Value of Hypermethylation (RASSF1A) Promoter Gene among Patients with Hepatocellular Carcinoma

INTRODUCTION

Hepatocellular carcinoma (HCC) is one of the most frequent and most common aggressive malignancies worldwide. ¹

Although most HCCs originate from the accumulation of genetic and epigenetic abnormalities induced by various risk factors, underlying mechanisms of hepatocarcinogenesis are mainly involving cellular signaling pathways and its regulatory molecules. ²

Aberrant DNA methylation is one of the most common epigenetic alteration characteristics of malignant cells. ³

However, the use of tumor tissues to test for promoter methylation is invasive and thus impractical for HCC screening in high-risk populations.³ 

The RAS association domain family 1A gene (RASSF1A ) is a tumor suppressor gene that is located in the 3p21.3 region and is an important member of the RAS signaling pathway. ⁴

The RASSF1A  gene has been concerned and studied intensively for its tumor suppression, and hypermethylation in the promoter region is suspected as the main mechanism of silencing that is observed widely in human malignancies, including HCC tissues. ¹

The detection of the promoter hypermethylation of RASSF1A  in peripheral blood DNA could be a valuable biomarker for early-stage diagnosis in populations at high risk of HCC. ⁵

 SUBJECTS AND METHODS

This study was performed on a total of 80 subjects, selected from Tropical Medicine Department at Theodor Bilharz Research Institute (T.B.R.I) Hospital. They were divided into following groups:

Group I (HCC): Comprised of 30 patients with HCC on top of cirrhotic liver with elevated serum AFP and typical imaging  findings (abdominal ultrasound, triphasic  CT) .

Group II (LC): Comprised of 30 patients with liver cirrhosis (LC) on top of chronic hepatitis (CH), with normal serum AFP.

Group III (Control): It included 20 patients  age and sex matched appararently normal healthy volunteers.

• Informed verbal consents were obtained from all subjects.
• This study was approved by local ethical committee.

All subjects  were subjected to  the following: Full History taking regarding age, smoking, schistosomasis, HCV/HBV and diabetes, clinical examination for patients only and imaging techniques (abdominal ultrasonography and triphasic CT) for  detection of liver cirrhosis,  hepatic focal lesions (HFL), presence of ascites and hepatosplenomegaly and laboratory investigations:

Routine laboratory tests: complete blood count (CBC), was done on automated cell counter Beckman Coulter AcT Diff* and routine liver function tests and  kidney function tests.  They were all assayed by the automatic chemical autoanalyzer   Beckman Coulter Synchron CX5 Pro.

Special investigations: determination of  serum HBsAg, and serum HCV Ab using ELISA method using kit purchased from Human (Cat. no. 51275) determination of serum AFP level was assayed by solid phase,  chemiluminescent immunometric assay on automated analyzer-ADVIA centaur using kit supplied by Siemens Diagnostics and determination of RASSF1A  methylation status using methylation specific polymerase chain reaction (MSP)  ⁶

Specimen collection and storage: ten ml venous blood  were withdrawn  from all subjects under complete aseptic conditions  and then each was  divided into 3 parts :

1)     Six  ml were collected on plain tubes , left for 10 minutes at room temperature to clot and then centrifuged at 800 xg (3000 rpm)  for 5 minutes, then the separated serum was then divided into three  separate aliquots .One aliquot used for  routine biochemical  laboratory tests,one for hepatitis (HBV,HCV) serological markers assay and the last one used for determination of serum AFP.

2)     Two ml were collected in a sterile vaccutainer containing ethylene diamine tetra-acetic acid "EDTA", which were used for DNA extraction. Samples were stored frozen at    (–20^oC) till the time of DNA extraction and RASSF1A  methylation study.

3)     Two ml were collected in a sterile vaccutainer containing EDTA for complete blood picture assay .

N.B: Prothrombin Time (PT), Prothrombin Concentration (PC) and International Normalized Ratio (INR) values were collected from patients’ data sheets.

Calculation of Child-Pugh score:

Table 1: Clinical and laboratory measures of liver disease employed in Child-Pugh score 

Methodology:

Extraction of genomic DNA and estimation of  RASSF1A  methylation status  using methylation specific PCR (MSP):  

The test was  done in 4 main steps: extraction of genomic DNA from EDTA anticoagulated whole blood, bisulfite modification of the extracted DNA, amplification of the modified extracted DNA and detection of PCR amplified products using  2%  agarose gel electrophoresis and ultraviolet light transillumination.

Extraction of genomic DNA   from peripheral blood  leucocytes of EDTA anticoagulant done using   QIAamp® DNA Blood Mini kit: by spin columns (Catalog no. 51104) supplied by QIAGEN*

Principle: with the QIAamp DNA Isolation method, genomic DNA is prepared from blood cells. The lysis is achieved by the incubation of whole blood in a lysis buffer in the presence of Proteinase K at 56°C. Appropriate conditions for the binding of DNA to the specialized silica-based membrane in the spin columns are created by the addition of ethanol to the cell lysate. Contaminations are removed by washing with 2 different ethanol-based wash buffers. Pure genomic DNA is finally eluted with elultion buffer.

DNA extraction

Bisulfite modification

Conversion of unmethylated cytosine residues into uracil, leaving the methylated cytosine residues unchanged

Detection of PCR amplification products using gel electrophoresis

Fig. 1:   Diagram of Extraction of genomic DNA and estimation of RASSF1A methylation status using methylation specific PCR (MSP)

Bisulfite modification of extracted DNA: this was done using EpiTect Bisulfite Kit (Catalog no. 59104) supplied by QIAGEN

Principle: the EpiTect Bisulfite Kit provides a fast and streamlined 6-hour procedure for efficient DNA conversion and purification of as little as 1 ng DNA. DNA fragmentation is prevented during the bisulfite conversion reaction by a unique DNA protect Buffer that contains a pH-indicator dye as a mixing control in reaction setup, allowing confirmation of the correct pH for cytosine conversion. Furthermore, the bisulfite thermal cycling program provides an optimized series of incubation steps necessary for thermal DNA denaturation and subsequent sulfonation and cytosine deamination, enabling high cytosine conversion rates of over 99%. Desulfonation, the final step in chemical conversion of cytosines, is achieved by a convenient on-column step included in the purification procedure.

DNA amplification using the polymerase chain reaction (PCR): enzymatic amplification was performed using Dream Taq Green PCR Master Mix (2X) (Catalog no, K0171)  (Thermo Fisher scientific, Fermentas, USA) and BIORAD thermal cycler(BIORAD).

Principle of PCR: it consists of repetitive cycles of DNA denaturation, primer annealing and extension by Tag DNA polymerase. Each cycle produces complementary DNA strands to which the primers bind before the polymerase affects another extension in the next cycle of amplification. Accordingly the products of each cycle are doubled, generating an exponential increase in the overall number of copies synthesized.

Reagents:

1) Dream Taq Green Master Mix (2X)

• (2X) PCR Master Mix (2x1.25 ml) composition:

i)Taq DNA Polymerase (recombinant) in reaction buffer: 0.05 units/ul

ii) MgCl₂: 4 mM

iii)dNTPs ( dATP, dCTP, dGTP, dTTP): 0.4 M of each.

• Nuclease free deionized water(2x1.25 ml)

2) Primers: supplied by Fermentas.

Primers were reconstituted to obtain a concentration of each primer of 10 pmol /amplification

Methylated RASSF1A  promoter;

RASSF1A-MS Sense: (5′-GGGTTTTGCGAGAGCGCG-3′) and RASSF1A -MAS Anti-sense: (5′-GCTAACAAACGCGAACCG-3′)

Unmethylated RASSF1A  promoter;

RASSF1A-US Sense: (5′- GGTTTTTGTGAGAGTGTGTT-TAG-3′). RASSF1A -UAS Anti-sense: (5′- CACTAACAAACACAAACC-3′) .

The PCR Protocol:

1-All reactions were performed in a total volume of 25ul as (Yeo  et al., 2005) ⁶ described. The following were pipetted with no delay on ice.

2-The PCR reaction mixture was mixed well by vortexing.

3-The PCR reaction tubes were closed and placed inside the heating block in the DNA thermal cycle , and the cap was tightly closed.

 4-The computerized thermal cycler was programmed for the following conditions

A. Initial denaturation step of 95°C for 12 minutes.

B. 40 PCR cycles of amplification; each cycle consisted  of

• Denaturation at 95°C for 45 seconds.
• Anneling for 45 seconds at 60°C for methylated and at  56°C for the unmethylated  primers.
• Extension at  72°C for 1 minute.

    C. Extension step of 72°C for 10 min.

    D. Detection of PCR amplification products using 2% agrose  gel electrophoresis and ultra-violet light transillumination.

RESULTS

This study was conducted on 80 subjects; divided into three groups:

Group I (HCC): Included 30 patients with HCC on top of cirrhotic liver with elevated serum AFP, they were 26 males and 4 females patients. Their ages ranged between 46 and 53.3 years with mean age (48.80±1.36) years.

Group II (LC): Included 30 patients with liver cirrhosis (LC) and normal serum AFP, they were 22 males and 8 females patients. Their ages ranged between 46 and 55 years, with mean age (49.7 ± 1.4) years.

Group III (Control):Included 20 -age and sex matched appararently healthy volunteers as the control group, They were 18 males and 2 females. Their ages ranged between 41.7 and 52.7 years, with mean age (47.6± 1.54) years.

*Data  presented as number ( % )                                    **Data  presented as mean ± standard deviation

P value

Table 2: Demographic data and principal characteristics of the studied groups.

Table 3: Ascites findings among patients groups

Table 4: Abdominal triphasic CT scan findings in group I (HCC) patients.

*Data expressed as Median  ,  (25th % - 75 th % )  percentiles .

**Data presented as mean ± SD.

* **P value ≤  0.05  is statistically different

****Groups bearing same initials are statistically indifferent  at  P value 0.05, while groups sharing different initials are statistically different at  P value 0.05 (by ANOVA testing).

Table 5: Comparison of laboratory tests among the studied groups.

Table 6: Descriptive comparison between the studied groups as regard HCV-Ab, HBsAg, RASSFIA-Methylated and Unmethylated

^* Data presented as mean ± SD     **Data presented as numbers (%) 

***Data expressed as Median ,  (25^th % - 75^th%) percentiles-    P Value ≤ 0.05, is statistically significant

Table 7: Relationship between RASSFIA-U/RASSFIA-M and clinicopathological parameters of all patients (n= 60) group I (HCC) and group II (LC).

Table 8: Risk estimate of RASSF1A  methylation status.

Table 9: Risk estimate among HCC (group I) and control (group III)