Polymorphisms in the coding region of the MT1a gene are associated with longevity in the Italian Central female population. Metallothioneins (MTs) play a crucial role in zinc-related cell homeostasis due to their high affinity for zinc, which is fundamental for immune response and antioxidant activity. MTs regulate zinc homeostasis by binding and releasing zinc when required for immune response. However, this release is limited in chronic inflammation and ageing. Certain polymorphisms of MT genes, particularly the MT1a sub-isoform, may affect this release, which is still an unresolved problem in ageing. 

The screening of the MT1a gene has revealed that the polymorphism corresponding to an A/C (Asp/Thr) transition at position 647 nt in the Mt1a coding region is more involved in longevity, at least in old women, rather than the other corresponding to A/G (Lys/Arg) transition at 1,245 nt position. In addition, old and very old females with Asp/Asp genotype (called C-carriers) display higher zinc release by MT (detected by Zinpyr-1 fluorescent probe in presence of NO donor), low MT levels and reduced IL-6 plasma concentrations. This suggests their involvement in longevity and lower inflammatory status. This fact is confirmed by the analysis of haplotypes in which the allele Asp is more involved in longevity.

Therefore, the +647 MT1a polymorphism affects MTs induction and zinc release more, which are indispensable to control inflammatory status and subsequently reach healthy longevity. This study aims to screen these two single nucleotide polymorphisms in the MT1a coding region from a healthy Italian old female population as a tool for better understanding genetic influences on successful ageing. Moreover, their allelic frequency distribution was studied in association with intracellular zinc ion bioavailability and zinc release by MTs using specific fluorescent zinc probe (Zinpyr-1) and NO donor (Dyethilamine-NOnoate acetoxymethylated) (AcOM-DEA/NO), because NO is involved in zinc release by MTs via s-nitrosylation.

We screened two single-nucleotide polymorphisms (SNPs) found in the dbSNP database (http://www.ncbi.nlm.nih.gov/SNP/) (PubMed Reference rs 8052394; rs 11640851) corresponding to an A/G transition at +1245 nt position and an A/C transition at +647 nt position in the coding region of the human MT1a gene. The first polymorphism corresponds to an amino acid transition from lysine in the MT1a-A(adenine) to arginine in the MT1a-G (guanosine), while the second one corresponds to an amino acid transition from asparagine in the MT1a-A (adenine) to threonine in the MT1a-C (cytosine). We performed a PCR-restriction fragment length analysis (PCR-RFLP) using the following primers: +1245 A/G MT1a sense 5-ATCTCCCATCTCCGACAGTG-3 antisense 5-AAAGAAAACCAGAGCCAG- and +647 A/C MT1a sense 5-CGGCTTGTTGTCCTTACTGG-3 antisense 5-TGGGCAAGGTTCTTGTTTCC-.

251 healthy old and very old females were enrolled for the study, and all subjects were judged to be in good health based on their clinical history and blood tests. The healthy old subjects were recruited according to the immunogerontological SENIEUR protocol, while nonagenarians were recruited according to their grade of activities of daily living. Amplification was accomplished by initial incubation at 94°C for 5 min, followed by 35 cycles at 94°C for 1 min, 60°C for 30 s, and 72°C for 1 min and a final extension at 72°C for 7 min. The products of PCR were digested with Pst-I at 37°C overnight (Roche, Germany), then separated on a 3% agarose gel and stained with ethidium bromide. The presence of adenine (A allele) at locus 1,245 within the coding region of MT1A produced one fragment of 390 bp, whereas the presence of guanine (G allele) generated two fragments of 199 bp and 191 bp after Pst-I digestion. 

In conclusion, the study highlights the importance of MT1a gene polymorphisms in relation to longevity and inflammatory status in ageing females. The +647 MT1a polymorphism affects MTs induction and zinc release more than the +1245 MT1a polymorphism, and the Asp/Asp genotype is associated with higher zinc release by MT, low MT levels and reducedBromide. The presence of adenine (A allele) at locus 647 within the coding region of MT1A produced one fragment of 187 bp, whereas the presence of cytosine (C allele) generated two fragments of 140 bp and 47 bp after Mnl-I digestion. Genomic DNA from peripheral blood leukocytes taken from old and nonagenarian healthy subjects, extracted by the phenol chloroform method, was amplified with the primers described above. Conditions for amplification were as follows. 

Haplotype analysis: Using the acquired allele data, +647 MT1a and +1245 MT1a, two loci haplotypes were estimated by the expectation-maximization algorithm using the Arlequin software package. To test for departures from Hardy-Weinberg a Markov-chain random walk algorithm was used (Guo and Thompson 1992) also implemented in Arlequin.

PBMC recovery and storage: Twenty-five microliters of PCR mixture was composed of genomic DNA samples (200 ng), 300 μmol/l dNTPs, 2 mM MgCl2, 1 Taq polymerase buffer, 10 pmol of each primer, 2.5 U of Taq DNA polymerase (Eppendorf, Germany). Individual blood samples from old healthy females were drawn in plastic syringe containing heparin. PBMCs were obtained by ficoll-hypaque (gradient density d = 1.077) (Biochrom AG, Berlin, Germany) and centrifuged at 1500g for 15 min at 20°C. All samples presented viability higher than 90% assessed by Trypan blue dye exclusion. Aliquots of cells (2x106 cells) were frozen in FCS containing 10% of DMSO under liquid nitrogen until use. Before using, the cells were gently defrosted and counted.