Molecular detection and DNA Methylation of HBV in a group of Iraqi patients with Hepatocellular Carcinoma

 

Dr. Hiba Sabah Jasim

PhD, Department of Microbiology, Medicine College, University of Baghdad

 

ABSTRACT:

Hepatitis B virus is the major cause of liver abnormalities, cirrhosis, and hepatocellular carcinoma, and can cause thousands infection in the year. Methylation of CpG in different islands of the virus genome considered the major epigenetic alteration of virus genes, the aim of this study is to estimate the distribution of the virus also the conduct the role of the methylation pattern of the virus genome in the prognosis of the disease, as methylation can be considered as biomarker for cancer prognosis. Materials and methods: A total of one hundred patients included in this study, a fresh tissue sample was taken from each patient for molecular technique to detect hepatitis B virus, and then to estimate the methylation pattern of the virus. Detection of the virus by conventional polymerase chain reaction using two specific primers sets by nested PCR. Then detection of the methylation pattern of HBV using methylation specific polymerase chain reaction by specific primer set for methylation of the hepatitis B virus. Results: High percentage of the virus was present in the hepatocellular carcinoma cases with (46/60) patients, which represent 76.7%, while the incidence of the virus in liver abnormalities group was (4/20) patients, which represented 20% of all patients in this group. The methylation state was present in high percentage in hepatocellular carcinoma group with 65.2%, while the percentage of methylation in liver abnormalities was zero%.

 

 

 

INTRODUCTION:

Hepatitis B virus infects about two billion patients around the world, nearly half of million deaths occur each year as a result of hepatitis B infections [1]. Chronic infection with this virus affects nearly high percentage of patients worldwide and the infection may progress to liver cirrhosis, hepatocellular carcinoma in 20% of the cases [2].

 

The virus is a member of hepadna family, which consists of a double stranded DNA. Integration between hepatitis B virus DNA and the host genome usually detected in chronic infection and cancer cases.

 

The mechanism of infection of this virus is complicated, indirect method suggested that this virus cause cell injury and liver regeneration may occur due to host immune response [3]. Severe cell proliferations during liver regeneration cumulate mutations, give a chance of mistakes during DNA replication. Cells that acquired suitable genetic mutations will suffer from clonal propagation and may develop into hepatocellular carcinoma [4].

 

Hepatitis B virus is the essential causative agent of hepatocellular carcinoma, this virus associated with hepatocellular carcinoma in humans usually present after several years of chronicity with this virus; while, the causes of huge gap in the time between infection with the virus and starting of carcinoma case and the exact molecular techniques by which the virus devise carcinogenesis of the liver is not obvious [5,6]. Transformation of serious cellular genes (due to combination between viral and host genome), the presence of x-protein in the DNA of the virus which has convert effect, atypical epigenetic anomaly in the host gene considered the potential factor give rise to improve hepatocellular carcinoma related to hepatitis B virus [7]. An epigenetic change like DNA methylation which has a significant function in silencing many genes may affect foreign genomes, new studies effort to check whether the virus DNA is likewise methylated by host genome. The virus genome contain three CpG islands I, II, III, the island I is the start site for surface antigen, the island II is the promoter of x-protein, the island III is the start site for polymerase gene. These islands found to be methylated in various hepatitis B virus correlate with hepatocellular carcinoma [8].

 

MATERIALS AND METHODS:

A total of one hundred patients included in this study, 80 of them with hepatocellular carcinoma and the other 20 were with liver abnormalities, a fresh tissue sample was taken from each patient from different hospitals in Baghdad for molecular technique to detect hepatitis B virus, and then to estimate the methylation pattern of the virus and the effect of this epigenetic change in the prognosis of the disease.

 

All tissue samples were histologically diagnosed and the diagnosis was taken from the histological laboratory in the hospital to detect the histological abnormalities in the tissue samples.

 

Detection of the virus by conventional polymerase chain reaction using two specific primer sets by nested PCR to increase the specify of the reaction, using the first primer set (P1b, S1-2), then the product of this reaction was further used in the second polymerase reaction using the second primer set (B2, BB1R) which is specific for hepatitis type B, the sequences of these two primers were demonstrated in table one.

 

Polymerase chain reaction master mix was prepared with total volume of 50 𝜇L per one reaction, containing 10 pmol forward and reverse primers, 1.5 U of Go Taq ® DNA Polymerase (Promega, USA), 1X of Green Go Taq ® reaction buffer Polymerase (Promega, USA) and 400 𝜇M of dNTPs Polymerase (Promega, USA), then ddH₂O was added until the volume reach to 48 𝜇L. 2 𝜇L of DNA was added to PCR mixture. No template control tube was prepared for each reaction, this tube contains all master mix components but instead of DNA, 2𝜇L of ddH₂O was added. Amplification was done using PCR thermal cycler (Eppendrof, Germany). The amplified products were electrophoresed in 1.5 % agarose gel. Presence of bands by gel electrophoresis means positive results and the virus is present.

 

Table one: Primer sequence for genotyping of hepatitis B virus:

Primer	Sequence of the primer	Position	Specificity	Polarity
P1b	5`-TCA CCA TAT TCT TGG GAA CAA GA-3`	nt 2823–2845	universal	sense
S1-2	5`-CGA ACC ACT GAA CAA ATG GC-3`	nt 685–704	universal	antisense
B2	5`-GGC TCM AGT TCM GGA ACA GT-3`	nt 67–86	type B specific	sense
BB1R	5`-CAG GTT GGT GAG TGA CTG GAG A-3`	nt 324–345	type B specific	antisense

 

After detection of the virus by nested PCR, the second step was modification of the DNA using bisulphite modification kit from Qiagen, then detection of the methylation pattern of HBV using methylation specific polymerase chain reaction by specific primer set for methylation of the hepatitis B virus, the primer sets used for methylation specific PCR were demonstrated in table two.

 

Polymerase chain reaction master mix with total volume of 25 𝜇L per one reaction was prepared, the master mix consist of 10 pmol forward and reverse primers, 2 unit of Go Taq ® DNA Polymerase enzyme (Promega, USA), 1X of Go Taq ® reaction buffer Polymerase (Promega, USA) and 200𝜇M of dNTPs Polymerase (Promega, USA), then ddH₂O was added until the volume reach to 23𝜇L. 2𝜇L of modified DNA was added to each tube of the reaction, just the tube of no template control there was an addition of 2𝜇L of ddH₂O instead of modified DNA. Amplification was done using PCR thermal cycler (Eppendrof, Germany). Then the products were electrophoresed in 2% agarose gel. Presence of bands means positive results and considered of methylated case.

 

Table two: Specific primer sets for methylation of HBV in three different CpG islands:

Name of CpG island	Sense	Antisense
CpG island I	5`-TTG TTG GTG GTT TTA GTT TTG-3`	5`TCC ACC ACT AAT CTA AAC TC-3`
CpG island II	5`-ATG GTT GTT AGG TTG TGT TG-3`	5`-AAA CRT TCA CTA TAA TCT CC-3`
CpG island III	5`TGT TTT GAG TAT TTG GTG TTT TTTG-3`	5`-TTT CCC ACC TTA TAA ATC AA-3`

 

RESULTS:

Hepatitis B virus genotyping and methylation pattern were analyzed by deriving the DNA of the virus from different hepatocellular carcinoma, and liver abnormalities sample tissues using polymerase chain reaction technique and analyzing of the results were done by gel electrophoresis.

 

 

Table three: Detection of hepatitis B virus in tissue samples of liver abnormalities by nested PCR:

Groups	Number	Positive for the virus	Percentage	Negative for the virus	Percentage
Control group	20	0	0 %	20	100 %
Liver abnormalities	20	4	20 %	16	80 %
Hepatocellular carcinoma	60	46	76.7 %	14	23.3 %
Total	100	50	50 %	50	50 %

 

Table four: Methylation pattern of hepatitis B virus derived from different liver tissue abnormalities by methylation specific PCR:

Groups	No. of patients	Methylated	Percentage	Non-methylated	Percentage
Liver abnormalities	4	0	0 %	4	100 %
Hepatocellular carcinoma	46	30	65.2 %	16	34.8 %
Total	50	30	60 %	20	40 %

 

Table five: Percentage of the methylation in the different three CpG islands of the HBV genome:

Groups	No. of patients	Methylated	CpG island I	%	CpG island II	%	CpG island III	%
Liver abnormalities	4	0	0	0%	0	0%	0	0%
Hepatocellular carcinoma	46	30	15	50%	5	16.7%	10	33.3%

 

 

High percentage of the virus was present in the hepatocellular carcinoma cases with (46/60) patients, which represent 76.7%, while the incidence of the virus in liver abnormalities group was (4/20) patients, which represented 20% of all patients in this group, these results were demonstrated in table three.

 

The methylation state was present in high percentage in hepatocellular carcinoma group with 65.2%, while the percentage of methylation in liver abnormalities was zero%; the results of methylation were demonstrated in table four.

 

DISCUSSION:

This study tries to manage a particular dissection of hepatitis B virus genome methylation in different cases of liver cancer and abnormalities. The results demonstrate that CpG sites of Island I is largely methylated in hepatocellular carcinoma when compared with other two islands II and III, this result is similar with other studies shows particular methylation of CpG sites in the hepatitis B virus DNA [9,10] and suggest that hepatitis B virus may be frequently methylated through the expansion of liver cancer. Anywise, hepatitis B virus genes in obscure hepatitis B virus associated with chronicity of the infection, when compared to hepatocellular carcinoma cases, was practically non-methylated, proposing that hepatocytes in a chronic infection little target the hepatitis B virus DNA for methylation and silencing of the virus genome in cells with chronic infection is not usually caused by increase methylation of the virus genome.

 

More investigations were used to check the connection of viral incidence, coding genes, and the methylation pattern in the hepatitis B virus genes. Epigenetic alteration such as methylation cause inhibition of coding genes which play a role in protection versus the sequence of the virus [6], the result of this study suggest that DNA methylation considered the essential epigenetic alteration causing silencing of hepatitis B genes.

 

The hepatitis B virus genome exhibited hypermethylation in both liver abnormalities and hepatocellular carcinoma samples when compared to normal liver samples of chronic cases, this indicate that methylation of hepatitis virus genes largely related to malignant cases. More studies are needed to estimate if these alterations are the causes of up normal type or considered the result of unregulated epigenetic alteration in convert hepatic tissues.

 

These findings indicate that methylation pattern of the hepatitis B virus may effected by non-regulated DNA methylation in malignant cells and this may participate in the disease phenotype, though it is distinct to be accountable for the obscure status.

 

Practical effect of the specific hypermethylation may demonstrate the monitoring of sever reduction in the output of viral genes in infected cells [11]. Thus, the results of this study support the thesis of the methylation of hepatitis B virus genes might play an essential role in host control mechanism in order to modify the output of these genes and that malicious conversion largely corroboration the fixing of epigenetic alteration in hepatitis B virus DNA.

 

REFERENCES:

1.      World health Organization (WHO). Hepatitis B. Fact sheet N 204. Revised August 2008.http://www.who.int/mediacentre/factsheets/fs204/en/index.

2.      Baig S, Siddiqui AA, Ahmed W, Qureshi H, Arif A. The association of complex liver disorders with HBV genotypes prevalent in Pakistan. Virol J 2007; 4: 128.

3.      Chisari FV, Ferrari C. Hepatitis B virus immunopathogenesis. Annu Rev Immunol 1995;13:29-60.

4.      Narimatsu T, Tamori A, Koh N, et al. p16 promoter hypermethylation in human hepatocellular carcinoma with or without hepatitis virus infection. Intervirology 2004; 47:26-31.

5.      Lupberger J, Hildt E. Hepatitis B virus-induced oncogenesis. World J Gastroenterol 2007; 13:74–81.

6.      Fernandez AF, Rosales C, Lopez-Nieva P, et al. The dynamic DNA methylomes of double-stranded DNA viruses associated with human cancer. Genome Res 2009; 19:438–451.

7.      Park IY, Sohn BH, Yu E, et al. Aberrant epigenetic modifications in hepatocarcinogenesis induced by hepatitis B virus X protein. Gastroenterology 2007; 132:1476–1494.

8.      Guo Y, Li Y, Mu S, Zhang J, Yan Z. Evidence that methylation of hepatitis B virus covalently closed circular DNA in liver tissues of patients with chronic hepatitis B modulates HBV replication. J Med Virol 2009; 81:1177–1183.

9.      Vivekanandan P, Thomas D, Torbenson M. Hepatitis B viral DNA is methylated in liver tissues. J Viral Hepat 2008;15:103–107.

10.   Vivekanandan P, Thomas D, Torbenson M. Methylation regulates hepatitis B viral protein expression. J Infect Dis 2009; 199:1286–1291.

11.   Hantz O, Parent R, Durantel D, Gripon P, Guguen-Guillouzo C, Zoulim F. Persistence of the hepatitis B virus covalently closed circular DNA in HepaRG human hepatocyte-like cells. J Gen Virol 2009; 90: 127–135.

 

 

 

 

Accepted on 26.06.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(9):4489-4492.