gether with a third unlabelled primer hybridising with the opposite strand. Fluorescent dyes were assigned in order to distinguish allele-specific amplification products (i.e. one allele primer highlighted in blue, the other in green). PCR products were detected directly in the gel during electrophoretic separation on an Applied Biosys- tems automatic sequencer 377A. A single one-colour peak represents homozygous genotypes, whereas two merged peaks of different colour are indicative of hetero- zygous genotypes. This allele-specific amplification methodology enabled us to analyse a total of 648 DNA samples from a French population (324 centenarians and 324 controls) by PCR amplification of the loci T C 2444 and T C. We observed heterozygosity values of 1011 26.85% and 48.46%for the T C and T C loci, re- 2444 1011 spectively, in the control population (total number of al- leles, n=648). In this latter population, the calculated polymorphism information content (PIC) values were 0.22 and 0.35 for the T C and T C variants, respec- 2444 1011 tively. Allelic frequencies observed for each population, at the loci T C and T C are presented in Fig. 3. We 2444 1011 observed a perfect identity of allele frequencies between the two populations for the T C locus (Fig. 3A), 2444 whereas there was a weak and non-significant difference χ ( 2= 1.705, P=0.2) at the T C locus (Fig. 3B). Hence, 1011 the comparison of allele frequency distributions between 324 centenarians and the same number of controls clear- ly revealed the absence of any significant genetic associ- ation with human longevity, both at the T C and the 2444 T C locus. Genotype distributions for the two popula- 1011 tions are summarised in Fig. 4. We checked that geno-

in Hardy-Weinberg equilibrium in the populations. The rarest genotype was homozygosity for allele C at both loci and for both populations (Fig. 4A, B). When we compared genotypes from the two populations, we did not detect any significant enrichment of any of the geno- types at any of the loci analysed (locus T C, 2444 χ ≥ χ 2=1.052, P 0.5; locus T C, 2=2.9, P=0.3). The lat- 1011 ter results suggest that there was no association between the long-lived phenotype and either of these two poly- morphic loci. We ruled out any sex-related difference both in the centenarian population (288 women, 36 men) and in the control sample (270 women, 54 men) regard- ing the allele and genotype frequencies for the two loci (data not shown).

Since we hypothesised that the T C transition could 2444 have a direct impact on PARP enzyme activity through the V762A point mutation, we focused the analysis on data concerning genotype distributions from individuals who had been previously tested for PARP activity [15]. None of the genotype combinations, at any locus studied, showed any association with a high or low level of maxi- mal PARP activity expressed in cpm/106 (by the Mann- Whitney test: locus T C; total n=95; T/T(n=69) versus 2444 T/C(n=19), P=0.89; T/T(n=69) versus C/C(n=7), P=0.89; T/C(n=19) versus C/C(n=7), P=0.88. Locus T C: total 1011 n=94 individuals; T/T(n=39) versus T/C(n=42), P=0.76; T/T(n=39) versus C/C(n=13), P=0.92; T/C(n=42) versus C/C(n=13), P=0.69). Moreover, no difference could be found between the values of specific PARP activity, as- sembled according to the three possible combinations of

Fig.

Allelic distribution at the loci T C (A) and

T C (B) analysed by allele-

specific amplification of

DNA samples from narians (n=

) and controls (n=

)

 FrequencyOonoOo     controls (n = 324)0,850,15  centenarians (n = 324)0,85 0,15  

  FrequencyOonoOo      Controls (n = 324) 0,85 0,150,85 0,15coCentenarians (n = 324)   Frequency   gControls (n = 324)0,660,34  OoCentenarians (n = 324)0,69 0,31   

  Frequen:   gControls (n = 324)0,660,34 OoCentenarians (n = 324) 0,69 0,31   Frequency     gControls (n = 324)0,270,01 OCentenarians (n = 324) 0,25 0,02    Frequency   gControls (n = 324)0,480,10   tO Centenarians (n = 324) 0,42 0,10   

  Frequency           eeccControls (n = 324) 0,72 0,27 0,01Centenarians (n = 324) 0,73 0,25 0,02  

 OCentenarians (n = 324) 0,73   0,25 0,02   0,8 Frequency   0,42 0,48 0,10gControls (n = 324)0,42 0,10ooCentenarians (n = 324) 0,48       

 

cited studies, Western blot analyses indicated that the as- sociations with longevity were due to differences in spe- cific PARP activity rather than PARP-1 abundance. We therefore hypothesised that qualitative differences in the PARP-1 protein could be mediated by genetic differ- ences at the level of the PARP-1 gene coding sequence. Consequently, for the present study, we selected 18 do- nor samples from the population tested in our previous study [15] with extremely high or low levels of PARP activity, respectively (nine centenarians and nine con- trols). Our objective was to find new polymorphisms in the PARP-1 coding region likely to be responsible for the higher specific PARP activity observed in the cell lines from centenarians. The complete 3,320 bp sequence of PARP-1 cDNA originating from these cell lines was am- plified in eight overlapping fragments and sequenced completely. We showed that polymorphisms indeed exist in the PARP-1 coding region, and one out of the four nu- cleotide variants identified causes a change in the prima- ry structure of the protein. The T C transition in exon 2444 17 leads to the V762A exchange in the catalytic region of PARP-1 protein sequence (Fig. 1). Some nucleotide variants have already been described in the PARP-1 cod- ing sequence, but none of the polymorphisms we de- scribe here matched the nucleotide positions that were differing in the cDNA sequences presented by Auer et al.[34], Uchida et al. [35], Kurosaki et al. [36] and Cherney et al. [32]. By contrast, van Gool et al. [37] did observe the T C transition in different sequences es- 2444 tablished from independent cDNA libraries, but they did not confirm that it was a true polymorphism. In the pres-