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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 31  |  Issue : 1  |  Page : 333-338

Theassociationbetween cyclin D1 G870A polymorphism and hepatocellular carcinoma in an Egyptian population


1 Clinical Pathology Department, Faculty of Medicine, National Liver Institute, MenoufiaUniversity, Shebeen El-Kom, Egypt
2 Clinical Pathology Department, National Liver Institute, MenoufiaUniversity, Shebeen El-Kom, Egypt

Date of Submission28-Jun-2016
Date of Acceptance21-Aug-2016
Date of Web Publication14-Jun-2018

Correspondence Address:
Esraa T A Allam
MBBCH, Menoufia, 32511
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_348_16

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  Abstract 


Objective
To study therelationshipbetween cyclin D1 G870A polymorphism and hepatocellular carcinoma (HCC) in Egyptian patients.
Background
Cyclin D1, encoded by the gene CCND1, is aregulatoryprotein in the cell cycle transition from G1 phase to S phase. Acommon polymorphism(G870A) in the exon 4 of CCND1 gene affects splicing of the CCND1 transcript and may cause uncontrollable cellular growth. Therefore, the CCND1 G870A polymorphism may influence an individual's susceptibility to the development of certain tumors, which may include HCC.
Patients and methods
This study was carried out on 100 participants: 60patients with HCC and 40 healthy age-matched and sex-matched volunteers. All participants underwent full history, liver profile, α-fetoprotein, and cyclin D1 G870A polymorphism assessment, which was done by PCR-restriction fragment length polymorphism assay.
Results
The CCND1 genotype distribution among patients with HCC was significantly different from that of healthy controls(P=0.000). Compared with the wild-typeGG genotype, both the variant AA and AA+GA genotype and the A allele were associated with risk of HCC[odds ratio(OR): 64; 95% confidence interval(CI): 6.67–614.2;P=0.000; OR: 19.67; 95% CI: 2.4–160.94;P=0.000; and OR: 4.03; 95% CI: 2.2–7.41;P=0.000, respectively]. Moreover, there is no significant correlation between the different cyclin D1 genotypes and clinicopathologic features of HCC.
Conclusion
Our results suggest that the CCND1 G870A polymorphism is associated with an increased risk of HCC in our Egyptian population.

Keywords: cyclin D1 gene, hepatocellular carcinoma, PCR-RFLP, polymorphism


How to cite this article:
El-Bassiounya MA, Gawishb EA, Eissaa ES, Tawfeeka GA, Allam ET. Theassociationbetween cyclin D1 G870A polymorphism and hepatocellular carcinoma in an Egyptian population. Menoufia Med J 2018;31:333-8

How to cite this URL:
El-Bassiounya MA, Gawishb EA, Eissaa ES, Tawfeeka GA, Allam ET. Theassociationbetween cyclin D1 G870A polymorphism and hepatocellular carcinoma in an Egyptian population. Menoufia Med J [serial online] 2018 [cited 2019 Apr 18];31:333-8. Available from: http://www.mmj.eg.net/text.asp?2018/31/1/333/234211




  Introduction Top


Approximately 700000 people die of hepatocellular carcinoma(HCC) each year worldwide, making it the third leading cause of cancer death [1]. Owing to the lack of efficient approaches in clinical management, the mortality of HCC remains very high. The 5-year survival for patients with HCC has poorly improved over the past decades. As a result, more and more attention has been paid to the discovery of potential biomarkers that are of diagnostic and prognostic value for HCC [2].

The main etiology of HCC is liver cirrhosis caused by chronic hepatitis B or C, alcohol, fatty liver diseases, or less commonly, autoimmune or genetic metabolic liver diseases [3].

Cyclin D1 is a key regulatory protein in the cell cycle, playing a critical role in the transition from G1 to S phase of the cell cycle [4].

The CCND1 gene, CCND1, is located on chromosome 11q13. The gene is polymorphic with a common A/G substitution at nucleotide 870(A870G, rs9344) in the conserved splice donor region of exon 4. The A870G has been shown to increase the frequency of alternative splicing and can lead to an increase in the half-life of the protein. The variant CCND1 corresponding to the A allele may have a longer half-life than the G allele, which may bypass the G1/Scheck point [5]. Several studies, including meta-analysis, indicated the association of CCDN1 870A genotype with a great variety of cancers such as colorectal, lung, esophageal, breast, oral, and squamous cell carcinoma of head and neck [6]. Therefore, in this study, we aimed to investigate its association with HCC risk in an Egyptian population.


  Patients and Methods Top


The present study was conducted at Clinical Pathology Department, Faculty of Medicine, Menoufia University, during the period from March 2014 to January 2015.

Patients

This study was carried out on 100 participants: 60patients with HCC as case group and 40 healthy age-matched and sex-matched volunteers, with no previous history of liver or malignant diseases and negative for hepatitis viral markers, as controls. Patients with HCC were excluded if they have inflammatory diseases, hematological malignancy, and cancer of any organ other than the liver. The diagnosis of HCC cases was based on the recommendations of the European Association for the Study of Liver using imaging technique(computed tomography or MRI) [7]. Awritten consent was obtained from each individual, and the protocol was approved by the Ethical Committee of Menoufia Faculty of Medicine.

Methods

All participants were submitted to history taking; clinical examination; Child–Pugh classification; radiological examination including abdominal ultrasound and triphasiccomputedtomography; and laboratory tests including CBC (Symex XT 1800; Menemeni, Thessaloniki, Greece), liver function tests (Rochintegra 400 plus; Basel, Switzerland), α-fetoprotein(Hitachi Cobas E 411; Basel, Switzerland), qualitative measurement of HCV-RNA and HBs Ag by PCR(CA, USA), and determination of CCND1 gene polymorphisms by PCR-restriction fragment length polymorphism.

DNA extraction and genotyping

Genomic DNA was isolated from ethylene diamine tetra acetic acid-preserved whole blood by standard proteinase K digestion ethanol precipitation using Thermo Scientific Gene JET Genomic DNA Purification Kit(Thermo Fisher Scientific, Massachusetts, USA).

After ethanol precipitation, the DNA was purified and dissolved in double distilled water and frozen at−20°C until use. The CCND1 G870A genotype was determined by PCR-restriction fragment length polymorphism assay. The PCR primers(Thermo scientific) were as follows: forward 5'-GTG AAG TTC ATT TCC AAT CCG C-3' and reverse 5'-GGG ACA TCA CCC TCA CTT AC-3'. PCR cycling conditions were 5min at 95°C, followed by 35cycles of 45 s at 94°C, 45 s at 55°C, and 45 s at 72°C, with a final elongation step at 72°C for 5min. For restriction fragment length polymorphism, the PCR products were digested with five units ScrFIenzyme(MA 01938-2723. United States.) at 37°C and visualized by electrophoresis on 3% agarose under ultraviolet illumination. The allele types were determined as follows: a single 167bp fragment for the AA genotype, two fragments of 22 and 145bp for the GG genotype, and three fragments of 22, 145, and 167bp for the AG genotype [8].

Statistical analysis

Statistical analysis of the present study was conducted with SPSS, version17.0(SPSS Inc., Chicago, Illinois, USA). Data were expressed into two phases: descriptive included mean, SD, and range and analytical study included χ2-test, t-test, Mann–Whitney test, Kruskal–Wallis test and Z test, Fisher's exact test, odds ratio(OR), and confidence interval(CI) test. P value more than 0.05 was considered statistically nonsignificant. P value of less than 0.05 was considered statistically significant. P value of less than 0.001 was considered statistically highly significant.


  Results Top


Baseline characteristics of the study patients

Baseline characteristics of the patients and controls showed no significant differences between the two groups in terms of age and sex[Table1]. Moreover, there was a statistically significant difference between cases and controls regarding alanine aminotransferase(ALT), aspartate aminotransferase(AST), total and direct bilirubin, serum albumin, and international normalized ratio(INR)[Table2].
Table 1: Comparison between studied patients regarding age and sex

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Table 2: Descriptive statistics of the studied patients regarding laboratory investigations

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CCND1 genotypes and allele distribution among study patients

The frequency distributions of the different genotypes for CCND1 polymorphism are shown in [Figure1] and [Table3]. There was a highly significant higher percentage of GG genotype among control group than cases group(25vs. 1.7%, P=0.001) and highly significant lower percentage of AA genotype among control group than cases group(12.5vs. 53.3%, P=0.000). Moreover, there is a highly significant lower percent of A allele among control group than cases group(75.8vs. 43.7, P=0.000).
Figure 1: (a) Agarose gel electrophoresis for cyclin D1 gene amplification bands correspond to ladder band size (167 bp). (b) Agarose gel electrophoresis showing PCR-restriction fragment length polymorphism analysis of cyclin D1 gene after addition of restriction enzyme: lanes (ladder) 2 and 3 (GG) band (145 and 22 bp), lanes 4–7 (AG) band (167 145 and 22 bp), and lanes 8, 9 and 10 (AA) band 167 bp.

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Table 3: Comparison between cases and control regarding genotypes and alleles

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CCND1 gene polymorphism and the risk for hepatocellular carcinoma

To evaluate the risk of HCC according to the CCND1 genotype using the GG genotype as the reference genotype, GA genotype significantly increased the risk of HCC by 10.08-fold (OR: 10.8; 95% CI: 1.29–90.56; P=0.02), whereas AA genotype significantly increased the risk of HCC by 64.0-fold(OR: 64; 95% CI: 6.67–614.2; P=0.000). Comparing A allele versus G allele, it was found that A allele significantly increased the risk of HCC by 4.03-fold compared with G allele(OR: 4.03; 95% CI: 2.2–7.41; P=0.000) [Table4].
Table 4: Odds ratio of genotypes and alleles between cases and control groups

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CCND1 genotypes and clinic pathological features of hepatocellular carcinoma

To assess the role of CCND1 gene polymorphism in clinic pathological features of HCC cases, the distribution of clinical and laboratory parameters among CCND1 genotypes was estimated [Table5], and there was no statistically significant difference between the different genotypes regarding clinical examination (spleen, liver, ascites, child classification, encephalopathy, and tumor size) or laboratory investigations(ALT, AST, albumin, total and direct bilirubin, INR, and α-fetoprotein).
Table 5: Clinical examination and laboratory investigation in relation to different genotypes among hepatocellular carcinoma cases

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  Discussion Top


HCC is one of the 10 most commonly occurring solid cancers worldwide. The most recent data indicate that its incidence is still increasing in many countries whereas the most effective way of reducing mortality owing to HCC is prevention [9].

Liver carcinogenesis is a complex and multifactorial process, in which both environmental and genetic features interfere and contribute to malignant transformation. The identification of host factors that may play an important role in HCC development may improve our understanding of the implications of the various biological pathways involved in liver carcinogenesis; such progress may as well help refine the selection of patients who could benefit from specific preventative measures or could be given adapted screening policies. Numerous candidate gene studies have reported associations between single nucleotide polymorphism and the presence of HCC [10].

CCND1, a key regulator of cell cycle progression, plays an important role in the transition from G1 to the S phase during cell division [11],[12]. The adenine-to-guanine(A/G) substitution at nucleotide 870(CCND1 G870A polymorphism) and excessive cyclin D1 activity are common in numerous human tumors, including breast cancer, lung cancer, head and neck cancers, gastric cancer, gynecological cancers, and blood-related cancers [13].

In this study, we investigated the association between CCND1 G870A polymorphism and the risk of HCC in Egyptian population. In the present study, the mean age of patients with HCC was 54.1±6.5years, a finding which may indicate that HCC is more commonly encountered in oldages. These results are in agreement with Luo etal.[13], who found that the mean age of patients with HCC was 54.1±10.5years. Moreover, Shaker etal. [14] foundthat the most frequent age category affected by HCC was between 51 and 60year. On the contrary, higher age incidence was reported by Giakoumidakis [15], who found that HCC is more frequent inpatientswith age of at least 65years.

In the present study, 83.3% of patients with HCC were males and 16.7% were females. This is in agreement with the result of the studyconductedby Rizk etal.[16], who found that mostpatientswith HCC were males. Moreover, Holah etal. [17] reported that most of patients with HCC were males.

De Lope etal. [18] suggestedthat this male predominance may be because of higher rates of exposure to liver carcinogens and hepatitis virus infections in men or to an estrogen-mediated inhibition of interleukin-6 production by Kupffer cells in females, leading to reduced liver injury and compensatory proliferation.

Considering cyclin D1 G870A polymorphism, our study shows statistically significant difference in distribution of the genotypes and allele frequencies between patients with HCC and healthy controls. It shows highly significant higher percentage of GG genotype among control group than cases group (25vs. 1.7%), nonsignificant difference regarding percentage of GA genotype, and highly significant lower percent of AA genotype among control group than cases group(12.5vs. 53.3%). Additionally, it shows highly significant higher percent of G allele among control group than cases group(56.3vs. 24.2) and highly significant lower percent of A allele among control group than cases group(43.7vs. 75.8).

Our results are similar to the results obtained by Akkız etal.[8], who found that the CCND1 G870A single nucleotide polymorphism is associated with an increased frequency of HCC in Turkish population. The data showed that CCND1 A allele was more frequently observed in the HCC group than control group with significant difference in the genotype frequency between the two groups(P=0.009).

In contrast with our study, Hu etal. [19] statesthat the CCND1 genotype distribution among patients with HCC was not significantly different from that among healthy controls in a Chinese population (P=0.08).

Our study also shows that GA genotype significantly increased the risk of HCC by 10.08-fold when comparing with the GG genotype(P=0.02), whereas AA genotype significantly increase the risk of HCC by 64.0-fold when comparing with the GG genotype(P=0.000).

Moreover, the present study show that GA+AA and GG+GA increase the risk of HCC by 19.6 and 8.0-fold, respectively.

Comparing A allele versus G allele, it was found that A allele significantly increase risk of HCC by 4.03-fold (P=0.000), and our results are similar to the results obtained by Akkız etal.[8], who found that both AA genotype and A allele increased the risk for HCC in Turkish population.

In the meta-analysis by Zhao etal.[20], which involvedleukemia and HCC, CCND1 G870A polymorphism was significantly associated with HCC risk under the recessive model(AA vs. AG+GG).

Found that the G870A genotype had no association with the susceptibility of hepatitis B virus-related HCC in the Chinese cohort study.

Hu etal. [19] stated thatcomparedwith the wild-type GG genotype, neither the variant AA genotype nor the A allele was associated with risk of HCC. However, stratification analysis by hepatitis B virus carrier status revealed that the variant genotypes containing the A allele were associated with a significantly increased risk of HCC among HBsAg-positive individuals(adjusted OR: 3.87; 95% CI: 1.12–13.30).

Studying the correlation between cyclin D1 G870A genotypes and clinical data shows that there was no statistically significant difference between the different genotypes and clinical parameters. This agrees with the results of Akkız etal.[8], and Hu etal.[20].

Moreover, there was no statistically significant difference between the different genotypes regarding laboratory investigations(ALT, AST, albumin, total and direct bilirubin, INR, and α-fetoprotein).

Our results suggest that this polymorphism may have role in liver carcinogenesis. These results may be limited by the low number of patients.

If our findings are confirmed in both larger series and other ethnic origins, genetic testing of CCND1 G870A polymorphism may be useful in detecting high-risk individuals such as hepatitis virus sero-positive patients who are at an even greater risk for HCC, and the results may encourage the higher risk population to have frequent medical examinations to detect early-stage HCC. Furthermore, the knowledge of the mechanisms involved in HCC carcinogenesis may help to identify targets for the development of chemoprevention or therapeutic strategies.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure1]
 
 
    Tables

  [Table1], [Table2], [Table3], [Table4], [Table5]



 

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