Confounding by Single Nucleotide Polymorphism rs117648444 (P70S) Affects the Association of Interferon Lambda Locus Variants with Response to Interferon-α-Ribavirin Therapy in Patients with Chronic Genotype 3 Hepatitis C Virus Infection

Abstract

Genome-wide association studies discovered interferon lambda (IFNL or IFN-λ) locus on chromosome 19 to be involved in clearance of chronic hepatitis C virus (HCV) infection in patients following interferon-α-ribavirin (IFN-RBV) therapy. Subsequent studies established a dinucleotide polymorphism rs368234815, as the prime causal variant behind this association. The ΔG allele of this variant gives rise to a new IFNL gene, IFNL4, coding for IFN-λ4 whose activity paradoxically associates with lesser viral clearance rates. A low-frequency, nonsynonymous single nucleotide polymorphism (SNP) rs117648444 within the 2nd exon of IFNL4 changes the 70th amino acid from proline to serine resulting in lower activity of the functional IFN-λ4 protein, thereby increasing HCV clearance rates. In the present study, we used a cohort of genotype 3 HCV-infected patients, drawn from different geographical regions of India who underwent IFN-RBV therapy, to examine the association of several important IFNL locus SNPs/variants with sustained virological response (SVR). Intriguingly, the causal variant rs368234815 did not show the best strength and significance of association with SVR, while further analysis revealed that a negative confounding effect of rs117648444 was responsible for this phenomenon. Our results indicate that IFNL locus SNPs are subject to either a positive or a negative confounding effect by rs117648444; the nature of confounding depends on the linkage of the IFNL SNPs with the low-activity IFN-λ4-generating minor allele of rs117648444. Thus, our work demonstrates that the linkage disequilibrium structure of the IFNL region may confound the results of association studies. These results have implications for the design and understanding of future case-control studies involving IFNL locus SNPs/variants.

Impact of IFNL4 Genetic Variants on Sustained Virologic Response and Viremia in Hepatitis C Virus Genotype 3 Patients

“…The fourth SNP that was tested (SNP rs117648444 [G>A]) is located in an exon of IFNL4 and substitutes a proline for a serine at position 70 (P70 and S70, respectively). It has been shown to reduce antiviral activity in HCV infected patients (Terczyńska-Dyla and others 2014) to be a confounder of IFN-induced SVR in HCV genotype 3 patients (Bhushan and others 2017).…”

Genetics of the Human Interferon Lambda Region

“…Because only the rs368234815-dG allele provides functional significance to rs117648444, this marker should not be tested alone. The combination of rs368234815 and rs117648444 improved and explained the association with viral clearance and expression of interferon-stimulated genes, providing support for the primary causal role of IFN-λ4 and its variants in these associations (Galmozzi and Aghemo 2014; Terczynska-Dyla and others 2014; Bhushan and others 2017; Bhushan and Chinnaswamy 2018; Galmozzi and others 2018).…”

Functional genetic variants of the IFN-λ3 (IL28B) gene and transcription factor interactions on its promoter

“…All the constructs were confirmed for correctness by DNA sequencing. The details of the HCV-infected patient cohort including ethics approvals are provided elsewhere [ 10 ]. All the DNA oligonucleotides used in the study were custom synthesized from IDT; the probes used for electrophoresis mobility shift assays (EMSA) were synthesized by Sigma-Aldrich (St. Louis, MO, USA).…”

“…The details of the 4 kb IFN-λ3 promoter construct (p4kbIFNL3) are described in Ref. [ 10 ]. The plasmid encoding p300 gene was purchased from Addgene, USA.…”