2.3. Statistical analysis

Hardy–Weinberg Equilibrium (HWE) was checked by the good- ness-of-fit test for biallelic markers. The differences in HP genotypes and allele distributions between the groups were evaluated by the Pearson's χ2 and the Armitage's trend test and the results were expressed as odds ratios (ORs) with 95% confidence intervals. These calculations were done using tests for association available at http:// ihg2.helmholtz-muenchen.de/cgi-bin/hw/hwa1.pl. The analysis of HP*1F/S polymorphism was also carried out by 2×2 contingency tables (available at http://faculty.vassar.edu/lowry/VassarStats. html) and applying Fisher's exact test (two-tailed) when an expected cell value was b5. Statistical significance was set at p-valueb0.05 for all the tests.

On the basis of these considerations, a possible association between HP polymorphisms and longevity has been investigated in the present work.

2. Materials and methods

2.1. Subjects

3. Results

Peripheral blood was obtained from 1072 (569 females and 503 males) unrelated individuals, 18–106 years old, randomly recruited from the same geographical area of Central Italy (Marche region) on the eastern side of the Apennines. The whole population studied is composed of Caucasian individuals and is ethnically homogeneous: the people are the descendants of an ancient pre-Roman Italian population called the Piceni [8]. The same donors provided informa- tion concerning their health condition so as to ascertain that no pathological condition existed (e.g. cancer, diabetes, heart diseases, hypertension, obesity, and chronic inflammatory diseases). Almost all subjects up to age of 65 were recruited in blood donor centres while those over 65 were recruited through family physicians or among home-guests for the elderly. The population under study was divided into three sex-specific age classes: for men [women], the first class consists of individuals b66 years old [b73 years old], the second class of individuals 66–88 years old [73–91 years old], and the third class of individuals N88 years old [N91 years old]. These gender-specific age classes were defined according to demographic information and account for the different survivals of men and women in the Italian population [9]. The study protocol was approved by the Joint Ethical Committee (JEC) University of Camerino-Azienda ASUR Marche ZT-10 Camerino, in accordance with the Declaration of Helsinki in its revised edition and with international and local regulatory requirements.

The allele and genotype frequency distributions of the HP 1/2 polymorphism were compared among the age classes (Table 1). No significant deviation from HWE expectation was detected among the gender-specific age classes except for females Age Class 2 (p=0.0251). Genotype distributions do not differ significantly between males and females within age classes.

In the whole population studied HP*1/*1 genotype was associated with increased probability of young subjects to attain increased life- span (Comparison 1: O.R. 1.709, 95% C.I. 1.138–2.566, p=0.0114) with a concomitant gain of HP*1 allele (Comparison 1: O.R. 1.273, 95% C.I. 1.039–1.558, p=0.0194) (Table 1). On the other side, carriers of HP*2 allele displayed an overall significant disadvantage in reaching Age Class 2 (O.R. 0.585, 95% C.I. 0.390–0.879, p=0.0092), also considering HP*2/*1 heterozygous or HP*2/*2 homozygous genotype singularly (Table 1). No differences were reported for Comparison 2, suggesting that HP 1/2 polymorphism exerts its effect on human survival acting earlier in life (Table 1).

Given the significant association of HP*1/*1 genotype and HP*1 allele with increased life-expectancy, we also performed HP*1F/S subtyping to check the possible existence for a preferential advantage of HP*1F or HP*1S allele. Since HP*1/*1 and HP*2/*1 genotypes express two distinct phenotypes, extremely different at molecular (stereo configuration, molecular weight and subunit composition) and genetic level, F/S genotype distributions comparison was conducted separately. No significant differences were noticed between age groups studied either considering total HP*1F and HP*1S allele frequencies (Table 2) or according to HP 1/2 genotypes (Table 3).

2.2. HP genotyping

Genomic DNA extraction was carried out from peripheral blood through standardized salting-out method and DNA was stored at −20 °Cuntilgeneanalysis.HP1/2genotypingwasperformedbyallele- specific PCR as described [10] with slight modifications. Genomic DNA was amplified by PCR with primers flanking the duplicated HP 1/2 polymorphic region.Sense primer:5′-AGCCCACCCCTCCACCTATGTGCC- 3′; antisense primer: 5′-GCTTAAGATCCCAGTCGCATACC-3′. The ampli- fication was performed in 20 μl reaction mix containing 80 ng template DNA,0.4 UPhusionHighFidelty-DNApolymerase(Finnzymes,Finland), 0.5 μMofeachprimerand200 μM(finalconcentration)ofdNTPsand1× Phusion HF buffer (Finnzymes). PCR was performed on a Biometra T- gradient thermal-cycler with initial denaturation at 98 °C for 3 min, followed by 30 cycles of a two-step protocol consisting of denaturation at 98 °C for 15 s and annealing/extension at 72 °C for 3 min and 45 s followed by a final extension of 3 min at 72 °C. HP 1/2 genotypes were identified by 0.8% agarosegelelectrophoresis. The HP*1and HP*2alleles yield a3.245-anda4.945-bpband,respectively. HP*1F/Ssubtypingwas performed by RFLP-PCR method according to Koch et al. [11] using primers A (5′-GAGGGGAGCTTGCCTTTCCATTG-3′) and B (5′- GAGATTTTTGAGCCCTGGCTGGT-3′), and restriction endonuclease DraI (Fermentas). All samples were genotyped in duplicate and the accuracy ofHP1/2genotypingwasassessedbycomparisonwiththeresultsofHP protein profiling performed with plasma samples of 65 young individuals.

4. Discussion