Development and Validation of a Simple, Rapid and Inexpensive PCRRFLP Method for Genotyping of Common IL28B Polymorphisms: A Useful Pharmacogenetic Tool for Prediction of Hepatitis C Treatment Response

Abstract

BackgroundIn 2009, 3 genome-wide association studies implicated IL28B single-nucleotide polymorphisms (SNPs) as the strongest genetic pretreatment predictor of sustained virological response (SVR) in hepatitis C infection. Recently, the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) included IL28B testing in their guidelines.ObjectivesThe main aim of this study was to develop and validate a simple, rapid, and inexpensive polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for genotyping of common IL28B polymorphisms (rs12979860 and rs8099917).Patients and MethodsTwo methods were developed to genotype common IL28B polymorphisms: 1) PCR-sequencing as a reference method and 2) PCR-RFLP as a rapid and inexpensive method. Both polymorphisms were genotyped in 104 Iranian hepatitis C patients by both methods simultaneously. To validate the PCR-RFLP method, the PCR-RFLP genotyping results should be 100% concordant with the PCR-sequencing results.ResultsGenotyping of rs12979860 and rs8099917 by PCR-RFLP was concordant with PCR-sequencing in 104 (100%) individuals. The analytical sensitivity and specificity of the PCR-RFLP method for genotyping of both SNPs are 100%. Among these 104 patients with chronic hepatitis C, the frequency of the rs12979860 CC, CT and TT genotypes were 40.4%, 47.1% and 12.5% and the frequency of the rs8099917 TT, GT and GG genotypes were 59.6%, 35.6% and 4.8%, respectively. Also, three IL28B haplotypes (rs12979860-rs8099917) were found among our patients including C-T, T-G and T-T with 63.9%, 22.6% and 13.5% frequency, respectively. C-G haplotype was absent in all of our patients.ConclusionsWe have developed a validated, fast, and simple PCR-RFLP method for genotyping of common IL28B SNPs that is more cost-effective than sequencing.

Distribution of IL28B Genotypes in Iranian Patients with Chronic Hepatitis C and Healthy Individuals

“…The IL28B rs12979860 and rs8099917 SNPs were genotyped by the PCR-RFLP method as formerly described (13) Briefly, genomic DNA of hepatitis C infected patients and healthy individuals were extracted from their peripheral blood using QIAamp® DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. 100-300 ng of genomic DNA was amplified using 10 pmol of primers 5’-GCGGAAGGAGCAGTTGCGCT-3’ and 5’-GGGGCTTTGCTGGGGGAGTG-3’ in the PCR amplification for genotyping of IL28B rs12979860 SNP and 100-300 ng of genomic DNA was amplified using 10 pmol of primers 5’-CCCACTTCTGGAACAAATCGTCCC-3’ and 5’-TCTCCTCCCCAAGTCAGGCAACC-3’ in a separate PCR for genotyping of IL28B rs8099917 SNP.…”

The interferon lambda 4 rs368234815 predicts treatment response to pegylated-interferon alpha and ribavirin in hemophilic patients with chronic hepatitis C

“…Genotyping of rs368234815, rs12979860, and rs8099917 SNPs was performed by DNA sequencing as previously described. [9]…”

Interleukin 28b Gene Polymorphisms as a Predictor of Treatment Response in Egyptians With Chronic Hepatitis C.

“…Olfat et al, 2011 on the other hand, found that, the frequencies of the IL-28B genotypes were as follows: TT, 46%; GT, 42%; and GG, 12%. Sharafi et al, 2012 (a) 24 recorded that TT, GT and GG genotypes were 59.6%, 35.6% and 4.8%, respectively. Sharafi et al, 2012 (b) 25 reported that the frequency of IL28B rs8099917 TT, GT, and GG genotypes in chronic hepatitis C patients was 58.3%, 37.1%, and 4.6% and in healthy individuals was 64.1%, 32.4% and 3.5%.…”