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A single-nucleotide polymorphism of IL12A gene (rs582537 A/C/G) and susceptibility to chronic hepatitis B virus infection among Iraqi patients
Egyptian Journal of Medical Human Genetics volume 23, Article number: 110 (2022)
Abstract
A case–control study (80 patients with chronic hepatitis B virus [HBV] infection and 96 controls) was performed to evaluate the association of an IL12A gene variant (rs582537 A/C/G) with HBV infection. Allele G showed a significantly lower frequency in patients compared to controls (31.2 vs. 46.9%; probability [p] = 0.009; corrected p [pc] = 0.027) and was associated with a lower risk of HBV infection (odds ratio [OR] = 0.49; 95% confidence interval [CI] = 0.29–0.83). A similar lower risk was associated with genotypes CG (17.5 vs. 29.2; OR = 0.25; 95% CI = 0.08–0.81; p = 0.02) and GG (10.0 vs. 16.7; OR = 0.25; 95% CI = 0.07–0.91; p = 0.036), but the pc value was not significant (0.12 and 0.126, respectively). Serum IL-35 levels showed significant differences between individuals of different genotypes (p = 0.007). The highest median was associated with CA genotype (286.5 pg/mL), followed by genotypes CG (227.0 pg/mL), GG (206.5 pg/mL), CC (169.0 pg/mL), AA (137.5 pg/mL) and finally AG (125.0 pg/mL). In conclusion, rs582537 appears to be an important genetic variant that may influence not only susceptibility to HBV infection but IL-35 levels.
Introduction
We recently demonstrated that interleukin (IL)-35 showed significantly lower levels in serum of patients infected with hepatitis B virus (HBV) compared to a healthy control group [1]. IL-35 is heterodimeric cytokine consisting of two subunits: IL-12α chain p35 (IL-12p35) and IL-27β chain Epstein–Barr virus-induced 3 (EBI3). IL-12p35 subunit is encoded by IL12A gene, which is located in the long arm of human chromosome 3 (3q25.33) [2]. Two single-nucleotide polymorphisms (SNPs) located in intron 2 of the IL12A gene (rs582054 and rs583911) were also studied by our group to assess their association with HBV infection. rs583911 showed no association, while A allele and AT genotype of rs582054 were significantly associated with the risk of HBV infection [3]. rs582537 is a SNP located between the SNPs rs582054 and rs583911, and studies have associated this SNP with susceptibility to primary biliary cholangitis (PBC) [4,5,6]. PBC, formerly known as primary biliary cirrhosis, is a chronic autoimmune disorder that results in progressive destruction of intrahepatic bile ducts resulting in cholestasis, which in turn leads to cirrhosis [7]. It has been reported that previous infection with HBV may exacerbate the severity of PBC and may lead to poorer outcomes [8]. Therefore, we hypothesized that SNP rs582537 may also be associated with the risk of HBV infection. To the best knowledge of investigators, this SNP has not been studied in HBV infection.
A case–control study (80 patients with chronic HBV infection and 96 controls) was performed during January−July 2020 to evaluate the association between rs582537 and susceptibility to HBV infection. Information for patients and controls was previously detailed [1, 3]. An allele-specific polymerase chain reaction (PCR) assay was used to amplify a 251-bp DNA region comprising rs582537 A/C/G (ancestral allele: C) using three forward primers (FA: 5'-TTTGGGCAATTGTCTGTCTCA-3′, FC: 5′-TTTGGGCACTTGTCTGTCTCA-3′ and FG: 5′- TTTGGGCAGTTGTCTGTCTCA-3′) and one reverse primer (5'-TTGCAGTGCACAGACGC-3′). Agarose gel electrophoresis was performed to detect genotypes of PCR products. These methods were previously detailed [3].
Genotype frequencies were tested for Hardy–Weinberg equilibrium (HWE) using Pearson’s chi-square goodness-of-fit test. Two-tailed Fisher’s exact test was used to assess significant differences between allele and genotype frequencies. Age- and gender-adjusted multinomial logistic regression was performed to calculate odds ratio (OR) and 95% confidence interval (CI). Serum levels of IL-35 were given as median and interquartile range (IQR). Significant differences between medians were assessed with Kruskal–Wallis test. A probability (p) value ≤ was considered significant. Bonferroni correction was applied to correct p value (pc) due to multiple comparisons. GraphPad Prism version 8.0.0 (San Diego, California, USA) and IBM SPSS Statistics 25.0 (Armonk, NY: IBM Corp.) were used to perform statistical analysis.
rs582537 was recognized by six genotypes (CC, CA, CG, AA, AG and GG) corresponding to three alleles (C, A and G). Genotype frequencies of rs582537 were in good agreement with HWE in HBV patients and controls (p = 0.529 and 0.127, respectively). Allele G showed a significantly decreased frequency in patients compared to controls (31.2 vs. 46.9%; p = 0.009; pc = 0.027) and was associated with a lower risk of HBV infection (OR = 0.49; 95% CI = 0.29–0.83). A similar lower risk was associated with genotypes CG (17.5 vs. 29.2; OR = 0.25; 95% CI = 0.08–0.81; p = 0.02) and GG (10.0 vs. 16.7; OR = 0.25; 95% CI = 0.07–0.91; p = 0.036), but the pc value was not significant (0.12 and 0.126, respectively) (Table 1).
Predetermined IL-35 levels [1] were examined in all participants (patients and controls) after stratification by rs582537 genotypes. Median IL-35 levels showed significant differences between individuals of different genotypes (p = 0.007). The highest median was associated with CA genotype (286.5 [IQR 169.0–523.0] pg/mL), followed by genotypes CG (227.0 [IQR 161.0–430.0] pg/mL), GG (206.5 [IQR 106.5–336.5] pg/mL), CC (169.0 [IQR 144.0–282.0] pg/mL), AA (137.5 [IQR 118.0–335.0] pg/mL) and finally AG (125.0 [IQR 67.0–210.0] pg/mL) (Fig. 1).
These data indicate that G allele may have protective effects against the development of HBV infection. Besides, IL-35 serum levels were influenced by rs582537 genotypes. Interestingly, genotypes comprising G allele ranked second and third among the highest level order of IL-35. Thus, the down-regulated levels of IL-35 in HBV patients [1] might be causally related to the observed lower frequency of G allele and GG genotype. Similar to our study, a strong association between rs582537 and PBC was reported and the SNP was considered a risk variant involved in the pathogenesis of disease [4,5,6].
In conclusion, rs582537 appears to be an important genetic variant that may influence not only susceptibility to HBV infection but IL-35 levels. As this study was the first, further studies in genetically different population groups [9, 10] are warranted to understand the role of rs582537 in the pathogenesis of HBV infection.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Abbreviations
- CI:
-
Confidence interval
- EBI3:
-
IL-27β chain Epstein–Barr virus-induced 3
- HBV:
-
Hepatitis B virus
- HWE:
-
Hardy–Weinberg equilibrium
- IL:
-
Interleukin
- IL-12p35:
-
IL-12α chain p35
- IQR:
-
Interquartile range
- OR:
-
Odds ratio
- p :
-
Probability
- PBC:
-
Primary biliary cholangitis
- pc :
-
Corrected probability
- SNP:
-
Single-nucleotide polymorphism
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Acknowledgements
We appreciate the kind help and cooperation of the medical staff at the Specialized Center for Gastroenterology and Hepatology and Central Blood Bank in Baghdad.
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This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.
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The three authors (RTM, RHA and AHA) contributed equally to data management, statistical analyzes and manuscript writing and reviewing. All authors read and approved the final manuscript.
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The Ethical Approval Committee at the University of Anbar approved the study (Reference: 23).
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Mohsen, R.T., Al-Azzawi, R.H. & Ad’hiah, A.H. A single-nucleotide polymorphism of IL12A gene (rs582537 A/C/G) and susceptibility to chronic hepatitis B virus infection among Iraqi patients. Egypt J Med Hum Genet 23, 110 (2022). https://doi.org/10.1186/s43042-022-00322-9
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DOI: https://doi.org/10.1186/s43042-022-00322-9