ORIGINAL INVESTIGATION

De Benedictis · E. Falcone · G. Rose · R. Ruffolo · Spadafora · G. Baggio · S. Bertolini · D. Mari · Mattace · D. Monti · M. Morellini · P. Sansoni · Franceschi

DNA multiallelic systems reveal gene/longevity associations not detected by diallelic systems. The APOB locus

Received: 27 November 1995 / Revised: 24 July 1996

Abstract To identify possible genetic factors affecting human longevity we compared allele pools at two candi- date loci for longevity between a sample of 143 centenar- ians (S) and a control sample of 158 individuals (C). The candidate loci were APOB and TPO, which code for apo- lipoprotein B and thyroid peroxidase, respectively. Both restriction fragment length (RFL) (XbaI and EcoRI ) 2488 4154 ¢ and variable number of tandem repeat (VNTR) (3 APOB- VNTR) polymorphisms were analysed at the APOB lo- cus; the TPO-VNTR polymorphism (intron 10) was ana- lysed at the TPO locus. The main result of the investiga- tion was that there is an association between the APOB locus and longevity that is revealed only when multiallelic polymorphisms are considered. In particular: (i) the fre- ¢ quency of 3 APOB-VNTR alleles with fewer than 35 re- peats is significantly lower in cases than in controls; (ii)

the linkage disequilibrium between the XbaI-RFLP and the EcoRI-RFLP is significantly different from 0 in cases but not in controls; (iii) the EcoRI-RFLP and XbaI-RFLP allele frequencies do not discriminate between cases and controls. The differences observed between case and con- trol allele pools are specific to the APOB locus, since no significant difference was observed at the TPO locus.

Introduction

Longevity can be regarded as a multifactorial trait, result- ing from the interaction between environmental factors and sets of epistatic alleles having pleiotropic age-depen- dent effects (Kirkwood and Franceschi 1992). A possible approach to the identification of these longevity alleles is the comparative analysis of polymorphic markers at can- didate loci between a long-lived group and a control group (Schachter et al. 1993). Indeed it is to be expected that if a certain gene affects lifespan, there will be a dif- ference between the allele pools of the long-lived and the control groups. So far significant longevity/allele associa- tions have been found at the HLA (Proust et al. 1982; Takata et al. 1987), ACE (Schachter et al. 1994), APOB (Kervinen et al. 1994), APOE (Kervinen et al. 1994; Louhija et al. 1994; Schachter et al. 1994) and APOC (Louhija et al. 1994) loci.

P. Spadafora Dept. of Cell Biology, University of Calabria, Rende, Italy G. Baggio Inst. of Internal Medicine, University of Padua, Padua, Italy S. Bertolini Atherosclerosis Prevention Centre, Dept. of Internal Medicine, University of Genoa, Genoa, Italy D. Mari Hemophilia and Thrombosis Centre, University of Milan, Milan, Italy R. Mattace Chair of Geriatrics, University of Reggio Calabria, Catanzaro, Italy D. Monti

C. Franceschi Dept. of Biomedical Sciences, University of Modena, Modena, Italy M. Morellini Dept. of Experimental Medicine and Pathology, University La Sapienza, Rome, Italy P. Sansoni Inst. of Clin. Med. Gen. and Terapia Medica, University of Parma, Parma, Italy

The information content of a genetic marker is a func- tion of the number and the relative frequencies of differ- ent alleles found at that locus in a population (Botstein et al. 1980). DNA markers resulting from a variable number of tandem repeats (VNTR) are highly informative multial- lelic systems (Harding 1992) that can be analysed by poly- merase chain reaction (PCR) amplification of the variable region. Because of their high information content, VNTR markers at candidate loci could be more efficient than sin- gle diallelic polymorphisms in revealing gene/longevity associations.

Hemophilia and Thrombosis Centre, University of Milan, Milan, Italy

On the basis of these considerations, we decided to verify whether VNTR markers at candidate loci were able to reveal significant differences between gene pools from

centenarians and controls. The first loci chosen for analysis were apolipoprotein B (APOB) and thyroid oxidase (TPO).

90 subjects, respectively). Seventy-seven were men and 81 were women, from 20 to 60 years old. The average age was 34.1 years with 13.5 years SD.

All the subjects gave their informed consent prior to their in- clusion in the study.

The APOB gene codes for apolipoprotein B, the main protein in low-density lipoproteins (LDL). A connection has been found between some polymorphisms of the APOB gene and atherosclerotic cardiovascular disease (Young ¢ 1990). The 3 APOB-VNTR, which is located less than ¢ 100 bp 3 of the second transcriptional termination signal of the APOB gene (2p24–p23) (Chan et al. 1985), is ana- lysable by PCR amplification and consists of a dimeric AT-rich core repeat sequence of 30 bp (basic repeat unit of 15 bp) (Boerwinkle et al. 1989; Ludwig et al. 1989). More than 15 alleles (from 21 to 55 repeats) are known, with the highest frequency peaks at 37 and 49 repeats (nomencla- ture according to Boerwinkle et al. 1989). The polymor- phic information content (PIC) (Botstein et al. 1980) is higher than 70%. Furthermore, significant linkage dise- ¢ quilibrium has been found between the 3 APOB-VNTR and diallelic polymorphisms of the APOB gene (Renges et al. 1992; De Benedictis et al. 1996).

Genotypic typing

DNAs were extracted from blood buffy coats following standard procedures.

The APOB-VNTR and TPO-VNTR typing were carried out on PCR-amplified DNA fragments according to the procedures de- scribed by Boerwinkle et al. (1989) and by Rose et al. (1993), re- spectively. In both cases, allelic designations refer to the number of repeats in the core sequence motif.