Experimental Gerontology 39 (2004) 263-268

Gene variations in SHC1 and human longevity
Simon P., Diana van Heemst, Suzan van der Spoel, Marian Beekman, Bernd W. Brandt, P. Eline Slagboom, Rudi G.J. Westendorp, on behalf of the Long Life study group

a Section of Gerontology and Geriatrics, Department of General Internal Medicine, Leiden University Medical Center, P.O. Box 9503, RC Leiden 2300, The Netherlands
b Section of Molecular Epidemiology, Department of Medical Statistics, Leiden University Medical Center, P.O. Box 9600, RA Leiden 2300, The Netherlands

Received 2 July 2003; received in revised form 30 September 2003; accepted 7 October 2003 

Abstract
Mice with a specific region of the SHC gene deleted live 30% longer without disease. These mice have lower levels of oxidative stress and apoptosis which are linked to old age survival in humans. This makes SHC1 an important candidate gene for human longevity. We found no variations in the p66 specific region of the SHC1 gene in 30 young and 30 extreme long-lived subjects. Thus in humans, no common sequence variations occur in p66 specific region of the SHC1 gene. In two independent cohorts of respectively 730 and 563 subjects aged 85 and over, we tested the only known non-synonymous polymorphism, Met410Val for association with longevity using a prospective follow-up design. In the first cohort, we found increasing valine allele frequency in three strata of increasing age at death (2.8-5.2%). Moreover, compared to Met/Met carriers, mortality rate was reduced by a factor of 0.71 (95% CI: 0.45-1.13) for Met/Val carriers in the combined cohorts, with similar risk estimates in both cohorts. Low valine allele frequency resulted, however, in low power to detect statistical significance. These data suggest that an association between the Met410Val polymorphism and longevity in humans may exist.

Keywords: Longevity; Polymorphism; Insulin signaling; Human; Oxidative stress; Apoptosis

Introduction
The SHC gene, named after its Src and Collagen homologous regions, has been related to longevity in mice, but the role of SHC1 gene variants with respect to human longevity has not yet been investigated. Mice in which the p66SHC specific region of the SHC gene is knocked out (p66SHC2/2) have a higher resistance to paraquat, which causes oxidative stress, and live up to 30% longer in the absence of other phenotypic abnormalities (Migliaccio et al., 1999). Cells derived from p66SHC2/2 mice show increased resistance to apoptosis in reaction to oxidative stress (Migliaccio et al., 1999), and intracellular oxidant levels were found to be decreased (Nemoto and Finkel, 2002). Apoptosis (Campisi, 2003) and reactive oxygen species (Martin et al., 1996) have been suggested to play a role in human longevity. Recently, it was shown in mice fed a high-fat diet that deletion of the p66SHC gene reduces vascular cell apoptosis and early atherogenesis (Napoli et al., 2003). Taken together, these findings implicate the p66SHC1 gene as an important candidate gene for human longevity.

Methods
p66SHC1 signaling is associated with apoptosis and is only seen in vertebrates (Luzi et al., 2000). The SHC1 gene contains one known coding variation, which corresponds to Met225Val in the p46 isoform, Met300Val in the p52 isoform and to Met410Val in the p66 isoform (Fig. 1) (Almind et al., 1999). No association of the Met300Val variation was found with birth weight and length, impaired insulin secretion, insulin resistance, and type 2 Diabetes Mellitus (Almind et al., 1999). However, these observations do not exclude the possibility that the variant specifically affects the function of p66SHC1 and not of p46SHC1 and p52SHC1. Neither the p66SHC1 specific part of the SHC1 gene nor its upstream promoter region have been investigated for variations in humans.

Subject selection
The Leiden 85-plus Study is a population-based study in which all inhabitants of Leiden, The Netherlands, aged 85 years and over were invited to take part. Between 1986 and 1988 a total number of 977 participants were enrolled in cohort87 (SlagProcessed Text:

The sequences were assembled using the STADEN Package (Staden, 1996). A normal shotgun assembly was used, and the result was compared to the previously published sequence (GenBank accession number: AF455141). All trace files were reviewed manually for mutations. The SAS system, RELEASE 8.0, was used for all calculations. The results are shown in Fig. 2. The 981 bp fragment contains a p66SHC1 specific promoter area, 50 UTR and CH2 encoding region, and a p66p52SHC1 joint region. In the sample of 30 young blood donors and the sample of 30 oldest old, we found no sequence variation in the p66SHC1 fragment. As each subgroup of 30 persons represents a sample size of 60 chromosomes, the upper limit of the confidence interval for 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.

To discriminate between the methionine (atg) and valine (gtg) genotype of codon 410 of p66SHC1, a TaqMan MGB assay was used. Frequencies of the valine allele in different strata of age at death in cohort87 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 heterozygous carriers increases with age at death, suggesting lower mortality among heterozygote carriers.