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ORIGINAL ARTICLE
Year : 2019  |  Volume : 32  |  Issue : 1  |  Page : 255-260

Association of HLA-G gene polymorphism with hepatocellular carcinoma in Egyptian population


1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Clinical Pathology, National Liver Institute, Menoufia University, Menoufia, Egypt

Date of Submission20-Apr-2017
Date of Acceptance18-Jun-2017
Date of Web Publication17-Apr-2019

Correspondence Address:
Mai I Elashmawy
Department of Clinical Pathology, National Liver Institute, Menoufia University, Shebein El Kom, Menoufia Governorate
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_293_17

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  Abstract 


Objective
The aim of this work was to study the association of human leukocyte antigen-G (HLA-G) gene polymorphism with hepatocellular carcinoma (HCC) in Egyptian population.
Background
HCC represents an international public health concern as one of the most common and deadly cancers worldwide. It is the third most common cause of cancer-related death worldwide. In Egypt, HCC is the second most common cancer among men and the sixth most common cancer among women. In most cases, HCC develops within an established background of chronic liver disease (70–90% of all patients). The worldwide heterogeneous incidence reflects variations in the main risk factors, which include cirrhosis, chronic infection of hepatitis B virus and hepatitis C virus, aflatoxin, exposure to pesticide, and genetic host factors.
Patients and methods
This study was conducted on 100 participants: 40 patients with HCC, 40 patients with chronic hepatitis C with no radiological evidence of HCC who presented to the Hepatology Department, National Liver Institute, Menoufia University, and 20 age-matched and sex-matched healthy control group during the period between February 2015 and February 2016. HLA-G gene polymorphism was determined using PCR.
Results
Our study revealed no statistical difference between patients with HCC and those with chronic hepatitis C, nor between patients with HCC and the control group as regards HLA-G gene polymorphism.
Conclusion
HLA-G gene polymorphism is not associated with an increased risk for HCC development.

Keywords: chronic hepatitis C, hepatocellular carcinoma, HLA-G gene, PCR, polymorphism


How to cite this article:
El Bassiouny MA, Elshaarawy AA, Tawfeek GA, Elashmawy MI. Association of HLA-G gene polymorphism with hepatocellular carcinoma in Egyptian population. Menoufia Med J 2019;32:255-60

How to cite this URL:
El Bassiouny MA, Elshaarawy AA, Tawfeek GA, Elashmawy MI. Association of HLA-G gene polymorphism with hepatocellular carcinoma in Egyptian population. Menoufia Med J [serial online] 2019 [cited 2024 Mar 29];32:255-60. Available from: http://www.mmj.eg.net/text.asp?2019/32/1/255/256097




  Introduction Top


Hepatocellular carcinoma (HCC) is considered as a global problem. In Egypt it is one of the health problems that face the health authorities [1]. It represents the second most common cancer among men and the sixth most common cancer among women [2].

Several risk factors of HCC have been identified, including chronic infection of hepatitis B virus and hepatitis C virus (HCV). In Egypt, HCV is the main risk factor for HCC [3]. Nevertheless, only a fraction of infected patients develop HCC during their lifetime, suggesting that genetic factors might modulate HCC development. Consequently, identification of additional contributing factors including genetic factors could help to select high-risk populations [4]. Continuous inflammation and hepatocyte regeneration in chronic hepatitis and subsequent progression to cirrhosis lead to chromosomal damage and initiate hepatocarcinogenesis [5].

Evasion from immune destruction has been characterized as an emerging hallmark of cancer, which could contribute to the development and progression of human malignancies [6].

Human leukocyte antigen-G (HLA-G) can interact with immunoglobulin-like transcript-2, which is expressed by T and B lymphocytes, natural killer cells, monocytes/macrophages, and dendritic cells, and with immunoglobulin-like transcript-4, which is expressed only by myeloid cells (i.e., dendritic cells, monocytes, and macrophages and neutrophils) [7].

HLA-G impairs T-cell function, by inhibiting proliferation and cytotoxicity and by inducing apoptosis and expansion of regulatory T-cells; in addition, it inhibits differentiation, proliferation, and cytokine production in B lymphocytes [8].

It was found that the expression of HLA-G is generally detected in tumors but not in normal tissues, and such expression positively correlated with tumor progression, metastasis, invasiveness, and worse prognosis of patients [9].

Both HLA-G mRNA and protein are detectable in human HCC cell lines [10].

The 14 base pair (bp) insertion/deletion (in/del) polymorphism of the 3' untranslated region of the HLA-G is known to regulate HLA-G expression. This led to studies of this polymorphism in HCC patients [11].


  Patients and Methods Top


This study was conducted on 100 participants: 40 patients with HCC, 40 patients with chronic hepatitis C with no radiological evidence of HCC, and 20 apparently healthy individuals as a control group. The three groups were age and sex matched.

The study protocol was approved by the local ethics committee of the Menoufia University. Informed consent was taken from both patients and controls before the beginning of the study.

Collection of blood samples

A volume of 10 ml of venous blood was drawn from all participants included in this study by means of clean venipuncture from the cubital vein and was divided into four aliquots:

  1. The first part (1.8 ml) was collected in vacutainer tubes containing 200 μl of 3.8% citrate solution for prothrombin time (PT) assay
  2. The second part (2 ml) was collected in vacutainer tubes containing EDTA for molecular testing of polymorphism using PCR
  3. The third part (2 ml) was collected in vacutainer tubes containing EDTA for complete blood count
  4. The fourth part, 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 measurement of liver function tests and the second one was used for measurement of HCV-antibody and α-fetoprotein (AFP).


For all participants, the following were carried out: collection of relevant clinical data and basic laboratory tests including the following:

  1. Complete blood count (Sysmex XT 1800; Sysmex, Kobe, Japan)
  2. Liver function tests (Integra 400 auto analyzer; Roche Diagnostics, Firmenstandort: Sysmex Deutschland GmbH, Aspelohe, Norderstedt, Germany)
  3. AFP (COBAS Elecsys immunoassay analyzer; Roche diagnostics)
  4. PT (Coagulometer CA-1500; Siemens AG, Wittelsbacherplatz 2, Munich, Germany)
  5. Hepatitis viral markers (hepatitis B surface antigen and HCV-antibody) (COBAS Elecsys immunoassay analyzer, Roche Diagnostics)
  6. Molecular testing for HLA-G gene polymorphism using PCR. Total DNA was extracted from EDTA-treated blood sample using Thermo Scientific Gene JET Genomic DNA Purification Kit (Thermo Fisher Scientific, Massachusetts, Waltham, MA USA).


The 14-bp fragment encompassing the polymorphic site in HLA-G gene region was amplified using the provided master mix (Thermo Fisher Scientific) and we used two outer primers (FO: 5'-GTGCTATGAGGTTTCTTTGACTTCA ATG-3'; RO: 5'-GGAATCTTCTCCTTTAAT TAACCCATC-3') and two specific primers [FI (insertion allele): 5'-GACTGAGTGGCAAGTATTTGT TCATG-3'; RI (deletion allele): 5'-GTCACAAAGGGACTTGCCACTC-3'] according to Eskandari-Nasab et al. [12].

The PCR amplification was performed with a total volume of 25 μl using the following:

  1. 12.5 μl of a ready for use master mix
  2. 1.5 μl of each forward primer (0.5 μmol)
  3. 1.5 μl of each reverse primer (0.5 μmol)
  4. 5.5 μl of template DNA (100–500 ng)
  5. 1 μl of sterile deionized water to reach a final volume of 25 μl.


The PCR amplification was performed on preprogrammed thermal cycler (Perkin Elmer GeneAmp PCR System 2400 Thermal Cycler, Midland, ON, Canada) under the following conditions: an initial denaturation step at 95°C for 5 min, followed by 30 cycles, with each cycle consisting of denaturation at 95°C for 30 s, annealing at 58°C for 25 s, and extension at 72°C for 30 s. Final extension at 72°C for 10 min was carried out. A volume of 5 μl of normal range (100 bp) ladder and 10 μl of PCR product were applied. The amplified PCR products were detected using agarose gel electrophoresis with 2% agarose. The gel was visualized on a 302 nm ultraviolet transillumination for three genotypes: ins/ins) genotype was detected by the presence of two bands of 436 and 350 bp, del/del genotype was detected by the presence of two bands of 436 and 158 bp, and in/del was detected by presence of three bands of 436, 350, and 158 bp [Figure 1].
Figure 1: A representative agarose gel picture of the PCR products of the 14 bp insertion/deletion (ins/del) of HLA-G gene. Lanes 2, 3, 4, 6, 7, 8: ins/del; lane 5: ins/ins; lane 9: del/del.

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Statistical analysis

Results were collected, tabulated, and statistically analyzed using statistical package SPSS, version 20 (IBM Corp, Armonk, New York, USA).


  Results Top


The present study showed no significant difference in distribution of the HLA-G genotypes and allele frequencies between the HCC and chronic hepatitis C groups and between the HCC and healthy control groups (P > 0.05) [Table 1].
Table 1: Comparison between the different studied groups according to HLA-G genotype and allele frequency distribution

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The study showed a highly significant statistical difference as regards PT, prothrombin concentration, aspartate aminotransferase (ALT), alanine aminotransferase, and albumin between the three studied groups (P < 0.001) [Table 2].
Table 2: Comparison between the three studied groups according to liver function

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On comparing the three studied groups, there was a significant statistical difference as regards encephalopathy and hepatomegaly, whereas there was no statistical difference as regards bleeding, portal hypertension, splenomegaly, and ascites [Table 3].
Table 3: Comparison between the two studied groups according to different clinical parameters

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On comparing the three studied groups, there was a highly significant statistical difference as regards platelet count, red blood cells, hemoglobin, and hematocrit (P < 0.001). However, there was no significant statistical difference in white blood cells (P > 0.05) [Table 4].
Table 4: Comparison between the three studied groups according to complete blood count

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On studying the relation between HLA-G genotypes and liver functions in each group, no statistical significance was found (P > 0.05) [Table 5].
Table 5: Relation between human leukocyte antigen-G genotypes and liver functions in each group

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  Discussion Top


This work aimed to study the association of HLA-G gene polymorphism with HCC in Egyptian population.

Considering HLA-G gene polymorphism, the present study showed statistically nonsignificant difference in the distribution of HLA-G genotypes and allele frequencies between the HCC and chronic hepatitis C groups and between the HCC and healthy control groups. This is in agreement with the results of the study by Zhang and Wang [13].

This study showed that the 14 bp deletion allele was 55% in HCC cases and 50% in controls, and hence the current study also agreed with the study performed by Coelho et al. [14].

The study by Jiang et al. [15] showed a greater frequency of 14 bp del/del (58.8%) compared with ins/ins (5.7%) and ins/del (35.5%) in HCC cases, whereas this study showed that the frequency of 14 bp in/del (50%) was greater than del/del (30%) and ins/ins (20%) in HCC cases.

A meta-analysis performed by Kim et al. [16] documented that the 14-bp ins/del polymorphism probably has no role in chronic hepatitis or HCC.

This study showed that international normalized ratio, alanine aminotransferase, ALT, alkaline phosphatase, γ-glutamyl transferase, total bilirubin, direct bilirubin, and AFP were significantly higher in HCC patients compared with the control group. However, hemoglobin, platelets, total protein, and albumin were significantly lower in HCC patients compared with controls; similar results were obtained by El-Garem et al. [17].

This study found a significant statistical increase in AFP and ALT among patients with HCC compared with patients with chronic hepatitis C. Akkiz [18] agreed with these results of the present study.


  Conclusion Top


In conclusion, this study demonstrates that the 14-bp HLA-G gene polymorphism probably has no significant association with HCC or chronic hepatitis C in Egyptian population, and hence it is not associated with an increased risk for HCC development, which stimulates other research toward different HLA-G polymorphisms or even other genetic factors, which could lead to the discovery of markers with more impact and better diagnostic potential.

Acknowledgements

The study is partially funded by National Liver Institute, Menoufia University.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Tilburgs T, Crespo AC, van Der Zwan A, Rybalov B, Raj T, Strangerd B, et al. Human HLA-G extravillous trophoblasts: immune-activating cells that interact with decidual leukocytes. Proc Natl Acad Sci USA 2015; 112:7219–7224.  Back to cited text no. 8
    
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König L, Kasimir-Bauer S, Hoffmann O. The prognostic impact of soluble and vesicular HLA-G and its relationship to circulating tumor cells in neoadjuvant treated breast cancer patients. Hum Immunol 2016; 77:791–799.  Back to cited text no. 9
    
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Zhang S, Wang HT. Association between HLA-G 14-bp insertion/deletion polymorphism and cancer risk: a meta-analysis. JBUON 2014; 19:567–572.  Back to cited text no. 13
    
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Coelho AV, Moura RR, Crovella S, Celsi F. HLA-G genetic variants and hepatocellular carcinoma: a meta-analysis. Genetics and molecular research: GMR. 2016; 15(3).  Back to cited text no. 14
    
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Jiang Y, Chen S, Jia S, Zhu Z, Gao X, Dong D, et al. Association of HLA-G 3' UTR 14-bp insertion/deletion polymorphism with hepatocellular carcinoma susceptibility in a Chinese population. DNA Cell Biol 2011; 30:1027–1032.  Back to cited text no. 15
    
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Kim HY, Kim M, Kim Y, Choi JY, Jung SW, Kim DG, et al. Diagnostic value of AFP-L3 and PIVKA-II in hepatocellular carcinoma according to total-AFP. World J Gastroenterol 2013; 19:339–346.  Back to cited text no. 16
    
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Akkiz H. Hepatocellular carcinoma pathogenesis: does peroxisome proliferation-activated receptors (PPAR) alpha polymorphism has a role in hepatocarcinogenesis associated with HBV and HCV infections ? Turk J Gastroenterol 2008; 19:231–233.  Back to cited text no. 18
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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