slightly higher percentage of A/A genotype; however, the difference between these two populations cannot be considered significant (w2=0.840, with P=0.657). Since the gender distribution was different in the group of centenar- ians, we have analyzed the genotype distribution in the subgroups of males and females. The number of males (six individuals) was clearly too low to allow any statistical analysis; in the case of females (44 donors), we found that the difference in the percentage of A/A subjects between centenarians and controls was more marked (Table 1B), even if such difference did not reach the level of statistical significance (w2=3.740, with P=0.154).

isms have been identified in the coding region, but two polymorphic sites exist in the second and third intron of the gene.26 The first one, named IVS2nt-124, is an A to G substitution in the nucleotide 124 upstream from the first base of the third exon. The second polymorphism, named IVS3nt169, is a deletion of a T at position 169 downstream from the last base of the third exon. It is still not known if these polymorphisms are associated with a different expression of the gene.

71377 polymorphism in the same groups is reported in Table 2A. The percentage of G/G homozygous subjects was quite high in control subjects (76.7%), and did not differ significantly from percentage we have found in centenarians (88.0%). Heterozygous subjects were about 21% of young population, and 12% in centenarians. Among centenarians, we could not find any subject who was homozygous for the allele A, while two subjects in the control group showed this genotype. Also in the subgroup of females, the genotypes and allele frequency do not differ significantly from controls (Table 2B).

genotype set in the promoter of Fas gene or in FasL introns may contribute to successful aging, we have analyzed the frequency of these polymorphisms in a group of centenar- ians and young donors living in Northern Italy (Emilia Romagna Region).

Finally, we have analyzed 71377 and 7670 genotypes in combination, and have estimated the haplotype frequen- cies in both populations. The frequency of combined genotypes and haplotypes are reported in Table 3A and B. The distribution of combined genotypes between centenarians and controls was not significantly different (P=0.550). Some combinations of genotypes were lacking, mainly because of the absence of A/A genotype at position 71377 in centenarians.

Results

To examine the possible association between the two polymorphisms (7670 and 71377) reported for Fas promoter and longevity, a total of 86 young controls and 50 centenarians were genotyped.

The haplotype estimation, analyzed by using a sensitive method, 27 did not evidence any significant difference between the two populations. The highest differences between haplotype frequencies were in those subjects who have the allele A in 71377 position; such differences were mainly due to the absence of centenarians with A/A genotype at position 71377. In both cases, alleles are not in linkage disequilibrium (P=0.44 for centenarians, P=0.099 for young donors).

young and centenarian subjects are shown in Table 1A. In both groups A and G alleles are in Hardy-Weinberg equilibrium; the heterozygous form of MvaI polymorphism was present in 48% of the centenarians; individuals who were homozygous for A and G were 34% and 18%, respectively. In comparison to young controls, we found a

1A Genotype and allele frequency of the –670 polymorphism in young controls and centenarian subjects

Genotype Allele frequency G/G G/A A/A G A Controls (n=86) 16 (18.6%) 47 (54.7%) 23 (26.7%) 45.9% 54.1% Centenarians (n=50) 9 (18%) 24 (48%) 17 (34%) 42% 58% w2=0.840 z=0.516 P=0.657 P=0.608

Genotype Allele frequency G/G G/A A/A G A Controls (n=86) 16 (18.6%) 47 (54.7%) 23 (26.7%) 45.9% 54.1% Centenarians (n=50) 9 (18%) 24 (48%) 17 (34%) 42% 58% w2=0.840 z=0.516 P=0.657 P=0.608 B Genotype and allele frequency of the –670 polymorphism in young controls and centenarian females Genotype Allele frequency G/G G/A A/A G A Controls (n=36) 6 (16.7%) 23 (63.9%) 7 (19.4%) 48.6% 51.4% Centenarians (n=44) 9 (20.4%) 19 (43.2%) 16 (36.4%) 42% 58% w2=3.740 z=0.726 P=0.154 P=0.468

B Genotype and allele frequency of the –670 polymorphism in young controls and centenarian females

Genotype Allele frequency G/G G/A A/A G A Controls (n=36) 6 (16.7%) 23 (63.9%) 7 (19.4%) 48.6% 51.4% Centenarians (n=44) 9 (20.4%) 19 (43.2%) 16 (36.4%) 42% 58% w2=3.740 z=0.726 P=0.154 P=0.468

Cell Death and Differentiation

Table 2A Genotype and allele frequency of the –1377 polymorphism in young controls and centenarian subjects

Genotype Allele frequency G/G G/A A/A G A Controls (n=86) 66 (76.7%) 18 (20.9%) 2 (2.4%) 87.2% 12.8% Centenarians (n=50) 44 (88%) 6 (12%) 0 (0%) 94% 6% w2=3.087 z=1.162 P=0.214 P=0.106

B Genotype and allele frequency of the –1377 polymorphism in young controls and centenarian females

Genotype Allele frequency G/G G/A A/A G A Controls (n=36) 26 (72.2%) 9 (25%) 1 (2.8%) 84.7% 15.3% Centenarians (n=44) 38(86.4%) 6 (13.6%) 0 (0%) 93.2% 6.8% w2=3.081 z=1.377 P=0.214 P=0.168

Table 3A Fas combined genotype frequency in centenarian and control subjects

Genotype Group 7670 71377 Controls Centenarians G/G G/G 12 (13.9%) 6 (12%) G/G G/A 3 (3.5%) 3 (6%) G/G A/A 1 (1.2%) 0 G/A G/G 32 (37.2%) 22 (44%) G/A G/A 14 (16.2%) 2 (4%) G/A A/A 1 (1.2%) 0 A/A G/G 22 (25.6%) 16 (32%) A/A G/A 1 (1.2%) 1 (2%) A/A A/A 0 0 w2=6.876 P=0.550

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

Haplotype Frequency in controls Frequency in centenarians AA 2.1+1.4% 1.3+1.4% AG 51.9+3.9% 56.6+5.0% GA 10.6+2.5% 4.7+2.3% GG 35.7+3.6% 37.3+4.9% P=0.456