The Study of APOA1, APOC3 and APOA4 Variability in Healthy Ageing People Reveals Another Paradox in the Oldest Old Subjects

Garasto1, G. Rose1, F. Derango1, M. Berardelli1, A. Corsonello2, E. Feraco2, V. Maletta 3, A. Bruni3, C. Franceschi4, L. Carotenuto5 and G. De Benedictis1,*

1Department of Cell Biology, University of Calabria (Rende, Italy) 2Italian National Research Center on Aging, INRCA (Cosenza, Italy) 3Regional Center of Neurogenetics, (Lamezia, Italy) 4Department of Experimental Pathology, University of Bologna (Bologna, Italy) 5Department of Electronics, Computer and System Sciences, University of Calabria (Rende,

of Cell Biology, University of Calabria (Rende, Italy) 2Italian National Research Center on Aging, INRCA (Cosenza, Italy)

4Department of Experimental Pathology, University of Bologna (Bologna, Italy) 5Department of Electronics, Computer and System Sciences, University of Calabria (Rende, Italy)

Summary

genes coding for apolipoprotein A1 (APOA1 ), apolipoprotein C3 (APOC3 ) and apolipoprotein A4 (APOA4 ) tandemly organised within a short region on chromosome 11q23-q24. Polymorphisms of these genes have extensively investigated in lipoprotein disorders and cardiovascular diseases, but poorly investigated in healthy The aim of this study was to describe possible modifications of the APOA1, APOC3, and APOA4 gene by cross-sectional studies carried out in a healthy ageing population whose ages ranged from 18 to 109 years subjects, 327 males and 473 females, free of clinically manifested disease, and with emato-chemical parameters −   the norm). APOA1-MspI-RFLP ( 75 nt from the transcription starting site), APOC3-SstI-RFLP (3 UTR, nt), and APOA4-HincII-RFLP (Asp /Ser ) were analysed according to age and sex. A significant age- 127 127 variation of the APOA1 gene pool was observed in males. An analysis of the allele average effect exerted by A/P alleles (Absence/Presence of the restriction site) on lipidemic parameters in 46–80 year old showed that allele A decreased, while allele P significantly increased, serum LDL-cholesterol. Unexpectedly, P allele was over-represented in the group of the oldest old subjects, thus giving evidence of another “genetic

Keywords: APOA1, APOC3, APOA4, human longevity

of the APOE 4 allele (Schachter et al. 1994; Jian-Gang et al. 1998; Gerdes et al. 2000; Carrieri et al. 2001), the importance of the variability of other APO genes, in particular APOA1, APOC3 and APOA4, on healthy ageing and mortality has yet to be assessed, although epidemiological and clinical evidence has clearly shown the significance of these genes in lipid metabolism and cardiovascular diseases (Groenendijk et al. 2001).

Introduction

Because of the role played by lipoproteins in predispos- ing to, or protecting from, common age-related diseases (Genest, 2002; Hegele, 2001; Mahley & Rall, 2000; Vrablik et al. 2001) the effect of the inherited vari- ability of apolipoprotein (APO) genes on the rate and quality of human ageing is a matter of interest. How- ever, except for the well established detrimental effect

The APOA1, APOC3 and APOA4 genes are tandemly organised in a cluster of 17 Kb DNA on the long arm of human chromosome 11 (11q23-q24). Shared regulatory elements for the expression of the three genes are interspersed throughout the cluster

Corresponding Author: G. De Benedictis, Department of Cell Biology, University of Calabria, 87030 Rende, Italy. Tel: 39 0984 492932; Fax: 39 0984 492911. E-mail: g.debenedictis@unical.it

46–80 years (median age 62 years), and 229 subjects aged 81–109 years (median age 101 years). The last age-class included 184 subjects older than 100 years (43 males and 141 females). These centenarians, classified in the categories A and B according to the criteria re- ported in Franceschi et al. 2000, were in fairly good health and did not suffer from major pathologies or disabilities.

(Haase et al. 1990; Kan et al. 2000). All three genes contain several polymorphisms, whose role has mainly been analysed in common diseases. An aim of the present study was to discover possible variations of the APOA1, APOC3 and APOA4 genes which may oc- cur as the population ages and mortality selection oc- curs, attempting to escape confounding factors which could bias cross-sectional association studies. Therefore, particular attention was paid to the homogeneity of the sample population and to an exact definition of the phenotype under study. With regard to the first point, all the samples were recruited from Calabria, a well defined area of southern Italy whose population is characterised, from a historical-demographic point of view, by a consistent level of isolation due to the geographic nature of the region. For the phenotype, only subjects free of clinically manifested diseases, and with emato-chemical parameters in the norm, were en- rolled in the study. Furthermore, since sex-specific ef- fects on the ageing phenotype are known (Ivanova et al. 1998; De Benedictis et al. 1999; Tan et al. 2001; De Luca et al. 2001) the study was carried out according to sex.

Two samples (40 subjects each) of 46–80 year old males affected by Cardiovascular Disease (CD), either with high levels of serum Low Density Lipoproteins (CDL group), or with low levels of serum High Den- sity Lipoproteins (CDH group), were also analysed. Di- agnostic criteria and lipidemic parameters of the two samples of CD patients are reported at the web site http://biologia.unical.it/labs/genetica.html. All the patients and their families were born and living in the same area as the healthy group.

All the subjects included in the study had given their informed consent.

Materials and Methods

Lipidemic Parameters

Samples