The authors have declared that no competing interests exist.
Current address: Experimental and Observational Rheumatology, Instituto de Investigación Sanitaria, Hospital Clínico Universitario de Santiago, Santiago de Compostela, Spain
Chronic hepatitis C (CHC) is a major cause of liver disease worldwide which often leads to progressive liver inflammation, fibrosis, cirrhosis and hepatocellular carcinoma (HCC). CHC displays heterogeneous progression depending on a broad set of factors, some of them intrinsic to each individual such as the patient's genetic profile. This study aims to evaluate the contribution of certain genetic variants of crucial interferon alpha and lambda signaling pathways to the hepatic necroinflammatory activity (NIA) grade of CHC patients.
NIA was evaluated in 119 CHC patients by METAVIR scale and classified as low (NIA = 0–2, n = 80) or high grade (NIA = 3, n = 39). In a candidate gene approach, 64 SNPs located in 30 different genes related to interferon pathways (
Seven SNPs located in
The identified genetic variants in interferon signaling pathways may constitute useful prognostic markers of CHC progression. Further validation in larger cohorts of patients is needed.
Hepatitis C virus (HCV) is a major cause of liver-related morbidity and mortality, affecting 170 million people worldwide [
On the other hand, the implication of inflammation in the progression of CHC and development of HCC is well established and several inflammatory signaling pathways link inflammation and cancer [
The study population included 119 patients of European descent from Hospital Universitario de La Princesa, Hospital Universitario San Cecilio de Granada and Hospital Universitario Central de Asturias. All patients gave their written informed consent to participate in the genetic analysis before their enrollment. This retrospective cohort study was approved by the local Ethics Committee of Hospital de La Princesa (Madrid, Spain) and good clinical practice guidelines were followed.
Included patients had previous diagnosis of HCV infection, which was confirmed by the presence of detectable HCV RNA in serum. HCV RNA levels and HCV genotype were determined using the COBAS AMPLICOR® assay (Roche Molecular Diagnosis GmbH, Mannheim, Germany) and the reverse-hybridization line probe assay (INNO-LiPAHCV; Innogenetics, Zwijndreht, Belgium), respectively. At Hospital Universitario San Cecilio, viral load was determined the by HCV Ampliprep TaqMan, Roche Molecular System (cutoff <15 IU/mL). Patients showed no evidence of HBV infection, HIV infection, alcoholism, autoimmune, or drug-induced liver disease. Patients’ serum samples were also subjected to routine laboratory tests in the biochemical laboratory of each center using common commercial methods, measuring alanine transaminase (ALT), aspartate aminotransferase (AST) and gamma glutamyl transferase (GGT).
Liver biopsies, obtained for diagnostic purposes, were analyzed histologically according to the METAVIR classification system [
DNA was isolated from whole blood samples from Hospital Universitario de La Princesa and Hospital Universitario San Cecilio by using a MagNA Pure DNA isolation kit (Roche Diagnostics, Mannheim, Germany) in accordance with the manufacturer’s instructions and stored at −80°C until assay. Genomic DNA was extracted from peripheral blood samples from Hospital Universitario Central de Asturias with the Magtration-MagaZorb DNA Common Kit-200 N using the Magtration 12GC system (Precision System Science Co., Ltd., Woerrstadt, Germany) and the Maxwell 16 Blood Purification Kit using the Maxwell 16 Instrument (Promega Corporation, Madison, Wisconsin, USA).
A total of 63 SNPs located in 30 interferon signaling pathway and interferon stimulated genes were selected by their tagSNP condition (tagSNPs capture most of the genetic variation in a region) or location in key regulatory regions as described in the HapMap project, Release no. 27, Phase II and III (
Hardy–Weinberg equilibrium and allele/genotype frequencies were calculated using SNPStats software [
Clinical variables were expressed as medians and 1st-3rd quantiles or number of patients and percentages, except for age (median and range). Associations between variables and NIA were assessed by Mann-Whitney U-test or Chi-squared test. Two-tailed p values below 0.05 were considered significant (SPSS version 15.0; SPSS Inc., Chicago, IL, USA).
Subsequently, significant clinical variables and SNPs were analyzed by multivariate logistic regression using a step-backward method, in which none of the variables was forced to be included into the model. ROC analyses were performed to discriminate the power of factors independently associated with inflammatory grade (SPSS version 15.0; SPSS Inc., Chicago, IL, USA).
Main clinical characteristics of the 119 patients included in the study are summarized in
Overall (n = 119) | NIA≤2 (n = 80) | NIA = 3 (n = 39) | p value | |
---|---|---|---|---|
Sex (W/M) | 36% / 64% | 34% / 66% | 41% / 59% | ns |
VG (1/non1) | 88% / 12% | 88% / 12% | 90% / 10% | ns |
Age (years old) | 45 (21–67) | 46 (21–67) | 45 (25–67) | ns |
VLx10-5 (IU/mL) | 11 (5–23) | 12 (5–30) | 7 (5–13) | 0.01 |
ALT (IU/L) | 72 (51–115) | 69 (48–102) | 82 (60–146) | ns |
AST (IU/L) | 47 (36–65) | 43 (33–58) | 58 (41–75) | 0.005 |
GGT (IU/L) | 45 (28–83) | 41 (27–91) | 50 (29–81) | ns |
Fibrosis |
73% / 27% | 77% / 23% | 64% / 36% | ns |
Data are shown as median and 1st-3rd quantiles except for age (median and range) and sex, viral genotype and fibrosis (percentage of patients).
1Only 104 data available.
NIA, necroinflammatory activity; VG, Viral Genotype; VL, Viral Load; W, women; M, men; IU, international units; ALT, alanine transaminase; AST, aspartate aminotransferase; GGT, gamma glutamyl transferase; FIB, fibrosis stage; ns, non significant.
Genotype frequencies of each SNP and its association with necroinflammatory activity are detailed in (
Seven SNPs located in
Locus | SNP | Model | Genotype | NIA≤2 | NIA = 3 | p value |
---|---|---|---|---|---|---|
rs12979860 | Additive | C/C | 20 (25.3%) | 15 (38.5%) | 0.031 | |
C/T-T/T | 59 (74.7%) | 24 (61.5%) | ||||
rs11576173 | Recessive | G/G-A/G | 70 (98.6%) | 34 (89.5%) | 0.034 | |
A/A | 1 (1.4%) | 4 (10.5%) | ||||
rs1497056 | Dominant | A/A | 60 (81.1%) | 21 (60%) | 0.021 | |
A/G-G/G | 14 (18.9%) | 14 (40%) | ||||
rs2057778 | Recessive | A/A-A/C | 74 (97.4%) | 34 (87.2%) | 0.036 | |
C/C | 2 (2.6%) | 5 (12.8%) | ||||
rs33932899 | Recessive | C/C-G/C | 75 (100%) | 34 (91.9%) | 0.034 | |
G/G | 0 (0%) | 3 (8.1%) | ||||
rs3738579 | Dominant | A/A | 33 (43.4%) | 9 (23.7%) | 0.036 | |
A/G-G/G | 43 (56.6%) | 29 (76.3%) | ||||
rs280519 | Recessive | G/G-A/G | 50 (71.4%) | 34 (91.9%) | 0.009 | |
A/A | 20 (28.6%) | 3 (8.1%) |
Main clinical characteristics and the seven SNPs found in association with NIA grade were included in a multivariate logistic regression analysis (
Factor | Univariate | Multivariate | |||
---|---|---|---|---|---|
OR | CI 95% | p | p | ||
Sex | W | 1 | - | - | |
M | 0.73 | 0.33–1.62 | ns | ||
Viral Genotype | 1 | 1 | - | - | |
non1 | 0.8 | 0.20–2.50 | ns | ||
Age (years old) | ≤40 | 1 | - | - | |
>40 | 0.63 | 0.28–1.41 | ns | ||
Fibrosis stage | FIB≤2 | 1 | |||
FIB = 3–4 | 1.96 | 0.79–4.89 | ns | - | |
Viral Load (IU/mL) | ≤6·105 | 1 | - | - | |
>6·105 | 0.76 | 0.34–1.74 | ns | ||
ALT (IU/L) | ≤40 | 1 | - | - | |
>40 | 0.76 | 0.23–2.70 | ns | ||
AST (IU/L) | ≤40 | 1 | - | 0.01 | |
>40 | 2.43 | 1.07–5.86 | 0.0003 | ||
GGT (IU/L) | ≤30 | 1 | - | - | |
>30 | 1.35 | 0.58–3.31 | ns | ||
C/C | 1 | - | ns | ||
rs12979860 | C/T-T/T | 0.51 | 0.27–0.96 | 0.031 | |
A/A | 1 | - | ns | ||
rs1497056 | A/G-G/G | 2.86 | 1.17–6.97 | 0.021 | |
G/G-A/G | 1 | - | ns | ||
rs11576173 | A/A | 8.24 | 0.89–76.53 | 0.034 | |
A/A-A/C | 1 | - | ns | ||
rs2057778 | C/C | 5.44 | 1.01–29.47 | 0.036 | |
A/A | 1 | - | 0.02 | ||
rs3738579 | A/G-G/G | 2.47 | 1.03–5.93 | 0.036 | |
G/G-A/G | 1 | - | 0.02 | ||
rs280519 | A/A | 0.22 | 0.06–0.80 | 0.009 | |
C/C-G/C | 1.00 | ns | |||
rs33932899 |
G/G | NA | NA | 0.034 |
aOR cannot be calculated as the value of one genotypic group is 0.
OR, odds ratio; CI, Confidence interval; W, women; M, men; IU, international units; ALT, alanine transaminase; AST, aspartate aminotransferase; GGT, gamma glutamyl transferase; FIB, fibrosis stage;
It must be noted that neither
ROC_AUC analysis of each variable associated with NIA in the multivariate analysis was performed to assess their putative predictive value (
AUC of the variables associated with NIA grade: AST serum levels >40IU/mL (AUC = 0.63), risk allele of
As known, HCV clearance, CHC progression and response to treatment is driven by multiple and complex interactions among host, viral and environmental factors [
Interestingly, in this study we have inspected multiple SNPs located at numerous IFN related genes finding an independent association of some of them with the NIA of CHC patients. In particular, rs280519 and rs3738579 showed a significant influence in the grade of the disease and displayed a considerable individual predictive performance, which was substantially improved when they were simultaneously considered in addition to AST serum levels (AUC-ROC = 0.74).
The upregulation of many ISGs has been associated with decreased HCV viral RNA levels in the liver of CHC patients as well as in infected hepatocytes [
Tyk2 signaling is shared by both type I and III IFNs and its altered expression as well as some Tyk2 genetic variants have been implicated in the pathogenesis of numerous autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, diabetes, ulcerative colitis and Crohn's disease [
Further validation of the above described genetic signature may entail a great interest for accurate prediction of CHC patients with higher risk of CHC progression, with special attention to the unexpected elevated occurrence/recurrence of hepatic and extrahepatic manifestations related to current IFN-free based therapies.
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Linkage disequilibrium plot of rs280519 was generated by using the SNP Annotation and Proxy Search tool (SNAP, version 2.2, Broad Institute [
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The authors wish to thank each of the patients who generously consented to participate. The authors also thank PhD. Manuel Gómez Gutierrez for his careful language assistance.