All calculations were performed with the SAS system, RELEASE 8.0.

3. Results

shown in Fig. 2. The 981 bp fragment includes a p66SHC1 specific promoter area, 50 UTR and CH2 encoding region, and a p66–p52SHC1 joint region. We found no sequence variation in the p66SHC1 fragment in the sample of 30 young blood donors, nor in the sample of the 30 oldest old. As each subgroup of 30 persons represents a sample size of 60 chromosomes, the upper limit of the confidence interval for

2.5. Allelic discrimination of p66SHC1 codon 410

To discriminate between the methionine(atg) and vali- ne(gtg) genotype of codon 410 of p66SHC1 a TaqMan MGB

~195 promoter 1 transcript 223 ATG-1 553 ATG-2p66 -for > | | iil CH2 PTB p66-rev 1       

Fig. 2. Map of the SHC1 fragment used for sequencing. The black arrows represent the primers. The p66 core promoter contains five separate promoter boxes at positions 2195, 2132, 2101, 278, and 257 (Ventura et al., 2002). Position 1 demarcates the beginning of the long transcript. Exon 1 encodes the p66 specific collagen homology region (CH2) and part of the phosphothyrosine binding (PTB) domain, shared by all SHC1 proteins.

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Table 2

   TaSsT2SsTSSeS a to 9oO oO oO oO[BATAAN dAQeNUINDTQSoTaoo9037287 88 89508 36886645Age 85692nat risk 585

Frequencies of the valine allele in strata of age at death in cohort’87 of the Leiden 85-plus Study

Strata of age death 85–90 90–95 95 þ p for years years years trend* Number in stratum 126 332 272 Number heterozygotes 7 24 25 % heterozygotes 5.6% 7.2% 9.2% 0.140 val(gtg) allele frequency 2.8% 3.6% 4.6% 0.192

*p-value calculated using Chi-square for difference in frequencies between strata and linear by linear association.

the observed frequency of zero was calculated to be 2.5% for the combined group of young and old persons, and 5% within both subgroups separately. This means that the maximum allele frequency of undetected polymorphisms is 2.5 and 5%, respectively.

Allele frequencies of the valine allele in different strata of age at death in cohort’87 of people aged 85 years and over are listed in Table 2. All frequencies were in Hardy–Weinberg equilibrium. No subjects homozygous for valine were found. The frequency of Met/Val hetero- zygous carriers increases with age at death, suggesting, although not statistically significant ðp ¼ 0: 14Þ; lower mortality among the heterozygote carriers.

Fig. 3. Actuarial survival curve for the Leiden 85-plus Study. The genotypes are indicated as follows: Met/Met, white dots; Met/Val, black dots. Age is depicted in years, n is number.

Survival in both cohorts of the Leiden 85-plus Study from age 85 to 90 years is shown in Fig. 3. The precision weighted pooled mortality risk was a factor of 0.71 (0.45–1.13) decreased for Met/Val carriers compared to Met/Met carriers based on the combined observations in both cohorts. Strikingly, the same effect is observed in both cohorts independently. In cohort’87 mortality risk was 0.63 (0.29–1.36). In this subgroup 126 of the 430 (29%) subjects had died. In cohort’97 mortality risk and 95% confidence interval for Met/Val heterozygous subjects is 0.75 (0.42– 1.34). After five years of follow-up 201 of the 563 (36%) subjects had died.

with longevity; however, no statistical significance was reached in our different analyses, probably due to low Val allele frequencies.

No common sequence variation was found in the 941 bp domain specifically coding for the p66SHC1 isoform and its core promoter. This, together with the fact that in the rest of the gene (1505 bp) only one coding variant was found at a low frequency, a methionine to valine change at position 410 of p66, suggests that major variations in the SHC1 gene may not be tolerated. This is in contrast to findings in the mouse, where removing the entire exon 1 of the SHC gene resulted in longer lived mice with lower levels of oxidative stress (Migliaccio et al., 1999), lower levels of cell apoptosis (Migliaccio et al., 1999) and reduced early atherogenesis when fed a high-fat diet (Napoli et al., 2003). Our findings implicate that such sequence changes with strong effects on gene functionality do not occur naturally at high frequency in the p66 specific part of the human SHC1 gene, thus raising the possibility that evolution may have selected against their occurrence.

Although neither the trend observed for enrichment of the Val allele with increasing age of death, nor the decreased mortality risk of Met/Val carriers compared to Met/Met carriers in the separate and combined cohorts reached statistical significance, these results all hint at a similar suggestion, namely that an association of the Met410Val polymorphism with longevity may exist.

4. Discussion

Our results also suggest that an association between the Met410Val polymorphism and longevity may exist. Heterozygote carriers had lower mortality in two indepen- dent cohorts with an average decrease in mortality of 29%. These findings suggest a beneficial effect of the valine- allele. The allele frequency of the valine allele is, however,

The key finding of this study is twofold. First, we did not detect common sequence variation in the p66 specific region of the SHC1 gene that would occur of at a population frequency of at least 5%. Second, our data suggest that the Met410Val polymorphism in SHC1 may be associated

S.P. Mooijaart et al. / Experimental Gerontology 39 (2004) 263–268