Angiotensinogen gene polymorphism of threonine/methionine at position 235-potential problems of the modified restriction endonuclease (Tth111-I) digestion method.

There are four methods for detecting the angiotensinogen gene Agt T235/M235; 1) allele-specific oligonucleotide hybridization (PCR-ASO); 2) mutagenically separated PCR (MS-PCR) using three primers to amplify all possible alleles in on PCR reaction; 3) restriction endonuclease Tth111-I; 4) SfaN-I digestion method using mismatched primer for the PCR (modified PCR-RFLP). Two of these four methods have been used in Japanese studies. The reported allelic frequencies of Agt T235/M235 in normal controls in the Japanese population are around 0.75-0.84/0.25-0.16 (as a whole 0.80/0.20) by PCR-ASO and 0.70-0.65/0.30-0.35 (as a whole 0.67/0.33) by the modified PCR-RFLP (Tth111-I). The present study tested how these methods contribute to the differences in Agt T235/M235. By PCR-ASO, the genotypes could be clearly determined. However, it is hard to complete every digestive reaction under the experimental conditions described for modified PCR-RFLP (Tth111-I). Thus, for studying Agt T235/M235, PCR-ASO or some method other than PCR-RFLP (Tth111-I) can be recommended. Our findings suggest that the allele frequency of Agt T235/M235 in the normal Japanese population is closer to 0.75-0.84/0.25-0.16. Although a strong association was reported between the Agt T235 allele and essential hypertension or myocardial infarction, using the modified PCR-RFLP (Tth111-I), two of three studies using PCR-ASO found no or only a weak association. The relationship between the Agt T235 allele and essential hypertension or myocardial infarction in the Japanese population needs to be assessed.