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J Clin Lab Anal. 2018 Sep;32(7):e22440. doi: 10.1002/jcla.22440. Epub 2018 Apr 16.

A facile PCR-RFLP method for genotyping of ITPA rs1127354 and rs7270101 polymorphisms.

Author information

1
Baqiyatallah Research Center for Gastroenterology and Liver Diseases, Baqiyatallah University of Medical Sciences, Tehran, Iran.
2
Middle East Liver Diseases (MELD) Virology Laboratory, MELD Center, Tehran, Iran.
3
Armin Pathobiology Laboratory, Tehran, Iran.
4
Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran.

Abstract

BACKGROUND:

Inosine triphosphate pyrophosphatase (ITPA) gene single nucleotide polymorphisms (SNPs), rs1127354 and rs7270101, may cause a functional impairment in ITPase 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.

KEYWORDS:

hepatitis C virus; inosine triphosphate pyrophosphatase gene; polymerase chain reaction; restriction fragment length polymorphism; sequencing; single nucleotide polymorphisms

PMID:
29660762
DOI:
10.1002/jcla.22440
[Indexed for MEDLINE]

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