Results

PON1 192 alleles and genotypes in young people and centenarians

Materials and methods

No significant difference was present between Northern and Southern Italians either in centenarians or in controls (data in http://biologia.unical.it/labs/genetica.html). Table 1 re- ports genotypes and alleles of the PON1 polymorphism at codon 192 in the whole Italian sample. In both the age- classes the observed genotypes were in agreement with those expected at Hardy-Weinberg Equilibrium (P40.05). Signifi- cant differences between young people and centenarians were evident when allele and genotype frequency distribu- tions were compared (w2=5.429, df=1, P=0.020 for allele frequency, and w2=6.798, df=2, P=0.034 for genotype frequency). In particular, the proportion of B+ subjects (AB+BB individuals) was increased in centenarians in comparison to young people (w2=6.765, df=1, P=0.011). The role of gender as a confounding variable was tested by MH test, and no significant result was found (data not shown).

Subjects

A sample of 579 Italian unrelated young individuals (347 males and 232 females, median age=40.5 years) ranging from 20 to 65 years of age and a sample of 308 Italian unrelated centenarians (241 females and 67 males) were analysed. The samples were recruited in both Northern Italy (368 young subjects and 216 centenarians) and Southern Italy (211 young subjects and 92 centenarians). Control subjects were apparently healthy at the time of blood collection judging from clinical examination and recent clinical history. All the subjects gave their informed consent.

Genotyping

DNA was extracted from blood lymphocytes by the salting- out method.13 Polymerase chain reactions were performed using primer sequences derived from published data and specific for the amplification of regions surrounding codon 192 and codon 55 sites.14 The amplification reaction for codon 192 locus was performed on a Perkin-Elmer Cetus 9700 thermal cycler with initial denaturation at 948C for 4 min, followed by 40 cycles, each one comprised of denaturation at 938C for 1 min, annealing at 648C for 40 s and extension at 728C for 1 min, with a final extension time of 10 min at 728C. The PCR products were digested with AlwI (New England Biolabs) for 4 h at 378C, and the samples were electrophoresed in 2% agarose gels. Amplification of the codon 55 polymorphism was performed on a Perkin-Elmer Cetus 9700 thermal cycler with initial denaturation at 968C for 3 min, followed by 35 cycles, each one comprised of denaturation at 958C for 1 min, annealing at 568C for 1 min and extension at 728C for 1 min, with a final extension time

PON1 55 alleles and genotypes in young people and centenarians

Again no geographic difference was observed either in centenarians or in young control subjects for PON1 codon 55 variants (data in http://biologia.unical.it/labs/geneti- ca.html). Table 2 shows genotypes and alleles in the whole Italian sample. In both centenarians and young people the observed genotypes were in agreement with those expected at Hardy-Weinberg Equilibrium (P40.05). Unlike what has been observed at codon 192, allele and genotype frequency distributions were not statistically different between young people and centenarians. Moreover, the M+ subjects (LM plus MM individuals) were present at the same proportion in the two age-classes (0.598 vs 0.607, respectively).

Table 1 Genotype and allele frequency distributions at PON1 192 locus

Combined analysis of PON1 192/55 loci in young people and centenarians

Since the haplotype estimation from population data is found on the assumption of homogeneity of the population, and is therefore very sensitive to possible stratification, haplotype analysis in young people and centenarians was carried out first in Northern and Southern Italians separately (data in http://biologia.unica- l.it/labs/genetica.html). As no significant geographic dif- ference was observed either in young people or in centenarians, the following analyses were carried out in the whole Italian sample. The results are shown in Table 3. A significant difference in estimated haplotype frequency distributions was found between young people and centenarians (Fisher Exact Test, P=0.012). The difference was predominately due to an increase of BM haplotype in centenarians (1.4%) in comparison to that found in young people (0.3%).

Young subjects Centenarians (n=579) n (%+s.e.) (n=308) n (%+s.e.) Genotypesa Observed Genotypesa Observed Genotypesa AA subjects 320 (0.553+0.021) 142 (0.461+0.028) AB subjects 212 (0.366+0.020) 137 (0.445+0.028) BB subjects 47 (0.081+0.011) 29 (0.094+0.017) B+ subjectsb 259 (0.447+0.021) 166 (0.539+0.028) Allelesc A allele 852 (0.735+0.013) 421 (0.683+0.019) B allele 306 (0.265+0.013) 195 (0.317+0.019)

aw2=6.798, df=2, P=0.034; bw2=6.765, df=1, P=0.011; df=1, P=0.020.

Table 2 Genotype and allele frequency distributions at PON1 55 locus

Young subjects Centenarians (n=579) n (%+s.e.) (n=308) n (%+s.e.) Genotypesa Observed Genotypesa Observed Genotypesa LL subjects 233 (0.402+0.020) 121 (0.393+0.028) LM subjects 262 (0.453+0.021) 155 (0.503+0.028) MM subjects 84 (0.145+0.015) 32 (0.104+0.017) M+ subjectsb 346 (0.598+0.020) 187 (0.607+0.028) Allelesc L allele 728 (0.629+0.014) 397 (0.644+0.019) M allele 430 (0.371+0.014) 219 (0.356+0.019) aw2=3.754, df=2, P=0.155; bw2=0.077, df=1, P=0.829; cw2=0.433,

combination of genotypes at codons 192 and 55 (Table 4), and we found that their distributions were significantly different between young subjects and centenarians (w2=11.525, d.f.=3, P=0.010). In particular, we found that B+ subjects (Arg +) could be sub-grouped on the basis of the 192 genotype at codon 55 as M+ and M-, and that only B+M+ individuals (AB.LM, AB.MM, BB.LM) were increased in centenarians (0.173 in young people vs 0.263 in centenar- ians), whilst the proportion of B+M- individuals

aw2=3.754, df=2, P=0.155; bw2=0.077, df=1, P=0.829; cw2=0.433, df=1, P=0.535.