New polymorphisms in the human poly(ADP-ribose) polymerase-1 coding sequence: lack of association with longevity or with increased cellular poly(ADP-ribosyl)ation capacity.

Received: 27 March 2000 / Accepted: 9 July 2000 / Published online: 30 August 2000 Springer-Verlag 2000.

Florence Cottet and Marie-Laure Muiras were graduate students at the Ecole Pratique des Hautes Etudes (EPHE, Paris, France) in collaboration with the Fondation Jean Dausset-Centre d'Etude du Polymorphisme Humain (Paris, France). Cottet worked on the genetics of human longevity and Muiras received her Ph.D. from the University Paris VI, France, in the same field. Muiras is now a postdoctoral fellow at the German Cancer Research Centre (Heidelberg, Germany), where she works on clinical trials related to poly(ADP-ribosyl)ation and its relation to genomic instability.

Abstract:
Poly(ADP-ribose) polymerase-1 (PARP-1), encoded by the PARP-1 gene, is a ubiquitous and abundant DNA-binding protein involved in the cellular response to various genotoxic agents. In a previous study, we showed that maximal oligonucleotide-stimulated poly(ADP-ribosyl)ation was significantly higher in permeabilized lymphoblastoid cell lines from a French population of centenarians compared with controls aged 20-70 years, supporting the notion that longevity is associated with a genetically determined high poly(ADP-ribosyl)ation capacity. Here, we describe four new genetic polymorphisms, three of which represent silent nucleotide variants (C>T, 402 T>C, G>A), and one of which leads to a valine-to-alanine exchange (T>C). We undertook an association study between two of these polymorphisms and human longevity or poly(ADP-ribosyl)ation capacity in permeabilized lymphoblastoid cells. By analyzing 648 DNA samples from a French population (324 centenarians and 324 controls) by fluorescent-allele-specific PCR, we showed the absence of any significant enrichment of any of the genotypes in the study of centenarians versus controls. Furthermore, we studied genotype distributions from individuals who had previously been tested for poly(ADP-ribosyl)ation capacity. None of the genotype combinations at any polymorphic site studied could be related to a high or low level of poly(ADP-ribosyl)ation capacity. Together, these results strongly suggest that the longevity-related differences in the poly(ADP-ribosyl)ation capacity of human lymphoblastoid cell lines cannot be explained by genetic polymorphisms in the PARP-1 coding sequence, and that other mechanisms have to be considered as potential factors.

Marie-Laure Muiras received her Ph.D. from the University Paris VI, France, in the field of genetics of human longevity. She is presently a postdoctoral fellow at the German Cancer Research Centre (Heidelberg, Germany). Her research interests include poly(ADP-ribosyl)ation and its relation to genomic instability. Florence Cottet was a graduate student at the Ecole Pratique des Hautes Etudes (EPHE, Paris, France) in collaboration with the Fondation Jean Dausset-Centre d'Etude du Polymorphisme Humain (Paris, France), where she was interested in the genetics of human longevity. She is now in charge of clinical trials in a pharmaceutical company.

Fondation Jean Dausset-Centre d'Etude du Polymorphisme Humain, 27, rue Juliette Dodu, 75010 Paris, France.

Keywords: Human longevity, Poly(ADP-ribose) polymerase, Centenarian, Allele-specific amplification, Genetic polymorphism.

A. Brkle, University of Newcastle, Department of Gerontology, Institute for the Health of the Elderly, Wolfson Research Centre, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne, NE4 6BE, UK.

Abbreviations: PARP: Poly(ADP-ribose) polymerase; ROS: reactive oxygen species; EBV: Epstein-Barr virus.

Introduction:
Poly(ADP-ribosyl)ation is a post-translational modification of nuclear proteins that facilitates the cellular recovery from DNA damage. This modification is catalyzed by the nuclear enzyme poly(ADP-ribosyl) polymerase-1 (PARP-1; EC 2.4.2.30), with NAD+ serving as substrate. PARP-1 is found in most eukaryotic species and accounts for about 85% of cellularThe pellet was resuspended in 3 L of loading buffer (formamide 83%, EDTA 4 mM, Dextran blue 8 mg/ml). Sequencing reaction products were loaded on 4.25% polyacrylamide/6 M urea gels and analyzed on an Applied Biosystems Gene Scanner 377A for 7 h (1.5 kV, 51°C). Signals were collected by the sequence analysis software (version 3.0, Applied Biosystems) and analyzed with Sequence Navigator (Applied Biosystems).

Materials and methods

Subjects and DNA samples

Centenarians were recruited with personal consent through the Chronos Project at the Fondation Jean Dausset-Centre d'Etude du Polymorphisme Humain (CEPH) from all regions of France. They were Caucasians born in France and were at least 99 years old on the day of blood collection. The mean age of the centenarians was 101 years. Controls were selected from unrelated Caucasian individuals who were born in France and matched with centenarians for birthplace. The mean age of controls was 51 years. The proportion of women was 88.9% in the total sample tested (n=648) and 83.33% in the centenarian and control population, respectively, reflecting the differential longevity of women and men in France.

Peripheral blood leukocytes were routinely isolated by Ficoll gradient centrifugation, EBV-immortalized by standard methods, and cryopreserved in liquid nitrogen until used. DNA was extracted from EBV-immortalized cell lines by standard methods [30], resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH7.6), and kept frozen until used as described below.

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