Experimental Gerontology 38 (2003) 1065-1070 www.elsevier.com/locate/expgero Variability of the SIRT3 gene, human silent information regulator Sir2 homologue, and survivorship in the elderly. Rose, S. Dato, K. Altomare, D. Bellizzi, S. Garasto, V. Greco, G. Passarino, E. Feraco, V. Mari, C. Barbi, M. BonaFe, C. Franceschi, Q. Tan, S. Boiko, a Department of Cell Biology, University of Calabria, Rende 87030, Italy b Italian National Research Center on Aging, INRCA, Cosenza, Italy c Department of Molecular Pathology, University of Bologna, Bologna, Italy d Department of Clinical Biochemistry, University of Southern Denmark, Odense e Max Planck Institute for Demographic Research, Rostock, Germany Received 16 April 2003; received in revised form 18 July 2003; accepted 25 July 2003 Abstract The human sirtuin 3 (SIRT3) gene encodes a putative mitochondrial NAD-dependent deacetylase (SIRT3), which belongs to the evolutionarily conserved family of sirtuin 2 proteins. Studies in model organisms have demonstrated that SIR2 genes control lifespan while no data are available regarding a possible role of SIRT3 in human longevity. By analyzing the genotype-specific survival function relevant to the G477T marker of SIRT3, we found that in males the TT genotype increases (p < 0.0272), while the GT genotype decreases (p < 0.0391) survival in the elderly. Since SIRT3 lies in a chromosomal region (11p15.5) where four genes potentially associated with longevity are located (HRAS1, Insulin-like Growth Factor 2, Proinsulin, and Tyrosine Hydroxylase), we tested for linkage-disequilibrium between G477T alleles and alleles of the above genes. The disequilibrium was not significant in any case, thus suggesting that SIRT3 itself, or a gene strictly linked to SIRT3, may have a role in human longevity. Keywords: SIRT3 gene; Human longevity; Silent Information Regulator 2 homologue. 

1. Introduction. The role played by Sir2p as NAD-dependent protein deacetylase links cellular metabolism, transcriptional silencing and aging (Guarente, 2000), and it has been proposed that SIR2 genes may regulate aging in many species, possibly by coordinating the pace of aging to the metabolic rate (Guarente and Kenyon, 2000; Guarente, 2001). The SIRT3 gene (www.ncbi.nlm.nih.gov/omim: MIM 604481; contig NT_035113) lies at the telomeric terminal on the 11p15.5 chromosome. Allelic association studies carried out in people older than 100 years showed that a relationship exists between longevity and polymorphism of four genes located in this region. In particular, by analyzing an STR marker of the Tyrosine Hydroxylase (TH) gene, as well as the haplotypes defined by this marker and RFLPs of Proinsulin (INS) and Insulin-like Growth Factor 2 (IGF2) genes, significant age-related variations of the allelic pool had been observed (De Luca et al., 2001; Tan et al., 2002); similar age-related variations were also found for the 30 VNTR marker of the HRAS1 gene (Bonafe` et al., 2002). The HRAS1 gene (contig NT_035113) lies at 0.665 Mb from the telomere, while the genes IGF2, INS and TH lie at 2.112, 2.140, and 2.145 Mb, respectively, from the telomeric terminal and are comprised within the contig NT_028310. Therefore, within a short piece of chromosome, five genes potentially involved in longevity are located.

2. Materials and methods. An appropriate campaign of recruitment was launched in Calabria (southern Italy) for genetic studies on aging in 1999, except for centenarians whose recruitment started beginning from 1994. The recruitment campaign was focused on university students and staff for 18-60 years old people, thermal baths and aged people-Academy for 60-80 years old people. As to the centenarians, they were identified through the birth registers of the Municipalities of Calabria and then contacted. Only subjects born and living in Calabria were invited to take part in the study and after a detailed explanation of the researchParticipating cohorts in the cross-sectional study were assessed for linkage disequilibrium D using the method described by Weir (1996). The null hypothesis D = 0 was tested between the G477T marker of SIRT3 and previously analyzed markers at the loci HRAS1 (Bonafè et al., 2002) and IGF2, INS, TH (De Luca et al., 2001). Haplotypic frequencies were estimated using Maximum Likelihood Estimation (MLE) with Arlequin 1.1 software. 

A 738 bp DNA fragment was extracted from the blood buffy coats of 50 randomly selected subjects. Variability in the evolutionary conserved domain of the SIRT3 gene (exon 2-exon 3) was searched for, identifying a silent G/T transversion at position 477 of the coding region (G477T corresponding to Ser159Ser; AF083108). This marker was used to investigate possible modifications of the SIRT3 gene pool in people of increasing age, including the oldest old. In males, the TT genotype increased (p = 0.0272), while the GT genotype decreased (p = 0.0391) the value of the survival function; the GG genotype did not significantly affect survival function (p = 0.871) (Figs.13). 

The association between SIRT3 variability and survival was found to be restricted to males, as previously observed for other loci (see Franceschi et al., 2000; De Luca et al., 2001 and references therein). Since the association between SIRT3 and survivorship could be due to allelic associations, we tested for linkage disequilibrium (LD) between the G477T marker of SIRT3 and markers of other genes spanning for 1.5 Mb about in the same region and associated with longevity (HRAS1, IGF2, INS, TH). LD was checked between the G477T polymorphism and HRAS1-30 VNTR; IGF2-Ava II-RFLP; INS-Fok I-RFLP, and HUMTHO.1-STR. Results are shown in Tables 1-4, respectively, where it can be seen that the null hypothesis D = 0 cannot be rejected in any case.