|Year : 2014 | Volume
| Issue : 2 | Page : 372-378
Cytotoxic T-lymphocyte antigen-4 gene polymorphisms in hepatocellular carcinoma patients in Egypt
Hala H El-Said1, Naglaa M Ghanayem2, Eman A Badr2, Ashraf Y El-Fert1, Ahmed K Gaballah1
1 Department of Clinical Biochemistry, National Liver Institute, Menoufia University, Menoufia, Egypt
2 Department of Medical Biochemistry, Faculty of Medicine, Menoufia University, Menoufia, Egypt
|Date of Submission||04-Jun-2014|
|Date of Acceptance||04-Jun-2014|
|Date of Web Publication||26-Sep-2014|
Ahmed K Gaballah
MBBCh, Yassin Abdel-Ghaffar St., National Liver Institute, Menoufia University, Shebin El-kom, Menoufia
Source of Support: None, Conflict of Interest: None
Our objective is to examine the genotypes at rs231775 in the cytotoxic T-lymphocyte antigen 4 gene (CTLA4) in an Egyptian population to detect the association between this single-nucleotide polymorphism (SNP) and susceptibility to hepatocellular carcinoma (HCC).
CTLA-4 is an important negative regulator in immune response. Its polymorphism +49G>A (dbSNP: rs231775) has been linked to an increased risk of T-cell-mediated autoimmune diseases, infectious diseases, and even carcinomas.
This study included 30 HCC patients (26 men and four women) and 20 healthy controls (18 men and two women). Laboratory investigations including complete blood picture, liver function tests, serum a-fetoprotein, and hepatitis viral markers (HBsAg, anti-HCV-Ab) were performed for all participants. The CTLA4 polymorphism at rs231775 was genotyped using the TaqMan allelic discrimination Assay technique.
The data showed a higher frequency of the A/A genotype (40 vs. 10%) and the A allele (55 vs. 27.5%) in patient groups (HCC) compared with the healthy controls. Our results also indicated a statistically significant difference in CTLA4 rs231775 between HCC patients and healthy controls in the AA genotype and the A allele (P = 0.04, 0.012), respectively.
Our results suggested that the A/A genotype and the A allele of rs231775 increase the risk of developing HCC in an Egyptian population, whereas the G allele appeared to have a protective effect in developing HCC.
Keywords: CTLA4, genotype, HCC, polymorphism
|How to cite this article:|
El-Said HH, Ghanayem NM, Badr EA, El-Fert AY, Gaballah AK. Cytotoxic T-lymphocyte antigen-4 gene polymorphisms in hepatocellular carcinoma patients in Egypt. Menoufia Med J 2014;27:372-8
|How to cite this URL:|
El-Said HH, Ghanayem NM, Badr EA, El-Fert AY, Gaballah AK. Cytotoxic T-lymphocyte antigen-4 gene polymorphisms in hepatocellular carcinoma patients in Egypt. Menoufia Med J [serial online] 2014 [cited 2019 Nov 19];27:372-8. Available from: http://www.mmj.eg.net/text.asp?2014/27/2/372/141711
| Introduction|| |
Hepatocellular carcinoma (HCC) ranks fifth in the frequency of cancers worldwide and is the third most common cause of cancer death . It is usually asymptomatic in the early stages and tends to be invasive. Therefore, most patients present with an incurable disease at the time of detection, which makes early diagnosis of HCC critical for a good prognosis. Surgical resection remains the treatment of choice for these tumors, but unfortunately, only 10-20% of primary HCCs are resectable at the time of diagnosis. Continuous researches are ongoing worldwide to determine and evaluate sensitive and specific new markers for the diagnosis of HCC .
The most common cause of HCC is chronic hepatitis or liver cirrhosis caused by hepatitis C virus (HCV) and hepatitis B virus (HBV) infection. Therefore, early detection of patients at risk, such as chronic carriers of HBV and HCV, is justified to improve the outcome of treatment of hepatocellular malignancy ,.
In Egypt, the incidence rate of HCC has increased markedly in the last decade . This could be because of HBV and HCV epidemic infections. Furthermore, improvements in diagnostic tools and healthcare led to increased survival rates among cirrhotic patients, allowing time for some of them to develop HCC . However, not all the individuals infected with HBV/HCV develop HCC, indicating the implication of other environmental and genetic risk factors in the multistage process of this complex disease .
T cells play an important role in immune response, and molecules that mediate the regulation of T-cell activity could influence susceptibility to cancer. Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is one of the most studied negative regulators of T cells. The molecule is homologous to CD28, but with the opposite effect, thereby suppressing antitumor immunity and increasing susceptibility to cancer [8,9].
The inhibitory role of CTLA-4 is executed through multiple mechanisms such as competitive binding with CD28 for B7 on the antigen-presenting cells, reducing both interleukin (IL)-2 and IL-2 receptor productions and arresting T cells at the G1 phase in the cell cycle [10-12].
Furthermore, antibody-mediated blockage of CTLA-4 in vivo has been shown to mediate cancer regression in patients with metastatic melanoma and renal cancer as a result of increased T-cell activation ,. These findings suggest that CTLA-4 may play an important role in cancer development and progression ,.
The CTLA4 gene has been mapped to human chromosome 2q33  and consists of four exons that encode a leader sequence, and extracellular, transmembrane, and cytoplasmic domains ,. Two main human CTLA-4 transcripts have been detected: a full-length isoform and a soluble form lacking exon 3 ,.
Various single-nucleotide polymorphisms (SNPs) in the CTLA4 gene have been implicated in susceptibility to autoimmune disorders including Graves' disease , type 1 diabetes mellitus , celiac disease , Addison's disease , autoimmune thyroid disease ,, systemic lupus erythrematosus , and rheumatoid arthritis . Other studies also correlated polymorphisms in the CTLA4 gene with neoplastic diseases, such as breast cancer , non-Hodgkin's lymphoma  as well as squamous cell carcinoma , and infections, such as hepatitis B . Therefore, the CTLA4 gene is a potential cancer susceptibility gene.
| Participants and methods|| |
This study was carried out on 50 individuals including 30 diagnosed untreated HCC patients. They presented to the Hepatology Department, National Liver Institute, Menoufia University, in the period from October 2011 to April 2012. The diagnosis was made on the basis of clinical examination, laboratory tests, ultrasound, and computed tomography; in addition, a control group of 20 age-matched and sex-matched individuals was studied.
All patients and control groups were subjected to the following:
(1) Full assessment of history.
(2) Complete clinical examination.
(3) Abdominal ultrasonography and/or computed tomography.
(4) Laboratory investigations were performed including the following:
(a) Complete blood picture.
(b) Liver function tests:
Aspartate transaminase (AST), alanine transaminase (ALT) , serum bilirubin, serum albumin (Alb), serum alkaline phosphatase (ALP), serum gamma-glutamyl transferase (GGT), serum total proteins, and prothrombin time and concentration (PT and PC%) were also determined.
- Viral markers were assessed (HBsAg, HCV-Ab) .
- Estimation of serum a-fetoprotein (AFP) was performed .
- Genotyping of CTLA4 +49 A/G SNP (rs231775) was performed.
A written consent was obtained from each individual and the protocol was approved by the ethical committee of Menoufia Faculty of Medicine.
A volume of 10 ml of venous blood was collected from all participants included in this study by venipuncture from the cubital vein and was collected as follows: 4 ml was collected into EDTA-containing tubes for complete blood count and genotyping of the CTLA4 gene. A volume of 2 ml was collected into a citrated tube for PT and PC. The remaining 4 ml was collected in another vacutainer tube (with no additives), left to coagulate for 15 min, and then centrifuged at 3000 rpm for 10 min; then, the sera were separated into aliquots for the measurement of liver function tests, viral markers (HBsAg, anti-HCV-Ab), and AFP.
DNA extraction and genotyping
Blood samples were collected in EDTA-containing tubes. Genomic DNA was extracted from whole blood using the Gene JET Whole Blood Genomic DNA Purification Mini Kit (Thermo Scientific, Vilnius, Lithuania).
CTLA4 rs231775 was genotyped using the TaqMan allelic discrimination Assay technique that detects variants of a single nucleic acid sequence. The presence of two primer/probe pairs in each reaction allows genotyping of the two possible variants at the SNP site in a target template sequence. The actual quantity of target sequence is not determined. The allelic discrimination assay classifies unknown samples as follows:
- Homozygotes (samples with only allele 1 or allele 2).
- Heterozygotes (samples with both allele 1 and allele 2).
Using the Maxima Probe qPCR Master Mix (2×), purchased from Thermo scientific, primers and probes were purchased from BIONEER. According to Hu et al. : the forward primer was 5Ͳ-CCTGAACACCGCTCCCAT-3Ͳ, and the reverse primer was 5Ͳ-GCTCCAAAAGTCTCACTCACCT-3Ͳ. Probes: allele A, 5Ͳ-FAM-AGCTGAACCTGGCTACCAGGACCT-BHQ-3Ͳ, and allele G, 5Ͳ-HEX-CTGAACCTGGCTGCCAGGACCT-BHQ-3Ͳ.
The genotyping reaction mix was prepared by mixing 12.5 ml master mix, 0.3 ml of each primer, 1.2 ml of each probe, and 4.5 ml of DNAse-free water. For each unknown reaction, 5 ml (0.1 mg/ml) of genomic DNA template was added and for the negative control reaction, 5 ml of DNAse-free water was added.
The cycling parameters were set as follows: initial denaturation step at 94°C for 4 min, 50 cycles of denaturation at 94°C for 30 s, annealing/collection at 50°C for 25 s, and extension at 72°C for 40 s, and a final extension step at 72°C for 3 min using Line gene 9660 (Bioer Technology Co. Ltd, Tokyo, Japan).
Data were statistically analyzed using the statistical package for social science program, version 13 for windows for all the analyses (Kirkwoodm, 1990). P-value less than 0.05 was considered statistically significant.
| Results|| |
[Table 1] shows that all the studied groups were homogenous in terms of age and sex as there was no statistically significant difference between the HCC and the control groups.
[Table 2] shows a highly statistically significant difference between the HCC and the control group in all the studied parameters, with a significant increase in ALT, AST, ALP, GGT, total bilirubin, and AFP in the HCC group compared with the control group; in contrast, there was a significant decrease in total protein, Alb, PC, platelets, and Hb in the HCC group.
|Table 2: Differences between HCC and control groups in all the variables studied|
Click here to view
[Table 3] shows the allele and genotype distribution of CTLA4 (rs231775) in the studied groups. The G allele frequencies of the control group were higher than that of the HCC group (72.5 vs. 45%). The data also showed that the frequency of the A/A genotype was higher in the HCC patients compared with the healthy controls (40 vs. 10%).
In [Table 4], a statistically significant difference in rs231775 was found between the HCC patients and the healthy controls in the AA genotype and the A allele (P = 0.04, 0.012), respectively. The data also showed that the frequencies of the A/A genotype and the A allele were higher in the HCC patients compared with the healthy controls (40 vs. 10%) and (55 vs. 27.5%), respectively.
|Table 4: Comparison between the HCC and control groups in CTLA4 genotypes and alleles|
Click here to view
In [Table 5], no statistically significant difference was found between CTLA4 genotypes and sex, ascitis, spleen, nodes, metastasis, focal lesion, and BCLC stage in HCC patients.
|Table 5: Comparison between CTLA4 genotypes in sex, ascitis, spleen, nodes, metastasis, focal lesion, and stage in the HCC group|
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[Table 6] shows a nonstatistically significant difference between CTLA4 genotypes and outcome in HCC patients.
|Table 6: Comparison between CTLA4 genotypes and outcome in the HCC group|
Click here to view
| Discussion|| |
The present study was carried out on 30 HCC patients recruited from the hepatology department and outpatients' clinic from the National Liver Institute in Menoufia University during the period from October 2011 to April 2012. The mean age of the HCC patients in this study was almost 44 years.
Sherman  reported that the incidence of HCC increases in patients older than 45 years of age. Similarly, EL-Serag  reported that the greatest proportional increase in cases of HCC has been observed between 45 and 60 years of age. Also, Bosch et al.  reported that liver cancer incidence rates increase after 20 years of age and peak at about 50 years of age. A center study over a decade carried out by El-Zayadi et al.  showed that the most predominant age group is between 40 and 59 years.
However, Leerapun et al.  reported a higher mean reaching 60 years of age.
The peak age tends to be related inversely to the frequency of the tumor; thus, in western countries (low-incidence regions), the median age at diagnosis is 65 years. However, in Asia and Africa (high-incidence areas), the age at diagnosis is considerably less, occurring in the fourth and fifth decades of life, respectively . This may be attributed to the emergence of HCV infection as well as the acquisition of both HBV and HCV infection at a younger age .
The present study shows that HCC was more predominant in men than women. The ratio was ~6 : 1.
This result was in agreement with the most recent World Health report (WHO) that indicated a total of 714 600 new cases of HCC worldwide, with 71% among men. Yeh and Chen (42); Kuske et al. (43); and EL-Serag (44) have also reported that male sex is an important risk factor for HCC.
This could be explained by differences in exposure to risk factors. In addition, sex hormones and other X-linked genetic factors may also be important . It has been speculated that men have a higher incidence of HCC than women because of the stimulatory effects of androgen and the protective effects of estrogen. This anticarcinogenic effect is probably because of estrogen-mediated inhibition of IL-6 production, a multifunctional cytokine largely responsible for the hepatic response to infections or systemic inflammation, leading to a reduction in the risk of HCC in women ,,.
From the hematological tests in the current study, platelet count, PC (%), and hemoglobin were significantly lower in HCC and cirrhotic patients compared with the control group.
This was in agreement with Franca et al. , who reported that there are various theories about thrombocytopenia in chronic liver diseases. These theories include decreased thrombopoietin levels, splenic sequestration of platelets because of portal hypertension, auto-antibody destruction of platelets, and bone marrow suppression because of underlying liver disease. They also attributed the decrease in PC and the increase in international normalized ratio to the decreased production of tissue factor, factor VII, and vitamin K-dependent factors, which are synthesized by hepatocytes, and coagulation factors in the common pathway (prothrombin, factors V and X, and fibrinogen). The associated anemia likely results from increased portal pressure generated from the resulting cirrhosis, which leads to a relative hypersplenism .
The liver biochemical profile, ALT, AST, GGT, and ALP, was significantly higher in HCC patients compared with the control group. In addition, serum Alb levels were significantly lower in the HCC group compared with the control group.
Our results are in agreement with those of Awadallah et al. , who reported a significant deterioration in the liver function in the HCC group compared with the control group.
The present study also found significantly higher levels of AFP in HCC patients compared with the control group.
This was in agreement with Spadaro et al. (49) and Anwar et al. , who reported a significant increase in serum AFP in the HCC group compared with the control group.
Increasing production of AFP in HCC patients is most probably because of certain genes that are reactivated as a result of malignant transformation of cells, but the explanation of the association of AFP with tumor progression is still being investigated. However, in the last decade, it was found that AFP, which is excreted into the circulation, can promote cell growth and has been defined as a growth regulator in oncogenic growth and tumor progression. Recently, it was reported that intracellular AFP may function as a signal molecule through binding key proteins involved in growth or apoptosis signal pathways .
In terms of the genotyping results, in this study, it was found that the frequency of the AA variant genotype of CTLA4 (rs231775) was higher in HCC patients compared with the healthy controls (40 vs. 10%).
This was in agreement with Hu et al. , who reported that the frequency of the AA genotype was higher in HCC patients compared with the healthy controls (12.4 vs. 9.3%).
Also, the results of Gu et al.  were in agreement with those of the current study as they reported that the AA genotype frequency was increased in HCC patients compared with the healthy participants (14 vs. 11%).
The results of Liu et al.  are in agreement with the results of this work as they reported that the AA genotype was increased in HCC patients (59%) compared with the control group (38.2%).
The genotyping results of this study show that the frequency of GG carriers was higher among the healthy controls compared with HCC patients (55 vs. 30%).
This was in agreement with Hu et al. , who reported that the frequency of the GG genotype was higher among the healthy controls compared with the HCC patients (46.7 vs. 43%).
Similarly, Gu et al.  obtained results that were in agreement with those of the current study as they reported that the GG genotype frequency was predominant in the control group compared with the HCC group (45 vs. 41%).
Also, Liu and Lu  reported a higher frequency of the GG variant in the control group compared with the HCC patients (61.8 vs. 40.3%), respectively.
In terms of the allele distribution in this study, the results showed that the A allele was increased in the HCC group compared with the control group (55 vs. 27.5%), whereas the G allele was the predominant allele in the control group compared with the HCC group (72.5 vs. 45%).
In agreement with the current study, Hu et al.  showed that the frequency of the A allele was increased in the HCC group compared with the control group (34.7 vs. 31.2%), whereas the G allele was higher in the control group (68.8 vs. 65.3%).
The results of Gu et al.  were also in agreement with these results as they observed a higher frequency of the A allele in the HCC group compared with the control group (37 vs. 33%), whereas the G allele was the predominant allele in the control group (67 vs. 63%).
A study carried out by Liu and Lu  on 155 HCC patients reported an increased frequency of the A allele in the HCC group compared with the control group (44.6 vs. 28.8%), whereas in control participants, the G allele was the predominant allele (71.2 vs. 55.4%).
In this study, a statistically significant difference in CTLA4 rs231775 was found between HCC patients and healthy controls in the AA genotype and the A allele (P = 0.04, 0.012), respectively.
These findings are in agreement with the results of a recent study carried out by Hu et al.  on 853 HCC patients; they reported a significant difference between the HCC group and the control group in the AA genotype (P = 0.022).
Similarly, a study carried out by Liu and Lu  on 155 HCC patients and 165 healthy controls found results that were in agreement with those of this study; they found a significant difference in the A allele and the AA genotype between the patient and the control group (P < 0.05).
The results of the study carried out by Gu et al.  on 375 HCC patients confirmed these results as they reported a weak trend for the relationship between the polymorphism and HCC susceptibility, although this was not significant. However, a significant difference in the AA genotype and the A allele was found in men among the HCC patients and the healthy controls (P = 0.034, 0.029), respectively.
Maurer et al.  explained that the A/G variant at CTLA4 rs231775 leads to a threonine/alanine change in amino acid position 17 of the leader peptide, which alters the intracellular distribution of CTLA-4 and IL-2 production, and then influences T-cell proliferation.
Anjos et al.  also reported that individuals with homozygous GG have one-third less CTLA4 on their T-cell surface than those with homozygous AA, and T cells carrying the AA genotype had significantly lower activation and proliferation rates compared with T cells carrying the GG genotype upon stimulation.
Furthermore, functional assays in vitro showed that the CTLA-4 encoded by the rs231775 A allele has enhanced interaction with the B7.1 molecule and increased inhibitory effect on T-cell activation and proliferation compared with that encoded by the G allele .
| Conclusion|| |
It can be concluded that rs231775 in the CTLA4 gene might be a candidate risk factor for development of HCC, presumably by downregulating T-cell activation and proliferation.
Individuals carrying the A/A genotype and the A allele of CTLA4 rs231775 have an increased risk of developing HCC, whereas individuals carrying the GG genotype and the G allele have a protective effect for developing HCC.
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]