In Romania, chronic HCV infection is a national health problem due to its potential progression to liver failure and hepatocellular carcinoma. In 2015 one million Romanians[1]were infected with hepatitis C. The prevalence indicated by the World Health Organization (WHO – 4.5%) is double compared with the rest of the countries in the European community. Nearly 10% of the 12 million people in Europe infected with HCV are in Romania.¹ Romania ranks as number 1 in terms of people infected with HCV and in fourth place in Europe in terms of mortality due to liver disease – a mean percentage of 44.5% of the deaths are due to liver failure, compared to the European average of 15%.^2,3,4 The locally predominant genotype among the infected individuals is 1b or a mixture of genotypes 1a and 1b.⁵ Antiviral therapy with pegylated interferon-alpha/ribavirin (PegIFN-α/RBV) leads to sustained virological response (SVR) in 40-50% of patients with HCV genotype 1b infection. The ultimate goal of anti-HCV therapy is achieving SVR, defined as HCV RNA remaining undetectable 24 weeks after the cessation of therapy.^6,7 A certain percentage of the treated patients may develop several serious side effects during therapy, requiring dose reduction or stopping treatment.⁸Many genome-wide association (GWA) studies have independently identified some host genetic factors that play an important role in the prediction of outcomes in chronic HCV patients.^9,10,11 Among these host genetic factors considered to play a role in the immunologic response process against viral aggression are the human leukocyte antigen (HLA) molecules.¹² HLA antigens present peptide epitopes to CD4+ T cells that have been proved to play a role in PegIFN-α/RBV response.¹³ Recent data have also shown a link in the immunological pathway involving CD4+ T-cells response for HCV patients presenting spontaneous viral clearance, that target mostly the non-structural proteins (NS3, NS4 and NS5) with the predominant response being against NS3.¹⁴ An epitope in the NS3 region shows high affinity for the HLA-DR locus, indicating the possible involvement of HLA class II region polymorphisms in virus clearance.^15,16 Several associations have been made in the past between HLA alleles with susceptibility and resistance to HCV infection, pathogenesis leading to liver failure, cirrhosis and the response to IFN therapy.^17,18,19 The results reported by previous studies are different, varying with the population ethnic groups studied in the geographical areas²⁰ shown in Table 1, and also the associated alleles’ interpretation has been inconsistent, limited by lack of external validation or small sample size. Although all these findings provide new insights into the genetic regulation of HCV clearance and its clinical approach, it is important to identify patients who are likely to be therapy-resistant, to avoid side effects and cost of PegIFN-α/RBV treatment, and the focus should be on personalized antiviral therapy.

 Table 1. HLA class II DQB1* association with HCV viral clearance and persistence across global population

 

 

The study included a group of 51 selected patients registered at the Infectious Diseases Department Clinical Hospital of Constanța  (Romania), diagnosed with HCV infection, and a control group of 102 healthy subjects registered in the Bone Marrow Donors Registry at the Immunogenetics and Virology Department, Clinical Institute Fundeni Bucharest. Patient enrollment in the study was performed during 2013-2015 based on the following criteria: the subjects included in the study group were of Caucasian origin, positive for anti-HCV/ HCV RNA, viral genotype 1b, with inflammatory activity and extent of liver fibrosis according to the METAVIR scale <4 showing no signs of cirrhosis. Patients with hepatic carcinoma or coinfection with HBV or HIV were excluded. The study was carried out according to hospital ethics obtaining the informed consent from all the patients before blood samples were collected. Patients infected with HCV received the PegIFN-α/RBV combination therapy for 48 weeks and were evaluated during this period clinically, biochemically and for HCV RNA at weeks 4, 12 and 24 in order to assess the response to therapy. According to their SVR (defined as undetectable HCV RNA at the end of treatment) the patients were classified as responders (SVR) or non-responders (non-R). Typing of the patient’s blood samples was performed in the Cell Molecular Genetics Department lab, Medicine Faculty, Ovidius University of Constanța (Romania). DNA extraction was carried out from whole blood sample, collected in EDTA vacutainers using QIAamp@ DNA Blood Minikit (Qiagen, Hilden, Germany) and the following protocol was followed. DNA quantification was done by measuring the absorbance at 260 nanometers (nm) and the purity assessed at 280 nm; the genomic DNA was analyzed by the SSO (sequence-specific oligonucleotide probe assays) method designating the corresponding genotype HLA-DQB1 for each subject. As typing method the “RELI HLA SSO systems” from Invitrogen (Carlsbad, California, United States) was used, an assay based on oligonucleotide probes corresponding to known polymorphic sequence motifs in the second exon of HLADQB1 genes immobilized on a nylon strip. The relevant regions of the HLA genes were amplified with biotinylated primers, denatured and hybridized to the immobilized probe array. The colored strips bands were manually introduced and analyzed in the computer with the UniMatch software, in order to designate  the corresponding allele at a medium to high (four digits) resolution.

Statistical analysis
The study was designed as an association study by comparing the frequency distribution of the subjects’ alleles in different groups. The assumption that there is no association between host HLA-DQB1 alleles and viral persistence or spontaneously/induced treatment viral clearance was made. Some HLADQB1 alleles will be present in significantly increased numbers in the non-R patients compared with the SVR or control group, representing alleles that confer risk for disease and non-response to therapy, while other alleles of the same gene will be better represented in SVR subjects or control group vs. non-R, representing protective alleles that will most likely confer spontaneous viral clearance or induced viral clearance by therapy achieving SVR. The control group subjects were chosen in order to provide a background estimate of the overall population of HLA alleles in the geographical area, under the assumption of zero recombination between the marker allele and the associated disease loci. The alleles frequencies were calculated by the diploid number of chromosomes in each group (2n), where “n” is the number of chromosomes. The experimental data were processed using IBM SPSS Statistics v.20 (Armonk, NY, USA). The  procedures used were: descriptive statistics (characterization variables defined in the database), graphs, nonparametric statistical tests (χ2 test for association, the link between the two categorical variables, by determining in some cases the risk ratio/odds ratio). HLA-DQB1 alleles’ frequencies for comparison inside groups for statistical significance, was analyzed by using a χ2 test, and p values <0.05 were considered statistically significant.

The patients in the study group received treatment with the classical combined therapy PegIFN-α/RBV (Peg-IFN α-2a 180 mg/week and ribavirin 15 mg/kg/day) for 12 months, in accordance with patients’ viral genotype 1. During treatment at week 4, 12, 24 and at the end of treatment, the patients were tested by RT-PCR for HCV viral loads to evaluate their response to therapy. Based on the therapeutic outcomes, patients were retrospectively qualified into two groups: sustained responders (SVR, n=32) – patients who remained negative for HCV-RNA during the evaluation period – and non-responders (non-R, n=19) – patients who failed to achieve a 2log₁₀IU/mL reduction in HCV-RNA at week 12. The age of participants enrolled in the study (controls and HCV patients) ranged from 13 to 75 years, the differences between the two genders in the two study groups being not significant (males 50.3%, female 49.7%), the same differences being observed in the SVR group (male 19.5%, female 22.4%) and controls (male 63.6%, female 69.7%). A larger difference regarding age was observed in the non-R group where more than a half of the non-responder patients (n=19) were males (n=13, 25.4%, mean age 43, SD 16.17 years) while only 6 of the non-R patients were female (11.7%) - Table 2.

  Table 2. Characteristics of the analyzed group (control and patients) based on therapy response, age and gender. Where n=number of subjects in the study

 

    

The DQB1 defined loci by high resolution (four digits) reverse line probe hybridization assay revealed a total number of 9 alleles from the DQB1 segment (DQ2*group with the DQB1* 0201 allele, DQ7*group with the DQB1*0301 allele, DQ8* group with the DQB1*0302 allele, DQ5* group with DQB1*0501,*0502,*0503, DQ6* group with *0602,*0603,*0604). Some of the alleles from the DQB1 cluster were not found, neither in patients, nor in the control group, showing a relatively small frequency in our population.

The corresponding frequencies of the HLA-DQB1 alleles in the SVR and non-R groups are shown in Table 3 and Figure 1. When the comparison inside the patient group was made according to therapy response – SVR vs. non-R – two significant statistical associations were highlighted. The first, HLA-DQB1*O201, was found to have high frequency in non-responders (16.7%), and was considered to be associated with risk for non-response to RBV/IFN therapy in this case (p<0.001, OR 25.09, 95%CI: 5.345-117.81), and the second HLA DQB1*0301 (19.6%) was increased in the patient group who achieved SVR, and was considered to be associated with protection and HCV viral clearance in this study (p = 0.017, OR 0.25, 95%CI: 0.08 -0.82).

 Table 3. HLA-DQB1 alleles’ distribution and risk association in the SVR and non-R groups

 

 

 

Figure 1.HLA –DQB1* frequencies according to therapy response

When each subgroup (SVR and non-R) was compared separately with the control group, other statistically significant associations were observed, with DQB1 *0201 present in low numbers in the SVR group compared with controls (SVR 2n=2, frequency 0.75% vs. controls 2n=39, frequency 14.69% p<0.001, OR 0.13 95%CI: 0.03-0.58).

Alleles from the DQ7 group (HLA-DQB1*0301) were found in the control and SVR groups in 15.2% and 14 % of cases, respectively, while lower levels were identified in the non-R group (5.6 %). HLA-DQB1* allele frequencies have shown that the DQB1*0301 allele was found in similar frequencies in the control (16.7 %) and SVR (17.7 %) groups, whereas in the non-SVR group it was found in only 6.5 %. The less common allele in the non–R group was *0602 (2n=2, frequency 0.8%) vs. control group (2n=30, frequency 12.4%). Alleles’ frequencies and the statistically compared data for HLA-DQB1* in control, SVR and non-R groups are shown in Table 4.

 Table 4. HLA –DQB1 alleles distribution in terms of therapy response showed in comparison: SVR vs. control group, non-R vs. control group.

 

 

In the SVR group, the most common alleles were *0301 (7.5 %) and *0501 (4.1 %) found in almost the same percentage as in the control group. In contrast, in the non-R group the most common alleles were *0201 (7.0%) and *0502 (2.5%). In controls, the highest level allele was DQB1 0301 (2n= 45, 16.8%), whereas the other common alleles were *0201 and *0602. Consulting bibliographical sources that have provided benchmark frequencies of HLA alleles in the Caucasian populations in Europe²¹ and in the Romanian population²², we noticed that the frequency of DQB1*alleles in the chosen control group were approximately within the reference values for the Caucasian population.

Among the HLA-DQB1 alleles genotyped in this study we found two statistically significant associations with different outcomes in terms of therapy response in patients with HCV infection. Significantly higher rates of SVR were achieved in patients with the DQB1*0301 alleles (19.6%) compared with non-R patients DQB1*0201 (16.7%) alleles. DRB1*0201 (p<0.001) was considered a risk allele, HCV patients genotyped with this allele having increased chances to be non-responders to PegIFN-α/RBV therapy. The protection allele is considered to be DQB1*0301 (p=0.017), patients presenting this allele being more likely to have response to therapy, achieving SVR more than the ones without the mentioned allele. In other words DQB1*0201, considered a susceptibility allele for the disease and viral persistence, was more represented in the non-R group, while DQB1*0301 from the DQ07* group confered viral clearance and protection during HCV infection. These observations are in accordance with other results in similar studies in Caucasian population regarding HLA alleles and HCV association outcomes, where those alleles are presented in linkage disequilibrium with HLA-DRB1 alleles as fallow: DQB1*0301 – haplotype HLA- DRB1*1104 - DQB1*0301 is associated with viral clearance and protection for disease and DQB1*0201 - haplotype DRB1*0701-DQB1*0201 and DRB1*0301-DQB1*0201^19,25,26,27is associated with non-response to PegIFN-α/RBV therapy, viral persistence chronicity and disease severity.

The high frequency of the protective allele DQB1*0301 (2n=45) found in the control group with little frequency differences in the patients group (SVR and non-R) could indicate a population with presence of a protective marker allele towards HCV that can develop also spontaneous clearance of the virus in certain cases. As this allele was found at a lower frequency in non-R patients, we can presume that this allele is probably responsible for treatment-induced viral clearance. Also the relatively small number of subjects included in this study made it difficult to interpret the statistical results; in some cases, DQB1*0503 was present only in SVR patients and not in the non-R group, this making the risk impossible to calculate. Ideally, the results validated by this study should be replicated in an independent study on a different sample of the same population. Some DQB1 alleles (*0502, *0302, *0604)^23,24 found to be associated with different HCV outcomes reported in others previous studies may have lower frequencies in the analyzed population, so the observations in that study are difficult to replicate on smaller samples like the present one. In the genome wide association studies, it is shown that only 15% of spontaneous HCV viral clearance was due to the DQB1*0301 allele, and the percentage is significantly increased with treatment-induced clearance, meaning that HLA-DQB1 alleles may activate the NK (natural killer) cells on IFN/RBV therapy, and the up-regulation degree may depend on HLA genotype. This fact underlines that other host, environmental, and viral factors (e.g., NK cells, IL28B, viral genotype, gender, age, therapy compliance etc.) are also involved in spontaneous HCV clearance, suggesting the complexity in understanding the factors with role in adaptive immunity such as HLA alleles. The importance of genotyping HLA DQB1 alleles increases when cumulated with other disease predictors such as virus genotype or interleukin IL28B. This may help in the selection of patients with the possible chance to respond to IFN/RBV therapy and achieve SVR. The results shown in this study have to be taken into account only for patients infected with HCV genotype 1b who were treated with PegIFN-α/RBV, and not with other types of new drugs. In this case, the HLA-DQB1 genotype can be useful in deciding the appropriate therapy plan for these patients.

In conclusion, the results show that there may be HLA-DQB1 alleles associated with PegIFN-α/RBV therapy, according to the nature of the host immune response, however other similarly designed larger studies are needed to confirm these findings.

1. Global surveillance and control of hepatitis C. Report of a WHO consultation organized in collaboration with the
Viral Hepatitis Prevention Board. J Viral Hepat 1999;6.35- 47.
2. Antipa C, Popescu A, Teleguță M, et al. [Seroprevalence of hepatitis C virus among the multiply transfused]. Rom J
Virol 1993;44:9-15.
3. Iancu LS, Pandele GI, Stanciu C, Luca V. 2002 Hepatitis C virus serotypes in Romanian patients with chronic hepatitis
and cirrhosis. Rev Med Chir Soc Med Nat 2002;106:79-82.
4. Naoumov NV. Hepatitis C virus infection in Eastern Europe. J Hepatol 1999;31 Suppl 1:84-7. [CrossRef]
5. Constantinescu I. [Genotyping of hepatitis viruses in Romania]. Paper presented at: The role of molecular
biology techniques in elucidating the mechanisms of HCV infection and developing new markers for assessing the
associated fibrosis and hepatocellular carcinoma. June 2008, Tîrgu Mureș, Romania.
6. Manns MP, McHutchison JG, Gordon SC, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet 2001;358:958-65. [CrossRef]

7. McHutchison JG, Lawitz EJ, Shiffman ML, et al. Peginterferon alfa-2b or alfa-2a with ribavirin for treatment
of hepatitis C infection. N Engl J Med 2009;361:580-93. [CrossRef]
8. Fried MW. Side effects of therapy of hepatitis C and their management. Hepatology 2002;36:S237-44. [CrossRef]
9. Duggal P, Thio CL, Wojcik GL, et al. Genome-wide association study of spontaneous resolution of hepatitis C virus infection: data from multiple cohorts. Ann Intern Med 2013;158:235-45. [CrossRef]
10. Alric L, Fort M, Izopet J, et al. Study of host and virus related factors associated with spontaneous hepatitis C virus clearance. Tissue Antigens 2000;56:154-8. [CrossRef]
11. Alric L, Fort M, Izopet J, et al. Genes of major histocompatibility complex class II influence the outcome of hepatitis C virus infection. Gastroenterology 1997;113:1675-81. [CrossRef]
12. Dring MM, Morrison MH, McSharry BP, et al. Innate immune genes synergize to predict increased risk of chronic disease in hepatitis C virus infection. Proc Natl Acad Sci U S A 2011;108:5736-41. [CrossRef]
13. Gheorghe L, Rugină S, Dumitru IM, Franciuc I, Martinescu A, Balaş I. HLA class II alleles in Romanian patients with chronic hepatitis C. Germs 2015;5:44-9. [CrossRef]
14. Kamal SM, Fehr J, Roesler B, Peters T, Rasenack JW. Peginterferon alone or with ribavirin enhances HCV-specific CD4 T-helper responses in patients with chronic hepatitis C. Gastroenterology 2002;123:1070-83. [CrossRef]
15. Lloyd AR, Jagger E, Post JJ. Host and viral factors in the immunopathogenesis of primary hepatitis C virus infection.
Immunol Cell Biol 2007;85:24-32. [CrossRef]
16. Fanning LJ, Levis J, Kenny-Walsh E, Whelton M, O’Sullivan K, Shanahan F. HLA class II genes determine the natural variance of hepatitis C viral load. Hepatology 2001;33:224–30. [CrossRef]
17. Fanning LJ, Kenny-Walsh E, Shanahan F. Persistence of hepatitis C virus in a white population: associations with
human leukocyte antigen class 1. Hum Immunol 2004;65:745-51. [CrossRef]