Abstract
Many people living with hepatitis C virus (HCV) infection will continue to rely on interferon-based regimens until effective strategies to minimize the cost of directly acting antivirals (DAAs) and to improve treatment access are implemented. Host single-nucleotide polymorphisms related to IFNL3 and IFNL4 are associated with spontaneous clearance of HCV, and pegylated interferon– and DAA-based treatment outcomes. We describe a simple and rapid genotyping method for IFNL rs12979860, rs8099917, and rs368234815 using high-resolution melting analysis for DNA extracted from whole blood, buffy coat, plasma, serum, and dried blood spots. This assay successfully detected all three polymorphisms on DNA extracted by the automated platform easyMAG from all samples when compared to sequenced amplicons. Analysis of 126 participants with recent HCV infection from the Australian Trial in Acute Hepatitis C study demonstrated the prevalence of favorable single-nucleotide polymorphisms were 62%, 51%, and 45% for rs8099917 TT, rs12979860 CC, and rs368234815 TT/TT, respectively. The genotyping assay described here provides a rapid and affordable IFNL3 and IFNL4 genotyping method for a range of clinical sample types. Until global access to DAAs is achieved, IFNL3 and IFNL4 genotyping could identify those likely to clear naturally and in whom treatment could be delayed, or help prioritize DAA treatment to those less likely to respond to interferon-containing regimens.
Directly acting antivirals (DAAs) have revolutionized hepatitis C virus (HCV) treatment. However, high associated costs and restricted access mean that many of those living with HCV may not receive the benefit of these new therapies. As such, affordable diagnostic tools are required to prioritize and select cost-effective HCV treatment. Diagnostic tools, such as IFNL3 and IFNL4 genotyping, can predict those most likely to spontaneously clear HCV, for whom treatment could be delayed. These tools may also stratify individuals and prioritize DAA treatment to those less likely to respond to interferon-containing regimens, or identify those likely to respond well to therapy with shortened treatment. A combination of strategies that improve harm reduction and prevention programs, increase HCV screening rates, and optimize treatment regimens are required to reduce the transmission and burden of disease in populations most affected by HCV infection.
Spontaneous HCV clearance occurs in 25% of individuals.1,2 Polymorphisms in IFNL3 [single-nucleotide polymorphism (SNP) rs8099917, located near IFNL3 gene and SNP rs12979860] or called IL-28B, are the strongest host factors predicting both spontaneous HCV clearance in acute infection3 and response to interferon-based treatment in chronic infection.2,4,5 Recently, Prokunina-Olsson et al6 described a new dinucleotide variant rs368234815 (previously designated as ss469415590) that creates (ΔG) or disrupts (TT) an open reading frame of a gene encoding the interferon λ-4 (IFN-λ4) protein. The dinucleotide variant rs368234815 is in high linkage disequilibrium with rs12979860, but is more strongly associated with both HCV clearance, and pegylated interferon-α and ribavirin treatment response in individuals of African ancestry than rs12979860.6 Recent findings showed that recombinant IFN-λ4 protein strongly stimulates Jak-STAT signaling and interferon-stimulated gene induction through binding to the IFN-λ receptor.7 The active IFN-λ4 protein may therefore be the driver of high baseline hepatic interferon-stimulated gene expression at the time of infection and consequently poor HCV clearance.8 Those with the polymorphism resulting in an impaired IFN-λ4 (IFNL4) variant respond better to treatment and have improved rates of spontaneous clearance compared with those who express the active IFN-λ4 variant.8
There is recent evidence to suggest that IFNL polymorphisms may retain clinical relevance for management of interferon-containing DAA therapy. In treatment-naive patients receiving interferon-containing DAA treatment, the IFNL3 genotype continues to be significantly associated with sustained virological response and treatment duration required to achieve this response.9 Recently, the IFNL3 rs12979860 genotype has been shown to be a strong predictor of tissue inflammation and fibrosis during chronic HCV infection,10 potentially broadening the clinical relevance of IFNL genotypes to disease progression. The authors conclude that the IFNL3 genotype may be an important part of the development of an individualized patient management algorithm.
Our aim was to develop a rapid, reliable, and inexpensive method to genotype IFNL3 and IFLN4 SNPs using real-time high-resolution melting (HRM) analysis11,12 on a range of clinical sample types. This assay was then used to estimate the prevalence of favorable IFNL3 and IFNL4 in recent HCV, using a well-characterized cohort of participants from the Australian Trial in Acute Hepatitis C (ATAHC) study.
Materials and Methods
Study Population and Study Design
Assay validation and comparison of sample types was completed on 10 healthy volunteers (St Vincent's Hospital, Sydney; ethics approval LNR/12/SVH/358). The prevalence of IFNL3 and IFLN4 genotypes in recent hepatitis C virus infection was assessed in the ATAHC study. The ATAHC study enrolled 163 participants with acute and early chronic HCV infection. The study design has been described previously in detail.13 Acute and early chronic infection was defined as either anti-HCV antibody seroconversion in the past 2 years, or clinically documented acute HCV infection. Acute clinical HCV infection was defined as a positive HCV antibody and symptomatic seroconversion illness or alanine transaminase level >400 IU/L, with exclusion of other causes of acute hepatitis, at most 12 months before the initial positive anti-HCV antibody. All participants with available peripheral blood mononucleocytes (PBMCs) were identified for the extraction of genomic DNA for this study (N = 126).
Assay Design and Optimization
Several primers were designed to amplify a 300-bp region encompassing each SNP, rs8099917, rs12979860, and rs368234815 (using NCBI Primer_BLAST; http://www.ncbi.nlm.nih.gov/tools/primer-blast, last accessed May 11, 2015) located in human on chromosome 19: 19q13.13 for IFNL3 (NCBI reference number NT_011109.16) and 19q13.2 for IFNL4 (NCBIJN806234). PCR conditions, including primer combinations, temperature, and magnesium gradients, were optimized to produce high-quality melting curves for HRM analysis. Final PCR conditions and HRM analysis methods are listed in Reaction Conditions and HRM Analysis.
Sample Preparation
Nine milliliters of EDTA and acid citrate dextrose whole blood were collected from healthy volunteers to prepare several sample types: EDTA whole blood and dried blood spots (DBS), EDTA-derived dry pellet buffy coats and plasma, and acid citrate dextrose–derived serum. DBS were generated by pipetting 50 μL of EDTA whole blood per spot on a Whatman 903 Protein saver card (GE Healthcare, Silverwater, NSW, Australia). EDTA whole blood was centrifuged at 700 × g for 15 minutes to collect buffy coat (resuspended in 1 mL of sterile PBS) and plasma (recentrifuged at 1500 × g for 20 minutes). Acid citrate dextrose whole blood was centrifuged at 400 × g for 10 minutes for serum separation. ATAHC PBMCs were collected from lithium heparin at the time of the study and aliquoted around 10 million cells per mL in cryopreservant solution and stored in vapor-phase cryogenic storage tanks.
DNA Extraction
DNA from one DBS spot and 100 μL of resuspended buffy coat was extracted using the easyMAG (bioMerieux, Baulkham Hills, NSW, Australia) and eluted in 50 μL for subsequent PCR. DNA was extracted from 200 μL of whole blood, plasma, or serum using both easyMAG (bioMerieux) and QIAamp DNA Blood Mini Kit (Qiagen, Chadstone Centre, VIC, Australia) and eluted with 50 μL or 200 μL of elution buffer, respectively. The DNA concentration was measured with a NanoPhotometer (Implen, Munich, Germany). PBMCs (200 μL or 2 million) of ATAHC samples were used to extract genomic DNA using Axyprep-96 blood genomic DNA kit (AP-96-BL-GDNA-1; Fisher Biotec Australia, Wembley, WA, Australia). This 96-well format genomic DNA extraction format has been previously tested and compared with the Qiagen column kit with satisfactory results.
Reaction Conditions and HRM Analysis
Amplification and HRM analysis was completed on a Roche LightCycler 480 System and Roche Gene Scanning software version 1.5.0.46 (Roche, Castle Hill, NSW, Australia). PCR reactions contained a final concentration of 2 μmol/L each primer, 2.5 μL DNA, and 1× Roche LightCycler 480 High Resolution Melting Master mix (Cat. No. 04 909 631 001) in a final volume of 20 μL. A final concentration of 2 mmol/L MgCl2 was chosen for rs8099917 and rs12979860 and 2.5 mmol/L MgCl2 for rs368234815. PCR cycling conditions were 95°C for 5 minutes, then 40 cycles of 95°C, 56°C, and 72°C for 30 seconds, 1 minute, and 1 minute, respectively. PCR was followed by melting curve conditions of 95°C for 1 minute, 60°C for 2 minutes, and a continuous increase to 98°C with 25 acquisitions per degree and a cooling step at 40°C for 5 minutes. Primers sequences were rs1297860 forward 5′-ATTCCTGGCGTGGATGGGTACT-3′, rs1297860 reverse 5′-AGGCTCAGGGTCAATCACAGAA-3′, rs8099917 forward 5′-AGTAAGTCTTGTATTTCACCTCCTGG-3′, rs8099917 reverse 5′-GCTGGGCCCTAACTGATACGCTATAAT-3′, IFNL4 forward 5′-TTTGGCTTCCCTGACGTCTC-3′, and IFNL4 reverse 5′-GCTCCAGCGAGCGGTAGTG-3′ (reverse primer kindly provided by Prof. David Booth, Westmead Milenium Institute for Medical Research, NSW, Australia).
Fluorescence was measured in real time to determine the number of cycles needed (Cp) for successful amplification and HRM analysis of each PCR product. The genotype success rate per sample type was evaluated compared to the sequencing genotype results derived from buffy coat samples. A threshold of amplification of maximum Cp number has been determined for each sample type from the lowest Cp value for which samples are 100% correctly genotyped by HRM. This threshold is estimated to provide an indication of the maximum number of cycle numbers for which amplification is considered adequate to provide a reliable genotype. Inadequate amplification of heterozygote genotypes, potentially to the result of low DNA concentration, may potentially amplify only one allele and therefore incorrectly call the sample as a homozygote. This highlights the importance of controls from all genotypes when testing samples that may generate low concentrations of DNA.
The cost of the assay for consumables and reagents is estimated at $3.12AUD per test result run in duplicate (based on a full 96-well plate, including three genotype controls, a PCR negative control, and 44 samples). The cost of extraction is $5.20AUD and $15AUD per sample with the Qiagen column system and easyMAG platform, respectively. The time estimated to perform the assay is similar to any PCR assay, including DNA extraction. Genotyping could be performed in less than a day.
Confirmatory Sequencing Reactions
IFNL3 and IFNL4 genotypes were identified from buffy coat–derived DNA by Sanger sequencing and used as the method of reference. The SNP for each polymorphism was confirmed by the presence of two different nucleotides on the chromatogram as two overlapping color peaks for heterozygotes or a single nucleotide/peak for homozygotes. Samples with genotypes confirmed by sequencing were used as references to assess other sample types and HRM methods.
Statistical Analysis
All analyses were performed using the statistical package Stata version 12.1 (StataCorp, College Station, TX). Prevalence of favorable allele for HCV clearance in rs368234815 (TT/TT), rs12979860 (C/C), and rs8099917 (T/T) was assessed in ATAHC participants with available SNP results. Distributions of selected demographic, clinical, and virological variables were compared between favorable and nonfavorable genotypes using χ2 or U-tests, as appropriate. Statistically significant differences were assessed at P < 0.05 (P values are two sided).
Results
IFNL3 and IFLN4 HRM Assay Development and Validation
Assay development demonstrated that IFNL3 and IFNL4 PCR amplification produced fluorescent curves and melting peaks suitable for HRM genotyping analysis of all SNPs under a range of conditions: with an annealing temperature from 54°C to 59°C, and within a 2 to 3.5 mmol/L MgCl2 concentration. Primers sequences, amplicon size, and melting temperature used to amplify IFNL3 rs1297860 and rs8099917 and IFNL4 rs368234815 are summarized in Table 1. High-resolution melting curves under the final assay conditions for the three genotypes of each polymorphism can be found in Figure 1. The homozygote curves appeared parallel and differentiated by 0.5°C in the normalized and temperature-shifted curve graphs (Figure 1), the heterozygote curves were identified with an altered curve shape because of a difference in the temperature at which hetero- and homoduplexes dissociate. The alternative visualization of HRM curves as normalized and temperature-shifted difference plots (Figure 1) facilitated genotype identification relative to reference samples.
Table 1.
Primer Sequence, Amplicon Size, and Melting Temperature for IFN-λ Amplicons Used for HRM
| Primers | Amplicon size | Primer sequences | Melting range temperature |
|---|---|---|---|
| rs1297860 4F | 184 bp | 5′-ATTCCTGGACGTGGATGGGTACT-3′ | 90.5°C to 91.5°C |
| rs1297860 4R | 5′-AGGCTCAGGGTCAATCACAGAA-3′ | ||
| rs8099917 1F | 207 bp | 5′-AGTAAGTCTTGTATTTCACCTCCTGG-3′ | 78.0°C to 79.0°C |
| rs8099917 5R | 5′-GCTGGGCCCTAACTGATACGCTATAAT-3′ | ||
| rs368234815 F2 | 151 bp | 5′-TTTGGCTTCCCTGACGTCTC-3′ | 92.5°C to 94.2°C |
| rs368234815 R1 | 5′-GCCTGCTGCAGAAGCAGAGAT-3′ |
HRM, high-resolution melting.
Figure 1.
High-resolution melting (HRM) analysis curves. HRM analysis enables homogeneous genotyping without probes, even when the sequence change is only a single base. With saturation dyes, the PCR product is labelled along its entire length so that all melting domains are detected. Single-base genotyping is identified by the difference of melting curves as shown by the normalized melting curve (left graphs) and temperature shift (right graphs) for rs8099917, rs12979860, and rs368234815, with favorable homozygotes as green curves (TT, CC, and TT/TT, respectively), heterozygotes in blue curves (GT, CT, and ΔG/TT, respectively), and unfavorable genotype as red curves (GG, TT, and ΔG/ΔG, respectively). The assay was performed using the Roche LightCycler 480 System and the HRM analysis performed with the Roche Gene Scanning software.
A comparison of genomic DNA extracted from each sample type, using either easyMAG or QIAamp columns, showed plasma and serum produced the lowest concentration (<2 ng/mL) (Table 2), which was also reflected by high Cp numbers between 35 to 40 cycles. With the elution volume adjusted, the yield of extraction from DBS was equivalent to whole blood with a final concentration of 5 and 20 ng/mL, respectively. Buffy coat produced the highest DNA concentration, 70 ng/mL, and amplified between 22 and 25 cycles (Table 2).
Table 2.
Extraction and Elution Volumes Used for Sample Types and Their DNA Concentration Measured by Nanophotometer (Implen)
| Sample type | Sample volume extracted | Elution volume by Qiagen column extraction | Elution volume by easyMAG extraction | Average DNA concentration (ng/mL) Qiagen/easyMAG |
|---|---|---|---|---|
| Plasma (EDTA) | 200 μL | 200 μL | 50 μL | 0.17/1.72 |
| Serum (ACD) | 200 μL | 200 μL | 50 μL | 0.83/1.50 |
| Whole blood (EDTA) | 200 μL | 200 μL | 50 μL | 19.15/19.65 |
| Dried blot spot (venous EDTA whole blood) | 50 μL | 50 μL | 5.35 | |
| Buffy coat cell pellet (EDTA whole blood)∗ | 50 μL | 68.60 |
ACD, acid citrate dextrose.
Buffy coat cell pellet issued from 1/10th of a 9-mL EDTA tube or estimated to be equivalent to 800 μL of whole blood.
The validation study of 10 volunteers demonstrated that the HRM genotype results from the buffy coat, whole blood, and serum extracted by the easyMAG method (Figure 2) matched sequencing data from buffy coat controls. DNA extracted from serum or plasma by Qiagen columns incorrectly identified some known heterozygotes as homozygotes (3 of 10). A Cp threshold for successful genotyping was estimated from these graphs at 37, 38, and 39 Cp for IFNL rs8099917, rs1297860, and rs368234815 respectively.
Figure 2.
The number of cycles (Cp) values and genotype success rate in clinical samples for single-nucleotide polymorphisms (SNPs) rs8099917, rs12979860, and rs368234815. Every sample type high-resolution melting analysis (HRM) result for genotyping were compared to the result from sequencing using buffy coat samples, and the calculated success rate is shown on top of each sample measurement distribution. The y axis shows the Cp needed for PCR amplification. Cp per sample type is inversely proportional to DNA concentration. A threshold of amplification has been estimated for every SNP for which every cohort samples would need to be amplified under this threshold for 100% success rate genotyping. The threshold has been estimated in our condition at 37.2 cycles for rs12980275, 38.4 cycles for rs8099917, and 39.7 cycles for rs368234815. DBS, dried blood spots.
The Distribution of IFNL3 and IFNL4 Genotypes in Individuals with Recent HCV Infection
A total of 163 participants were enrolled in the ATAHC study (Figure 3). Among 126 PBMCs samples available for extraction, 16 participants were not enrolled into the ATAHC protocol and were excluded from this study. One-hundred and ten participants were included (Figure 3).
Figure 3.
Characteristics of Australian Trial in Acute Hepatitis C (ATAHC) participants among those with available genotypes for IFNL single-nucleotide polymorphisms (SNPs) rs12980275, rs8099917, and rs368234815. Data for IFNL rs8099917, rs12979860, and rs368234815 polymorphisms were available for 109, 106, and 109 individuals, respectively. The genotype frequency is shown for each SNP. The ethnicity distribution of the ATAHC participants tested is 93% Caucasian (102 participants of 110), 4% Asian (4 of 110), 2% indigenous (2 of 110), 1% African ancestry (1 of 110), and 1% defined as mixed or other (1 of 110). PBMCs, peripheral blood mononucleocytes.
Data for IFNL rs8099917, rs12979860, and rs368234815 were available for 109, 106, and 109 individuals, respectively. The genotype frequency in this population was as follows for rs8099917: 62% T/T (n = 68), 33% G/T (n = 36), 5% G/G (n = 5); rs12979860: 51% C/C (n = 54), 40% C/T (n = 42), 9% T/T (n = 10); and rs368234815: TT/TT 45% (n = 49), 45% TT/ΔG (n = 45), and 10% ΔG/ΔG (n = 11) (Figure 3). The ethnicity distribution of the ATAHC participants tested is 93% Caucasian (102 participants of 110), 4% Asian (4 of 110), 2% indigenous (2 of 110), 1% African ancestry (1 of 110), and 1% defined as mixed or other (1 of 110).
Discussion
For many people with hepatitis C infection, the cost and restrictions associated with interferon-based and DAA-based treatment limits access to effective therapy. IFNL3 and IFNL4 genotypes are known to be associated with spontaneous clearance, and interferon-based treatment outcomes.9 Here, we described a rapid and affordable method for the detection of IFNL3 and IFNL4 SNPs in a range of HCV clinical sample types. Application of this IFNL3 and IFNL4 genotyping assay could be used to reduce the overall costs associated with HCV treatment by helping predict spontaneous clearance and select effective treatment options and durations of treatment for individuals infected with HCV.14,15
The HRM assay described in this paper provides a rapid, reliable, and affordable alternative to current methods, such as sequencing or hydrolysis probe-based detection,11 to distinguish genotypes for rs12979860, rs8099917, and rs368234815.16 Many new thermal cycling instruments provide platforms that are now capable of HRM. This assay has been successfully tested on different clinical samples such as whole blood, buffy coat, and DBS. Serum or plasma could be used for genotyping but would require collection from blood with low centrifugation (400 × g) and the use of an automated extraction platform to increase DNA yield. Altogether, this study shows that this novel assay could be easily adopted by research laboratories or virological laboratories requiring a rapid and affordable IFNL3 and IFNL4 genotyping assay.
The distribution of IFNL3 favorable genotypes in this current study of individuals with recent HCV infection in Australia (51% rs12979860 CC and 62% rs8099917 TT) is higher than that reported in other Australian cohorts of genotype 1 chronic HCV infection (36% rs12979860 CC and 54% rs8099917 TT).17 This result could be explained by the fact that participants with the favorable genotype are more likely to spontaneously clear the virus1,3,18 and therefore not develop chronic infection. Consequently, participants with recent HCV infection recruited into acute studies are more likely to carry the favorable genotype than those with chronic HCV infection. This distribution of IFNL4 rs368234815 among individuals with recent HCV infection in this current study is similar to that in a German study of HIV-positive patients with acute and chronic hepatitis C.19
This study has a few limitations. The HRM assay relies on amplification of reference samples of known genotype, and interpretation relative to the control is improved if the reference sample is processed in an identical method to the unknown. Likewise, genotyping coverage is also improved by using high-quality nucleic acids. Samples from the ATAHC cohort, used in this study, were collected 7 to 10 years earlier, and the success rate is likely to be higher with samples from more recent cohorts.
In conclusion, this study describes a rapid, simple genotyping assay for IFNL rs12979860, rs8099917, and rs368234815 for a range of clinical sample types. This assay may assist in the clinical management of people infected with hepatitis C by predicting the likelihood of HCV clearance and response to interferon-based therapy.
Acknowledgments
We thank all the members of the ATAHC study group, including participants, nurses, investigators, coordinators and research staff, and Prof. David Booth for providing the sequence of the IFNL4 reverse primer.
Footnotes
Supported by National Health and Medical Research Council grant HIV and HCV vaccines and immunopathogenesis #510448, NHMRC career development award 1035383 (J.G.), NHMRC fellowship 1051859 (G.M.), and NHMRC practitioner research fellowship 1028432 (G.D.). The Australian Trial in Acute Hepatitis C study was funded by NIH grant R01 DA 15999-01. The Kirby Institute for Infection and Immunity in Society is funded by the Australian Government Department of Health and Aging and is affiliated with the Faculty of Medicine, University of New South Wales.
Disclosures: None declared.
References
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