Human peroxisome proliferator-activated receptor-c (PPAR-c), the most extensively studied isoform of the nuclear receptor superfamily (PPAR-a, PPAR-d, and PPAR-c), has a key role in regulating lipid and glucose homeostasis, adipo- cyte differentiation, and fatty acid storage. The PPAR-c gene (PPARG, OMIM 601487), located on chromosome 3p25, con- tains 9 exons. Two protein isoforms are produced by alter- native mRNA splicing. PPARG2 is encoded by exons 1–6 and exon B.10 A polymorphic site in exon B, Pro12Ala (rs1801282), has been widely investigated in relation to various disorders, including type 2 diabetes, insulin sensitivity, obesity, cardio- vascular disease, cancer, and AD.10–13 There is wide agree- ment on a protective role of the PPARG 12Ala allele against type 2 diabetes.12 PPARG has been also suggested to be a longevity determinant in the mouse,14 and a male gender- specific association of the Pro/Ala heterozygous genotype with longevity has been previously observed.15

Allelic frequencies were determined by the gene-counting method. Agreement between the observed genotype distri- butions and those expected according to Hardy–Weinberg equilibrium was verified by an exact test. Linkage disequi- librium (D) between the two FSHR SNPs and haplotype fre- quencies were estimated by the maximum likelihood method using the EH program (http://linkage.rockefeller.edu/ott/ eh.htm/).20 The differences in allele and genotype frequencies between long-lived subjects and non-survivor controls were analyzed by a chi-squared test. The probability of surviving to age over 90 years ( ‡90 years) or not associated with the FSHR and PPARG genotypes was estimated by the odds ratios (ORs) adjusted for other variables calculated by logistic re- gression. Statistical analyses were performed using Statistix 8.0 Analytical Software.

The aim of this study was to verify a possible association between longevity and both FSHR and PPARG. We were interested to see whether the protective action FSHR exerted against AD in women, a disease of advanced age, could extend to survival, predisposing to longevity. We also wanted to examine PPARG, which is involved in suscepti- bility to numerous degenerative diseases of advanced age, AD included, for its effect on a longevity association ob- served only in men. The association between FSHR and PPARG polymorphisms and longevity, defined as survival to age 90 years and older, was investigated by means of a follow-up study.

Results

In a previous study on FSHR,9 we observed an almost complete linkage disequilibrium between the polymor- phisms at codon 307 (alleles Thr and Ala) and at codon 680 (alleles Asn and Ser). In the present study, after having ascertained in a subsample (n =96) that the two FSHR polymorphisms were in total linkage disequilibrium (FSHR 307-680 haplotype frequencies: ThrAsn =0.521, AlaAsn =0, ThrSer = 0.005, AlaSer = 0.474; D= 100% of Dmax, p < 0.0001), we typed the total sample only for polymorphism FSHR Asn680Ser. In the total sample, the observed FSHR 680 ge- notype frequencies agreed with those expected according to Hardy–Weinberg equilibrium ( p= 0.94) and the FSHR 680 Asn allele frequency was 0.531 –0.022.

Materials and Methods

The sample was recruited in 2000 for the multidisciplinary LONCILE (Longevity of Cilento) study on the anthropolog- ical and biological characteristics of the elderly population of the Cilento area in the district of Salerno, southern Italy.16 It consisted of 277 unrelated individuals (43.7% males) born between 1900 and 1930 (mean age, 82.9–5.7 years– standard deviation [SD]), enrolled without selection criteria, except age (> 70 years) and birthplace. Mortality data on 267 sub- jects were collected in 2009. In 2000, 14.5% were aged 90 years old or older. During the 9-year follow-up period, the mortality rate was 62.5% (51.5 % men); 55.1% of the subjects died before reaching age of 90 years. As the mean life ex- pectancy in this Italian geographic area in the year 2000 for subjects 83 years old is 7 years for women and 6 years for men (ISTAT, http://demo.istat.it/unitav/index.html/), we defined as long-lived those subjects who survived to over age 90 years ( ‡ 90 years). The sample of the long-lived (n =114) comprised subjects aged 90 years or older in 2000 (regardless of whether the participants were still living or

Table 1 reports the distribution of the FSHR 680 genotypes in the long-lived and the controls of both sexes. A marginally significant increase ( p= 0.07) in FSHR 680 Ser/Ser genotype frequency (0.25) was observed in the long-lived women as compared to the control women (0.08). The crude OR for the probability of surviving to age 90 years (‡ 90 years) or not associated with the FSHR 680 Ser/Ser genotype, calculated only for the women, was 3.75 (95% CI 1.003–14.03, p= 0.039), suggesting that carrying the FSHR 680 Ser/Ser genotype favors the survival of women to 90 years and older. In contrast, no difference in the FSHR 680 genotype frequencies was observed between the long-lived and the control males.

Table Estimates of the Effects of

PPARG Pro la Genotypes on the Probability

A of Surviving to Age over Years in Men

Table and Pro la Genotype 1. FSHR 680 PPARG 12A Distribution in Long-Lived Subjects and Controls

Males Females Long-lived Controls Long-lived Controls FSHR 680 No. (%) No. (%) No. (%) No. (%) genotypes Asn/Asn 8 (22) 8 (19) 22 (37) 13 (36) Asn/Ser 20 (54) 24 (59) 22 (37) 20 (56) Ser/Ser 9 (24) 9 (22) 15 (25) 3 (8) p 0.92 0.07 PPARG genotypes Pro/Pro 45 (92) 41 (79) 57 (88) 37 (93) Pro/Ala 3 (6) 11 (22) 7 (11) 3 (8) Ala/Ala 0 0 1 (2) 0 p 0.03 0.62

Variable OR 95% CI p value PPARG Pro/Ala genotype 0.13 0.02–0.79 0.03 ESR1 PP genotype 4.78 0.99–23.7 0.05 CYP19 T carrying genotypes 1.67 0.56–4.96 0.35 Fertility level 1.55 0.50–4.79 0.44

OR, Odds ratio; CI, confidence interval.