Lack of Association of Polymorphisms in IL22 and IL22RA1 Genes with Fibrosis Severity in Patients with Chronic Hepatitis C

Abstract

The IL-22 pathway has been shown to play an important role in the pathogenesis of liver fibrosis. However, little is known about the role of single-nucleotide polymorphisms (SNPs) in IL-22-related genes in relation to the severity of liver fibrosis. This study aimed to investigate the association of polymorphisms in IL22 and IL22RA1 genes with the severity of liver fibrosis in patients with chronic hepatitis C. A total of 326 patients (165 with mild fibrosis and 161 with severe fibrosis) were included. Four SNPs in IL22 (rs1179251, rs2227473, rs1012356, and rs2227485) and two in IL22RA1 (rs4648936 and rs3795299) were evaluated by real-time PCR. No significant association was observed between the polymorphisms studied and the severity of liver fibrosis. The SNPs rs1179251, rs2227473, rs1012356, and rs2227485 in IL22 and rs4648936 and rs3795299 in IL22RA1 may not be involved in the pathogenesis of liver fibrosis in patients with chronic hepatitis C.

Introduction

Hepatitis C is a severe global public health problem. It is estimated that around 71 million people worldwide (1% of the population) live with the hepatitis C virus (HCV), and that ∼400,000 people die annually as a result of disease complications, mainly due to cirrhosis and hepatocellular carcinoma (9).

Liver inflammation caused by HCV involves various mechanisms that include the host's regulatory immune response, mediated by cytokines that play important roles against viral infections and viral polypeptides, which interact with cells involved in innate and adaptive immunity (2,10,18). A previous study has demonstrated that polymorphisms in the IL22RA2 gene are involved with the severity of liver fibrosis in patients with chronic hepatitis C and schistosomiasis (17). In line with this, we recently demonstrated that IL-22R1 levels decrease as fibrosis increases, and that patients with liver cirrhosis have higher IL-22BP/IL-22 ratios, in liver tissue from patients with chronic hepatitis C (6). This protective role of IL-22 in liver fibrosis has been confirmed in other diseases and animal models (5,13,20). Moreover, intravenous administration of IL-22 in a phase 2b trial improved the Model for End-stage Liver Disease (MELD) score, promoted reduction in markers of inflammation, and increased markers of regeneration in patients with alcohol-associated hepatitis (1).

Single-nucleotide polymorphisms (SNPs) in the IL22 and IL22RA1 genes have been associated with infectious diseases such as hepatitis B, tuberculosis, and cerebral malaria in previous studies (8,14,25). However, there is still no evidence of an association between polymorphisms in these genes and chronic hepatitis C. Therefore, the aim of this study was to evaluate the association of polymorphisms in IL22 and IL22RA1 genes with the severity of liver fibrosis in patients with chronic hepatitis C from the Northeast Region of Brazil.

Materials and Methods

Patients

A total of 326 patients from the Gastrohepatology Service of the Oswaldo Cruz University Hospital, Liver Institute of Pernambuco (Recife, Northeast Region of Brazil), were selected from August 2010 to May 2018. Patients were included in this study if they had persistent anti-HCV antibodies and HCV RNA positivity. Positive samples were genotyped by the line probe assay (INNO-LiPA HCV 2.0, Innogenetics, Ghent, Belgium). The presence of hepatitis A, hepatitis B, and HIV antibodies was considered exclusion criteria.

Written informed consent was obtained from all patients, and a profile with clinical, biochemical, and HCV genotype information was generated by means of a questionnaire. The present study was approved by the Ethics Committee in Research of the Federal University of São Francisco Valley under protocol number 0005/190913, and it was conducted in accordance with the provisions of the Declaration of Helsinki and Good Clinical Practice guidelines.

Study groups

Liver biopsies were evaluated by a single expert pathologist and assessed according to the METAVIR scoring system, in which fibrosis is scored as F1 (portal fibrosis), F2 (portal fibrosis with few septa), F3 (septal fibrosis), and F4 (cirrhosis) (4). Patients with METAVIR score F0 or F1 were classified as the mild fibrosis group, while those with F3 or F4 were classified as the severe fibrosis group. The presence of hepatocellular carcinoma was considered an exclusion criterion in both groups. Given that F2 is considered an intermediate phenotype between mild and severe fibrosis, we decided not to include this group in this study to ensure more homogeneous phenotypes.

DNA extraction and genotyping

Genomic DNA was extracted from whole blood using the QIAamp DNA Blood Kit (QIAGEN, Inc., Chatsworth, CA) following the manufacturer's instructions. The extracted DNA was stored at −20°C until further analysis. A QuantStudio 5 Real-Time PCR System (Applied Biosystems, Foster City, CA) performed IL22 (rs1179251, rs2227473, rs1012356, and rs2227485) and IL22RA1 (rs4648936 and rs3795299) genotyping with TaqMan Genotyping Assays in 96-well plates.

Statistical analysis

Statistical analysis of the data was performed using SPSS statistical software package version 22.0 (SPSS, Inc., Chicago, IL). Categorical variables were compared using the chi-squared test or Fisher's exact test when appropriate. The Kolmogorov–Smirnov test was used to check for normal distribution of continuous variables. Two-group comparisons were performed using Student's t test or Mann–Whitney U-test for parametrically or nonparametrically distributed data. Haploview 4.2 software was used to generate the haplotypes and to determine the Hardy–Weinberg equilibrium. The differences were considered statistically significant when p < 0.05.

Results

Patients' characteristics

Among the 326 HCV-infected patients, 165 cases were classified as mild fibrosis and 161 as severe fibrosis. The baseline characteristics of the population are displayed in Table 1. Patients with severe fibrosis were significantly older than those with mild fibrosis (mean age 58.1 ± 8.9 vs. 54.1 ± 11.9, respectively; p < 0.0001). Diabetes was also more prevalent in the severe group (26.7% vs. 13.3%, respectively; p = 0.003). The distribution of sex and body mass index (BMI) was homogeneous between the groups (p > 0.05).

Table 1.

Baseline Characteristics of Patients with Chronic Hepatitis C Virus According to Severity of Liver Fibrosis

Variable	Mild fibrosis (n = 165)	Severe fibrosis (n = 161)	p
Clinical
Age (years)	54.1 ± 11.9	58.1 ± 8.9	<0.0001
Male/female	69/96 (41.8/58.2)	72/89 (44.7/55.3)	0.597
BMI (kg/m²)	25.7 ± 4.0	26.7 ± 5.1	0.093
Diabetes	22 (13.3)	43 (26.7)	0.003
Biochemical
Total bilirubin (mg/dL)	0.60 (0.00–6.30)	0.90 (0.00–7.22)	<0.0001
AST (U/L)	42.00 (8.00–175.00)	77.00 (12.00–438.00)	<0.0001
ALT (U/L)	56.00 (12.00–561.00)	82.00 (7.00–578.00)	<0.0001
GGT (U/L)	48.50 (10.00–682.00)	113.50 (14.00–883.00)	<0.0001
ALP (U/L)	69.50 (12.00–410.00)	99.00 (23.00–456.00)	<0.0001
Platelets	1.99x10⁵ (6.20 × 10^4–4.85.10⁵)	1.23 × 10⁵ (4.1 × 10^4–3.70 × 10⁵)	<0.0001
Viral
Genotype 1	105 (63.6)	104 (64.6)	Reference
Genotype 2	7 (4.2)	4 (2.5)	0.539
Genotype 3	40 (24.2)	49 (30.4)	0.402
Undetermined	13 (7.9)	4 (2.5)	—

Values are presented as mean ± standard deviation, median (range) or n (%).

ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; GGT, gamma-glutamyl transpeptidase; HCV, hepatitis C virus.

Regarding biochemical characteristics, total bilirubin, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transpeptidase, and alkaline phosphatase were all significantly higher in the severe group (p < 0.0001). The platelet count was higher in patients with mild fibrosis (p < 0.0001). Differences in the HCV genotypes between the groups were not significant (Table 1).

Polymorphism analysis

With the exception of rs1179251 (p = 0.0058) and rs2227485 (p = 0.0008) of IL22, all other SNPs were in agreement with the Hardy–Weinberg equilibrium expectations.

The allelic and genotypic frequencies of the polymorphisms in the IL22 and IL22RA1 genes according to the severity of fibrosis are summarized in Table 2. No association was found between polymorphisms in IL22 (rs1179251, rs2227473, rs1012356, and rs2227485) and IL22RA1 (rs4648936 and rs3795299) and the severity of liver fibrosis (Table 2). When analyzing the association of these SNPs with clinical variables, such as age, sex, BMI, diabetes, and viral genotype, only the rs4648936 was significantly associated with the occurrence of diabetes (p = 0.001) (Supplementary Table S1).

Table 2.

Genotyping Distribution of IL22 and IL22RA1 Single-Nucleotide Polymorphisms According to Severity of Liver Fibrosis

Gene	SNP	Allele/genotype	Mild fibrosis (n = 165/330)	Severe fibrosis (n = 161/322)	p
IL22	rs1179251	C	280 (84.8)	267 (82.9)	0.502
		G	50 (15.2)	55 (17.1)
		C/C	122 (73.9)	115 (71.4)	0.810
		C/G	36 (21.8)	37 (23.0)
		G/G	7 (4.2)	9 (5.6)
	rs2227473	C	266 (80.6)	257 (79.8)	0.799
		T	64 (19.4)	65 (20.2)
		C/C	109 (66.1)	100 (62.1)	0.295
		C/T	48 (29.1)	57 (35.4)
		T/T	8 (4.8)	4 (2.5)
	rs1012356	T	170 (51.5)	156 (48.4)	0.433
		A	160 (48.8)	166 (51.6)
		T/T	42 (25.5)	36 (22.4)	0.726
		T/A	86 (52.1)	84 (52.2)
		A/A	37 (22.4)	41 (25.5)
	rs2227485	G	246 (74.5)	231 (71.7)	0.418
		A	84 (25.5)	91 (28.3)
		G/G	98 (59.4)	89 (55.3)	0.747
		G/A	50 (30.3)	53 (32.9)
		A/A	17 (10.3)	19 (11.8)
IL22RA1	rs4648936	A	186 (56.3)	190 (59.0)	0.494
		G	144 (43.7)	132 (41.0)
		A/A	50 (30.3)	55 (34.2)	0.752
		A/G	86 (52.1)	80 (49.7)
		G/G	29 (17.6)	26 (16.1)
	rs3795299	G	197 (59.6)	200 (62.1)	0.527
		C	133 (40.4)	122 (37.9)
		G/G	56 (33.9)	61 (37.9)	0.759
		G/C	85 (51.5)	78 (48.4)
		C/C	24 (14.5)	22 (13.7)

SNP, single-nucleotide polymorphism.

No association was observed in the IL22 and IL22RA1 haplotypes with the severity of liver fibrosis (Supplementary Table S2).

Discussion

This study investigated the association of polymorphisms in the IL22 and IL22RA1 genes with the severity of liver fibrosis in patients with chronic hepatitis C. The results showed no significant association of the SNPs rs1179251, rs2227473, rs1012356, and rs2227485 of IL22 and rs4648936 and rs3795299 of IL22RA1 with the severity of liver fibrosis.

The first evidence that polymorphisms in the IL-22 pathway could be associated with the outcome of HCV infection was demonstrated by Hennig et al., who identified two SNPs in IL22, rs1012356 and rs1179251, associated with viral clearance and response to treatment with pegylated interferon-alpha and ribavirin, respectively. No association was found between IL22 polymorphisms and severity of liver fibrosis (11).

A few years later, Sertorio et al. demonstrated that the carriage of a genetic variant in IL22RA2, the physiological inhibitor of IL-22, increased the risk of developing severe fibrosis in four different cohorts (two cohorts with HCV and two with schistosomiasis) (17). Since then, there have been no new studies investigating the role of polymorphisms in the IL-22 pathway in the pathogenesis of chronic hepatitis C.

Asadi et al. failed to demonstrate an association of IL22 rs1179251 polymorphism with hepatitis B virus infection in Iranian patients (3). On the contrary, this SNP has been shown to be associated with gastric cancer in patients with Helicobacter pylori (15,22).

The IL22 rs2227473 and rs2227485 SNPs have been shown to influence susceptibility to pulmonary tuberculosis in the Chinese population. The rs2227473 is located within a putative binding site for the transcription factor AhR-ARNT complex, where carriers of TT or TC genotypes produce more IL-22 than patients with a homozygous CC genotype (25). The mechanisms involved are still unclear, but IL-22 could stimulate the production of β-defensin by lung epithelial cells, acting as a barrier against the establishment of a Mycobacterium tuberculosis infection (23,25).

On the contrary, the T allele at rs2227473, associated with increased IL-22 production, was associated with childhood cerebral malaria in two independent African populations. The authors suggest that IL-22, acting in synergy with IL-17A, may have a deleterious effect in cerebral malaria, through the direct or indirect promotion of blood–brain barrier permeability and brain inflammation (14). Regarding the pathogenesis of liver fibrosis, Gao et al. found no association between IL22 SNPs (rs2227473 and rs2227485) and clinical outcomes of hepatitis B virus, such as chronic hepatitis B, liver cirrhosis, or hepatocellular carcinoma, in Chinese patients (8).

Few studies in the literature have assessed polymorphisms in the IL22RA1 gene. The SNPs rs4648936 and rs3795299 have been associated with severe chronic rhinosinusitis (7) and preeclampsia risk (16), respectively. In the present study, they were not significantly associated with liver fibrosis, but interestingly, the rs4648936 was associated with diabetes occurrence. The IL-22R1 is highly expressed in the pancreatic α and β cells (19). Previous studies have shown that IL-22 acts through the IL-22 receptor, playing a protective role in diabetes by promoting β cell regeneration, reducing chronic inflammation, and decreasing oxidative stress (12,21,24). Although our primary objective was not to assess the role of IL22 and IL22RA1 SNPs in diabetes, our findings may pave the way for future studies to assess the relationship of rs4648936, as well as other SNPs of the IL22 pathway, with the occurrence of diabetes.

This study found no association of the rs1179251, rs2227473, rs1012356, rs4648936, rs3795299, and rs2227485 polymorphisms in IL22 and IL22RA1 with the severity of liver fibrosis in patients with HCV. Our study has some limitations, such as the small sample size and the number of SNPs investigated. Although the SNPs studied have been associated with other infectious diseases or are located in regulatory regions, it is possible that other polymorphisms may be involved with the severity of liver fibrosis in patients with HCV. Further studies are needed to evaluate the role of polymorphisms in IL22 and IL22RA1 in the outcome of chronic hepatitis C in other populations.

Footnotes

Acknowledgment

This work is based, in part, on research conducted as part of Dr. de Brito's doctoral thesis.

Authors' Contributions

R.J.V.C.B.: investigation and writing—original draft preparation; R.F.C.: conceptualization, validation, and writing—review and editing; B.M.S.S.: investigation and formal analysis; W.L.B.J.: investigation and formal analysis; L.R.S.V.: data curation and validation; L.M.M.B.P.: resources; P.M.: conceptualization, supervision, and writing—review and editing.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

Supplementary Material

Supplementary Table S1

Supplementary Table S2

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