Study on the IFNL 4 gene ss 469415590 variant in Ukrainian population

Aim. To determine genotype and allele disribution for the IFNL4 gene ss469415590 and examine it for linkage with the IL28B gene rs12979860 in Ukrainian population. Methods. The studied group consisted of 100 unrelated donors of Eastern European origin representing the population of Ukraine. Genotyping for the IFNL4 gene ss469415590 was performed using the amplification-refractory mutation system PCR. Genotyping for the IL28B gene rs12979860 was performed by the PCR-based restriction fragment length polymorphism assay. Results. Genotype frequencies for both studied variants showed no significant deviation from those expected according to Hardy-Weinberg equilibrium. Allelic distribution for ss469415590 was: TT – 0.665, G – 0.335. Allelic frequencies of rs12979860 were: C – 0.655, T – 0.345. The results of likelihood ratio test indicated a linkage disequilibrium between the studied variants (p > 0.0001), the major alleles ss469415590 TT and rs12979860 C were in phase. The genetic structure of Ukrainian population in terms of two studied polymorphic variants is similar to the European population presented in the «1000 genomes» project. Conclusions. Considering a tight linkage revealed in Ukrainian population between the ss469415590 variant and rs12979860, a crucial genetic marker of chronic hepatitis C treatment efficiency, this polymorphism might be a promising target for further investigation as a pharmacogenetic marker.


Introduction.
For the past several years wide-range studies have proven the association of the IL28B gene rs12979860 with the antiviral therapy efficiency in patients with chronic hepatitis C (virus genotype 1) as well as with the spontaneous viral clearance [1,2].However, the exact molecular mechanism for such association remained unclear.According to one of the hypotheses the rs12979860 was expected to be linked to unknown at that moment causal variant [2].
The progress in the field has been achieved recently due to the discovery of previuosly unknown transcript which expression in hepatocytes was activated by hepatitis C virus exposure [3].It appeared that a new dinucleotide polymorphic variant ss469415590 caused a frame-shift mutation creating an open reading frame -the IFNL4 (interferon lambda 4) gene [3].This polymorphism was shown to be in a linkage disequillibrium with rs12979860 in some populations [4] and hence is being extensively studied now as a genetic marker of sustained virological response in chronic hepatitis C patients [1][2][3][4].
The aim of the study presented was to determine genotype and allele disribution for ss469415590 and examine it for the linkage with rs12979860 in Ukrainian population.
Matherials and methods.The studied group consisted of 100 unrelated donors of Eastern European origin representing the population of Ukraine.The informed consent was obtained from all participants prior to enrollment in the study.The study has been approved by The Bioethical Committee of Institute of Molecular Biology and Genetics of NAS of Ukraine.
The material of the study was genomic DNA extracted from peripheral blood samples using standard phenol-chloroform technique.Genotyping for the IFNL4 gene ss469415590 was performed using the amplification-refractory mutation system (ARMS) PCR.Additional mismatches were introduced in the primers to avoid the dimer formation.
The primers sequences were IFNL4DG: TCC TTT ACA CGG TGA TCG CAG C; IFNL4TT: TCC TTT ACA CGG TGA TCG CAG AA; and IFNL4com: TGA TTG ACC CTG AGC CTG CG.The conditions for amplification were as follows: initial denaturation at 95 °C for 5 min, 30 cycles of 30 s at 95 °C, 30 s at 62°C, and 30 s at 72 °C, followed by 5 min final extension at 72 °C.The amplification products of 299 bp were visualized on 2 % agarose gel with ethidium bromide staining.Genotyping for the IL28B gene rs12979860 was performed by the PCR-based restriction fragment length polymorphism assay as described previously [5].
Statistical analysis has been performed using Gene Pop statistical package [6].The c 2 test was used to detect deviations from Hardy-Weinberg equilibrium in genotype distribution.
The likelihood-ratio test has been performed to estimate the linkage disequilibrium between ss469415590 and rs12979860.P < 0.05 was regarded as a significant value.
Results and discussion.The results of genotyping for both studied polymorphic variants are presented in Table 1.Genotype frequencies for both studied variants showed no significant deviation from those expected according to Hardy-Weinberg equilibrium.The c 2 values for ss469415590 and rs12979860 equaled 0.91 and 0.42 respectively (df = 2).Allelic distribution for ss469415590 was: TT -0.665, DG -0.335.Allelic frequencies of rs12979860 were: C -0.655, T -0.345.
The likelihood ratio test was performed to estimate the genotypic linkage disequilibrium between ss469415590 and rs12979860.The results indicated that the studied variants are tightly linked (p > 0.0001), the alleles ss469415590 TT and rs12979860 C were in phase.
The recent data show substantial variation in the ss469415590 and rs12979860 allele distributions between different populations.
Therefore, a comparative analysis of the previously reported ss469415590 and rs12979860 variant allele frequencies [7] and the results obtained in this study was performed (Table 2).
There was no difference between the ss469415590 and rs12979860 allele distribution reported for European population and that obtained in this study.Respective distributions for both polymorphic variants in Eastern Asian, African, and Ad Mixed American populations were significantly different from the Ukrainian one.Conclusions.In this study we have presented the genotype and allele distribution for the recently discovered ss469415590 in the IFNL4 gene in Ukrainian population, obtained using ARMS-PCR.The genetic structure of Ukrainian population in terms of two studied polymorphic variants is similar to the European population presented by the «1000 genomes» project.
Taking into account a tight linkage revealed in Ukrainian population between the ss469415590 variant and rs12979860, a crucial genetic marker of chronic hepatitis C treatment efficiency, this polymorphism might be a promising target for further investigation as a pharmacogenetic marker. Funding.

Table 1
Genotype frequency for studied polymorphic variants *Calculated between respective population and Ukrainian populations.
This work was supported by the National Academy of Sciences of Ukraine (grant number 0112U 002108); and the State of Ukraine (grant number 0113 U006253).