Tetra-Primer ARMS PCR Optimization for Detection of IVS-II-I (G-A) and FSC 8/9 InsG Mutations in β-Thalassemia Major Patients in Isfahan Population.

Background: β-thalassemia, a monogenic autosomal recessive disorder, is prevalent in Middle East, particularly in Iran. In Iran, near to 20 mutations in the β-globin gene are introduced as common mutations with varying incidence frequencies in each city. Therefore, detection and screening for couples at high risk can help to solve the problems of this disease. In this study, optimized genotyping of two common mutations in Isfahan Province, IVSII-I (G-A) and FSC-8/9 insG, was performed using the T-ARMS method.

Methods: In this case-control study, 10 healthy individuals and 30 patients affected by β-thalassemia major with a mean 24.76 ± 4.5 years were selected from Omid Hospital in Isfahan Province. After designing tetra primers for two prevalent mutations IVSII-I (G-A) and FSC-8/9 insG, samples were genotyped using tetra-primers ARMS PCR technique.

Results: We have developed a sensitive single tube tetra-primers PCR assay to detect both IVSII-1 (G-A) and FS8-9 insG mutations. Moreover, we have distinguished homozygous and heterozygous forms of these mutations successfully. The frequency of IVSII-1 (G-A) mutation from 30 patients in Isfahan was 86.6% (33.3% heterozygote, and 53.3% mutant homozygote) and for FS8-9 insG mutation was 16.6% (13.3% heterozygote, and 3.3% mutant homozygote).

Conclusion: Tetra-primers ARMS PCR could be a reliable, accurate and simple technique for genotyping SNP and different mutations. So far, no study was done on optimization methods for genotyping mutations in β-thalassemia by T-ARMS. Here, we successfully adjusted and enhanced this method for recognizing two common mutations (FSC-8/9 insG and IVSII-I (G-A)) of β-thalassemia in Isfahan population.