Polymorphism rs368234815 of Interferon Lambda 4 Gene and Spontaneous Clearance of Hepatitis C Virus in Haemodialysis Patients: A Case Control Study

doi:10.21203/rs.2.18966/v1

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Polymorphism rs368234815 of Interferon Lambda 4 Gene and Spontaneous Clearance of Hepatitis C Virus in Haemodialysis Patients: A Case Control Study

https://doi.org/10.21203/rs.2.18966/v1

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published 22 Jan, 2021

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Background

In non-uremic subjects, IFNL4 rs368234815 predicts the HCV clearance. We investigated whether rs368234815 is associated with spontaneous HCV clearance in haemodialysis patients and whether it is a stronger predictor of HCV resolution than the IFNL polymorphisms already associated with HCV clearance in haemodialysis subjects. An association of rs368234815 with survival and alterations in transcription factor binding sites (TFBS) caused by IFNL polymorphisms were also evaluated.

Methods

Among 161 haemodialysis patients with positive anti-HCV antibodies, 68 (42.2%) spontaneously resolved HCV infection, whereas 93 remained HCV RNA positive. Patients were tested for near IFNL3 rs12980275, IFNL3 rs4803217, IFNL4 rs12979860, IFNL4 rs368234815, and near IFNL4 rs8099917. IFNL4 rs368234815 polymorphism (TT/TT, ΔG/TT, ΔG/ΔG) was genotyped by restriction fragment length polymorphism analysis; other IFNL polymorphisms - by high resolution melting curve analysis. The Kaplan-Meier method with the log-rank test was used for survival analysis. In silico analysis included the use of ENCODE TFBS ChIP-seq data, HOCOMOCO, JASPAR CORE, and CIS-BP databases, and FIMO software.

Results: The probability (OR, 95%CI, P) of spontaneous HCV clearance for rs368234815 TT/TT patients was higher than for the ΔG allele carriers (2.63, 1.38–5.04, 0.003). This probability for other major homozygotes varied between 2.80, 1.45–5.43, 0.002 for rs12980275 and 2.44, 1.27–4.69, 0.007 for rs12979860. In the additive model, rs368234815 TT/TT was the strongest predictor of HCV clearance (6.38, 1.69–24.2, 0.003). Survival analysis suggested an association of the ΔG allele with mortality due to neoplasms (log-rank P = 0.005). The rs368234815 ∆G allele caused TFBS removal for PLAGL1.

Conclusions

In haemodialysis patients, the association of rs368234815 with the spontaneous HCV clearance is better than that documented for other IFNL3/IFNL4 polymorphisms only in the additive mode of inheritance. However, an identification of the homozygosity in the variant ∆G allele of rs368234815 means a more potent prediction of persistent HCV infection in haemodialysis subjects that it is observed in the case of the variant homozygosity of other tested IFNL3/IFNL4 polymorphisms. Removal of PLAGL1 TFBS in subjects harboring the rs368234815 ∆G allele may contribute to cancer susceptibility. The association of rs368234815 with cancer-related mortality needs further studies in HCV-exposed subjects.

Infectious Diseases

haemodialysis

hepatitis C virus

interferon-λ4 gene

spontaneous viral clearance

transcription factors

Since 2002, single nucleotide polymorphisms (SNPs) located in the interferon-λ genetic region (IFNL) on chromosome 19q13 are associated with antiviral protection [1,2]. In humans, polymorphisms of the interferon-λ3 gene (IFNL3) or located near IFNL3 (mainly rs12979860, rs4803217, rs8099917, rs12980275) became established genetic markers of spontaneous and PEGylated interferon-based antiviral drug-induced hepatitis C virus (HCV) clearance [3-8].

In 2013, Prokunina-Olsson et al. [9] discovered the interferon-λ4 gene (IFNL4) located on chromosome 19q13.2 upstream of IFNL3 that harbours the dinucleotide frame-shift variant rs368234815 (TT/ΔG), originally named ss469415590. The polymorphism rs368234815, located within the first IFNL4 exon, is classified as a deletion/insertion genetic variation (TT/ΔG). The IFNL4 rs368234815 polymorphism controls a generation of the functional protein – interferon-λ4 (IFN-λ4) [9]. The IFNL4 ΔG allele creates an open reading frame for IFN-λ4, whereas the alternative IFNL4 TT variant eliminates IFN-λ4 [9]. IFNL4 rs368234815 influences the signalling pathway between interferon-λ3 receptor 1 (IFN-λR1) and interferon-stimulated response element (ISRE). Overexpression of IFNL4 suppresses IFNL3 induction and promoter activation [10]. Transient IFNL4 overexpression in a hepatoma cell line induced signal transducer and activator of transcription (STAT)1/STAT2 phosphorylation and expression of interferon-stimulated genes (ISG) [9]. Low intracellular expression of IFN-λ4 induces IFN-λ expression, leading to the pre-activation of the Janus activated kinase (JAK)-STAT signalling and expression of ISG [11]. The phenotype of a favourable outcome of HCV infection correlates with the inability to encode IFN-λ4 [9,10].

There is a high linkage disequilibrium (LD) between rs368234815 and rs12979860, previously designated IFNL3 rs12979860 [12]. At present, we know that rs12979860 lies in the intron 1 of IFNL4 and has to be related to IFNL4. Since 2013, it is known that rs12979860 is associated with HCV spontaneous or therapeutic eradication due to its high LD with IFNL4 rs368234815 (TT/ΔG) [9]. In the study by Ferraris et al. [11], IFNL4 ∆G showed 100% correlation with the TT genotype of rs12979860. Compared to rs12979860, rs368234815 appeared similarly [9,13] or more strongly [14-16] associated with HCV clearance. IFNL3 rs4803217, altering IFNL3 mRNA stability [3], is also in a substantial LD with IFNL4 rs368234815. However, rs4803217 seems to be less strongly associated with HCV clearance compared to IFNL4 rs368234815 [17].

An association between IFNL polymorphisms and HCV clearance was studied mainly in non-uremic subjects [4-10,15-18]. The meta-analysis by Xie et al. [19] revealed that the rs368234815 TT/TT genotype was correlated with the sustained virological response in HCV-1/4-infected Caucasian patients but not in HCV-2/3-infected Caucasian patients. Conversely, the rs368234815 ΔG/ΔG genotype was linked to treatment failure in Caucasian patients, regardless of the HCV genotype. Reports concerning such associations in patients suffering from haemodialysis (HD)-dependent end-stage renal disease (ESRD) are scarce [20-23], and, to our knowledge, were not conducted for IFNL4 rs368234815, currently designated as the most promising predictor of HCV resolution [17]. Uremic milieu changes the expression of genes [24]. Therefore, it is worthy to investigate whether the impact of IFNL polymorphic variants on HCV clearance is comparable in HD patients to that observed in non-uremic populations.

We aimed to investigate whether IFNL4 rs368234815 is associated with spontaneous HCV clearance in ESRD patients on regular HD treatment. If so, whether genotyping of rs368234815 in HD subjects can be more useful as a predictor of spontaneous HCV resolution than other tested IFNL3/IFNL4 polymorphisms (near IFNL3 rs12980275, IFNL3 rs4803217, IFNL4 rs12979860, near IFNL4 rs8099917). Additionally, a retrospective survival analysis was performed concerning IFNL4 rs368234815 polymorphism. For a better understanding of genetic mechanisms underlying differences in analysed phenotypes possible attributed to IFNL (spontaneous HCV clearance, survival), we used in silico methods for prediction of alterations in transcription factor (TF) binding sites (TFBS) caused by the tested polymorphisms.

Patients

The study included 161 HD subjects with persistently positive anti-HCV antibodies. They were enrolled between January 2009 and November 2018 in the Wielkopolska region of Poland. The same patients were also tested in this study for near IFNL3 rs12980275, IFNL3 rs4803217, IFNL4 rs12979860, and near IFNL4 rs8099917. Some of them were included in our previous studies indicating associations of these SNPs with spontaneous HCV clearance in HD subjects [20,22]. The current data of rs12980275, rs4803217, rs12979860, and rs8099917 were used to compare predictivity of spontaneous HCV clearance between the newly tested rs368234815 and previously established genetic markers of HCV resolution.

Enrolled patients were never treated with anti-HCV medications. Among them, 93 (57.8%) were persistently HCV RNA positive and composed the case group, whereas 68 (42.2%) spontaneously resolved HCV infection (the control group).

Demographic, clinical, and baseline laboratory data of HD patients were obtained from physicians of HD facilities.

Survival studies

All anti-HCV positive patients genotyped for IFNL4 rs368234815 were included in the retrospective longitudinal survival study. In this group, survival probability since the onset of renal replacement therapy (RRT) to the last data collection (22^nd September – 7^th October 2019) was analysed concerning IFNL4 rs368234815 genotypes.

Genotyping

Chromosomal localization of tested IFNL3 and IFNL4 polymorphisms is shown in Supplementary Fig. 1A. of the Additional file 1.

IFNL4 rs368234815 polymorphism (TT/TT, ΔG/TT, ΔG/ΔG) was genotyped by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method as recently described by Pouryasin et al. [25]. Genotyping of rs4803217, rs12980275, rs8099917, and rs12979860 polymorphisms was carried out by a high-resolution melting curve analysis (HRM) as in our previous studies [20,22]. Supplementary Table 1 in the Additional file 1 shows conditions for the identification of polymorphisms genotyped by PCR-RFLP or HRM.

For quality control, approximately 10% of the randomly chosen samples were re-genotyped using the same genotyping method; the concordance rate was 100%. Among 161 tested samples, two (1.2%) failed the genotyping of rs12980275 and rs4803217 each, four (2.5%) – rs12979860, and one (0.6%) - rs8099917. Samples that failed the genotyping were excluded from statistical analyses.

Prediction of transcription factor binding sites

Potential regulatory impacts of the rs4803217, rs12980275, rs8099917, rs12979860, and rs368234815 through modifications of the TFBS motifs were assessed with the experimental ENCODE TFBS ChIP-seq data [26] and in silico prediction of DNA-binding sites collected in HOCOMOCO version 9 [27], JASPAR CORE version 5.0 ALPHA 2016 [28], and CIS-BP version 1.02 [29] databases with FIMO software version 4.11.1 [30].

We performed the computational analysis on the GenBank DNA sequences (contig NT_011109.17) [31] adjacent to SNP positions. FASTA sequences, one per each SNP allele, were used as an input for the FIMO. To minimize both false positive and false negative rates, the background file was calculated directly from input sequences with MEME-suit fasta-get-Markov script.

Results were analysed via a self-developed Python script to find locations of ENCODE ChIP-seq validated TFBS and FIMO predicted motif BS overlapping SNP positions. A p-value < 0.0005 and q-value < 0.05 were selected as the cut-off values for reliable predictions. Motifs matched in both orientations were also analysed concerning perfect reverse complement forming, thus increasing the true positive match probability. We obtained identified TF annotation information from the SwissProt database [32] and filtered statistically significant differentially bound hits of Homo sapiens motifs.

Statistical methods

The results are presented as a percentage for categorical variables, or medians (range) for continuous variables because they were usually non-normally distributed as determined by the Shapiro–Wilk test.

Demographic, clinical and laboratory data were compared in anti-HCV positive HD subjects stratified by results of HCV RNA testing. Continuous variables were compared using the Mann-Whitney test. The Chi-squared test or exact Fisher`s test was used for comparison of categorical variables, as appropriate.

The Hardy–Weinberg equilibrium (HWE) was analysed to compare the observed genotype frequencies to the expected ones using the Chi-square test (P > 0.05 with df = 1 for equilibrium).

Polymorphisms were tested for trends in association using Fisher`s test if two groups were compared or the Cochran-Armitage test if three groups were compared (P_trend). Genotype (P_genotype) and allelic (P_allelic) distributions were compared between the tested groups using Pearson's Chi-squared test or Fisher’s exact test, as appropriate.

Odds ratios (OR) and 95% confidence intervals (CIs) for OR were calculated to quantify how strongly the presence or absence of the tested allele or genotype is associated with the presence or absence of selected phenotypes of HD patients. Pearson's Chi-squared test was used for statistical evaluation of OR. All probabilities were two-tailed. Logistic regression was applied to assess the significance of the IFNL4 genotype among other possible determinants of HCV clearance. A ROC curve was plotted to show the AUC as a measure of the accuracy of the model.

The statistical power of this study was estimated by Quanto 1.2.4, under the “unmatched case-control” study design, with the present sample sizes and genetic effects that were observed in the study. All analyses were performed with a type I error rate of 0.05.

Statistical analyses of the above-mentioned data were performed using Statistica version 12 (Stat Soft, Inc., Tulsa, OK, USA). P-values less than 0.05 were considered significant. However, a Bonferroni correction was used for significance in detailed association analyses involving rs368234815, because such associations were not previously evaluated in HD patients.

Pair-wise LD between IFNL3 and IFNL4 polymorphisms was computed as both D’ and r² using the genotype data from the tested sample and the Haploview 4.2 software [33] (Supplementary Fig. 1B in the Additional file 1). Haplotypes were estimated using the mentioned Haploview 4.2 software (sliding windows method) and statistically analysed if their incidence in the examined group was over 1%. Statistical significance was assessed using the 1000-fold permutation test. Only P-values corrected by the test rules were considered significant.

Patients

The characteristics of HD patients exposed to HCV are shown in Table 1. Patients showing HCV RNA positivity compared with HCV RNA negative subjects were younger at RRT onset, had longer RRT duration, and more frequent chronic glomerulonephritis as a cause of ESRD. Liver enzyme activities were higher in HCV RNA positive patients.

IFNL4 rs368234815, other IFNL polymorphisms, and spontaneous HCV resolution

In all tested groups, the observed genotype frequencies were in concordance with HWE (Supplementary Table 2 in the Additional file).

In HCV exposed HD patients, IFNL4 rs368234815 was significantly associated with spontaneous HCV elimination. The TT/TT genotype subjects benefited with a 2.6-fold higher frequency of spontaneous HCV clearance compared with patients harbouring the ∆G allele (Table 2). In the dominant model of inheritance, the association of rs368234815 with the outcome of HCV infection was similar to that documented for other tested IFNL3/IFNL4 polymorphisms. In the additive model, rs368234815 TT/TT was the strongest predictor of HCV clearance. All these analyses were performed with the sample power equal to or exceeding 93% (Table 3).

IFNL4 rs368234815 as a predictor of spontaneous HCV clearance among other clinical data

Together with the TT/TT genotype of rs368234815, age at RRT onset, RRT duration, and chronic glomerulonephritis were chosen as possible explanatory variables for HCV spontaneous clearance. Among tested variables, only the TT/TT genotype remained a significant predictor of HCV resolution (OR 2.526, 95% CI 1.274 – 5.009, P = 0.008). The accuracy of the model in the receiver operating characteristic (ROC) curve analysis indicated the area under the curve (AUC) of 0.710 (Fig. 1).

Linkage disequilibrium

We examined LD between the rs12980275, rs4803217, rs12979860, rs368234815, and rs8099917 SNPs (Supplementary Fig. 1B in the Additional file 1). IFNL4 rs368234815 was the most strongly associated with rs12979860 (r² = 0.81); the weakest association was shown between this SNP and rs8099917 (r² = 0.31).

IFNL3 and IFNL4 haplotype analysis

We analysed the haplotypes of IFNL3 and IFNL4 concerning the outcome of HCV infection (Table 4). If all haplotypes other than the tested one were pooled together and used as the reference, spontaneous HCV clearance was associated with 10 haplotypes composed of at least two major alleles but without any variant allele of tested IFNL3/IFNL4 SNPs. The strongest association with spontaneous HCV clearance was revealed for the haplotype rs4803217_rs12979860_rs368234815_rs8099917 C_C_TT_T, whereas persistent HCV infection was attributed to the haplotype rs12979860_rs368234815 T_∆G.

In silico analysis

Table 5 summarizes data of the in silico analysis.

ENCODE TFBS ChIP-seq dataset from 187 TFs tested in 125 cell lines reported no peaks overlapping positions of rs4803217 and rs12980275. ENCODE ChIP-seq peaks for the ZNF263, GATA1, POLR2A and ARID3A TFs overlapped the position of the rs8099917. Positions of the rs12979860 and rs368234815 overlapped with the EZH2 ENCODE ChIP-seq peaks. In our in silico analysis, none of the predicted ENCODE ChIP-seq peaks for the TFBS overlapped positions of these SNPs directly. Therefore, they are not shown in Table 5.

The computational analysis revealed no direct differential motif binding for rs12980275 and rs8099917. Differentially bound transcription factors (TFs) were revealed for rs4803217 [HES2, DNA (cytosine-5)-methyltransferase 1 (DNMT1), Homeobox protein NKX3-2, POU domain, class 6, transcription factor 1 (POU6F1)], rs12979860 [E2F7, Oxysterols receptor LXR-alpha : Retinoid X receptor alpha (Nr1h3::Rxra), MAX Dimerization Protein (MGA), Oestrogen Related Receptor Alpha (ESRRA), T-box brain protein 1 (TBR1)], and rs368234815 (PLAG1 like zinc finger 1 identified also as M6422_1.02) (Table 5).

Survival and IFNL4 rs368234815

During the analysed period (an RRT duration) lasting in individual cases from 0.32 year to 34.0 years (median 10.9 years), there were 87 all-cause deaths of HD patients. Cardiovascular, infection-related, neoplasm-related, and other reasons of deaths comprised 56.3% (n = 49), 12.6% (n = 11), 10.3% (n = 9), and 20.7% (n = 18) of all deceased patients, respectively. Survival analysis of anti-HCV positive patients concerning rs368234815 SNP suggested an association of the ∆G/∆G genotype with mortality due to neoplasms (log-rank P = 0.005 in the recessive mode of inheritance) (Fig. 2).

To further explore a possible association between the IFNL4 rs368234815 ∆G/∆G genotype and mortality due to neoplasm, we genotyped not HCV-exposed HD patients who died from cancer in the analysed period and their DNA samples were stored. We gathered 80 samples (P-value for HWE concerning rs368234815 SNP = 0.752). In this group, the ∆G/∆G genotype was not associated with mortality due to cancer disease (log-rank P >0.05 for all three models of inheritance).

IFNL4 rs368234815 polymorphism is at present recognized as the most potent predictor of HCV infection outcome in the general population [17]. Our paper for the first time describes associations between IFNL4 rs368234815 polymorphism and spontaneous HCV clearance in HD patients who substantially differ from the general population due to persistent uremic status and altered immune competence [34]. In this study, we applied the PCR_RFLP method for genotyping of rs368234815 polymorphism which was recently described by Pouryasin et al. [25]. Among Iranian non-uremic patients with chronic HCV infection tested with this method, the rs368234815 ΔG/ΔG frequency was 17.3% [25]. Caucasian HD patients of Polish origin with persistently positive testing for HCV RNA revealed a similar frequency of the ΔG/ΔG genotype (16.1%).

Similarly, as in non-uremic subjects [14,15], rs368234815 was associated with spontaneous HCV clearance also in HD patients. On the same group of anti-HCV positive HD patients, we were able to show that major homozygosity in rs368234815 (TT/TT) similarly predicts spontaneous HCV clearance in the dominant model of inheritance as other tested IFNL3/IFNL4 polymorphisms already associated with HCV clearance in HD subjects. All tested IFNL polymorphisms (including rs368234815) indicated 2.4 – 2.8-fold higher frequency of spontaneous HCV clearance in HD patients characterized by the major homozygosity in analysed polymorphisms.

Predictivity of the IFNL4 rs368234815 TT/TT genotype concerning spontaneous HCV clearance was superior to other tested IFNL polymorphisms only in the additive inheritance model. In non-uremic subjects, the rs368234815 TT/TT genotype indicated 3.6-fold (Swiss Caucasians [15]) – 12-fold (American white women [14]) frequency of spontaneous HCV clearance compared with the ∆G/∆G genotype. In HD patients, the TT/TT genotype predicted a 6.4-fold higher probability of spontaneous HCV clearance. Therefore, uremic conditions do not seem to influence substantially IFNL4 rs368234815 expression concerning spontaneous HCV resolution. Additionally, an identification of the homozygosity in the variant ∆G allele of rs368234815 in HD subjects means a more potent prediction of persistent HCV infection that it is observed in the case of the variant homozygosity of other tested IFNL3/IFNL4 polymorphisms.

In HD patients, haplotypes containing IFNL4 rs368234815 alleles are also strongly associated with the outcome of HCV infection. Spontaneous HCV clearance was associated with 10 haplotypes composed of at least two major alleles but without any variant allele of tested IFNL3/IFNL4 polymorphisms. The strongest association with HCV clearance was revealed for the haplotype rs4803217_rs12979860_rs368234815_rs8099917 C_C_TT_T, whereas persistent HCV infection was mostly attributed to the rs12979860_rs368234815 T_∆G. Both these haplotypes include rs368234815 alleles. Although the major allele of rs4803217 was considered as the favourable allele [35,36], the haplotype including this allele and the IFNL4 ∆G allele in African American individuals has associated with a poor day 28 anti-HCV response compared with the association shown for the haplotype composed of minor alleles of IFNL3 rs4803217 and IFNL4 rs368234815 [17]. The haplotype data of O'Brien et al. [17] and ours indicate the importance of IFNL4 rs368234815 alleles in HCV clearance, although haplotypes including only favourable or only non-favourable alleles appeared stronger predictors of HCV outcome than those composed simultaneously of major and minor alleles, at least in HD patients.

We identified that minor alleles of the rs368234815, rs12979860, and rs4803217 cause statistically significant changes in TFBS. Therefore, the individual or combined effects of those changes may contribute to the differential IFNL3 and IFNL4 expression. As IFNL3/IFNL4 polymorphisms are well-established predictors of HCV clearance, removal or addition of the host TFBS in the presence of the minor alleles of tested polymorphisms might be at least partial explanation of HCV persistence in the IFNL3/IFNL4 minor allele bearers. Our computational analysis provides a list of candidate differentially bound TFBS in tested variants, for which the experimental ChIP-seq analysis could be applied to verify their impact exerted by the minor alleles. The following example shows how important is a need for such verification. In silico analysis revealed that the host TFBS for DNA DNMT1 is removed in the IFNL3 rs4803217 minor allele bearers who are predisposed for persistent HCV infection [17,35,36, this study]. However, CpG islands for DNMT1-induced DNA methylation were not shown in the interferon-λ genetic region [37], so removal of TFBS for DNMT1 may have no clinical relevance concerning HCV clearance.

In our study, the IFNL4 rs368234815 ∆G/∆G genotype appeared to be associated with cancer mortality in HCV-exposed HD patients. We consider this finding as a preliminary one due to a small number of HD patients who died from cancers in this group. Additionally, such a survival analysis in not HCV-exposed subjects did not reveal any relationship between neoplasm-related mortality and IFNL4 rs368234815 SNP. IFN-λ has a dual role in cancer. Direct antitumor effects of IFN-λ include inhibition of cell proliferation, promotion of cell apoptosis and cell cycle arrest. Indirect antitumor effects of IFN-λ include immune cell activation and angiogenesis inhibition. However, new evidence indicates that IFN-λ can also promote oncogenesis [38]. IFNL polymorphisms may differentiate their role in cancer antitumor effects or oncogenesis. Our in silico analyses demonstrated that neoplasm-related mortality may be due to the removal of TFBS for PLAGL1 zinc finger 1 protein which is associated with anti-proliferative activities and tumour suppression. PLAGL1 gene is often deleted or methylated and silenced in cancer cells [39]. This interesting aspect needs further elaboration.

In HD patients, the association of IFNL4 rs368234815 with the spontaneous HCV clearance is better than that documented for other IFNL3/IFNL4 polymorphisms (IFNL3 rs12980275, IFNL3 rs4803217, IFNL4 rs12979860, and near IFNL4 rs8099917) only in the additive mode of inheritance.
An identification of the homozygosity in the variant ∆G allele of rs368234815 means a more potent prediction of persistent HCV infection in HD subjects that it is observed in the case of the variant homozygosity of other tested IFNL3/IFNL4
Removal of PLAGL1 TFBS in subjects harboring the rs368234815 ∆G allele may contribute to cancer susceptibility.
The association of rs368234815 with cancer-related mortality needs further studies in HCV-exposed HD subjects.

AUC - area under the curve, CI - confidence interval, DNA – deoxyribonucleic acid, DNMT1 - DNA (cytosine-5)-methyltransferase 1, ESRD - end-stage renal disease, ESRRA - Estrogen Related Receptor Alpha, HCV- hepatitis C virus, HD - hemodialysis, HRM - high-resolution melting curve analysis, HWE - Hardy-Weinberg equilibrium, IFNL - interferon-λ genetic region, IFNL3 - interferon-λ3 gene, IFNL4 - interferon-λ4 gene, IFN-λ – interferon-λ, INF-λR1 – interferon-λ receptor 1, ISG - interferon-stimulated genes, ISRE - interferon-stimulated response element, JAK - Janus activated kinase, LD - linkage disequilibrium, MGA - MAX Dimerization Protein, mRNA – messenger ribonucleic acid, Nr1h3::Rxra - Oxysterols receptor LXR-alpha : Retinoid X receptor alpha, OR - odds ratio, PCR-RFLP - polymerase chain reaction-restriction fragment length polymorphism, POU6F1 - POU domain, class 6, transcription factor 1, ROC - receiver operating characteristic, RNA – ribonucleic acid, RRT - renal replacement therapy, SNP – single nucleotide polymorphism, STAT - signal transducer and activator of transcription, TBR1 - T-box brain protein 1, TF – transcription factor, TFBS – transcription factor binding sites.

Ethics approval and consent to participate: The study design was approved by the Institutional Review Board of Poznan University of Medical Sciences, Poland. Written informed consent was obtained from all HD patients included in the study or their parents, as appropriate. All methods were performed in accordance with the ethical standards of the Helsinki Declaration (1964, amended most recently in 2008), which was created by the World Medical Association.

Consent for publication: Not applicable.

Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests: The authors declare that they have no competing interests.

Funding: This work was supported by the Poznan University of Medical Sciences, Poznań, Poland [grant numbers 502-01-02225363-03679, 502-01-01124182-07474]. The study sponsor was not involved in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the manuscript for publication.

Authors' contributions: AEG conceived the study, designed the research, was involved in the data collection, analysed the data, wrote the manuscript, and participated in funding for the project. MKŚ was involved in the data collection and analysed the data. WM was involved in the data collection. AM was responsible for the genotyping and haplotype analysis. IS and MF did in silico analyses. PPJ participated in funding for the project. All authors edited and approved the final version of the manuscript.

Acknowledgements: Selected results of this study has previously been presented at 56^th ERA-EDTA CONGRESS. Budapest, Hungary, June 13^th-16^th 2019. Abstr. FP679. Nephrol. Dial. Transplant. 2019:34(suppl)1, i278-i279.

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Polymorphism rs368234815 of Interferon Lambda 4 Gene and Spontaneous Clearance of Hepatitis C Virus in Haemodialysis Patients: A Case Control Study

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