duplicate HP*2 homozygous individuals (Table 1). No significant differences were observed between age groups either considering total HP*1F and HP*1S allele frequencies or according to HP 1/2 genotypes. 

3. Discussion

The present study investigated the association between HP polymorphisms and longevity in a Central Italy population. Our results showed that individuals carrying the HP*1/*1 genotype have an increased probability of attaining longevity, whereas carriers of the HP*2 allele have a significant disadvantage in reaching Age Class 2.

The role of HP in aging process is warranted by its multiple functions, including antioxidant, anti-inflammatory, and immunomodulatory activities. The ability of HP to scavenge free hemoglobin and to modulate immune response makes it an attractive candidate gene for human longevity.

Our findings are in agreement with previous studies suggesting a positive association between HP*1/*1 genotype and longevity [10-12]. However, the molecular mechanism underlying this association remains unclear. It has been suggested that the protective effect of the HP*1/*1 genotype may be related to its ability to scavenge free hemoglobin more efficiently than other genotypes [13].

In addition, our results show that carriers of the HP*2 allele have a significant disadvantage in reaching Age Class 2. This observation is consistent with previous reports suggesting that the HP*2 allele may be associated with increased susceptibility to infections and inflammatory diseases [14-16]. The presence of duplicated exons in the HP*2 allele may also affect its structure and function, leading to altered scavenging and immune-regulatory properties.

In conclusion, our study provides evidence for an association between HP polymorphisms and human longevity. Further studies are needed to elucidate the molecular mechanisms underlying this association and to explore the potential therapeutic implications of targeting HP in age-related diseases.

4. Materials and methods

4.1. Subjects

Peripheral blood was obtained from 1072 unrelated healthy individuals (569 females and 503 males) aged 18-106 years, randomly recruited from the Marche region of Central Italy. 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. All participants provided informed consent.

4.2. Genotyping

DNA was extracted from peripheral blood leukocytes using standard protocols. HP genotyping was performed by PCR amplification of genomic DNA using specific primers and restriction enzyme digestion. HP*1F/S subtyping was performed by PCR amplification followed by sequencing analysis.

4.3. Statistical analysis

The allele and genotype frequency distributions were compared between age classes using Pearson's 2 test and Armitage's trend test. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to assess the association between HP polymorphisms and longevity. Hardy-Weinberg equilibrium (HWE) was checked using the goodness-of-fit test. Statistical significance was set at p-value < 0.05.

5. Conclusion

Our study provides evidence for an association between HP polymorphisms and human longevity in a Central Italy population. Individuals carrying the HP*1/*1 genotype have an increased probability of attaining longevity, whereas carriers of the HP*2 allele have a significant disadvantage in reaching Age Class 2. The pleiotropic functions of HP make it an attractive candidate gene for age-related diseases and further studies are needed to explore its therapeutic potential.The HP*2/*2 homozygous genotype was observed singularly (Table 1). No differences were observed for Comparison 2, suggesting that the HP 1/2 polymorphism exerts its effect on human survival by 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 for a possible preferential advantage of HP*1F or HP*1S allele. Since HP*1/*1 and HP*2/*1 genotypes express two distinct phenotypes, which are extremely different at the molecular (stereo configuration, molecular weight, and subunit composition) and genetic level, F/S genotype distribution 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