A straightforward genotyping of the relevant IL28B SNPs for the prediction of hepatitis C treatment outcome.
A sustained virological response is not achieved by a significant proportion of chronic hepatitis C patients treated with interferon-based regimens. Due to the associated side effects and high costs, therapy response markers have been thoroughly sought. Two Single Nucleotide Polymorphisms (SNPs), rs12979860 and rs8099917, which are located upstream from the IL28B gene, have been remarkably described to have a strong association with treatment efficacy. The aim of this study was to develop a straightforward method for genotyping such polymorphisms. A Polymerase Chain Reaction (PCR) followed by enzymatic restriction of amplicons was established for SNPs genotyping. Online computation resources were employed for retrieving reference sequences, such as the selection of oligonucleotides and restriction enzymes. Two pairs of primers were designed and validated for the amplification of segments encompassing rs12979860 (694bp) and rs8099917 (496bp) with common thermocycling parameters. The endonucleases Hpy166II and BsrDI were selected and used for allelic discrimination related to rs12979860 (C/T) and rs8099917 (T/G), respectively. The expected electropherotypes were confirmed for all possible genotypes in 75 blood samples. In addition, the results were validated by sequencing. The method constitutes a simple and reliable assay, which may be readily available for genotyping of rs12979860 and rs8099917 in laboratories that support hepatitis C treatment centers.
Department of Medicine of Universidade da Região de Joinville, Paulo Malschitzki, 10, Campus Universitário, Zona Industrial, 89219-710 Joinville, Santa Catarina, Brazil. simonebioq@hotmail.com
Simone
S
Moreira
Raquel Francine Liermann
RF
Garcia
Andréia
A
Gutberlet
Bruna Cristina
BC
Bertol
Leslie Ecker
LE
Ferreira
Mauro de Souza Leite
Mde S
Pinho
Paulo Henrique Condeixa
PH
de França
D000998:Antiviral Agents / Q000008:administration & dosage*; D017931:DNA Primers / Q000235:genetics; D005838:Genotype; D019698:Hepatitis C, Chronic / Q000188:drug therapy*; D006801:Humans; D007372:Interferons / Q000008:administration & dosage*; D007378:Interleukins / Q000235:genetics*; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D011379:Prognosis; D016896:Treatment Outcome; D000095022:Interferon Lambda
D000998:Antiviral Agents; D017931:DNA Primers; D007372:Interferons; D007378:Interleukins; C000624353:interferon-lambda, human; D000095022:Interferon Lambda