Association between IL7R polymorphisms and severe liver disease in HIV/HCV coinfected patients: a cross-sectional study

Interleukin-7 (IL-7) is a critical factor for T cell development and for maintaining and restoring homeostasis of mature T cells. Polymorphisms at α-chain of the IL-7 receptor (IL7R or CD127) gene are related to evolution of HIV-infection, but there are no data concerning the evolution of hepatitis C virus (HCV) infection. The aim of this study was to analyze the association between IL7R polymorphisms and severe liver disease in HCV/HIV coinfected patients. We performed a cross-sectional study in 220 naïve patients who underwent a liver biopsy. IL7R polymorphisms (rs6897932, rs987106 and rs3194051) were genotyped using the GoldenGate® assay. The outcome variables were: (a) liver biopsy: advanced fibrosis (F ≥ 3), severe activity grade (A3); (b) non-invasive indexes: advanced fibrosis (APRI ≥1.5 and FIB-4 ≥3.25). Logistic regression analysis was used to investigate the association between IL7R polymorphisms and outcome variables. This test gives the differences between groups and the odds ratio (OR) for liver disease. Patients with rs6897932 CC genotype had higher likelihood of having A3 than patients with rs6897932 CT/TT (adjusted odds ratio (aOR) = 4.16; p = 0.026). Patients with rs987106 TT genotype had higher odds of having F ≥ 3 (aOR = 3.09; p = 0.009) than rs987106 AA/AT carriers. Finally, patients with rs3194051 AA genotype had higher odds of having severe liver fibrosis (F ≥ 3; APRI ≥1.5, and FIB4 ≥3.25) than patients with rs3194051 AG/GG genotype [aOR = 2.73 (p = 0.010); aOR = 2.52 (p = 0.029); and aOR = 4.01 (p = 0.027); respectively]. The CTA haplotype (comprised of rs6897932, rs987106, and rs3194051) carriers had higher odds of having F ≥ 3 (aOR = 1.85; p = 0.012), APRI ≥1.5 (aOR = 1.94; p = 0.023), and FIB4 ≥3.25 (aOR = 2.47; p = 0.024). Conversely, the CAG haplotype carriers had lower odds of having F ≥ 3 (aOR = 0.48; p = 0.011), APRI ≥1.5 (aOR = 0.48; p = 0.029), and FIB4 ≥3.25 (aOR = 0.29; p = 0.010). The presence of IL7R polymorphisms seems to be related to severe liver disease in HIV/HCV coinfected patients, because patients with unfavorable IL7R genotypes (rs6897932 CC, rs987106 TT, and rs3194051AA) had a worse prognosis of CHC.


Background
Interleukin-7 (IL-7) is required for T cell development and for maintaining and restoring homeostasis of mature T cells. IL-7 is a critical factor in maintaining or inducing an effective antiviral CD4+ and CD8+ T cell responses [1]. IL-7 is implicated in the proliferation and survival of early T and B cells in the thymus and bone marrow, respectively. Besides, in the periphery, IL-7 enhances survival and proliferation of naïve and memory T cells, regulating the T cell homeostasis [2]. The responsiveness of IL-7 is dependent on expression of the IL-7 receptor, which is composed of the high-affinity α-chain (IL7Rα or CD127) and the common cytokine receptor gamma chain (CD132) [3].
In human immunodeficiency virus (HIV) infection, the interaction between IL-7 and CD127 has impact on development, survival, and proliferation of CD4+ T cells [4]. During HIV infection, IL-7 regulatory pathway is activated and IL-7 levels are increased, but it is not enough to maintain T cell homeostasis due to progressive destruction of CD4+ T cell [5]. In hepatitis C virus (HCV) infection, hepatocytes stimulated by type I interferon (IFN) are able to produce IL-7 and eventually lead to viral clearance and disease resolution in the liver [6]; and the early expression of CD127 on HCV-specific T cells predicts the HCV clearance during acute HCV infection [7]. However, IL-7 and CD127 levels are significantly diminished during chronic HCV infection, leading to an impaired HCV-specific cytotoxic T cell reactivity [8,9].
The IL7Rα (CD127) is encoded by IL7R gene, which plays an essential role in several human diseases. Thus, IL7R polymorphisms have been studied in several infectious diseases [10,11] and autoimmune diseases [12], showing a key role in the evolution of patients. In HIV infection, IL7R polymorphisms have been associated with the decline in the CD4+ cell count in untreated HIV-infected subjects [13], and CD4+ T-cell recovery in HIV-infected patients on combination antiretroviral therapy (cART) [14]. In HCV infection, we have recently reported an association between IL7R polymorphisms and virological response to HCV therapy with pegylated interferon alpha plus ribavirin (pegIFNα/ribavirin) [15]. Thus, IL7R polymorphisms seem to play a crucial role in the physiopathology of these diseases.
The cART has made of HIV infection a chronic manageable disease in high income countries [16]. In this setting, chronic hepatitis C (CHC) has turned into an important comorbidity and a major cause of death in HIV/HCV coinfected patients [17][18][19], since HIV infection accelerates the natural history of CHC and increases liver-related morbidity and mortality [20][21][22]. Interestingly, uncontrolled HIV replication and low CD4 counts are both associated with accelerated liver fibrosis progression in HIV/HCV coinfected patients [23], suggesting that earlier use of antiretroviral therapy could ameliorate this harmful effect [24].
The aim of this study was to analyze the association between IL7R polymorphisms (rs6897932, rs987106, and rs3194051) and severe liver disease in HCV/HIV coinfected patients.

Study design and patients
We carried out a cross-sectional study in HIV/HCV coinfected patients that underwent a liver biopsy at Hospital Gregorio Marañón (Madrid, Spain) between September 2000 and November 2008. All patients were of European ancestry.
Liver biopsies were performed on patients who were potential candidates for anti-HCV therapy and had not received previous interferon therapy (naïve for HCVtreatment). Selection criteria were: no clinical evidence of hepatic decompensation, detectable HCV RNA by polymerase chain reaction (PCR), negative hepatitis B surface antigen, availability of DNA sample, CD4+ lymphocyte count higher than 200 cells/µL, and stable cART for at least 6 months before study entry or no need for cART according to treatment guidelines used in the study period [25,26]. Patients with active opportunistic infections, active drug addiction, and other concomitant severe diseases were excluded. Thus, from our cohort of 361 HIV/HCV coinfected patients with liver biopsy data, only 220 patients had data available of IL7R genotypes.
The study was conducted in accordance with the Declaration of Helsinki and patients gave their written consent for the study. The Institutional Review Board and the Research Ethic Committee of the Instituto de Salud Carlos III approved the study.

Epidemiological and clinical data
Clinical and epidemiological data were obtained from medical records. Consumption of more than 50 g of alcohol per day for at least 12 months was considered as a high intake. Body mass index (BMI) was calculated as the weight in kilograms divided by the square of the height in meters. The duration of HCV infection for patients with a history of intravenous drug use (IDU; 87.7% of patients) was estimated starting from the first year they shared needles and other injection paraphernalia, which are the most relevant risk practices for HCV transmission [27]. The duration of HCV infection was not calculated when the date of initiation of their HCV infection could not be determined with certainty (n = 19).
Biochemistry panel was measured using an autoanalyzer Hitachi 912 (Boehringer Mannheim, Germany), while patients were fasting. The degree of insulin resistance was estimated for each patient using the homeostatic model assessment (HOMA) [28]: fasting plasma glucose (mmol/L) times fasting serum insulin (mU/L) divided by 22.5 [28]. Non-invasive liver fibrosis indexes were calculated according to the formula originally described for the aspartate aminotransferase to platelet ratio index (APRI): aspartate aminotransferase (AST) [U/L]/upper limit of normal for AST (ULN)/ (platelet [10 9 /L]) × 100 [29] and the FIB-4 index: age  [30]. Although these indexes may have a poor ability to identify patients with intermediate stage of fibrosis [31], we have previously reported that the diagnostic performance of APRI and FIB-4 indexes were acceptable for diagnosis of advanced fibrosis in HIV/HCV-coinfected patients [32].

HCV assays
HCV infection was documented in all patients by enzyme-linked immunosorbent assay and PCR test. HCV genotype was determined by hybridization of biotinlabeled PCR products to oligonucleotide probes bound to nitrocellulose membrane strips (INNO-LiPA HCV II, Innogenetics, Ghent, Belgium). Plasma HCV-RNA viral load was measured by PCR (Cobas Amplicor HCV Monitor Test, Branchburg, NJ, USA) and real-time PCR (COBAS AmpliPrep/COBAS TaqMan HCV test); and results were reported in terms of international units per milliliter (IU/mL), with a lower limit of detection of 10 IU/mL.

Liver biopsy
Liver biopsies were performed as we described previously [32]. The samples were always evaluated by the same pathologist, who was unaware of the patients' clinical or laboratory data. Liver fibrosis and necroinflammatory activity were estimated according to Metavir score as follows [33]: F0, no fibrosis; F1, mild fibrosis; F2, significant fibrosis; F3, advanced fibrosis; and F4, definite cirrhosis. The degree of necroinflammation (activity grade) was scored as follows: A0, no activity; A1, mild activity; A2, moderate activity; A3, severe activity.

Genotyping of IL7R polymorphisms
We have analyzed the most common single nucleotide polymorphisms (SNPs) including at IL7R gene, using the databases of HapMap Project (http://snp.cshl.org/ cgi-perl/gbrowse/hapmap_B35/) and NCBI (dbSNP) (http://www.ncbi.nlm.nih.gov/entrez/). The selection criteria were: (1) SNPs located at a putative regulatory region, although their effect has not been tested or demonstrated. (2) Allelic frequency greater than 20% in European people, because it is easier to detect a modest individual effect with a high SNP frequency >20% in the population when small sample sizes are used [34]. (3) A tag-SNP was selected when more than 1 SNP per gene was found and they had high linkage disequilibrium (LD). In summary, three SNPs [rs987106 (intronic region), rs6897932 (exon 6) and rs3194051 (exon 8)] were used for the genetic association study.

Outcome variables
We analyzed several outcome variables related to severity of liver disease [31]: (a) liver biopsy: advanced fibrosis (F ≥ 3), severe activity grade (A3); (b) non-invasive indexes: advanced fibrosis (APRI ≥1.5 and FIB-4 ≥3.25). These CHC-related outcomes were developed after a minimum follow-up time of 10 years with HCV infection.

Statistical analysis
For the description of the study population, p values were estimated with Chi square test for categorical variables.
The genetic analysis was carried out according to additive, recessive and dominant models, selecting the model that best fitted the outcome variable analyzed in each case. The genetic association study was performed via logistic regression analysis. This test gives the differences between groups and the odds ratio (OR) for liver disease. Each regression analysis was always adjusted by the most significant co-variables associated with each one of the outcome variables, avoiding the over-fitting of the regression. The co-variables were selected by "Stepwise" algorithm (at each step, factors were considered for removal or entry: a p value for entry and exit of 0.15 and 0.20, respectively), including gender, age, alcohol intake, BMI, HOMA, nadir CD4+ T-cells, AIDS, undetectable HIV-RNA (<50 copies/ml), CD4+ T-cells, time of HCV infection, time on cART, type of cART, HCV-RNA ≥500,000 IU/ml, and HCV genotype. The percentage of patients that were excluded in each of the multivariable analyses due to incomplete data of covariates was always <5%.
These analyses were performed by using the IBM SPSS Statistics for Windows, Version 21.0 (IBM Corp, Chicago, Armonk, NY, USA). In addition, pair-wise LD and Hardy-Weinberg equilibrium (HWE) analyses were computed by Haploview 4.2 software. Haplotype-based association testing was performed using Plink software (http://pngu.mgh.harvard.edu/~purcell/plink/) and each haplotype was compared with the rest of haplotypes (there was no reference category). All p values were twotailed and statistical significance was defined as p < 0.05. Table 1 shows the epidemiological and clinical characteristics of 220 HIV/HCV coinfected patients.

Characteristics of IL7R polymorphisms
A strong LD (non-random association of alleles at different loci) among IL7R polymorphisms was found (D′ ≥ 0.999), meaning that there is no evidence for recombination between these SNPs. However, the r-square among SNPs was low (r-square <0.50), meaning that the IL7R polymorphisms did not provide exactly the same information and the IL7R polymorphisms cannot substitute one for another ( Figure 1a). Additionally, all SNPs had a minimum allele frequency (MAF) >5%, displayed missing values <5%, and were in HWE (p > 0.05) (Figure 1b).

IL7R polymorphisms and severe liver disease
For rs6897932 polymorphism (Table 2A), patients with rs6897932 CC genotype had higher frequency of A3 in liver biopsy (p = 0.023) and had higher likelihood of having A3 than patients with rs6897932 CT/TT [adjusted odds ratio (aOR) = 4.16; p = 0.026].

Discussion
To our knowledge, this study is the first description of the relation between IL7R polymorphisms and severity of liver disease in HIV/HCV-coinfected patients. The major findings of our study were: (1) IL7R rs6897932 CC was associated with higher odds of having severe activity necroinflammatory grade; (2) IL7R rs987106 TT and rs3194051 AA genotypes were related to increased odds of having severe fibrosis; and (3) IL7R CTA haplotype was associated with severe fibrosis while IL7R CAG haplotype had a protective nature. Moreover, these relationships were less evident when patients were stratified by HCV genotype (data not shown), perhaps due to a smaller sample size of the groups. The rs6897932 C/T polymorphism is a missense variant located in the alternatively spliced exon 6 of IL7R gene, causing a substitution of threonine with isoleucine in the transmembrane domain of the IL7R gene. This polymorphism is implicated in splicing regulation, where the C allele leads to increased skipping of exon 6 by putatively disrupting an exonic splicing motif [36], increasing the production of sCD127 (soluble isoform) and decreasing CD127 levels (membrane-bound isoform). In this setting, rs6897932 CC genotype has been associated to higher plasma levels of sCD127 in Caucasian HIVinfected patients [14,37], which binds to circulating IL-7 and decreases the IL-7 bioavailability, limiting its effects [38]. Thus, rs6897932 C allele has been related to slower CD4+ T-cell recovery in European HIV infected patients on cART [39]. In our study, patients with rs6897932 CC genotype had higher odds of severe necroinflammatory activity grade. Given that the IL7R rs6897932 CC genotype is related to increased risk of autoimmune diseases [36,40], it is possible to speculate that higher level of sCD127 may confer worse immunological control of HCV infection in liver, with a higher necroinflammatory activity in liver biopsy. However, further studies investigating the underlying mechanisms are warranted.
In our study we also found that rs987106 TT and rs3194051 AA genotypes were related to higher odds of having severe liver fibrosis. The rs987106 polymorphism is an intronic variant between exons 6 and 7 of IL7R gene and rs3194051 is a missense polymorphism located at exon 8. These polymorphisms are predicted    to be involved in post-transcriptional regulation and besides, rs3194051 is also predicted to be implicated in proximal transcriptional regulation of CD127, according to an in silico analysis by using rSNPBase software [41]. In regards to rs987106, previous articles have described an association between rs987106 TT genotype and higher sCD127 levels [14] and rapid AIDS progression [13]. The rs3194051 polymorphism, a tag SNP of IL7R haplotype 4 in European populations, has been associated with highest CD127 on CD4+ T-cell, higher frequencies of recent thymic emigrants, and lower plasma sCD127 levels in patients with multiple sclerosis [42]. Thus, the rs3194051 A allele ancestral would be associated with lower CD127 on CD4+ T-cell, lower frequencies of recent thymic emigrants, and higher plasma sCD127 levels. Furthermore, rs3194051 A allele has been associated with slower CD4+ recovery in HIV infected patients on cART [14] and lower odds of achieving sustained virological response (SVR) after pegIFNα/ribavirin in HIV/HCV coinfected patients [15]. Moreover, as noted in the preceding paragraphs, unfavorable alleles of these three IL7R polymorphisms (rs6897932 C, rs987106 T, and rs3194051 A) have been related to low CD4+ T-cells and rapid AIDS progression in Caucasian naïve HIV infected patients [13] and slower CD4+ recovery in patients on cART [14,39]. Besides, uncontrolled HIV replication and low CD4 counts are both associated with accelerated liver fibrosis progression in HIV/HCV coinfected patients [23]. Thus, unfavorable IL7R genotypes (rs6897932 CC, rs987106 TT, and rs3194051 AA) could lead to an increased risk of severe liver disease, by decreasing CD4+ cells count and enhancing AIDS progression in HIV/HCV coinfected patients [43].
In our study, IL7R haplotypes (comprised of rs6897932, rs987106, and rs3194051) were also investigated to verify whether they could improve the association of severe fibrosis compared to IL7R polymorphisms alone. We found that CTA haplotype was associated with higher odds of having severe liver fibrosis; while CAG haplotype (comprised of one allele associated with higher sCD127 levels and two alleles related to lower sCD127 levels) was associated with lower odds of having severe liver fibrosis. However, OR values were similar to those obtained for individual SNPs. In this regard, we also recently reported that IL7R CTA haplotype (three alleles associated with higher sCD127 levels) was related to lower odds of achieving a SVR [15].
According to what we have discussed until now, IL7R polymorphisms are related to increased odds of having severity of liver disease, possibly due to regulation of sCD127 levels. Previous reports have suggested that IL-7R polymorphisms may have an influence on T-cell development and homeostasis, and thereby might contribute to an altered immune regulation [42,44]. Moreover, the CHC progression, which includes cirrhosis, end-stage liver disease, and hepatocellular carcinoma, is more rapid in HIV-infected patients, possibly because a damaged immune system may increase the risk for CHC progression among persons infected with HIV [45][46][47]. Therefore, one should not rule out the possible influence of IL7R polymorphisms in the development of end-stage of liver disease and hepatocellular carcinoma in HIV/HCV coinfected patients.
In our study, we compared the IL7R genotype frequencies depending on the presence of each of the outcome variables (independent events), which generated a high number of comparisons. There is a considerable controversy about adjusting the "p value" after multiple tests on clinical-orientated studies [48,49]. In our study there was a hypothesis supported by theory and previous reports in several infectious diseases [10,11], including HIV infected subjects [13,14] and HIV/HCV coinfected patients [15], and showing a key role of IL7R polymorphisms in the evolution of patients. Therefore, we were not literally doing a random search of a meaningful result, and our results should not be affected by the fact of carrying out a high number of statistical tests. Moreover, since p value is depending on the sample size, only big effects would be detected in small populations and it may be possible that we did not find any significant adjusted p value in some comparisons due to our size-limited population. We should take into account the fact that the effect size of our study is low since complex human diseases are under the control of many genes, each one of them contributing with modest individual effects [50].
There are other aspects to be taken into account for the correct interpretation of the results. Firstly, this is a cross-sectional study with data collected retrospectively. Secondly, the study contains a limited number of patients, which could limit achieving statistically significant p values in some groups and it may lead to potentially large percentage fluctuations in the proportion. Thirdly, all selected patients met a set of criteria for starting HCV treatment and this may have introduced a selection bias. Fourthly, this study was performed on patients with European ancestry, and it would be interesting to perform these analyses on different ethnic groups. Fifthly, our study only included HIV/HCV-coinfected patients and it would be interesting to know the role of studied IL7R polymorphisms in HCV monoinfected patients, but we did not have access to a cohort of HCV monoinfected patients.