- Open Access
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)
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.
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 . 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) . 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 . 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 . It has been reported that previous infection with HBV may exacerbate the severity of PBC and may lead to poorer outcomes . 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 .
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  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  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.
IL-27β chain Epstein–Barr virus-induced 3
Hepatitis B virus
IL-12α chain p35
- p :
Primary biliary cholangitis
- pc :
Mohsen RT, Al-Azzawi RH, Ad’hiah AH (2020) Serum level of interleukin-35 in patients with chronic hepatitis B virus infection. Iraqi J Sci 61:2860–2865. https://doi.org/10.24996/ijs.2020.61.11.9
Song M, Ma X (2016) The immunobiology of interleukin-35 and its regulation and gene expression. Adv Exp Med Biol 941:213–225. https://doi.org/10.1007/978-94-024-0921-5_10
Mohsen RT, Al-azzawi RH, Ad’hiah AH (2020) Single nucleotide polymorphisms of interleukin-35 subunit genes predict host susceptibility to chronic hepatitis B virus infection among Iraqi patients. Meta Gene 25:100735. https://doi.org/10.1016/j.mgene.2020.100735
Juran BD, Hirschfield GM, Invernizzi P, Atkinson EJ, Li Y, Xie G et al (2012) Immunochip analyses identify a novel risk locus for primary biliary cirrhosis at 13q14, multiple independent associations at four established risk loci and epistasis between 1p31 and 7q32 risk Variants. Hum Mol Genet 21:5209–5221. https://doi.org/10.1093/hmg/dds359
Hitomi Y, Ueno K, Kawai Y, Nishida N, Kojima K, Kawashima M et al (2019) POGLUT1, the putative effector gene driven by rs2293370 in primary biliary cholangitis susceptibility locus chromosome 3q13.33. Sci Rep 9:31. https://doi.org/10.1038/s41598-018-36490-1
Qiu F, Tang R, Zuo X, Shi X, Wei Y, Zheng X et al (2017) A genome-wide association study identifies six novel risk loci for primary biliary cholangitis. Nat Commun 8:14828. https://doi.org/10.1038/ncomms14828
Fejfar T, Vaňásek T, Hůlek P (2020) Chronic cholestatic liver diseases—primary biliary cholangitis and primary sclerosing cholangitis. Vnitr Lek 66:287–300. https://doi.org/10.36290/vnl.2020.084
Zhang Y, Shi Y, Wu R, Wang X, Gao X, Niu J (2018) Primary biliary cholangitis is more severe in previous hepatitis B virus infection patients. Eur J Gastroenterol Hepatol 30:682–686. https://doi.org/10.1097/MEG.0000000000001100
González-Galarza FF, Takeshita LYC, Santos EJM, Kempson F, Maia MHT, Da Silva ALS et al (2015) Allele frequency net 2015 update: New features for HLA epitopes, KIR and disease and HLA adverse drug reaction associations. Nucleic Acids Res 43:D784–D788. https://doi.org/10.1093/nar/gku1166
Norhalifah HK, Mat NFC, Edinur HA (2018) Cytokine gene polymorphisms in cancer and inflammatory disorders. Curr Immunol Rev 14:81–93. https://doi.org/10.2174/1573395514666180724121419
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.
This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.
Ethics approval and consent to participate
The Ethical Approval Committee at the University of Anbar approved the study (Reference: 23).
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
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
- Hepatitis B virus
- Single-nucleotide polymorphism