Identification of genetic mutations is important in diagnosis of disease before emergence of symptoms to distinguish the treatment. In this study, the sensitivity and specificity of PCR-RFLP and DHPLC techniques compared in mutation detection of C677T in the MTHFR gene and the serum homocystein changes in 677CC, TT and CT genotypes were evaluated.
A 271 bp DNA fragment covering the 677 site of MTHFR gene was amplified by PCR using the purified DNA from whole blood as template DNA. The PCR product digested by Hinf1 restriction enzyme and analyzed for detection of CC, TT and CT genotypes in 677 position by RFLP. The PCR products were mixed by references samples, In order to form heteroduplex and homoduplex and directly injected to Helix DVB column.
The C677T mutation distinguished by DHPLC technology as a multiple peak on chromatogram in mutant samples whereas only single peak in non mutant cases. Our data demonstrated that DHPLC has all the necessary factors for a screening technique such as sensitivity, specificity, speed, safety, and the ability to perform automatically. The number of normal, heterozygous and homozygous cases were 100, 40 and 14 respectively in 154 samples. The homocystein level estimated in the serum sample of these cases. The average amounts of homocystein were 13.14, 15.6 and 25.62 mmol/l respectively in normal, heterozygous and Homozygous cases.
These findings indicate the relationship between mutations in the MTHFR gene and increased levels of blood homocysteine. There are some more interfering factors in the increase of blood homocystein like gender, smoking as well as other genes such as MTRR gene where in this review has not been considered. It is better to consider these interfering factors in further studies to get a more reliable result.