Home About us Editorial board Search Ahead of print Current issue Archives Submit article Instructions Subscribe Contacts Login 


 
 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 35  |  Issue : 2  |  Page : 406-411

STAT4 gene polymorphism as a risk factor for hepatocellular carcinoma on top of chronic hepatitis C


1 Department of Lab Medicine, National Liver Institute, Menoufia University, Menoufia, Egypt
2 Department of Hepatology and Gastroentrology, National Liver Institute, Menoufia University, Menoufia, Egypt

Date of Submission16-Dec-2021
Date of Decision22-Jan-2022
Date of Acceptance30-Jan-2022
Date of Web Publication27-Jul-2022

Correspondence Address:
Mai Elashmawy
Menoufia
Egypt
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_297_21

Rights and Permissions
  Abstract 


Background
Single-nucleotide polymorphism in the STAT4 gene was strongly associated with the risk of hepatocellular carcinoma (HCC) progression in patients chronically infected with hepatitis C virus (HCV).
Objective
To study the effect of STAT4 gene (rs7574865) polymorphism on HCC in complicating chronic hepatitis C.
Patients and methods
One hundred and fifty individuals were divided into three groups. The studied participants were divided into three groups. Group I: 50 apparently healthy individuals served as controls. They were completely free clinically with normal laboratory investigations, normal abdominal ultrasonography, and no history of liver and renal disease. Group II: 50 chronic HCV patients confirmed by HCV-antibodies and PCR HCV RNA analysis. Group III: 50 HCV-induced HCC patients confirmed by computed tomography and serum alpha-fetoprotein. Most of the patients are males with a mean age of 54 years. Real-time PCR analysis to determine STAT4 (rs7574865) gene polymorphism was performed on DNA extracted from all the studied participants.
Results
Comparing the three studied groups, there was highly significant statistical difference regarding genotype and allele frequency distribution with a GG genotype of 20% in the control group, 46% in the HCV group, and 56% in the HCC group with a P value less than 0.001. GT genotype is 50% in the control group, 44% in the HCV group, and 38% in the HCC group. There were statistically significant differences in STAT4 (rs7574865) allele frequencies between the patient group (HCC and HCV as one group) and the control group (P < 0.001) [GG: odds ratio (OR)=9.562, 95% confidence interval (CI)=3.204–28.541, GT: OR = 3.075, 95% CI = 1.141–8.290]. Allele association study revealed a significant risk association between allele type and cancer development (OR = 3.066, 95% CI = 1.861–5.053).
Conclusion
The results suggested that STAT4 (rs7574865) GG genotype is associated with HCC development in Egyptians with chronic HCV infection.

Keywords: gene polymorphism, hepatitis C virus, hepatocellular carcinoma, real-time PCR, signal transducer and activator of transcription


How to cite this article:
Elashmawy M, Moneim AA, Abo-Raia G, Abo-Zeid M, Diab K. STAT4 gene polymorphism as a risk factor for hepatocellular carcinoma on top of chronic hepatitis C. Menoufia Med J 2022;35:406-11

How to cite this URL:
Elashmawy M, Moneim AA, Abo-Raia G, Abo-Zeid M, Diab K. STAT4 gene polymorphism as a risk factor for hepatocellular carcinoma on top of chronic hepatitis C. Menoufia Med J [serial online] 2022 [cited 2024 Mar 29];35:406-11. Available from: http://www.mmj.eg.net/text.asp?2022/35/2/406/352191




  Introduction Top


Primary liver cancer is the sixth most commonly diagnosed cancer and the fourth leading cause of cancer mortality worldwide, with an estimated 841 000 cases (9.3 cases per 100 000 person-years) and 782 000 deaths (8.5 deaths per 100 000 person-years) in 2018. It is more common in men than in women. Outcomes are poor with an estimated 5-year net survival of 19% (2009–2015) and an average of 19 years of life lost per death in the United States[1].

Hepatitis C virus (HCV) is one of the major etiologic agents that causes hepatocellular carcinoma (HCC) by generating an inflammatory, fibrogenic, and carcinogenic tissue microenvironment in the liver[2].

STAT protein family comprises seven members, including STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6[3].

STAT4 is a latent cytosolic factor that encodes many transcription factors transmitting signals stimulated by cytokines (i.e. interferons, interleukin-12, and interleukin-23)[4].

STAT4 plays a key role in liver inflammation and cancer, which have gained considerable attention[5].

There is an single-nucleotide polymorphism (SNP) (rs7574865 G/T) located in the third intron of the STAT4 gene that is worthy of being noted. Although it is not located in the promoter or 3'-untranslated region, it can influence STAT4 expression[6].

The aim of the study was to study the effect of STAT4 gene (rs7574865) polymorphism on HCC in complicating chronic hepatitis C.


  Patients and methods Top


A total of 150 participants involving 100 patients and 50 healthy controls with matched age and sex were included in this study. They were randomly selected from Menoufia University Hospitals between April 2019 and May 2020. The studied participants were divided into three groups: group I: 50 apparently healthy individuals served as controls, group II: 50 chronic HCV patients confirmed by HCV-antibodies and PCR HCV RNA analysis (may be cirrhotic or not), and group III: 50 HCV-induced HCC patients. The study was approved by the ethics committee of the National Liver Institute, Menoufia University, and written informed consents were obtained from all participants before study entry.

All participants were submitted to history taking and clinical examination, abdominal ultrasound, triphasic computed tomography abdomen, Child–Pugh classification, and laboratory investigations. A measure of 10 ml of venous blood was withdrawn from all those included in this study by clean venipuncture from the cubital vein, and was divided into four aliquots: the first portion (1.8 ml) was added to 200 μl of 3.8% citrate solution for prothrombin time. The measurement of prothrombin time is done by BFT II Analyzer1, which operates according to the optomechanical measuring principle. With this coagulation method, the control value for prothrombin time was 11.5 s that equals to 100% concentration and international normalized ratio of 1. The second and third portions (2 ml each) were added to EDTA for molecular testing and complete blood count (Sysmex Corporation, Kobe, Japan). The fourth portion: the remaining volume was collected in a plain vacutainer without additives and was allowed to clot at 37°C for 30 min and then centrifuged at 3000 rpm for 5 min. The clear supernatant sera were separated and divided into two sterile tubes; the first aliquot was used for the measurement of liver function tests and the second one was used for the measurement of HCV antibodies and alpha-fetoprotein (AFP).

Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, total bilirubin, direct bilirubin, total protein, and albumin of liver function tests were carried out on an autoanalyzer AU 480 (Beckman Coulter, AU Chemical Analyzer, California, United States) according to the manufacturer's protocol.

Measurement of serum AFP was determined by the two-step sandwich solid phase enzyme immunoassay based on electrochemiluminescence immunoassay on a COBAS Elecsys immunoassay analyzer (Elabscience Biotechnology Research Houston, Texas, USA).

Genomic DNA was isolated from a buffy coat using Gentra Pure Blood kit (Qiagen, Hilden, Germany) according to manufacturer's recommendations. The purity and concentration of extracted DNAs were observed with a spectrophotometer (Implen NanoPhotometer N60 UV/VIS Spectrophotometer, München, Germany). The extracted DNA was aliquoted and stored at −80°C. Genotyping of SNP rs1012068 was performed using real-time PCR TaqMan allelic discrimination assay purchased from Applied Biosystems (assay ID: C_27100398_10; Applied Biosystems, Foster City, California, USA). Two allele-specific fluorescent-labeled probes (VIC/VAM) TATGAAAAGTTGGTGACCAAAAT (G/T) AATAGTGGTTATCTTATTTCAGTGG were used to perform the assay using the ABI Prism 7500 Sequence Detection System, according to the manufacturer's protocols (Applied Biosystems) [Figure 1].
Figure 1: Allelic discrimination for STAT4 SNPs (rs7574865) assay sample no. 6, no. 7, and no. 8. The patient was genotyped as homozygous TT. Reaction at the green channel (FAM dye for allele T). No reaction at the yellow channel (VIC dye for allele G). Sample no. 20 was the negative control. SNP, single-nucleotide polymorphism.

Click here to view


Statistical analysis

IBM SPSS software package, version 20.0 (IBM Corp., Armonk, New York, USA) was used for statistical analysis. χ2 test, Monte Carlo, analysis of variance test, Student's t test, and Mann–Whitney tests were used. The significance of the obtained results was judged at the 5% level.


  Results Top


Regarding age, there was no statistically significant difference between groups regarding the sex of individuals in each group; there was no statistically significant difference between groups. The mean age of HCC patients was 54.26 ± 6.79 years. The mean age of patients with HCV was 53.44 ± 8.01 years and that of the control group was 52.58 ± 2.79 years. In this study, 74% of HCC patients were males and 26% were females. In the group of patients with HCV, 54% were males and 46% were females. Among the healthy control group, 86% were males and 14% were females.

Comparing the three studied groups, there was a highly significant statistical difference regarding aspartate aminotransferase, alanine aminotransferase, total bilirubin, direct bilirubin, gamma-glutamyl transferase, AFP, albumin, and total protein [Table 1].
Table 1: Comparison between the three studied groups according to liver function

Click here to view


Comparing the two studied groups (HCV and HCC groups), there was highly significant statistical difference regarding hepatomegaly, encephalapathy, and portal hypertension. There was no statistical difference regarding bleeding, splenomegaly or ascites [Table 2].
Table 2: Comparison between the two studied groups according to different clinical parameters

Click here to view


Comparing the three studied groups: there was highly significant statistical difference regarding genotype and allele frequency distribution with a GG genotype of 20% in the control group, 46% in the HCV group, and 56% in the HCC group with a P value less than 0.001. GT genotype is 50% in the control group, 44% in the HCV group, and 38% in the HCC group [Table 3].
Table 3: Comparison of genotype and allele frequency of STAT4 gene between hepatocellular carcinoma, hepatitis C virus, and control group

Click here to view


Comparing between the control group and (HCC and HCV as one group) according to the genotype and allele frequency distribution, there was highly significant statistical difference regarding genotype and allele frequency, P value less than 0.001 with a GG genotype of 20% in the control group and 51% in the patient group, while the G allele is 45% in the control group and 71% in the patient group [GG: odds ratio (OR)=9.562, 95% confidence interval (CI)=3.204–28.541, GT: OR = 3.075, 95% CI = 1.141–8.290]. The allele association study revealed a significant risk association between allele type and cancer development (OR = 3.066, 95% CI = 1.861–5.053) [Table 4].
Table 4: Comparison between control group and hepatocellular carcinoma and hepatitis C virus as one group according to genotype and allele frequency distribution

Click here to view



  Discussion Top


In Egypt, it is the most common cause of mortality-related and morbidity-related cancer. Many hospital-based studies reported increasing the incidence of HCC. The reason for increased incidence could be attributed to increasing the incidence and complications of HCV, which is the most important risk factor in developing liver cancer including HCC in Egypt[7].

In Egypt, HCC ranks the second and the sixth cancer in men and women, respectively. Biological reason for the variation of HCC incidence in women is explained by the level of estrogen hormone. It partially plays a role in the suppression of interleukin (IL)-6-mediated inflammation that reduces both compensatory proliferation and liver injury[8].

STAT4 is a pivotal mediator in inflammation and tumor development. The balance of IFN-γ activation through STAT4 can affect both antiviral and antitumor activities[9].

STAT4 was reported to be expressed at lower levels in HCC than in normal liver tissues and to be associated with tumor number, tumor size, and severity of HCC. STAT4 may modulate the pathological progression of HCC by inhibiting HCC cell proliferation, growth, and apoptosis, by acting as a tumor suppressor[10].

This work aimed to study the significance of STAT4 gene SNP (rs7574865) as a risk factor for HCC in chronic hepatitis C patients. The present study is the first to study STAT4 gene polymorphism as a risk factor of HCC in chronic hepatitis C patients in Egypt.

The study showed statistically significant difference in the distribution of genotypes and allele frequencies between HCC and hepatitis groups and also between HCC and healthy control groups. This agrees with the results of the study that studied STAT4 as a risk factor for HCC in chronic hepatitis B patients done by Zhang et al.[11].

This study showed that the G allele was 75% in HCC cases and 25% in controls. Similar allele frequency distributions were found in other reports such as Vietnamese Clark et al.[12], and in Korean cohorts as that of Kim et al.[13].

This study showed that the GG genotype was 56% in HCC cases and 20% in controls, so the current study also agreed with the study performed by Chanthra et al.[14], which demonstrated a positive association of rs7574865 with HCC risk when compared with healthy controls under an additive model (GG vs. TT: OR = 2.07, 95% CI = 1.06–4.03). This correlation remained significant under allelic and recessive models (OR = 1.46, 95% CI = 1.09–1.96 and OR = 1.71, 95% CI = 1.13–2.59, respectively).

Therefore, we can consider that the G allele in this SNP as a risk allele for HCC development, and also significantly associated with lower mRNA levels of STAT4 in HCC tissues compared with adjacent nontumor tissues, which is similar to the results obtained by Zhao et al.[15] meta-analysis, which suggested a significantly reduced risk of HBV-induced HCC associated with rs7574865 T allele in Asian populations.

The present study showed a significant statistical difference between different genotypes in chronic hepatitis C patient groups: the GG genotype is 46% and TT is 10% in agreement with El Sharkawy et al.[16], who demonstrated that the STAT4 rs7574865 genotype was associated with hepatic inflammation (OR = 1.42, 95% CI = 1.07–2.06) and advanced fibrosis (OR = 1.83, 95% CI = 1.19–2.83).

This study demonstrates that the GG genotype at rs7574865 in the STAT4 gene confers susceptibility to more severe liver injury, fibrosis, and tumors, which can be explained by the fact that those with the risk GG genotype have lower STAT4 mRNA expression in the liver and NK cells, so their NK cells display decreased phosphorylated STAT4 upon stimulation with IL-12 and IL-18, with consequent functional deficiency in STAT4-dependent IFN-γ production causing decreased HCV clearance, increased HCV persistence, increased liver fibrosis, and increased incidence of liver tumor development.

Finally, proving these points was the aim of our study and we hope to stimulate other research toward different STAT4 polymorphisms or even other genetic factors, which could lead to the discovery of markers with more impact and better diagnostic potential. Further association analysis and review are needed to pinpoint such genetic variants, potentially revealing data that could be useful for diagnostic procedures.


  Conclusion Top


The results suggested that STAT4 (rs7574865) GG genotype is associated with HCC development in Egyptians with chronic HCV infection. We can consider that the G allele in this SNP is a risk allele for HCC development.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Dasgupta P, Henshaw C, Youlden DR, Aitken JF, Sullivan A, Irving H, et al. Global trends in incidence rates of childhood liver cancers: a systematic review and meta-analysis. Paediatr Perinat Epidemiol 2020; 34:609–617.  Back to cited text no. 1
    
2.
Yang M, Parikh ND, Liu H, Wu E, Rao H, Feng B, et al. Incidence and risk factors of hepatocellular carcinoma in patients with hepatitis C in China and the United States. Sci Rep. 2020; 10:1–10.  Back to cited text no. 2
    
3.
Subramaniam A, Shanmugam MK, Perumal E. Potential role of signal transducer and activator of transcription (STAT) 3 signaling pathway in inflammation, survival, proliferation and invasion of hepatocellular carcinoma. Biochim Biophys Acta 2013; 1835:46–60.  Back to cited text no. 3
    
4.
Lamana A, López-Santalla M, Castillo-González R. The minor allele of rs7574865 in the STAT4 gene is associated with increased mrna and protein expression. PLoS One 2015; 10:e0142683.  Back to cited text no. 4
    
5.
Wang Y, Qu A, Wang H. Signal transducer and activator of transcription 4 in liver diseases. Int J Biol Sci 2015; 11:448–455.  Back to cited text no. 5
    
6.
Shi H, He H, Ojha SC. Association of STAT3 and STAT4 polymorphisms with susceptibility to chronic hepatitis B virus infection and risk of hepatocellular carcinoma: a meta-analysis. Biosci Rep 2019; 39:BSR20190783.  Back to cited text no. 6
    
7.
Rashed WM, Kandeil MA, Mahmoud MO, Ezzat S. Hepatocellular carcinoma (HCC) in Egypt: a comprehensive overview. J Egypt Natl Canc Inst 2020; 32:1–11.  Back to cited text no. 7
    
8.
Abd-Elsalam S, Elwan N, Soliman H, Ziada D, Elkhalawany W, Salama M, et al. Epidemiology of liver cancer in Nile delta over a decade: a single-center study. South Asian J Cancer 2018; 7:24.  Back to cited text no. 8
    
9.
Yang C, Mai H, Peng J, Zhou B, Hou J, Jiang D. STAT4: an immunoregulator contributing to diverse human diseases. Int J Biol Sci 2020; 16:1575.  Back to cited text no. 9
    
10.
Nguyen HN, Noss EH, Mizoguchi F, Huppertz C, Wei KS, Watts GF, et al. Autocrine loop involving IL-6 family member LIF, LIF receptor, and STAT4 drives sustained fibroblast production of inflammatory mediators. Immunity 2017; 46:220–232.  Back to cited text no. 10
    
11.
Zhang L, Xu K, Liu C, Chen J. Meta-analysis reveals an association between signal transducer and activator of transcription-4 polymorphism and hepatocellular carcinoma risk. Hepatol Res 2017; 47:303–311.  Back to cited text no. 11
    
12.
Clark A, Gerlach F, Van Tong H, Hoan NX, Song LH, Toan NL, et al. A trivial role of STAT4 variant in chronic hepatitis B induced hepatocellular carcinoma. Infect Genet Evol 2013; 18:257–261.  Back to cited text no. 12
    
13.
Kim LH, Cheong HS, Namgoong S, Kim JO, Kim JH, Park BL, et al. Replication of genome wide association studies on hepatocellular carcinoma susceptibility loci of STAT4 and HLA-DQ in a Korean population. Infect Genet Evol 2015; 33:72–76.  Back to cited text no. 13
    
14.
Chanthra N, Payungporn S, Chuaypen N, Piratanantatavorn K, Pinjaroen N, Poovorawan Y, et al. Single nucleotide polymorphisms in STAT3 and STAT4 and risk of hepatocellular carcinoma in Thai patients with chronic hepatitis B. Asian Pac J Cancer Prev 2016; 16:8405–8410.  Back to cited text no. 14
    
15.
Zhao X, Jiang K, Liang B, Huang X. STAT4 gene polymorphism and risk of chronic hepatitis B-induced hepatocellular carcinoma. Cell Biochem Biophys 2015; 71:353–357.  Back to cited text no. 15
    
16.
El Sharkawy R, Thabet K, Lampertico P, Petta S, Mangia A, Berg T, et al. A STAT 4 variant increases liver fibrosis risk in Caucasian patients with chronic hepatitis B. Aliment Pharmacol Ther 2018; 48:564–573.  Back to cited text no. 16
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
Abstract
Introduction
Patients and methods
Results
Discussion
Conclusion
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed592    
    Printed16    
    Emailed0    
    PDF Downloaded89    
    Comments [Add]    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]