Open AccessA facile PCR ‐ RFLP method for genotyping of ITPA rs1127354 and rs7270101 polymorphisms
Author(s) -
Alavian Seyed Ehsan,
Sharafi Heidar,
Shirmast Paniz,
Alavian Seyed Moayed,
Behnava Bita,
Pouryasin Mohammad,
Keshvari Maryam,
Pouryasin Ali
Publication year - 2018
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/jcla.22440
Subject(s) - itpa , genotyping , restriction fragment length polymorphism , genotype , single nucleotide polymorphism , biology , polymerase chain reaction , microbiology and biotechnology , snp genotyping , virology , gene , genetics , ribavirin
Background Inosine triphosphate pyrophosphatase ( ITPA ) gene single nucleotide polymorphisms ( SNP s), rs1127354 and rs7270101, may cause a functional impairment in ITP ase enzyme, resulting anemia protection in patients with chronic hepatitis C virus ( HCV ) infection undergoing ribavirin ( RBV )‐dependent regimens. The main purpose of this study was to provide and validate a simple, rapid, and inexpensive polymerase chain reaction‐restriction fragment length polymorphism ( PCR ‐ RFLP ) technique for genotyping of ITPA rs1127354 and rs7270101 polymorphisms in chronic HCV ‐infected patients. Methods In the current study, 100 Iranian patients with chronic hepatitis C were examined and genotyped for ITPA rs1127354 and rs7270101 gene polymorphisms. To genotype rs1127354 and rs7270101 polymorphisms, PCR ‐ RFLP technique and sequencing technique were performed on these samples. To validate the PCR ‐ RFLP method, the PCR ‐ RFLP genotyping results should be 100% concordant with the PCR ‐sequencing results. Results The rs1127354 and rs7270101 polymorphisms of ITPA gene were genotyped by PCR ‐ RFLP technique and sequencing simultaneously, and the results of both techniques were 100% concordant in all 100 patients. Both PCR ‐ RFLP and sequencing techniques indicated that the genotypic frequency of rs7270101 was 80% AA , 19% AC and 1% CC , and for rs1127354 was 79% CC , 20% CA and 1% AA , respectively. Conclusion We developed and validated a rapid and inexpensive PCR ‐ RFLP technique for the detection of ITPA rs1127354 and rs7270101 gene polymorphisms.