The 11p15.5 area is of particular interest as it encloses five genes (HRAS1, SIRT3, TH, INS and IGF2), the variability of which was found to be associated with human longevity.1–7 Most importantly, the above genes are homologous of genes that are known to modulate lifespan in model organisms (http://genomics.senescence. info/). As for HRAS1, an earlier study of a central-northern Italian sample showed that the frequency of a particular VNTR allele decreases in centenarians compared with that in young people, and it was estimated that, during ageing, carriers of this allele have a relative mortality risk of 1.126, with a 95% CI of 1.044–1.213.2 Likewise, by analyzing the genotype-specific survival function relevant to the G477T marker of SIRT3 in a southern Italian population, we found a male-specific association between the marker and survival in the elderly.7 A subsequent study showed that the association was because of linkage disequilibrium between the G477T marker and a VNTR allele-specific enhancer located in intron 5 of SIRT3. In particular, the 2b allele was absent in male centenarians, whereas it was significantly more frequent in younger males.1 Similarly, in both southern and northern Italian males, the functional microsatellite polymorphism (TCAT)n located in intron A of the TH gene showed association with life extension.4,8 In addition, the entire TH-INS-IGF2 region was found to be associated with longevity in southern Italians; in particular, the frequency distributions of TH-INS haplotypes and of TH-IGF2 haplo- types were significantly different between centenarians and younger controls in both females and males.5

Results

A number of positive associations were observed and filtered through the different analysis approaches, to correct for multiple testing and to take into account the different powers of the analysis methods.12 However, the results were not concordant in the three populations (Central Italy, South Italy and Germany).

No significant results (Po0.05) were found for of the earlier associated loci (ie TH, IGF2, INS HRAS1).1–4,6–8,13–18

An exception was SIRT3, for which different markers showed significant association signals in the populations, that is, the association was present in Italian females and German males.

Among the different genes, a specific interest was thus focused on the SIRT3 locus, given the extensive literature, suggesting an association of sirtuin genes with longevity19 and mitochondrial functionality,16 an important topic in ageing and longevity.20

A replication study was then designed for SIRT3: eight SNPs were tested in a French sample (546 cases–age range 99–106 years old, mean 103.1, and 315 controls–age range 18–70 years old, mean 51.2) at CEPH in Paris. None of the earlier findings were confirmed in this analysis.

To increase the power of the analysis and to gain a deeper insight into the genotype data, a meta-analysis was carried out on the SIRT3 SNP data. A total number of 2461 samples were included by using the inverse variance weighting for pooling in the presence of a fixed-effects model,21,22 and the Der Simonian–Laird estimation in the random-effects model.23 The analysis was carried out at the level of the rare and frequent genotypes, and at the heterozygous and allelic level. No positive association was found except for one SNP having a significant effect (rs939915); unfortu- nately, the genotyping of this marker in central Italians resulted in a considerable number of dropouts and the false discovery rate (q-value of 0.145) indicates that this result is not robust enough (Table 1).

On the whole, the above findings indicated that, at least in the investigated populations, and in some cases in a sex-specific manner, the 11p15.5 region harbors gene variants associated with life extension.

Therefore, instead of focusing on candidate genes, the aim of this study was to extend the earlier analyzes by investigating the entire 11p15.5 region (2.4Mb) with a set of suitable single-nucleotide polymorphisms (SNPs).

The same meta-analysis approach was also applied to the other markers for a better understanding of the Italian and German population results. A meta-analysis of all the 239 11p15.5 markers on 1600 samples was then carried out.

Materials and methods

A set of 239 SNPs (tagSNPs and htSNPs) were selected on the basis of HapMap data from CEPH trios that were found

The results were filtered by applying a false discovery rate analysis24–26 to the P-values resulting from the model adopted for each marker in the meta-analysis.

This experience, although unable to confirm the earlier findings, highlights all the common difficulties of such studies in human longevity.

Six SNPs were found to be significant for the frequent genotypes (rs4073591, DEAF1-rs4073590, KRTAP5-6-rs11040489, rs4930001, TSPAN32-rs800140 and rs16928120) at a q-value p0.36, confirming a few of the positive associations obtained in the original first three analysis approaches. Data on KRTAP5-6_rs11040489, rs4930001, TSPAN32_rs800140 and rs16928120 were significant in the allelic analysis too (Table 2).

tions) and statistical (multiple analysis correction, false-positive rate, meta-analysis criteria, among others) problems that become apparent when a complex trait such as longevity is addressed represent a hot topic in this field. Our choice was focused on combining different methods rather than discarding useful information. Instead of compromising on power, we filtered the signals through the consistency of the different analyses and by applying a false discovery rate approach to the final results.

Two findings were considered interesting: DEAF1 encodes a transcription factor binding the retinoic acid response element and activating the proenkephalin gene; when secreted, it is a suppressin, and stops cells in G0/G1. TSPAN32 codes for a tumor-suppressing fragment, and is located in the imprinted region of 11p15.5.