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Phase 4 (2009 Deadline)
Hepatitis B Virus-Associated Hepatocellular Carcinoma in Pakistan
Aleem Siddiqui, University of California, San Diego
Hajra Sadia (beginning January 2012) (previous PI Ishtiaq Qadri, until October 2011), National University of Sciences and Technology
Pakistani Funding (Department of State): $358,906
US Funding (Department of State): $311,990
Project Dates on US Side: November 15, 2010 - November 14, 2013 (Completed)
Project Overview
Human hepatitis B virus (HBV) infection is one of the leading causes of chronic hepatitis, which increases the risk for the development of hepatocellular carcinoma (HCC). There are an estimated 350 million chronic carriers of hepatitis B worldwide, and in Pakistan the incidence of HBV infection is alarmingly high. HBV is classified into eight genotypes, each with a distinct geographical distribution, which can be used in tracing the evolution and transmission of the virus. Differences between genotypes affect the disease severity, course and likelihood of complications, and response to treatment. The researchers in this collaborative project will characterize the HBV genotypes in Pakistan and determine the prevalence of specific genetic mutations that place hepatitis B chronic patients at high risk of liver failure and HCC. Early diagnosis for HCC has significant advantages for preventing the incidence of full-blown HCC. This epidemiological study should benefit the future prognosis of patients suffering from HCC and will also provide valuable training opportunities for Pakistani students, research fellows, and clinicians in molecular studies of infectious diseases.
Quarterly Update
The project is aimed at establishing the associations between HBV genotype(s), genetic mutations and the risk of HCC in chronic hepatitis B patients. The work done so far led to the isolation of about 90 serum samples from different regions of Pakistan and molecular characterization by serology, PCR amplification methods, genotyping, molecular cloning etc. The results showed that HBV genotype D is the most common in Pakistan. Mutations were identified within HBV genome as noted for liver cancer patients. Overall, these results suggest that HBV mutations may be one of the principal characteristics in the pathogenesis of HCC, as has been predicted. More work is however needed to establish firmly the underlying mechanism(s) by which mutations and the genotype play in the genesis of liver cancer associated with HBV infection.
The Pakistani side is in process of purchasing the Gradient PCR (Polymerase Chain Reaction) and High Speed Ultracentrifuge. The Ultracentrifuge will not only be used for isolation of HBV particles for this particular project, but also for isolation of other viruses by other researchers and students in the school. Four undergraduate students have completed their designated projects. Five Graduate students are currently working on different projects, four of which have completed their M.Phil and now continuing into PhD. A number of consultant gastroenterologist and hepatologist from various institutions across Pakistan are collaborating in this project (Aga Khan University (Karachi), Dow University of Health Sciences (Karachi), Shifa School of Medicine (Islamabad), Pakistan Institute of Medical Sciences (Islamabad), Poly Clinic Hospital (Islamabad), Holy Family Hospital (Rawalpindi), Nuclear Medicine Oncology and Radiation Institute (Islamabad). On the US side, four postdoctoral fellows carried out the project on HBV X protein’s role in mitochondrial metabolism. They have not been able to clone the HBx gene from Pakistani patients’ sera to continue to explore its functional relevance to HCC, mainly due to the departure of the original collaborator Dr. Qadri.
Progress Reports
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2011 Show summary || Hide summary
(Note: This project was suspended from October 5, 2011, until January 24, 2012, in connection with Dr. Qadri's departure from NUST. A new principal investigator has now been authorized so that the planned research and training activities may continue. The following is the most recent update summary covering activities on the project prior to its suspension. New information will be added in April 2012 when these researchers submit their next quarterly report.) Following up on initial discussions between the US and Pakistani PIs at the March 2011 S&T conference in Dubai, work on the project intensified once NUST received its grant funds from HEC in April. A prospective study was conducted in 10 HBV-infected patients identified randomly in different tertiary care hospitals of Islamabad, which led to the characterization of the hepatitis B virus sequence variations in hepatitis B virus surface antigen (HBsAg). Several novel mutations were identified after sequencing of the cloned surface gene ORF. In addition, other reported mutations affecting the binding of the antigen to antibodies were also detected. To sequence the pre-core and core regions of the hepatitis B virus and investigate the prevalence of point mutations of hepatitis B virus DNA in the infected Pakistani population, blood samples of 9 chronic hepatitis B patients were studied. Sequence analysis revealed high prevalence of point mutations in the pre-core and core regions. Three point mutations were reported for the first time in chronic HBV-infected Pakistani patients, with one of these mutations appearing to be associated with high morbidity and mortality. Further study will be done in the coming months to explore the significance of these and other mutations, and additional patient samples are being collected by the Pakistani team on an ongoing basis. Meanwhile, Dr. Qadri visited Dr. Siddiqui at UCSD for two weeks in late July and early August 2011 to collaborate on data analysis and plan future experimental activity.
2012 Show summary || Hide summary
During the second quarter of 2012, sera from HBV-infected HCC and non-HCC patients in Pakistan are being collected in collaboration with various hospitals, clinics, and diagnostic labs in Karachi, Islamabad, and Rawalpindi. The team at NUST obtained the clinical data and recorded the viral load, and samples with high viral titer were processed for further study. Genes from five samples have been cloned and sequenced. The team has obtained some preliminary data from the analysis and is waiting for the U.S. team to send some HBV clones to serve as wild type controls to compare the mutated sequences in Pakistani HBV-positive sera. In the U.S. laboratory, the team has initiated work on HBx and extensively utilized confocal microscopy to walk down the pathway of mitophagy. Tentatively next year, Dr. Siddiqui is planning to host a Pakistani researcher after he or she first completes training from Dr. Sadia.
2013 Show summary || Hide summary
During the second quarter of 2012, sera from HBV-infected HCC and non-HCC patients in Pakistan are being collected in collaboration with various hospitals, clinics, and diagnostic labs in Karachi, Islamabad, and Rawalpindi. The team at NUST obtained the clinical data and recorded the viral load, and samples with high viral titer were processed for further study. Genes from five samples have been cloned and sequenced. The team has obtained some preliminary data from the analysis and is waiting for the U.S. team to send some HBV clones to serve as wild type controls to compare the mutated sequences in Pakistani HBV-positive sera. In the U.S. laboratory, the team has initiated work on HBx and extensively utilized confocal microscopy to walk down the pathway of mitophagy. Tentatively next year, Dr. Siddiqui is planning to host a Pakistani researcher after he or she first completes training from Dr. Sadia.
On Pakistani side, since last quarter Zaira Rehman, faculty member has been focusing on mutational analyses of preS1 region with the aim of identifying mutations in various functional domains of the region, in order to identify mutations in various functional domains of the region. This region of HBV DNA serves many important functions in the HBV liver disease pathogenesis including hepatocellular carcinoma. The team has proven a high rate of mutation in basal core promoter region with nucleotide insertions. In addition, they determined the sequences of different HBV genome regions other than preS1 region (i.e. pre-S2, basal core promoter, and polymerase gene). The Pakistani team is currently working on compiling the characterization of preS1 region in a manuscript form for further publication, which will be the team’s goal for the next three months. The US team has been focusing on investigating the relevant of HBV X protein in inducing mitochondrial dynamics. Their recent work suggests that HBx induces mitophagy (elimination of damaged mitochondria) and fission activities. Furthermore, these effects prevent apoptosis or cell death thus facilitating chronic hepatitis. Avoiding cell death will pave the way to initiate liver oncogenesis (cancer). The data was obtained by silencing a mitochondrially-translocated gene called Parkin. When silenced for Parkin, cells initiated the cell death program. This important finding and other data will be compiled in a manuscript form in the coming three months.
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