Allele-specific amplification of the PARP-1 gene at the T1011C and T2444C loci

We analyzed 648 DNA samples (324 centenarians and 324 controls), matched for birthplace and available in the DNA bank at Fondation Jean Dausset-CEPH, by allele-specific amplification of two regions comprising the TC and TC transitions, respectively. Each site was amplified in the presence of three primers, two of which differed only in their 3-most nucleotide (marked in bold). The TC locus: PexC GCT CCT CCA GGC CAA CGC; 2444 PexT GCT CCT CCA GGC CAA CGT; Pex19 ACC CTG TTA CCT TAA TGT CAG TTT T; the TC locus: Pex6T TGC 1011 AGA TCT TGG ACC GAG TAG CT; Pex6C TGC AGA TCT TGG ACC GAG TAG CC; and P2cb GGT TGG GTG TCT GTG TCT TG. Each one of the two competing, allele-specific primers for the nucleotide variation locus (i.e., PexT/PexC and Pex6T/Pex6C for TC and TC, respectively) was modified at the 5 end with a fluorescent dye (C6-FAM, TET; Applied Biosystems dye/Eurogentec), so that genotypes could easily be distinguished. PCR conditions were as follows: 40 ng genomic DNA, 0.2 M each primer, 4 mM MgCl2, 10 mM Tris-HCl pH8.3, 50 mM KCl, 200M each dNTP, and 2.5 U Stoffel fragment (Perkin Elmer) in a final volume of 50 l. Samples were denatured for 5 min at 97°C and subjected to 30 cycles consisting of 30 s at 95°C, 30 s at 64°C (TC) or 65°C (TC), 30 s at 72°C. PCR products (expected sizes 148 bp and 159 bp for TC and TC, respectively) were analyzed on an Applied Biosystems automatic sequencer A377. Sizing of the products and collection of numbers of alleles were performed as described by the manufacturer (Genescan Users manual, Applied Biosystems). Statistical analysis of results was done with the Chi-square test.

Sequence analysis of PARP-1 cDNA

EBV-immortalized cell lines from 18 individuals (nine centenarians, nine controls) were selected according to the level of PARP activity previously measured [15] (i.e., either extremely high or low PARP activity, respectively). Polyadenylated RNA was extracted from up to 107 cells with the QuickPrep micro mRNA purification kit (Pharmacia Biotech), resuspended in RNase-free water, and used as a template for reverse transcription with the Ready-to-go T-primed first strand kit (Pharmacia BiHomozygous T/T (EM) 18 B:186F72M1 / T 18 G:18F72M1 / C42002100, homozygous C/C (I198:19-v45m1 / T (BM) 9G:19-va5m1), together with a third unlabelled primer hybridizing with the opposite strand. Fluorescent dyes were assigned 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 Biosystems automatic sequencer 377A. A single one-color peak represents homozygous genotypes, whereas two merged peaks of different colors indicate heterozygous genotypes. This allele-specific amplification methodology enabled us to analyze a total of 648 DNA samples from a French population (324 centenarians and 324 controls) by PCR amplification of the loci TC2444 and TC. We observed heterozygosity values of 26.85% and 48.46% for the TC and TC loci, respectively, in the control population (total number of alleles, n=648). In this latter population, the calculated polymorphism information content (PIC) values were 0.22 and 0.35 for the TC and TC variants, respectively. Allelic frequencies observed for each population at the loci TC and TC are presented in Fig. 3. We observed a perfect identity of allele frequencies between the two populations for the TC locus (Fig. 3A), whereas there was a weak and non-significant difference (χ2=1.705, P=0.2) at the TC locus (Fig. 3B). Hence, the comparison of allele frequency distributions between 324 centenarians and the same number of controls clearly revealed the absence of any significant genetic association with human longevity, both at the TC and the TC locus. Genotype distributions for the two populations are summarized in Fig. 4. We checked that genotypes were 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 genotypes at any of the loci analyzed (locus TC, χ2=1.052, P>0.5; locus TC, χ2=2.9, P=0.3). The latter results suggest that there was no association between the long-lived phenotype and either of these two polymorphic 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) regarding the allele and genotype frequencies for the two loci (data not shown).