Author + information
- Latheef Kasala,
- Rajasekhar Durgaprasad,
- Vanajakshamma Velam,
- Sarma PVGK,
- Aparna R. Bitla,
- Abhijit Chaudhury and
- Vasudeva Chetty Pakala
Elevated homocysteine (Hcys) levels (also called hyperhomocysteinemia) has an increased risk for atherosclerosis, which could eventually results in myocardial infarction. The common methylenetetrahydrofolate reductase (MTHFR) C677T single nucleotide polymorphism (SNP) has been associated with raised serum levels of homocysteine, an established risk factor for coronary artery disease (CAD). The association of MTHFR gene and the level of Hcys in the pathogenesis of CAD have been extensively studied in various ethnic groups but limited data is available from South-Indian subset. The present study therefore investigated the association of MTHFR polymorphisms and Hcys levels in young CAD patients of South-Indian subset.
A total of 100 subjects (aged <50 years) were recruited which included 50 CAD patients diagnosed on coronary angiography and 50 controls. This study was approved by the institutional ethics committee of our institute and a written informed consent was obtained from all the study participants. Serum Hcys levels were estimated by ELISA method in both patient and control groups. SNP analysis of exon-4 of MTHFR gene involved: 1) genomic DNA isolation from whole blood with QIAamp DNA Mini spin-column (Qiagen India Pvt Ltd., India) DNA extraction kit, 2) PCR amplification of exon-4 of MTHFR gene, 3) Sanger's sequencing of PCR products/amplicons, 4) Comparison of patient DNA sequences against to control/wild type sequence by using Clustal-X (v1.83) tool for multiple sequence alignment, and 5) translation of DNA sequence into amino acid sequence with ExPASy online tool (http://web.expasy.org/translate/) to find out the functional changes at protein level.
Mean age of the study and control groups was 40.1±8.7 years (range 20-48 years) and 31.6±7.2 years (range 24-50 years). Mean serum Hcys levels were 19.3±9.2 μmol/L and 16.8±1.2 μmol/L in study and control cohorts respectively (P=0.006). Hyperhomocysteinemia (Hcys>15 μmol/L) was observed in 62% (n=31) of patients. MTHFR 677 C > T polymorphism was observed in 10% (n=5, p=0.056; all of whom were identified with Hyperhomocysteinemia) of the study population whose mean Hcys levels were 35.8±4.6 μmol/L, whereas normal genotypic patients had mean Hcys levels of 17.5±7.7 μmol/L (p<0.0001). ExPASy results showed that a single amino acid change at position 222 replacing alanine (Ala) with valine (Val) in MTHFR 677 C > T genotypic samples. This change in single amino acid residue produces a thermolabile enzyme which has reduced activity at higher temperatures and is incapable of reduction of 5,10-methylenetetrahydrofolate to 5-MTHF resulting in the elevated levels of Hcys (Hyperhomocysteinemia).
There was a significantly higher mean homocysteine levels between MTHFR 677 C>T genotypic and normal genotypic Hyperhomocysteinemia patients. No novel mutations of MTHFR in this ethnic subset, but found C677T polymorphism in 10% of the study population. Hence, MTHFR 677 C >T polymorphism can be a possible genetic risk factor for premature coronary artery disease in South-Indian ethnic population.