Sorry, there is no online preview for this file type. X GENE VARIABILITY AND GENOTYPING OF HEPATITIS B VIRUS (HBV) IN SUDANESE. PDF | Hepatitis B and related viruses that infect mammalian hosts encode the “X” protein that has been shown to contribute Sorry, there is no online preview for this file type. . The Enigmatic X Gene of Hepatitis B Virus. The importance of the conserved third start codon in the HBV X gene became apparent in isolate w4B. Sorry, there is no online preview for this file type.
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Hepatitis B virus HBV is one of the most widespread human pathogens causing chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma HCC. This study investigated the clinical impact of single and combinational mutations in HBx gene on the pathogenesis of HCC during progressive stages of liver disease.
The patients filetupe categorized into inactive HBV carriers, active carriers, cirrhosis and HCC groups based on disease severity. H94Y and KM mutations were also significantly associated with severe liver disease. Several single and filety;e mutations in HBx correlating with severity and filetye clinical phases of HBV infection were identified. The mutational combinations may have a synergistic effect in accelerating the progression to HCC.
These specific patterns of HBx mutations can be useful in predicting the clinical outcome of HBV-infected patients and may serve as early markers of high risk of developing HCC.
Hepatitis B virus HBV is the major causative agent of hepatitis worldwide. HBV infection has been reported to result in liver cirrhosis, hepatic failure, or hepatocellular carcinoma HCCleading to aboutdeaths each year globally [ 1 ].
Additionally, it is predicted that the risk of HCC increases by 5- to fold in individuals who are chronically infected with HBV [ 4 ]. HBV is a small virus with a genome consisting of a circular ciletype double-stranded DNA of about 3. The genome is organized into four overlapping fildtype reading frames that encode xgens viral core protein capsidsurface proteins envelopereverse transcriptase, and non-structural X protein HBx [ 5 ]. Compared to the other three HBV proteins, a limited number of studies have examined the role of HBx mutations in the oncogenic potential of the virus.
HBx protein amino acids has functionally distinct domains with a strong negative regulatory N-terminus and a transactivation region mapped to its C-terminus [ 6 ]. HBx is a pleiotropic regulatory molecule exerting its effects on a wide range of cell processes, including cell cycle, cell proliferation, and apoptosis [ 8 ].
Interestingly, previous studies have shown that the majority of HBx detected in HCC tissues had mutations that may alter the function of HBx [ 1011 ]. HBx mutants harboring both point mutations and deletions, especially C-terminal truncations, have been frequently detected in HCC patients [ 101213 ].
Taken together, these studies suggest that HBx plays a crucial role in the pathogenesis and progression of HBV-related complications. Similarly, a study xgenf the Chinese population has found that the mutation at amino xgenr 88 of HBx was highly frequent in HCC tumor samples compared to non-tumor samples [ 16 ].
The functional role of HBx mutations in the development of liver complications was filety;e as well. In the present study, HBV-infected Saudi patients were investigated.
HCC patients had the highest average age followed by liver cirrhosis LC patients, whereas the inactive IC and active carrier AC groups showed similar values of average age. Different stages of HBV infection showed a predominance of ybv patients. Additional analyses were performed for female patients only and the results are shown in Supplementary Table 1.
VI mutation was more prevalent in HCC Another significant mutation at position 88 substitution of V to F was present in all the HCC samples, while only A combinational mutation with substitutions of K to M at position and V to I at position was present in Another triple mutation with substitution of K to M, V to I, and F to Y at positions, and fileetype, respectively, was present in Univariate and multivariate filtype regression analyses were performed to assess the association between HBx mutations and progression of the disease.
In addition, further analyses were performed for males and females separately Supplementary Tables 3 and 4. Two xgdne methods were used to evaluate residue conservation see methods and variability in the HBX protein described in methods. Both provided very similar results.
None of these mutations changes any residue that is consistently conserved across all sequences, suggesting that the effect on protein structure itself is minimal. However, these may change increase or decrease binding to multiple different proteins. The effect of these mutations on interactions with any particular protein is unknown, as the same regions of HBX have been implicated in binding multiple targets e.
Residue positions mutated in this work are indicated by arrows at the top.
The Enigmatic X Gene of Hepatitis B Virus
Consurf analysis results, with color coding ranging from most variable to most conserved residues are shown See scale at bottom left. Below the ConSurf results, secondary structure predictions from PsiPred server are shown helices and sheets. A multiple sequence alignment of 35 highly diverse HBX proteins from multiple species is shown. A sequence logo is placed right above the alignment. Below the alignment, the six regions A—E defined by a previous analysis [ 6 ] are shown.
Shown at the bottom green rectangles are regions of HBX that have 3D structural information. The PDB codes and chain identifiers, and the sequences are shown. Efforts to model the complete 3D structure of the HBX protein were not successful.
These hits however remain small, and unreliable. Sliding window analysis was used to assess the amino acid diversity between the different groups of patients in progressive stages of HBV infection. The dashed line indicates the p value of 0.
HBV infection is one of the most important causes of human liver disease and continues to remain a medical concern in Saudi Arabia. There is a significant number of cases as the incidence rate of HBV infection was There is an emerging interest to explore various aspects of HBV infection and related liver complications within the Saudi population. Many studies have shown that genetic variations in HBV genome influence clinical manifestations and progression of the HBV infection [ 2223 ].
HBx is a small regulatory protein with a plethora of activities influencing both viral and cellular genes. HBx is known to elicit an oncogenic potential by disrupting cell cycle regulation [ 724 ].
HBx detected in HCC patients is known to frequently harbor mutations, which may play a role in the progression of HBV infection [ 12 ]. The present study identified specific HBx mutations and investigated the clinical correlation of these mutations and the progression of HBV infection. Eight positional single mutations within the HBx protein playing a role in advancement of liver disease were identified in this study. Two mutations S42P and A47T present within the B-cell epitope region did not show any significant association with the clinical stages of HBV infection.
This was inconsistent with a previous Indian study reporting a significant association of mutation at codon 47 of HBx with increased disease severity [ 15 ]. Similarly, mutation at position 88 of HBx protein was associated with liver cirrhosis in Indian population [ 15 ], while a Chinese study has found a mutation at amino acid 88 more frequent in HCC samples [ 16 ]. As suggested by Malik et al. The results in our study also showed a significant association of these mutations with severity of liver disease.
However, some studies in the Indian population have reported that H94Y mutation was present at high frequencies in both LC and HCC groups, suggesting that population may increase or decrease the effect of this mutation on liver disease [ 2527 ]. Therefore, these mutations may be responsible for modulating the transactivation property of HBx that is known to be associated with hepatocarcinogenesis [ 3031 ]. The present study also investigated the association of combinational mutations in the HBx protein with the progression of HBV infection.
The combinational mutations analyzed could act synergistically to increase severity of liver disease. This double mutation was present at high frequency in the HCC For instance, it has shown an increase in frequency with the advancing clinical phases in Taiwanese [ 18 ], Chinese [ 28 ], and Indian populations [ 14 ].
The prevalence of this double mutation in the Saudi HCC patients This double mutation is present within the C-terminal of HBx protein, and several studies related mutations in C-terminal to cell proliferation and transformation contributing to hepatocarcinogenesis [ 31 ].
However, this mutation was observed at high frequency only in HCC patients As also stated by Iavarone et al. This triple mutation was present at very low frequency in IC 0. This suggested that the existence of triple mutations might burden the life cycle of HBV and accelerate the development of HCC. Similar observations were made by Lee et al. Multivariate logistic regression analysis was informative as it confirmed our finding that H94Y, IT and FY mutations in HBx can be useful as independent prognostic markers of HBV-associated liver disease evolution and advancement, especially when other contributing factors, such as viral load, exist.
Such finding could yield better management of HBV-infected patients before progression to liver complications. However, these findings should be interpreted with caution as the number of subjects who showed LC and HCC is limited in this study. In conclusion, the present study confirmed that mutations in HBx are frequent and found in patients exhibiting different manifestation of HBV-associated liver complications.
By comparing the full-length HBx sequences, several point and combinational mutations correlating with severity and progressive clinical phases of HBV infection were identified.
The Enigmatic X Gene of Hepatitis B Virus
Although the significant identified single mutations correlated well with the development of HCC, the synergistic effect of combinational mutations may have a role in accelerating progression to HCC. These mutations can be useful in predicting the clinical outcome of HBV-infected patients and may serve as early markers of high risk of developing HCC.
Prospective studies exploring the functional mechanism of the mutations identified in this study can improve the understanding of the progression of liver disease in HBV-infected patients in the Saudi population. A total of Saudi patients infected with HBV were analyzed in this study. The study protocol was approved by the institutional review boards of all bbv and conformed xgeme the Declaration of Helsinki. All subjects that participated in this study signed an informed consent prior to the enrollment in hbf study.
Patients were grouped into four different categories based on disease severity as follows: A nested PCR protocol was used xene amplify and detect mutations using primers hvb the entire region of the X gene Supplementary Table 5.
Nested primers were tagged with M13 sequence for direct sequencing after amplifications. The PCR conditions used were the same as in the first round with only the number filetyppe cycles reduced to A negative control was included in the amplification process. Purified PCR products were directly sequenced with the tagged M13 sequence forward and reverse primers in both directions.
The HBx protein sequence was submitted to the ConSurf server [ 38 ] hgv As no 3D structure is available for this protein, the analysis was run in ConSeq mode. A total of sequences were retrieved and a multiple sequence alignment was built using MAFFT [ 40 ]. Approximately sequences were identified as significant hits. Sequences with N- or C-terminal overhangs were removed and a final set of sequences were realigned. The alignment was filtered for the most diverse sequences using the program hhfilter available from the hhsearch package [ 41 ].
Secondary structure prediction was performed using the PsiPred server [ 42 ]. To predict the three dimensional 3D structure of the HBx protein, the amino acid sequence was submitted to the Phyre2 server www.