Genetic polymorphisms of Fas (CD95) and FasL (CD178) in human longevity: studies on centenarians. M Pinti, L Troiano, M Nasi, L Moretti, E Monterastelli, A Mazzacani, C Mussi, P Ventura, F Olivieri, C Franceschi, G Salvioli, and A Cossarizza conducted a study on apoptosis in aging. Aging causes changes in the balance between cell proliferation/activation and cell loss, which leads to immunosenescence. The study aimed to investigate the genetic regulation of Fas and FasL in aging and longevity. CD95 (Fas/Apo-1) is a type 1 transmembrane protein belonging to the tumor necrosis factor (TNF) receptor superfamily that plays an essential role in apoptosis pathways. Fas regulates immune processes such as selection of T cell repertoire and deletion of self-reactive cells. It can be bound by its natural ligand called FasL (CD178), a protein member of TNF superfamily. Cross-linking of Fas by FasL leads to trimerization of death domain (DD) of Fas in the inner membrane surface; the trimeric Fas recruits an adapter protein called the Fas associated DD (FADD), forming a death-inducing signaling complex (DISC). Procaspase 8 is autolytically cleaved into active caspase 8, which activates caspase 3 and starts a complex signaling pathway that leads to apoptosis.

The study analyzed the polymorphisms of 50 centenarians and 86 young donors living in Northern Italy for both Fas and FasL. The first Fas polymorphism at position 7670 has a heterozygosity of 51% in Caucasians. The second polymorphism at position 71377 has a wild type allele (G) with a frequency of 83% compared to the mutant allele. The study found that genotype and allele distribution for both polymorphisms were similar in controls and centenarians. Similar results were found for two FasL polymorphisms (IVS2nt-124 and IVS3nt169). The data suggest that Fas and FasL polymorphisms, as well as their haplotypes, are unlikely to be associated with successful human longevity.

The expression of Fas increases with age in CD4+ and CD8+ cells. This increase is present in both nave (CD45RA+, CD62L+) or memory T lymphocytes (CD45R0+, CD62L7), suggesting that the shift from nave to memory is not solely responsible for this phenomenon. The upregulation of Fas, along with reduced expression of bcl-2, has been correlated to the existence of an age-related increased susceptibility to apoptosis in CD4 and CD8 T cells. However, little is known about the molecular mechanism that modifies the expression of Fas in lymphocytes of elderly subjects or its transcriptional regulation, and it is not known whether and how aging influences the activity of this gene.

In conclusion, the study suggests that genetic polymorphisms of Fas and FasL are unlikely to be associated with successful human longevity.subjects

Genotype Group IVS2nt-124 IVS3nt-169 Controls Centenarians T/T T/T 46 (60.5%) 26 (61.9%) T/T delT 23 (30.3%) 12 (28.6%) delT T/T 6 (7.9%) 4 (9.5%) delT delT 1 (1.3%) 0 w2=0.298 P=0.586

We conducted a study on the possible association between two Fas promoter polymorphisms (7670 and 71377) and longevity in a group of 86 young controls and 50 centenarians. We estimated haplotype frequencies using a sensitive method and found no significant differences between the two populations, although some combinations of genotypes were lacking due to the absence of A/A genotype at position 71377 in centenarians.

We also analyzed the frequency of FasL genotypes and haplotypes in both groups and found no significant differences between the whole groups or between female subgroups.

In conclusion, our study found no evidence of an association between these polymorphisms and longevity in centenarians, suggesting that other factors may play a role in successful aging.Genotype Group IVS2nt-124 IVS3nt169 Controls Centenarians A/A T/T 29 (38.2%) 23 (54.7%) A/A T/delT 12 (15.8%) 6 (14.3%) A/A delT/delT 6 (7.9%) 1 (2.4%) A/G T/T 26 (34.2%) 10 (23.8%) A/G T/delT 3 (3.9%) 1 (2.4%) A/G delT/delT 0 1 (2.4%)

G/G T/T 0 0 G/G T/delT 0 0 G/G delT/delT 0 0

w2=6.083 P=0.298

B Haplotype frequency estimation for FasL polymorphisms in control and centenarian subjects. For each frequency, the calculated mean+standard deviation is reported

Haplotype Frequency in controls Frequency in centenarians 
A T 63.1+2.9% 74.4+7.5% 
A delT 17.7+2.7% 11.3%+5.7% 
G T 19.1+2.9% 12.5%+4.4% 
G delT 0 1.7+1.7%

P=0.326

There was an unexpected increase in parameters linked to inflammation, such as the capacity to produce proinflammatory cytokines, including TNF, which is involved in the induction of apoptosis in a variety of cell types that could confer an advantage in reaching a far advanced age.

The 71377 polymorphism is situated in the binding site for SP-1 transcription factor, and the presence of A in both alleles could significantly modify the transcription of Fas, with important consequences for the equilibrium of Fas/FasL apoptotic pathway. We are tempted to speculate that the presence of the homozygous form of this mutation could be harmful, and so the occurrence of A/A genotype in 71377 position is strongly negatively selected. However, young controls who have this genotype apparently do not show any disorder that can be associated with impaired expression of Fas. This point needs further investigation and can be clarified by direct quantitation of Fas mRNA in lymphocytes or other cells obtained from these subjects, as well as by in vitro analysis of Fas promoter activity with A or G in position 71377. Electro-mobility shift assays could further elucidate the eventual modifications occurring in SP-1 binding when A allele is present in both copies of the Fas gene. Finally, it will be crucial to investigate these polymorphisms in subjects of other age-groups.