human longevity, show less evidence of oxidative stress in plasma [9] and have a lower prevalence of carotid plaques [10] than aged control subjects, suggesting a possible overlap in the aging mechanisms between humans and mice. Based on these observations, we hypothesized that the SHC1 gene, the human homologue of shc1, may have some association with exceptional longevity in humans. In this study we examined 230 Japanese centenarians and 180 healthy younger controls and looked at the SHC1 locus as a candidate region that may be associated with longevity.

Identification of single nucleotide polymorphisms

Genomic DNA and mRNA sequences of SHC1 were obtained from GenBank (accession numbers AL451085 and U73377, respective- ly). PCR primer sets were designed to amplify the SHC1 gene, including DNA 1 kb upstream of the first exon and 0.5 kb downstream of the last exon, with some overlap of PCR amplicons (Table 1). Following PCR amplification genomic DNA was sequenced using the BigDye Terminator cycle sequencing kit and an ABI Prism 3700 DNA analyzer (Applied Biosystems, Foster City, Calif., USA). In total five partially overlapping PCR fragments, each about 2 kb in length, were sequenced using 23 primers. Polymorphisms were identified by comparing sequences from 108 chromosomes from 30 Japanese centenarians and 24 younger controls, using the Sequencher program (Gene Codes, Mich., USA). Polymorphisms were confirmed by reamplifying and resequencing both strands using the BigDye Terminator cycle sequencing kit or real-time pyrophosphate DNA sequencing [13, 14] using a PSQ 96 system (Pyrosequencing, Uppsala, Sweden) and an single nucleotide polymorphism (SNP) reagent kit (Pyrose-

Table 1 Oligonucleotide sequences for polymerase chain reaction amplification of SHC1; all primer sequences are given in the 50 to 30

Table 1 Oligonucleotide sequences for polymerase chain reaction amplification of SHC1; all primer sequences are given in the 50 to 30 direction

Fragment ID Forward primer Reverse primer Amplified region of AL451085 1 AGAACTGTTTCTTCCACCTCCTC ACCTCCACACAACCCATGTACTAA 100154–97867 2 CTTCTAGCCTGCCTTTTCTTTTTC ACTACAACTCCACCATGAGCAAAC 98084–95884 3 GAGCAGCTCTTGGATTAGGATTAG GAAGGAAGAGGGATACACTGTAGG 96086–93905 4 CCACCTTTCTACTTGGTTCTGG AGGAAGAAGGTCAATTCAGGTG 94352–92384 5 CCTTTGCCTTTTCTCACTTCAC CGTACACAATAGGCTTCCCAGA 92583–90696

123° 45 67 SHCI/-—    8 910 9L

quencing), according to the manufacturer’s instructions. DNA from the remaining subjects was sequenced only across regions contain- ing confirmed polymorphisms.

Statistical analysis

The (cid:1)2 test was performed between centenarians and control subjects for each allelic and haplotype frequency. Statistical significance was inferred at P<0.05. Pairwise linkage disequilib- rium (LD) was estimated as D=x (cid:2)p p , where x is the frequency of ij i j ij haplotype A B , and p and p are the frequencies of alleles A and 1 1 1 2 1 B at loci A and B, respectively. A standardized LD coefficient, r, 1 is given by D/(p p q q ) where q and q are the frequencies of the 1 2 1 2 1 2 other alleles at loci A and B, respectively. Haplotype frequencies for multiple loci were estimated by the expectation-maximization method. Computations were performed using the SNPAlyze software (Dynacom, Mobara, Japan).

Fig. 1 SNP map of the genomic region containing SHC1. Open rectangles Exons; horizontal lines introns. SNPs are indicated above the lines according to number

tions were more prevalent (9/10) than transversion substitutions (1/10). Only one insertion/deletion polymor- phism was detected in SHC1. All SNPs were submitted to the dbSNP database in the NCBI. Accession numbers are listed in Table 2.

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 centenar- ians. SNP8 was present in only one of the 820 chromo-

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, 6 within introns, and 1 within the 30 untranslated region (Fig. 1, Table 2). Among these SNPs transition substitu-

SNP ID Location Position in Nucleotide Function dbSNP ss AL451085 major/minor SNP 1 Exon 1 98491 C/T P158P ss12586030 SNP 2 Intron 1 98441 C/T  ss12586031 SNP 3 Intron 1 98440 C/T  ss12586032 SNP 4 Intron 2 97743 G/-  ss12586033 SNP 5 Intron 2 97555 C/G  ss12586029 SNP 6 Exon 3 97225 C/T A205V ss12586028 SNP 7 Intron 3 97115 G/A we ss12586027 SNP 8 Exon 9 94624 A/G M410 V ss12586026 SNP 9 Exon 11 94177 C/T S476L ss12586034 SNP 10 Intron 11 93825 C/T  ss12586025 SNP 11 Exon 12 92274 G/A L573L ss12586024 SNP 12 Exon 12 90925 C/T 30 UTR ss12586023

Table 3 Association analysis of SHC1