The philosophy of the Olivetti Heart Study is that the application of advanced technology for large-scale screening of the human genome cannot be the sole approach to a comprehensive understanding of the genetic bases of multifactorial diseases (such as CVD) and that two steps are essential in this regard:
1) tracking the mechanistic pathways whereby genetic variation is translated into functional changes and disease susceptibility;
2) considering gene-environment interaction with particular regard to the role of nutritional and metabolic factors.

General Methods
Genomic DNA of participants was isolated from leukocytes with a non- enzymatic, salting- out procedure (Lahiri et al, 1991).
The DNA, free of RNA and protein contamination as shown by 260/280 absorbance ratios, was amplified by the polymerase chian reaction (PCR), carried out according to Widén et al (1995) with specific modifications about protocol for every polymorphisms.
The PCR amplicons were checked on agarose gel electrophoresis using a Gel Electrophoresis Apparatus GNA- 200 (Pharmacia Biotech, Milan, Italy). Restriction fragment length polymorphism (RFLP) analysis was carried out by the addition of specific restriction endonucleases specific for every polymorphism. The digested samples were separated by electrophoresis on agarose gel and analyzed by UV light.

CHARACTERIZATION OF POLYMORPHISM OF:
Candidate genes of the Renin-Angiotensin- Aldosterone System (RAAS):
Angiotensin- converting enzyme, ACE, I/D (performed at Mario Negri SUD Institute,Dr. Licia Iacoviello);
Angiotensinogen, AGT, M235T
Angiotensin II type 1 receptor, AT1R, A1166C
Aldosterone synthase,CYP11B2, C-344T

Candidate genes for sodium-dependent hypertension :
Adducin, alpha, beta and gamma-chains: ADD1, alpha subunit; ADD2, beta subunit; ADD3, gamma subunit (performed at University of Milan Vita e Salute, Prof. G. Bianchi and Prof. D. Cusi);
Glucagon receptor gene: GCGR G40S;
Dopamine receptors: DR1, A-48G, DR2 A1/A2;
G-protein related receptor kinase: GRK4, R65L, A142V, A486V

Candidate genes for metabolic disorders associated with hypertension and disorders of mineral metabolism:
Beta 2 adrenoreceptor gene: ADRB2, codon 16, codon 27;
Beta 3 adrenoreceptor gene: ADRB3, codon 64;
Atrial natriuretic peptide: NPRC, promoter (performed at University of Ancona, Prof. R. Sarzani); ANP and NPRA,microsatellite marker (performed at University of Roma La Sapienza, Dr. S. Rubattu);
Vitamin D receptor: VDR, exon 2, allele aT/aT; allele b/B;
Calcium sensing receptor: CASR, exon 7;
Plasminogen activator inhibitor: PAI 1 G4/G5 (performed at Mario Negri SUD Institute,Dr. Licia Iacoviello);

Candidate genes for disorders of lipid metabolism:
Lipoprotein lipase gene: LPL, promoter transvertion, intron 6 transition, intron 8 transvertion, exon 2, exon 3, exon 6, exon 8, exon 9.

Candidate genes for disorders of metabolism :
LEPR, leptin receptor gene, Gln223Arg
LEPR, leptin receptor gene, Lys109Arg
LEPR, leptin receptor gene, Pro1019Pro

Polymorphism of Y Chromosome (for gender- related differences in BP and other metabolic phenotypes): HindIII +/-

Statistical handling of genetic data
Genetic data were analyzed for Hardy- Weinberg equilibrium and for allelic frequencies using the Tools for Population Genetic Analysis version 1.3 (available at http://bioweb.usu.edu/mpmbio/index.htm).
Statistical analysis was performed using the Statistical Package, for Social Sciences (SPSS- PC; SPSS Inc., Chicago, Illinois, USA).