Association between Human Leukocyte Antigen-DQ Polymorphisms and Treatment Response in Chronic Hepatitis B Egyptian Population: A Prospective Study

Department of Biochemistry, National Hepatology & Tropical Medicine Research Institute, Cairo, Egypt Tropical Medicine Department, Tanta University, Tanta, Egypt Hepatology, Gastroenterology and Infectious diseases, Kafr-Elsheikh University, Kafr el-Sheikh, Egypt Tropical Department, National Hepatology & Tropical Medicine Research Institute, Egypt Department of Microbiology, Faculty of Pharmacy, Cairo University, Egypt Department of Microbiology, Faculty of Pharmacy, Egyptian Russian University, Egypt Department of Clinical Pathology, Faculty of Medicine, Minia University, Minia, Egypt Department of Biochemistry, Modern University for Technology and Information, Egypt Internal Medicine, Faculty of Medicine, Minia University, Minia, Egypt


1INTRODUCTION
Hepatitis B Virus (HBV) is one of the most common causes of liver diseases. It is estimated by the World Health Organization to infect 257 million people worldwide [1]. As a consequence, it results in an estimated 600,000 deaths every year through complications of end-stage liver disease and Hepatocellular Carcinoma (HCC) [2]. Clinical outcomes, after Hepatitis B virus (HBV) exposure, vary extremely from spontaneous clearance to chronic hepatitis B and often progresses to Liver Cirrhosis (LC) and Hepatocellular Carcinoma (HCC) [3]. However, factors determining the chronicity of primary HBV infection and its disease progression to LC and HCC are not clearly understood. It has been shown that the outcomes of the HBV infection are not because of the variation in the virulence of viral strains [4], but rather due to host-immune response [5]. Human Leukocyte Antigen (HLA)-DQ genetic polymorphisms have been associated with chronic Hepatitis B Virus (HBV) outcomes. Several studies on different populations have focused on the role of HLA-DQ gene polymorphism in the pathogenesis of chronic HBV infection. However, these findings are still controversial [6 -10]. This study aimed to determine the impact of HLA-DQ polymorphism on chronic HBV patients and their response to antiviral therapy.

Patients
This study was carried out on a total number of 188 participants, they were subdivided as follows: Group I (patients' group): included 97 patients with chronic hepatitis B viral infection that was further subdivided according to response to treatment into responder and non-responder subgroups, Group II (Control group): included 91 normal healthy subjects who were matched to the patient group by sex and age. The patients were admitted to Tanta and Kafr-el Sheikh University hospital, Egypt, from April 2016 to January 2018. All patients with HBV infection, selected for this study, were further confirmed by being HBsAg (hepatitis B surface antigen) positive, HbcAb (hepatitis B virus core antibody) positive and HBeAg (hepatitis B e antigen) or HBeAb (hepatitis B e antibody) positive for at least 6 months. Patients were excluded from the study if they were known to have a disease other than HBV, such as HCV co-infection, renal insufficiency, proteinuria, suspected infections, clinically overt diabetes mellitus, thyroid dysfunction, other endocrine disorder or history of alcohol abuse.
Informed consent was obtained from all participants before the study. This study was approved by the Tanta University Faculty of Medicine research ethical committee. All patients were naïve and received lamivudine treatment.

Blood Sampling and Biochemical Assays
Fasting venous blood samples (10 ml) were collected by trained laboratory technicians. A portion of blood was allowed to clot and then centrifuged at 3500g for 5 min to separate the serum used for the assessment of Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), total bilirubin and Albumin by Beckman Automatic Chemistry Analyzer. Another portion of blood was collected in vacutainer tubes containing EDTA for Complete Blood picture (CBC), which was done by phoenix machine.
AFP was measured using Abbott, Axyam (USA, Supplied by Al-Kamal company Cairo, Egypt).

PCR Testing
HBV-DNA was extracted from serum using a kit (Cobas TaqMan HBV Test Rouche Diagnostics, Manheim, Germany) and was used as a confirmatory approach for HBV positivity for all subjects under the present study.
Blood aliquots were stored at -80 o C until assayed and thawed immediately before the gene polymorphism measurement of HLA DQ.

Genotyping Assay
HLA DQB2 polymorphism or Single Nucleotide Polymorphism (SNP) was assayed using the ABI Taq Man allelic discrimination assay. Genomic DNA was extracted from peripheral blood samples by protease K digestion, phenolchloroform extraction, ethanol precipitation, and DNA concentration was tested on Nano Drop 2000 spectrophotometer (Thermo Scientific, DE). According to the relevant HLA Class II SNP literature, HLA-DQ rs 7453920 SNP was selected as candidate site, as it is an HLA-DQ which shows minor allele frequency AA, AG, and GG. The SNP was genotyped using the Taq-Man allele identification assay on the ABI PRISM 7900HT system (Applied Biosystem, Foster City, CA, USA).

Statistical Analysis
All data analysis was operated with SPSS (V.20 for Windows). Genotype and allele frequencies were determined by direct counting and Hardy-Weinberg equilibrium was evaluated by the chi-square test. Comparisons between individual demographic characteristics were analyzed as appropriate with either a Student's t-test (for continuous variables) or a χ2 test or Fisher's Exact tests (for categorical variables) with a two-tailed p-value. Each SNP was analyzed using co-dominant, dominant, and recessive genetic models. The co-dominant model considers homozygous type versus wild type and hybrid type versus wild type, respectively. The dominant model considers the homozygous type and heterozygous type together versus the wild type, and the recessive model considers the homozygous type versus the heterozygous type and the wild type together.

RESULTS
Socio-demographic data of chronic HBV infection group and controls were obtained. The average age of the 97 HBV patients was 38.9 ± 9.04 years and there were a total of 70 (72.2%) males. The control group was matched for age (mean age 37.54±9.79) and sex [56(44.4%) were males].
In a general analysis for the SNP rs7453920, the overall genotypes frequencies were 37% for A/A, 60.6% for A/G, and 37% for G/G ( Table 1).
The participating patients were further subdivided into two subgroups according to the response to treatment. The baseline demographic and laboratory characteristics of the 97 enrolled patients are shown in Table 2.
A total of 77 (79.4%) patients were responders. Among this group, 72.7% were male, and the average age was 38.59 ±9.15 years.
There was no difference in gender and age between the responder group and non-responder group (p>0.05). Moreover, smoking, alcohol consumption, BMI, drinking coffee, or residency showed no significant difference between the two groups (p>0.05). In addition, the baseline levels of hemoglobin, ALT, AST, total bilirubin, INR, serum albumin, serum creatinine, blood glucose, and alpha-fetoprotein were similar in the two groups (p>0.05) ( Table 2).
Association between polymorphisms in HLA-DQ gene and treatment response.
SNP was in Hardy-Weinberg equilibrium in allele frequency in the non-responder group. When the association between polymorphisms in HLA-DQ gene and treatment response was evaluated, the results indicated that response to treatment declined when patients were carrying more unfavorable rs 7453920 GG with a response rate of 64%. Patients carrying the mutant allele AG, or the wild type allele AA were more likely to achieve a higher rate of response (84.8% and 83.3%, respectively) ( Table 3).

DISCUSSION
The outcome of HBV infection is determined by host factors, including immunological and viral factors, as well as viral load and viral genotypes [11 -15].
Variations in HLA class II strongly correlated with persistence or spontaneous clearance of HBV infection [16,17]. In addition, these variations may influence treatment efficacy and response to vaccination [18]. The host genetic background influences the immune processing of APC cells, which is necessary for an effective immune response. SNP rs7453920 affects the gene expression of the HLA-DQB2 molecule and it was found to be pivotal in the recognition and processing of viral particles [19]. However, these findings are still controversial.
This study was conducted to investigate the association between HBV chronic infection and HLA-DQB2 SNP (rs7453920) and clarify its effect on the treatment response. It was found that the G alleles of HLA-DQ rs7453920 significantly increased in chronic HBV infection patients as compared to controls. This was in accordance with Tao Xu et al. [11] who found that HLA-DQ rs7453920-A polymorphism was associated with decreased risk of HBV infection. All patients were treatment-naive and all received Lamivudine as treatment, then they were divided into two subgroups according to their response to treatment. No significant difference was found in basic characteristics including (age, gender, smoking, alcohol consumption, BMI, drinking coffee, PCR of HBV DNA) between two groups although these factors were proved to influence the risk of HBV infection persistence in previous studies [20,21].
Our study showed that patients carrying the more favorable rs 7453920 genotypes AG and AA had a significantly higher rate of response to treatment. Several previous clinical trials attempted to clarify the effect of the HLA gene variant on HBV treatment response. Chang et al., who studied the effect of HLA variants and response to interferon therapy in male Han Taiwanese patients with CHB, concluded that "G-C" haplotype of the five SNPs, including rs9277535 (HLADPB1), rs9276370 (HLA-DQA2), rs7756516 and rs7453920 (HLA-DQB2), and rs9366816 near HLA-DPA3, is associated with sustained therapeutic response to IFN-α treatment [22]. In addition, results of a meta-analysis performed by Xu et al. concluded that the HLA-DQ rs7453920-A allele was a protecting factor against chronic HBV infection, while rs 7453920-G serves as a risk factor for HBV infection [23].
On the other hand, Zhang et al. studied a different HLA-DQ locus and concluded that rs9275572 can be considered as a predictor of response to Lamivudine therapy [24].
To the best of our knowledge, this the first study to investigate HLA-DQB2 rs 7453920 polymorphism in chronic HBV Egyptian patients, but this study has its own limitation due to the small sample size. Therefore, further studies on a larger cohort are needed to validate these results.

CONCLUSION
The presence of HLA-DQB2 rs 7453920-G serves as a risk factor for chronic HBV infection and treatment failure in the Egyptian population.

ETHICS APPROVAL AND CONSENT TO PARTI-CIPATE
The research was approved by the Tanta University Faculty of Medicine research ethical committee, 31412/03/16 Egypt .

HUMAN AND ANIMAL RIGHTS
No animals were used in this research. All human research procedures were followed in accordance with the ethical standards of the committee responsible for human experimentation (institutional and national), and with the Helsinki Declaration of 1975, as revised in 2013.

CONSENT FOR PUBLICATION
Written informed consent was obtained from all the participants.