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ORIGINAL ARTICLE
Year : 2017  |  Volume : 30  |  Issue : 2  |  Page : 619-625

Study of Apa-I vitamin D receptor gene polymorphism in patients with hepatocellular carcinoma


1 Department of Clinical Pathology, Faculty of Medicine, National Liver Institute, Menoufia University, Shebein El-Kom, Egypt
2 Pathology Department, National Liver Institute, Menoufia University, Shebein El-Kom, Egypt

Date of Submission13-Dec-2016
Date of Acceptance05-Mar-2017
Date of Web Publication25-Sep-2017

Correspondence Address:
Yasmen A El-Hag Ali
Department of Clinical Pathology, National Liver Institute, Menoufia University, Shebein El-Kom, Menoufia Governorate, 32512
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_673_16

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  Abstract 

Objective
The aim of this study is to study vitamin D receptor(VDR) Apa-I gene polymorphism in patients with hepatocellular carcinoma(HCC) infected with chronic hepatitis C.
Background
HCC is a global problem. In Egypt, it is one of the major health problems facing the health authorities. Several major risk factors of HCC have been identified, including chronic infection of hepatitis B virus and hepatitis C virus. Nevertheless, only a fraction of infected patients develop HCC during their lifetime suggesting that genetic factors might modulate HCC development. Consequently, identification of additional genetic factors affecting transcription of specific regulatory genes could help to select high-risk populations.
Patients and methods
This study was conducted on 120patients between February 2015 and February 2016:40patients with HCC and 40patients with liver cirrhosis without any radiological evidence of HCC, who presented to the Hepatology Department, National Liver Institute, Menoufia University, along with 40 age-matched and sex-matched healthy controls. Vitamin D receptor Apa-I gene polymorphism was determined by PCR-restriction fragment length polymorphism.
Results
Our study revealed significant statistical difference between patients with HCC and those with liver cirrhosis also between patients with HCC and control group regarding VDR gene Apa-I polymorphism. In HCC group, there was also significant statistical difference between the studied Apa-I VDR genotypes and α-fetoprotein, tumor size, and serum albumin.
Conclusion
VDR Apa-I gene polymorphism could be associated with increased risk of HCC development in chronic hepatitis C virus-infected patients.

Keywords: hepatocellular carcinoma, polymorphism, vitamin D receptor Apa-I gene


How to cite this article:
El-Edel RH, Mostafa MS, Montaser BA, El-Hag Ali YA. Study of Apa-I vitamin D receptor gene polymorphism in patients with hepatocellular carcinoma. Menoufia Med J 2017;30:619-25

How to cite this URL:
El-Edel RH, Mostafa MS, Montaser BA, El-Hag Ali YA. Study of Apa-I vitamin D receptor gene polymorphism in patients with hepatocellular carcinoma. Menoufia Med J [serial online] 2017 [cited 2017 Nov 21];30:619-25. Available from: http://www.mmj.eg.net/text.asp?2017/30/2/619/215475


  Introduction Top


Hepatocellular carcinoma(HCC) is a global problem. In Egypt, it is one of the major health problems facing the health authorities[1]. It represents the second most common cancer in men and the sixth most common cancers in women[2].

Several major 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 the 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].

Specific recognition of cis-regulatory regions is essential for correct gene regulation. Such DNA sequences are recognized by transcription factors that recruit the transcriptional machinery[6].

The genomic actions of vitamin D are mediated through its binding to the vitamin D receptor(VDR), which allows it to modulate the transcription of genes in a cell-and tissue-specific manner[7].

1,25(OH) 2D3-VDR-regulated genes are composed of those encoding factors affecting cell life/cancer, the immune system, and metabolism. VDR likely reduces risk for many cancers by inducing the p53 and p21 tumor suppressors, as well as DNA mismatch repair enzymes[8].

The VDR directly or indirectly regulates the expression of more than 200 genes that influence cell proliferation, differentiation, and apoptosis, as well as immunomodulation and angiogenesis[9].

The human VDR gene located on chromosome 12q is composed of promoter and regulatory regions(1a–1f) and exons 2–9, which encode six domains(A–F) of the full-length VDR protein[10].

It is known that the functionality of VDR is affected by single-nucleotide polymorphisms(SNPs) in VDR gene. The most frequently investigated VDR gene polymorphisms are Taq-I(rs731236), Bsm-I(rs1544410), Fok-I(rs2228570), and Apa-I (rs7975232) [11].

The experimental studies support the tumor inhibitory effects of vitamin D on HCC cells, suggesting that vitamin D dysfunction may play an important role in the development and progression of HCC[12].


  Patients and Methods Top


This study was conducted on 120patients: 40patients with HCC, 40patients with liver cirrhosis, with no radiological evidence of HCC, and 40 apparently healthy subjects 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 consents were taken from both the patients and control group individuals before the beginning of the study.

For all patients, the followings were done: collection of relevant clinical data, basic laboratory tests including(complete blood count, liver function tests, α-fetoprotein and prothrombin time), hepatitis viral markers(hepatitis B surface antigen and HCV-antibody) and molecular testing for Apa-I VDR gene polymorphism was done by PCR based on restricted fragment length polymorphism. Total DNA was extracted from EDTA-treated blood sample using Thermo Scientific Gene JET Genomic DNA Purification Kit(Thermo Fisher Scientific, MA, USA).

The 745-base pair(bp) fragment encompassing the C to A polymorphic site in Apa-I VDR gene region was amplified using the provided master mix(Thermo Fisher Scientific, Waltham, Massachusetts, USA) and the following primers: forward in exon 8(5′-CAGAGCATGGA CAGGGAGCAA-3′) and reverse in exon 9 (5′-GCAACTCCTCATGGCTGAGGTCTC-3′). The PCR amplification was performed on preprogrammed thermal cycler (Perkin Elmer Gene Amp PCR System 2400 Thermal Cycler, SPSS-IBM corp., Armonk, NY, USA) under the following conditions: an initial denaturation step at 95°C for 2min, followed by 40cycles, each cycle consisted of denaturation at 94°C for 45 s, annealing at 67°C for 45 s, and extension at 72°C for 60 s. Final extension at 72°C for 2min was carried out. Confirmation of successful PCR amplification was done by 2% agarose gel electrophoresis for 20min at 150 V. Overall, 5 μl of normal range(100bp) ladder and 10 μl of PCR product were applied[Figure 1]. After PCR, the products are digested with Apa-I restriction enzyme, so the following reaction components were mixed gently at room temperature: 7 μl nuclease-free water, 2 μl 10×fast digest green buffer, 1 μl Apa-I restriction enzyme(Thermo Fisher Scientific), and 10 μl of DNA amplification product. The mixture was incubated at 65°C in a heat block for 5min. The reaction mixture was loaded directly and electrophoresed on 2% agarose gel containing ethidium bromide and visualized under ultra violet illumination. PCR products with C at the polymorphic site were digested into two fragments, 531 and 214bp, where those with A were not because of the absence of Apa-I enzyme restriction site. Samples yielding 531-bp and 214-bp fragments were scored as C/C, those with single 745-bp fragment as A/A, and 745-bp, 531-bp and 214-bp fragments as C/A[Figure 2].
Figure 1: A representative agarose gel picture showing vitamin D receptor Apa-I gene amplification bands of 745 bp DNA ladder (lanes 2–10) and 100 bp DNA ladder (lane 1).

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Figure 2: A representative agarose gel picture showing PCR-RFLP analysis of Apa-I genotypes in genomic DNA of study patients with Apa-I restriction enzyme. Lane 1 100-bp DNA ladder; lanes 2, 4, 6, 7, 8, and 9 C/A heterozygous (745, 531, and 214 bp bands), lane 5 C/C homozygous(531 and 214 bp bands); and lanes 3 and 10 A/A homozygous (745 bp).

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Statistical analysis

Results were collected, tabulated, and statistically analyzed by statistical package for social studies, version20.


  Results Top


The present study showed a highly significant statistical difference in Apa-I VDR(CC) genotype with increased C-allele frequency in patients with HCC than the cirrhotic group(P<0.001)[Table1].
Table 1: Comparison between the cirrhotic and hepatocellular carcinoma groups according to Apa-I vitamin D receptor genotype and allele frequency distribution

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The study showed also highly significant statistical difference in Apa-I VDR(CC) genotype with increased C-allele frequency distribution in patients with HCC than the control group(P<0.001)[Table2].
Table 2: Comparison between the hepatocellular carcinoma and control groups according to Apa-I vitamin D receptor genotype and allele frequency distribution

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Alanine aminotransferase(ALT), aspartate aminotransferase (AST), and α-fetoprotein(AFP) levels were significantly higher in HCC group compared with cirrhotic group, with no significant statistical difference in international normalized ratio(INR), alkaline phosphatase(ALP), albumin, total bilirubin, direct bilirubin, and total calcium [Table3].
Table3: Statistical analysis of laboratory results of the studied groups

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On comparing HCC with the control group, there were a significant increases in INR, AST, ALT, ALP, total bilirubin, direct bilirubin, and AFP and significant decreases in platelet count, serum albumin, and total calcium[Table3].

There was significant higher difference in tumor size with CC genotype than CA genotype also with CC genotype than AA genotype. The study showed also significant higher difference in AFP with CC genotype than CA genotype[Table4].
Table 4: Relation between Apa-I vitamin D receptor genotypes and different parameters in hepatocellular carcinoma group

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  Discussion Top


This work aimed to study the association between vitamin D receptor Apa-I gene polymorphism and HCV-related HCC development in Egyptian population.

Considering Apa-I vitamin D receptor gene SNP, the present study showed statistically significant difference in the distribution of the genotypes and allele frequencies between HCC and cirrhotic groups and also between HCC and healthy control groups. This agrees with the results of the study by Hung etal.[13].

In this study, Apa-I gene SNP in HCC group represent CC genotype(70%), CA genotype(20%), and AA genotype(10%), so most patients with HCC were CC genotype [Figure 3] and [Figure 4]. These results were similar to those obtained by Hung etal. [13] who found statistically significant association between Apa-I CC VDR gene SNP and the risk of HCV-related HCC, as it shows that in this groupApa-I CC genotype represents 71%, CA 26%, and AA 3%.
Figure 3: Comparison between the different studied groups according to genotype distribution. HCC, hepatocellular carcinoma.

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Figure 4: Comparison between the different studied groups according to allele frequency distribution. HCC, hepatocellular carcinoma.

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Falleti etal. [14] also found that VDR genetic polymorphisms are significantly associated with the occurrence of HCC in patients with liver cirrhosis.

Our study of the relationship between Apa-I genotypes and laboratory characteristics in HCC group showed significantly lower albumin levels with C/C genotype compared with other genotypes. In addition, patients with C/C genotype had significantly higher AFP level and larger tumor size when compared with other genotypes. Also, there was no statistical difference between Apa-I genotype and Child-Pugh grades, INR, liver enzymes, total bilirubin, direct bilirubin, total calcium, and phosphorus.

Similarly Hung etal. [13] found no statistical difference between Apa-I genotypes and AST and ALT.

Dundar etal. [15] and Yokoyama etal. [16] agreed with the results of the present study as they found no statistical difference between Apa-I genotypes and ALP and phosphorus level.

Also, Gezen etal. [17] found no statistical difference between Apa-I genotypes and ALP, phosphorus, and total calcium levels.

On the contrary, Dundar etal. [15] showed that A/A genotype had significantly higher total calcium level than the patients with C/C genotype. This may be because of multiple factors besides vitamin D action that could affect calcium level in the body.

Finally, the results in the present study suggest a significant association of VDR Apa-I polymorphism with the development of HCC in chronic HCV infection.


  Conclusion Top


VDR Apa-I gene polymorphism could be associated with increased risk of HCV-related HCC development in Egyptian population, but the definite association between them needs to be validated in other large multicenter cohort studies.

Acknowledgements

This study was 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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DundarU, SolakM, KavuncuV, OzdemirM, CakirT, Yildiz H, etal. Evidence of association of vitamin D receptor Apa I gene polymorphism with bone mineral density in postmenopausal women with osteoporosis. Clin Rheumatol 2009; 28:1187–1191.  Back to cited text no. 15
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table1], [Table2], [Table3], [Table4]



 

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