2.2. HP genotyping

Genomic DNA extraction was carried out from peripheral blood through a standardized salting-out method, and DNA was stored at -20°C until gene analysis. HP 1/2 genotyping was performed by allele-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 amplification was performed in a 20 μl reaction mix containing 80 ng template DNA, 0.4 U Phusion High-Fidelity DNA polymerase (Finnzymes, Finland), 0.5 M of each primer, and 200 M (final concentration) of dNTPs and 1 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% agarose gel electrophoresis. The HP*1 and HP*2 alleles yield a 3245- and a 4945-bp band, respectively. HP*1F/S subtyping was 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 of HP1/2 genotyping was assessed by comparison with the results of HP protein profiling performed with plasma samples of 65 young individuals.

4. Discussion

The results of the present study indicate that HP 1/2 polymorphism affects the probability of reaching extreme old age by acting early in adult life (Comparison 1). To our knowledge, this is the first report relating the HP genetic polymorphisms and human in a large group of elderly individuals. However, previous studies examined the age distribution of HP 1/2 polymorphism in subjects from three other populations, Chinese Fujian Han Jordanians [13] and Polish [14], respectively, which are very distinct for In our study, the frequencies of HP*1/*1 genotype and HP*1 allele were found to be significantly higher in the elderly groups than in the younger group, suggesting that possessing this genotype could confer a survival advantage for attaining longevity. Furthermore, this evidence is sustained by the above-mentioned studies, reporting significant age-dependent increase of HP*1 allele, with Polish study evidencing a considerable increase of HP*1/*1 genotype in subjects aged 81-91 years [14]. On the other side, both HP*2/*1 and HP*2/*2 carriers display a significant deleterious effect compared to HP*1/*1 homozygous.

Table 1

HP 1/2 polymorphism: genotype and allele frequencies in age classes studied. Values showing significant differences are shown in bold. O.R. = odds ratio. *Assuming HP*2 as the risk allele.

HP Groups Age Class 1 Age Class 2 Age Class 3 Males (b66 y, N=144) Males (66-88 y, N=261) Males (N88 y, N=98) Females (b73 yComparison 2: Age Class 3 vs. Age Class 2 vs. Age Class 1 

Age Class 1 Age Class 2 Age Class 3 

O.R. 95% C.I. p-value O.R. 95% C.I. p-value O.R. 95% C.I. p-value 

1*F 93 (42.5%) 178 (42.5%) 55 (37.7%) 

1.001 0.71-0.1393 1.000 0.818 0.556-1.205 0.330 

1*S 126 (57.5%) 241 (57.5%) 91 (62.3%) 

0.999 0.718-1.391 1.222 0.830-1.799  

Table 3 

HP*1 F/S polymorphism in carriers of HP*1/*1 and HP*2/*1 genotype.

Groups Comparison 1: Age Class 2 vs.

 Comparison 2: Age Class 3 vs.

Age Class 1 Age Class 2 Age Class 3 

Armitage's trend test p-value Armitage's trend test p-value Armitage's trend test p-value 

N=37 N=96 N=33 O.R.a O.R.a 

F*/F*  	8(21.6%)  	27(28.1%)  	7(21.2%)  	0.955  	0.917  	1.173  	0.558 

F*/S*  	15(40.6%)  	28(29.2%)  	11(33.3%) 

S*/S*  	14(37.8%)  	41(42.7%)  	15(45.5%) 

HP*2/*1 Age Class 1 Age Class 2 Age Class 3 

O.R. 95% C.I. P value O.R. 95% C.I. P value O.R. 95% C.I. P value 

N=145 N=227 N=80 

F*  	62(42.8%)  	96(42.3%)  	30(37.5%)  	0.981  	0.644-1.495  	1.000 0.819 0.485-1.382 0.509 

S*  	83(57.2%)  	131(57.7%)  	50(62.5%)  	1.019  	0.669-1.554  	1.221 0.724-2.062  

a Assuming HP*1S as risk allele.

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