Evaluation of IL28B Gene Sequence Variations in the Diagnosed Null-Response to DAAs Therapy by Intra PBMCs Nested HCV RNA PCR

INTRODUCTION

Introduction of first and second generations DAAs therapies markedly improved HCV resistance to treatment and paved the way to evaluate impacts of host related factors that affect HCV induced liver pathology and therapy response outcomes ^1,2.Positive association of certain IL28B-gene alleles(e.g. T-allele) was found in cirrhotic patients with CHCV-G1 infection in Caucasianpopulations. However, sequencing IL28B-gene in limited numbers of CHCV population from Asia, Latin America or the Middle East with hepatic-cirrhosis was inconclusive ^3-6. The TC IL28B-gene SNP was mainly reported in CHCV liver cirrhosis followed by the TT, while the wild CC genotype was the least reported sequences. ^7-8 The IL28B-

geneallele changes or SNPin liver cirrhosis was denied by others .⁹

Spontaneous HCV clearance in addition to favorable response to the INF based therapy were associated with wild IL28B CC gene sequence[10-13]. Contrarily, the wild CC IL28B-gene sequences were reported in association with a higher prevalence of hepatic cirrhosis ^17,18, and raised serum markers of necro-inflammatory hepatic responses to CHCV-infection. ^6,8,10,14,15 The primary immune response to HCV-infection is mediated predominantly through IFN-λ cytokines and may be influenced by IL28B genotype . ^16,17 There may be an association between frequent presence of IL28B T-allele and developing HCC. ¹⁸

Association of IL28B-gene TT SNP with post-DAAs therapy HCV relapse or resistance, and the wild CC IL28B-gene with SVR was reported in 2019 ¹⁹ ; other researchers found that SVRswere seen more often in C compared to T allele ²⁰. The high cure rate (up to 98%) that was reported in many studies is based on clearance of the virus only from serum, not from cells. Positive HCV PBMCs-PCR at 12weeks after end of treatment (EOT) with DAAs was addressed in < 60% [21-23].The disparity in SVRs outcomes evaluation after testing sera versus PBMCs for HCV-RNA excuses researchers from holding off the IL28B-gene studying that was requested by other investigators ²⁴.

Goals of the current research included evaluation of IL28B-gene SNPs impacts on response to the direct acting antivirals (DAAs) in the SVR subjects with negative serum and PBMCs-PCR, solitary intra-PBMCsHCV viral infections, and positive serum viral RNA-PCR. The results of IL28B-gene sequencing in association with DAAs therapy outcomes were correlated to parenchymal hepatic changes.

Subjects AND MATERIALS

Patients were recruited from the outpatient’s clinic of hepatology, gastroenterology, and infectious diseases at Al-Azhar university specialized hospital. Twelve weeks after end of treatment (EOT) with DAAs, 45 subjects were screened by both serum and PBMCs-HCV SRT-PCR and divided into the following equal groups (n=15 each): group I had negative both serum and PBMCs SRT-PCR,group II with positive PBMCs with negative serum SRT-PCR, and group III had positive serum SRT-PCR.All study subjectsreceived Sofosbuvir at 400 mg + Daclatasvir at 60 mg ± Ribavirin for 12 weeks according to national guidelines of HCV management at Egypt.

  II. SVR status assessment by SRT-PCR for detection of HCV-RNA: HCV-RNA was extracted from serum by using QIAamp Viral RNA Mini Kit (Qiagen artus, Hildenm, German). Total cellular RNA was extracted from the isolated PBMCs using the single-step method that was first published by Chomczynski and Sacchi ²⁵ and modified later [26-29] to detect HCV-RNA in PBMCs. Nested SRT-PCR was performed as confirmatory test by using 25 reactions mixture as described before ³⁰.

III- IL28B Screening Tests:The SNPs on IL28B were identified using a real-time PCR protocol based on the pre-validated TaqMan MGB™ probe for allelic discrimination assay (Applied Biosystems). Allelic discrimination plots were produced in Statistical Package for The Social Sciences (SPSS version 16.0; SPSS, Chicago, IL).

IV. Evaluation of hepatic parenchymal changes (early fibrosis and late cirrhosis): CHCV induced liver tissues changes were examined by ultrasonography (US) images and were correlated with Fib4 scoring system in all study populations³¹.

V. Statistics: SPSS 16.0 (IBM; NY, USA) is used to analyze the current data set. P

RESULTS

Evaluation of HCV replication within PBMCs by detecting intracellular antisense RNA-strand.

Table 1: Recognition of viral RNA sense and antisense strands within PBMCs of viral relapsers regardless to viremic status

Significant viral replication within PBMCs of HCV relapses regardless to the status of serum SRT-PCR (P<0.05).Intracellular-antisense strands presents a solid prove of viral replication in group II and III.

Correlation of IL28B-gene SNPs with liver parenchymal changes as described by hepatic ultrasonographic image and Fib4-scores.

Table 2: Single nucleotide polymorphism (SNPs) of IL28-gene in relation to ultrasonographic hepatic image variations

The wild type sequences (CC) of IL28B-gene was seen more often in association with normal hepatic parenchyma in group I compared to group II and III (P<0.01). The CT SNP of IL28B gene were equally detected in both group I and II (P>0.3)with significant detection on comparison with group III (respectively, P=0.02391 and 0.055) when liver tissues had normal US image. The TT SNP was only recognized in viremic patients (group III) in association with liver cirrhosis on comparison with Group I (P=0.02256) and group II (P=0.08647).

Table 3: Single nucleotide polymorphism (SNPs) of IL28-gene in relation to various ranges of Fib4-scores values

Only TT SNP of IL28B-gene showed close to significantly increased frequency in viremic patients who had Fib4 score >3.25 when compared to group I (P=0.054). In the rest of Fib4-score results, wild and SNPs sequences of the same gene had no correlation with all grades of hepatic parenchymal changes in all groups (P>0.09).

Correlation of IL28B-gene alleles with liver parenchymal changes as evaluated by hepatic ultrasonographic imageand Fib4-scores.

Table 4: IL28B-gene alleles nucleotide polymorphism in relation to ultrasonographic hepatic image variations

In normal hepatic parenchyma C allele was more seen in group I compared togroup II (P=0.000692) and group III (P=0.000003); the T allele wasdetected more often in intracellular HCV RNA relapserscompared to serologic relapse (P=0.012655). In cirrhotic patients the T allele was dominant in serologic relapses (group III) compared with cellular relapse (P=0.004308) and SVR subjects (P=0.000130); the C allele was recognized (27.35%) in intracellular relapsers compared to SVR subjects (P=0.005285).

Table 5: IL28B-gene alleles nucleotide polymorphism in relation to various ranges of Fib4-scores values

T allele was found in group III in association with high (Fib4:>3.25) or low (Fib4:<1.45) scores when respectively compared to group II (P=0.006369) and group I (P=0.039783). The group I was associated with frequent identification of C-allele within the intermediate score (Fib4:1.45-3.25) when compared with group III (p=0.010796).