Study Population. This study was designed as a case-control study. After informed written consent, we enroled 40 patients with essential hypertension and 40 normo­tensive subjects. Essential hypertension was diagnosed at the Department of Nephrology in Skopje, Republic of Macedonia. Exclusion criteria were secondary causes of hypertension.

Inclusion criteria were as follows: 1) family history of hypertension; 2) systolic blood pressure (SBP) >145 mm Hg and/or diastolic blood pressure (DBP) >90 mm Hg (mean values of three measurements made with a sphyg­momanometer) or previous anti-hypertensive treatment. The questionnaire required data on age, sex, height, weight, blood pressure (BP), and age at which high BP was first recorded.

Normotensive subjects were recruited from healthy, normotensive blood donors, with no history of hyperten­sion in the family. They were selected according to the following criteria: 1) no history of hypertension; 2) SBP <140 mm Hg and/or DBP <90 mm Hg; 3) matched by age and sex to the study group. Genomic DNA was isolated by standard phenol-chlorophorm extraction procedure from whole blood drawn into tubes containing EDTA [6].

Genotyping for the M235T Polymorphism of the Angiotensinogen Gene. This polymorphism was investi­gated by polymerase chain reaction (PCR) amplification of genomic DNA followed by restriction endonuclease activity. A rapid method for the detection of M235T was used [7]. The T®C transition at nucleotide 704 in exon 2 does not alter a restriction site, but produces a “half-site” for Tth111 I. Introduction of a corresponding site was achieved by a PCR primer with two mismatches in the downstream primer: 5'-CAG GGT GCT GTC CAC ACT GGA CCC C-3'; upstream primer: 5'-CCG TTT GTG CAG GGC CTG GCT CTC T-3'. The amplification yields a product of 165 bp. The presence of C at position 704 cleaved by Tth111 I generates a fragment of 141 bp.

The PCR was performed in a total volume of 100 mL containing 2.5 mM dNTPs, 0.5 mL of primers and 0.5 mL Taq polymerase (Gold; Applied BioSystems, Foster City, CA, USA). Cycling conditions were set as follows: initial denaturation at 94°C for 5 min., 40 cycles at 94°C for 1 min., 68°C for 1 min., 72°C for 1 min., and final extension at 72°C for 10 min. Five mL of unpurified product were diluted in the recommended restriction buffer containing 6 U of Tth111 I and digested overnight. Restriction frag­ments were separated by electrophoresis on 2.5% agarose gel (Fig. 1).

Statistical Analysis. Standard statistical methods such as proportions, T-test and c² test were applied. Statistical analyses were made using standard statistical packages, Statistic for Windows and Serigraphic Plus for Windows.

The difference of prevalence of the angiotensinogen polymorphism M235T between the group of patients with essential hypertension and the control group has been tested. Separate tests were performed for the prevalence of M235T in the group of patients with regard to age, sex, anti-hypertensive therapy and acquired risk factors.