Genetic Profiling of Interleukin Variants and their Correlation with Hepatitis C Viral Response to Different Therapeutic Strategies Administered in Pakistani Patients

Abstract

This research study aimed to investigate the response of patients infected with the hepatitis C virus (HCV) toward direct-acting antivirals (DAAs) therapy. The research included a cohort of HCV-infected patients from different regions of Pakistan. Experimental studies were performed for the detailed analysis of virological response recorded at specific time intervals during the whole course of treatment. Sustained virological response (SVR) was explicitly defined as undetectable HCV RNA at 12 weeks post-completion of therapy (SVR12), ensuring alignment with international clinical standards. The standard genotypes analyzed for the selected interleukin (IL) variants were: IL-4 rs2243250 (C/C, C/T, T/T), IL-17 rs612242 (A/A, A/G, G/G), and IL-22 rs2064501 (A/A, A/G, G/G). The genetic profiling of these variants highlighted the significant role of cytokines in modulating the patient's immune response. The research also focused on the association between single-nucleotide polymorphisms (SNPs) of IL genes and categorization based on the outcomes of treatment by sequencing analysis. Our study found that the IL-4 rs2243250 SNP was predominantly heterozygous as CT in 52% SVR and 37% relapse patients. The IL-17 rs612242 SNP was mainly identified as AG in 54% SVR and 39% relapse patients, and the IL-22 rs2064501 SNP was prevalent in 32% relapse and 49% SVR patients as AG genotype. A homozygous genotype T/T was also analyzed for polymorphism rs2243250 in a relatively small percentage (16%) of non-responders. These research findings showed that C/T, C/C, and A/G genotypes were dominant in patients who cleared the infection after treatment. It confirmed the influence of single-nucleotide polymorphisms on the success rate of DAAs therapy, determined by observing the baseline characteristics and genetic factors of selected patients after comparing with healthy controls. Due to the limited genotyping of healthy controls (n = 4), comparative results involving the control group were interpreted with caution.

Graphical Abstract

This is a visual representation of the abstract.

Keywords

direct-acting antivirals hepatitis C virus interleukins single-nucleotide polymorphisms

Introduction

Hepatitis C virus (HCV) comprises of a single-stranded RNA genome, which is known to be highly genetically variable. It belongs to the family of viruses known as Flaviviridae [Martinez and Franco, 2020]. Research scientists are aware that nearly 3% of the world’s population is affected by HCV, meaning that almost 165 million people get infected with hepatitis around the globe [Guntipalli et al., 2021; Salari et al., 2022]. A huge public health concern has progressed among the world’s population because HCV can result in outcomes such as hepatocellular carcinoma (HCC) and liver failure if prompt treatment is not initiated [Kishta et al., 2020].

Diagnosis of HCV is possible by various methods, such as determining their subtypes, antibody testing, measuring HCV RNA expression levels, and viral genotype. The modern development of direct-acting antivirals (DAAs) has revolutionized the treatment strategies of HCV infections. The success rate of DAAs in treating HCV infections is more than 90% [Hayes et al., 2022]. Additionally, DAAs therapy was also observed to have longer-lasting impacts than other treatment plans for patients suffering from HCV [Suda and Sakamoto, 2021].

A novel treatment approach involved the use of DAAs given in combination with ribavirin, administered over a duration of 12–72 weeks [Chebli et al., 2018]. Different responses were exhibited by different categories of patients due to this DAA therapy. These responses were given names according to the outcomes of treatment. ETR term is given to individuals when HCV RNA becomes undetectable (≤50 IU/mL), which is also called the end-of-treatment response because it is reported after the end of the treatment plan [Cabibbo et al., 2019]. If a patient’s data suggests that the viral load is undetectable after a follow-up time period of 6 months, i.e., the patient has completed the 6-month medication course, then this treatment response is termed SVR (sustained virological response). If the patients have a reoccurrence of HCV and RNA viral load is detected in their serum after the completion of therapy, then such patients are considered as a relapse. Non-responders (NR) are the patients affected with HCV in whom RNA viral load levels remain measurable throughout the whole treatment [Akram et al., 2011; Zarębska-Michaluk et al., 2023].

Cytokines are molecules involved in modulating the immune response, comprising interleukins (ILs) and interferons. We conducted a genetic analysis of SNPs of three IL genes: IL-4, IL-17, and IL-22 in this research study. IL-4 is a crucial cytokine that significantly influences the differentiation of naive TH cells into T-helper 2 effectors [Prout et al., 2018]. A novel family of cytokines has been recently investigated by researchers known as IL-17 [McGeachy et al., 2019]. This cytokine plays a pivotal role in initiating a distinct signaling pathway activated by binding of ligand and receptor, which drives the potential modulation of specific immune response [Morales et al., 2020]. IL-22 has an important role in protecting and preventing damage to the tissues, enhances the production of antimicrobial proteins, and releases chemokines [Zenewicz, 2021].

Materials and Methods

Selection of study population, ethical compliance, and blood sampling

Samples were obtained from the blood of patients affected with hepatitis C. These blood samples were gathered from two collection centers named the Association of Fatimah Jinnah Old Graduates and Akhtar Mubarak Referral Center, located in Lahore, Pakistan. The study protocol was approved by the Institutional Review Board of the Center of Excellence in Molecular Biology. Written informed consent was obtained from all participants prior to enrollment in accordance with the institutional guidelines. Blood sampling was performed for 124 HCV-infected patients, out of which 39 were males and 85 were female patients aged between 20 and 80 years. These samples were proceeded for further experimentations. The participants had three types of treatment outcomes. These three categories were Responders (SVR), NR, and relapse patients. Furthermore, blood of 30 healthy controls was also drawn from selected individuals. Written consent was taken from each patient prior to starting any experiment. The collected blood samples were stored at −20°C for long-term stability and then used for further experimental studies. For RNA extraction, serum was isolated from whole blood. For this purpose, lavender cap-Ethylenediaminetetraacetic acid (EDTA) tubes and gel-containing serum separator tubes with yellow tops were utilized. Different factors were brought into concentration to correlate with treatment outcomes. The major variables included biochemical, virological, and demographic profile.

Treatment regimen and clinical assessment

The patients selected for this study plan were administered with DAAs [Shalaby et al., 2012]. Participants were administered a standardized Sofosbuvir-based DAA regimen (e.g., Sofosbuvir/Daclatasvir or Sofosbuvir/Velpatasvir) for a duration of 12 weeks. The primary efficacy endpoint was SVR12. To address potential confounders, baseline clinical parameters including viral load, HCV genotype, and fibrosis status were documented for each patient.

RNA extraction and quantitative analysis of HCV RNA by RT-PCR

RNA expression levels of HCV were quantified by Real-time polymerase chain reaction (RT-PCR) from the RNA extracted from patient’s sera. RNA of HCV was isolated and extracted from serum samples (200 µL) by using a commercial kit available in the molecular virology lab, which was the Ambion RNA Isolation kit by Life Technologies. Quantification of extracted RNA was performed by using the HCV Real-^TM Quant (Sacace Biotechnologies, Como, Italy) kit. The values were set in specific FAM and Cy3 standard columns to generate Standard Curves for analyzing the results. The fluorescent signals were observed as graphs in real-time.

RT-PCR was carried out in a final reaction volume of 25 µL. Each reaction mixture contained RT-PCR master mix (2X), HotStart DNA polymerase, Moloney Murine Leukemia Virus reverse transcriptase, and nuclease-free water, along with the extracted RNA template. For each run, HCV quantification standards and an internal control standard were included in parallel to perform a comparative amplification curve analysis. Tris-EDTA buffer served as the negative control. Amplification was performed in a thermocycler programmed as follows: Initial Hold at 50°C for 30 min, followed by enzyme activation at 95°C for 15 min, and then 42 amplification cycles consisting of denaturation at 95°C for 20 s and annealing/extension at 60°C for 40 s. Standard concentrations provided with the HCV quantification data card were entered for FAM (6-carboxyfluorescein) and Cy3 fluorescent dye channels to generate amplification peaks calibrated to produce calculated and efficient results. Real-time signal acquisition was performed throughout the reaction, with the Cy3 channel representing HCV RNA detection and the FAM channel corresponding to the internal control.

Genomic DNA extraction

The genomic DNA was extracted successfully from the blood samples of patients by following the instructions of the manual provided with the Invitrogen PureLink Genomic DNA Mini Kit. The DNA was not only quantified by the Nanodrop Spectrophotometer but also confirmed by utilizing the Agarose gel electrophoresis technique.

PCR-based DNA amplification

To amplify the genomic regions of interest, specific primer sets corresponding to IL-4 single-nucleotide polymorphism (SNP) rs2243250, IL-17 SNP rs612242, and IL-22 SNP rs2064501 were designed and utilized in calculated concentrations. Each primer pair was carefully optimized for annealing temperature and extension time to achieve high specificity and efficiency of amplification. The PCR assays were conducted under these optimized thermal cycling conditions, ensuring reproducible amplification of the target loci.

The PCR products were subsequently analyzed through agarose gel electrophoresis (2%), prepared in 1X Tris-acetate-EDTA Buffer buffer to provide optimal resolution of DNA fragments. Ethidium bromide (0.5 µg/mL) was incorporated into the gel as a nucleic acid intercalating dye, enabling visualization of amplified DNA bands under ultraviolet light. A molecular DNA ladder was run in parallel to estimate the fragment sizes and confirm the accuracy of amplification. Following electrophoresis, the gels were visualized using a Gel Documentation (Gel Doc) imaging system under a Ultraviolet (UV) transilluminator. The presence of discrete DNA bands was compared to the expected product sizes to verify the successful amplification of the selected SNPs.

Purification of PCR products and sequencing of bands of interest

After confirming the size of the PCR products, the bands of interest were purified and cleaned by using the Thermo Scientific GeneJET PCR Purification Kit. The purified PCR products were added to the 96-well PCR plate and mixed with their specific primers. This plate containing PCR products was utilized in the process of DNA sequencing. An automated sequencing analyzer was used for the evaluation of the results of these samples. Sequences were generated in the form of peaks and analyzed with the help of Chromas v.2.6.6.

Statistical evaluation of experimental data

All biological, demographic, and virological data were initially compiled and organized using Microsoft Excel spreadsheets for preprocessing and data management. Genotyping definitions were standardized for rs2243250 (C/T), rs612242 (A/G), and rs2064501 (A/G), and all non-canonical or inconsistent labels were removed from the dataset. Statistical evaluations were performed using IBM SPSS (v25.0). Chi-square tests were employed for genotype distribution comparisons, while logistic regression models were utilized to calculate odds ratios (ORs) with 95% confidence intervals (CIs). GraphPad Prism (version 10) was utilized for graphical representations of SNPs. Descriptive statistics were applied to summarize continuous and categorical variables, while appropriate inferential tests were conducted to assess associations between genetic variants and clinical or virological outcomes.

The t-test and one-way Analysis of Variance were employed to compare group differences, and correlation analyses were performed to evaluate the relationship between demographic parameters and treatment response.

In addition, results obtained after sequencing were analyzed using specialized bioinformatics tools such as Chromas and Mutation Surveyor to identify mutations and confirm the presence of SNPs. For all statistical evaluations, a P value < 0.05 was considered statistically significant. Graphs and figures were generated using GraphPad Prism to provide a clear visual representation of the results.

Results

Patient demographics

The participants were characterized into three groups on the basis of their outcomes of treatment. The three categories were Responders (SVR), NR, and relapse patients. The majority of the patients in this research study were females, i.e., 68%, while 32% male patients were involved. Furthermore, the collected data about patients indicated that a higher percentage of females of the age group 20–50 years were infected with HCV, whereas almost an equal percentage of males were affected in both age groups, i.e., 20–50 and 51–80 years, as shown in Figure 1(a) and (b).

FIG. 1.

(a) Distribution of selected patients on the basis of gender (b) Distribution of selected patients on the basis of age groups.

Confirmation of bands of interest by gel electrophoresis

Agarose gel electrophoresis was performed to visualize and confirm the presence of DNA of the specific SNPs under study. The 1 kb DNA ladder was loaded in the first lane along with the genomic DNA to determine the size of the bands. The bands of interest were clearly seen under a UV transilluminator in a Gel Doc system, which verified the authenticity and efficiency of the experiment. The quantification of genomic DNA was also performed by using a Nanodrop Spectrophotometer, and the results were obtained in ng/µL.

PCR amplification curve analysis

The extracted DNA was amplified by PCR, which was run in a thermocycler. Specific primers of IL-4 SNP rs2243250, IL-17 SNP rs612242, and IL-22 SNP rs2064501 were used in calculated quantities to amplify the product. The product sizes of IL-4, IL-17, and IL-22 gene primers were 254, 347, and 390 bp, respectively, which were confirmed by performing agarose gel electrophoresis as shown in Figure 2(a)–(d).

FIG. 2.

(a) Lane 1: 100 bp DNA ladder, Lane 2–6: Purified PCR products of IL-4 SNP rs2243250 showing product size of 254 bp (b) Lane 1: 100 bp DNA ladder, Lane 2: Positive control Lane 3: Negative control Lane 4–15: Purified PCR products of IL-22 SNP rs2064501 showing product size of 390 bp (c) Lane 1: 100 bp DNA ladder, Lane 2: Positive control Lane 3: Negative control, Lane 4–20: Purified PCR products of IL-17 SNP rs612242 showing product size of 347 bp (d) Lane 2–14: Purified PCR products of IL-17 SNP rs612242 showing product size of 347 bp. IL, interleukin; PCR, real-time polymerase chain reaction; SNP, single-nucleotide polymorphism.

Categorization of the patients under study

Results were analyzed by forming a relation between gender and age group of HCV-infected patients and treatment responses. These patients were eventually categorized according to the outcomes of treatment. Out of 124 patients, 74 were categorized into SVR, which included 28 males and 46 females. Second, 36 were categorized into relapse patients, which included 11 males and 25 females. The third category, i.e., NR, included 14 patients, which involved 8 males and 6 females, as shown in Figure 3. Out of 86 patients who were in the age group of 20–50 years, 52 patients were in the SVR category, 26 were relapse patients, and 10 patients were NR. Moreover, out of 35 patients who were in the age group of 51–80 years, 19 patients were in the SVR category, 9 were relapse patients, and 7 patients were NR, as illustrated in Figure 4. The P value was calculated by statistically analyzing this data. A confidence interval of 95% was set for recording the results, i.e., a P value of ≤ 0.05 was considered significant.

FIG. 3.

Graphical representation of percentage distribution of categories (SVR, relapse, non-responders) on the basis of gender and treatment response. SVR, sustained virological response.

FIG. 4.

Graphical representation of percentage distribution of categories (SVR, relapse, non-responders) on the basis of age and treatment response.

Sequencing of 124 individuals was performed in order to determine the genotypes of different SNPs. Out of which 120 were HCV-infected patients, including 27% patients for IL-4 SNP, 24% patients for IL-17, 45% patients for IL-22 SNP, and 4 healthy controls (one for each IL gene) were selected for genotyping. Some SNPs are shown in Figure 5, illustrated by colored peaks in a chromatogram.

FIG. 5.

Chromatogram representing SNPs rs2243250, rs612242, and rs2064501 of interleukin variants (IL-4, IL-17, IL-22) in the genomic sequence of chronic HCV-infected patients. HCV, hepatitis C virus.

Correlation of genotype distribution among the study patients

The genotype distribution of alleles for IL-4 SNP rs2243250 demonstrated that 52% patients were heterozygous for C/T genotype in patients with sustained virological response, and 16% patients were homozygous for T/T genotype in NR. The T/T genotype in the IL-4–590 C/T (rs2243250) SNP is associated with increased serum IL-4 levels, often indicating a predisposition to enhanced Th2 immune responses and higher susceptibility to allergic diseases or, conversely, a protective effect against certain cancers. It indicates a homozygous mutation, frequently leading to altered cytokine regulation compared to the wild-type C/C genotype. In HCV-infected patients, particularly in studies focused on Egyptian patients with genotype 4, the IL-4–590 C/T (rs2243250) T/T genotype is associated with a higher risk of treatment failure (NR) and disease progression. Specifically, the T/T genotype was found to be more prevalent (17.6%) in NR compared to (7.6%) in responders. [Shalaby et al., 2012] For IL-17 SNP rs612242, the genotype distribution among patients included majorly the heterozygous genotype 54% A/G and 44% C/C genotype in SVR patients, whereas only 18% relapse patients showed the C/C genotype. Moreover, 27% SVR individuals were homozygous for A allele for rs612242. The genotype distribution of alleles for IL-22 SNP rs2064501 was determined as follows: The majority of the patients had heterozygous genotype A/G, i.e., 49% individuals had a genotype carrying one A allele and one G allele in individuals who exhibited SVR; however, 32% relapse patients were heterozygous for A/G genotype. Furthermore, 10% were homozygous for the G allele in NR. A comprehensive summary of this genotypic profile is shown in Table 1 and illustrated by Figure 6. The graphical representation of the statistical analysis is illustrated by the graphs shown in Figure 7(a) and (b).

FIG. 6.

Statistical evaluation of the genetic profile of healthy controls and chronic HCV-infected patients, showing the association between the percentage distributions of genotypes in IL variants.

FIG. 7.

(a) Correlation analysis of chronic HCV-infected patients performed between genotype distributions and outcomes of DAAs’ treatment. (b) Comparison of categories of HCV-infected patients (SVR, relapse, non-responders) with healthy controls to evaluate the response toward treatment due to interleukin variants. * P < 0.05; ** P < 0.01.

Table 1.

Genotype Distribution and Allelic Frequencies of SNPs in SVR, Non-Responders, and Relapse Patients

	SVR (n = 74)		Relapse (n = 36)		NR (n = 14)		P value
SNPs	Count	Proportion	Count	Proportion	Count	Proportion	P value
rs2243250
C/T	38	52	13	37	4	25	0.041
T/T	11	15	4	10	3	16
T/C	13	18	7	20	3	19
C/C	10	17	5	15	2	14
rs612242
A/G	40	54	14	39	4	27	0.006
C/C	30	41	6	18	2	17
T/C	17	23	4	10	2	14
A/A	20	27	8	22	1	6
C/A	30	41	6	18	2	17
rs2064501
A/G	36	49	12	32	3	24	0.027
A/A	24	32	11	31	4	29
G/G	16	21	4	10	3	19
C/T	19	26	5	13	2	16
G/G	16	21	4	10	3	19

P value < 0.05 = significant.

NR, non-responders; SNP, single-nucleotide polymorphism; SVR, sustained virological response.

Discussion

Hepatitis C is a chronic disease, which affects millions of people all around the globe and can be lethal if left untreated. Many effective therapeutic strategies have been used to cure this disease [Mohamed et al., 2020]. However, no treatment is 100% effective for treating hepatitis C. Antivirals are given to patients suffering from HCV infections to clear the viral load and achieve a SVR after completion of treatment [Manns and Maasoumy, 2022]. Interferon treatment was not completely effective against HCV; therefore, research studies were performed to devise an efficient strategy for treatment [Zoratti et al., 2020]. In recent years, DAAs have been prescribed to patients infected with chronic hepatitis C. Furthermore, SNPs have been identified for the analysis of different outcomes of treatment [Negro, 2020].

In this research study, the correlation between IL SNPs and treatment response has been evaluated in detail. The categorization of patients was done on the basis of their response toward treatment, among which three groups were selected for the study, i.e., SVR, NR, and relapse patients. A cohort of 124 HCV-infected patients was selected for sequencing analysis. The demographic profile, medical history, and baseline characteristics such as age and gender of these patients were documented for thorough analysis. The genotyping of patients was performed, and sequences were analyzed for the identification of SNPs. The study plan was designed to observe the association of SNPs rs2243250, rs612242, and rs2064501 with treatment response.

The results of our study showed that in the case of IL-4 rs2243250 SNP, the highest percentage of patients were heterozygous for CT genotype, observed in SVR (52%), a relative percentage was analyzed in relapse patients (37%), and a low percentage was observed in NR (25%). The homozygous C/C or T/T genotype was less frequently observed in patients for this polymorphism. This showed that CT genotype was more prevalent in SVR individuals, who responded effectively toward DAAs treatment, as compared to relapse and NR patients. A novel heterozygous genotype TG was also analyzed in 18% NR patients having this SNP in their genome. A research study found the contribution of polymorphism of IL-4-589C/T in the majority of patients susceptible to HCV-related HCC [Peiffer and Zeuzem, 2022], coinciding with the results of our research study. However, there was no significant difference in the percentage of homozygous (C/C) genotype observed between the healthy controls and the categories of selected HCV-infected patients in our research. In contrast, that research study was on the Egyptian population, whereas our research experiments were carried out on the blood samples collected from Pakistani patients.

In case of IL-17 SNP rs612242, the majority of chronic HCV-infected patients had a heterozygous genotype AG; the highest percentage (54%) of this genotype distribution was found in SVR patients. Whereas, for relapse patients, the percentage for AG genotype was 39%. Another genotype that was highly observed in chronic HCV-infected patients was C/C genotype, majorly in SVR patients, comprising of a percentage of 41%, while seen in only 18% relapse patients. Genetic analysis of this SNP showed that C/C genotype was more likely to be prevalent in individuals who cleared the infection efficiently after completion of treatment. The less frequently observed genotype was A/A (homozygous) in these patients, especially in NR (6%). The results of our research study aligned with another study model in which researchers evaluated the role of this IL-17 polymorphism in the formation of the cryoglobulinemic syndrome in patients infected with chronic hepatitis C [Abd-Elfattah et al., 2021]. They concluded that C/C genotype was the most commonly identified genotype in both healthy individuals and patients infected with chronic hepatitis C, who then cleared the infection after treatment. The SNP that encodes this pro-inflammatory cytokine is located in the IL-17A gene, which has a significant influence on the immune response of patients [Kalashnyk and Riabokon, 2019]. The genotype distribution among these patients can be better understood to find the correlation between risk of disease and response to treatment [Wang et al., 2016].

In this research study, we also observed the genetic profile of patients having IL-22 SNP rs2064501. The predominant percentage, comprised of the AG heterozygous genotype among the individuals infected with chronic hepatitis C, was observed especially in SVR (49%). However, in relapse patients, the percentage for AG genotype was 32%. The alleles A and G were also observed in homozygous genotype in a majority of SVR patients. This confirmed that AG genotype was effective in patients to cure the disease. For instance, AA genotype was present in 32% of individuals showing SVR. The least majority of patients showed a heterozygous CT genotype; only 13% relapse patients had this genotype. A research study observed the role of IL-22 and IL-22 binding protein in the regulation of fibrosis and cirrhosis in chronic HCV-infected patients [Mostafa et al., 2021]. Our results also coincided with their research study, depicting that AG and GG genotypes were more frequently identified in both healthy controls and patients.

Statistical analysis was performed to determine all these percentages of genotype distribution of SNPs. The variations were observed by applying the correlation tests, and eventually the P values were calculated for each variant. A P value of ≤ 0.05 was considered significant [Sertorio et al., 2015] for evaluating the association between genotype distribution and response towards treatment. The P values for SNPs rs2243250, rs612242, and rs2064501 were all significant, i.e., 0.041, 0.006, and 0.027, respectively. This proved that there was a significant correlation between the presence of genetic variants and treatment response as reported in SVR, relapse, and NR patients.

Genotypic distribution analysis of SNPs rs2243250, rs612242, and rs2064501 showed different patterns between SVR and relapse groups. Notably, the T/C genotype of rs2243250 was significantly associated with treatment outcomes (P = 0.0003), suggesting it may influence the risk of HCV relapse or protection depending on allelic dominance. This finding aligns with previous research linking rs2243250 in the IL4 gene to immune response modulation and antiviral therapy success in hepatitis C [Di Leo and Sardanelli, 2020].

Similarly, the C/A genotype of vrs612242 was significantly associated with treatment response (P = 0.01), indicating a role for this SNP in genetic susceptibility and interferon therapy effectiveness. In contrast, rs2064501 did not show significant differences across groups, consistent with earlier studies suggesting its impact on HCV clearance is limited or varies by population [Gao et al., 2017], as given in Table 2.

Table 2.

Dominant and Recessive Model Analysis of Allelic Frequencies of SNPs in the SVR and Relapse Groups

SNPs	Model	Genotype	SVR (n = 74)		Relapse (n = 36)		OR (95% CI)	P value
rs2243250	(Co-dominant)	C/T	38	52	13	37	1.62 (0.32–6.33)	0.69
	(novel Dominant)	T/G	11	15	4	10	1.39 (0.43–4.24)	0.76
	(novel Dominant)	T/C	13	18	7	20	0.08 (0.02–0.30)	0.0003
	(Dominant)	C/C	10	17	5	15	0.88 (0.25–3.01)	>0.99
	(Recessive)	T/T	12	16	4	11	0.64 (0.21–2.01)	0.57
rs612242	(Co-dominant)	A/G	40	54	14	39	0.57 (0.19–1.68)	0.43
	(Dominant)	C/C	30	41	6	18	0.54 (0.20–1.50)	0.34
	(Co-dominant)	T/C	17	23	4	10	2.38 (0.75–6.94)	0.19
	(Dominant)	A/A	20	27	8	22	0.50 (0.14–1.52)	0.36
	(Co-dominant)	C/A	30	41	6	18	3.40 (1.28–9.01)	0.01
rs2064501		A/G	36	49	12	32
		A/A	24	32	11	31
		G/G	16	21	4	10
		C/T	19	26	5	13
		G/G	16	21	4	10

P value < 0.05 = significant.

CI, confidence interval; OR, odds ratio.

The dominant and recessive model analysis of SNPs (rs2243250, rs612242, and rs2064501) between the SVR and NR groups revealed no statistically significant associations (P > 0.05), though some genotypes showed notable trends. Specifically, the rs2243250 T/T recessive model showed a slightly higher odds ratio (1.16, 95% CI: 0.25–5.74), suggesting a weak, non-significant association with treatment response. Similarly, rs612242 C/C (dominant model) exhibited an elevated OR (4.09, 95% CI: 0.87–19.18, P = 0.07), indicating a potential but not definitive role in influencing therapy outcome. These trends may reflect minor allelic effects that did not reach statistical significance due to limited sample size, as given in Table 3.

Table 3.

Dominant and Recessive Model Analysis of Allelic Frequencies of SNPs in the SVR and Non-Responder Groups

SNPs	Model	Genotype	SVR(n = 74)		NR(n = 14)		OR (95% CI)	P value
rs2243250	(Co-dominant)	C/T	38	52	4	25	2.63 (0.75–8.10)	0.15
	(novel dominant)	T/G	11	15	3	18	0.64 (0.16–2.43)	0.68
	(novel dominant)	T/C	13	18	3	19	0.78 (0.21–2.92)	0.71
	(Dominant)	C/C	10	17	2	14	0.93 (0.19–4.71)	>0.99
	(Recessive)	T/T	12	16	2	12	1.16 (0.25–5.74)	>0.99
rs612242	(Co-dominant)	A/G	40	54	4	27	2.94 (0.84–9.02)	0.14
	(Dominant)	C/C	30	41	2	17	4.09 (0.87–19.18)	0.07
	(Co-dominant)	T/C	17	23	2	14	1.78 (0.42–8.62)	0.72
	(Dominant)	A/A	20	27	1	6	4.81 (0.75–53.67)	0.17
	(Co-dominant)	C/A	30	41	2	17	4.09 (0.87–19.18)	0.07
rs2064501		A/G	36	49	3	24
		A/A	24	32	4	29
		G/G	16	21	3	19
		C/T	19	26	2	16
		G/G	16	21	3	19

Comparable studies have reported that rs2243250 and rs612242 polymorphisms in cytokine-related genes can modulate antiviral therapy response in HCV-infected patients, with the T and C alleles often linked to altered immune regulation [Gauthiez et al., 2017]. The findings suggest a significant association between these specific IL polymorphisms and the likelihood of achieving SVR12. The prevalence of the IL-4 CT genotype in SVR patients may indicate a specific immune modulation profile that favors viral clearance under DAA administration [Gopalakrishnan et al., 2018]. However, these results represent associations rather than functional proof of causality.

These results emphasize the role of immune-related genetic variations in influencing the likelihood of achieving SVR or experiencing relapse, supporting the idea that host genetics contribute to differences in hepatitis C treatment outcomes.

Conclusion

This research study showed that the most prevalent heterozygous genotype for IL-4 SNP rs2243250 was C/T in individuals with sustained virological response, and for IL-17 SNP rs612242, AG genotype was heterozygous in the majority of SVR patients. Similarly, the most commonly observed heterozygous genotype for SNP rs2064501 of IL-22 was A/G, and majorly found individuals who demonstrated SVR. A homozygous genotype T/T was also analyzed for polymorphism rs2243250 in a relatively small percentage of NR. This detailed profiling of sequences of patients confirmed that C/T, C/C, and A/G genotypes were found to be most predominant in individuals who completely responded toward DAAs therapeutic plan, for rs2243250, rs612242, and rs2064501, respectively.

Study limitations

To ensure transparency and balance, the following limitations are acknowledged:

The relatively small size of the NR and healthy control cohorts may limit the statistical power to detect minor allelic effects. The study was conducted at a single center in Lahore, which may restrict the generalizability of the results to broader populations. More functional experiments (e.g., protein expression levels or in vitro assays) should have been performed to confirm the biological impact of the identified SNPs. Only four healthy controls were successfully genotyped, rendering comparisons with the control group underpowered.

Future perspectives

In future, the genetic profiling of SNPs of IL-4, IL-17, and IL-22 variants will play a significant role in identifying the biomarkers of chronic HCV in affected patients. This can prove to be beneficial for the early prediction of infections caused by HCV. Emerging studies and research experiments performed for the analysis of these polymorphisms will help in the evolution of precision medicine. This research study can enhance the development of modern therapeutic plans according to the unique genetic profiles of patients by particularly focusing on the IL SNPs. Specific genetic markers can be identified by genotyping that act as predictors for treatment responses, which will lead to more efficient and targeted therapies. This research can provide insights into genetic factors in order to avoid the risk of the occurrence of lethal infections or HCC caused by HCV.

Footnotes

Authors’ Contributions

S.A. conceived the idea of study. I.T. performed experimental work and wrote the manuscript. H.N. performed statistical analysis. I.A. and M.S. helped in sampling and data acquisition. M.I. and S.A. helped in trouble shooting and proofread and finalized the manuscript.

Author Disclosure Statement

All authors declare no conflicts of interest.

Funding Information

This work was supported by University of The Punjab, The HEC Pakistan, MEAE and The MESRI as part of Franco Pakistani Hubert Curien Partnership.

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