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Variations in the NS5A gene of hepatitis C virus in response to interferon alpha therapy
Başlık:
Variations in the NS5A gene of hepatitis C virus in response to interferon alpha therapy
Yazar:
McKechnie, Victoria Margaret, author.
ISBN:
9780438060388
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (305 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 76-08C.
Advisors: Liz McCruden.
Özet:
Hepatitis C virus (HCV) is a major cause of chronic liver disease with most infected individuals becoming long-term carriers of the virus with the increased risk of developing cirrhosis and hepatocellular carcinoma. The only approved treatment for chronic HCV infection is interferon-alpha (IFN-alpha), an expensive, parenterally administered drug with relatively poor efficacy and almost universal side effects. It would therefore, be of great importance if those patients most likely not to respond to IFN treatment could be predicted prior to the start of the therapy. Demographic and viral factors have been suggested as possible predictive markers of treatment outcome although no single factor could accurately identify who would respond to treatment. The first evidence for such a marker was provided by a Japanese group (Enomoto et al., 1995, 1996) who identified a 40 amino acid interferon sensitivity determining region (ISDR) between amino acids 2209 and 2248 (numbering according to HCV-J; Kato et al., 1990) in the NS5A gene of genotype lb HCV. All patients with an ISDR amino acid sequence identical to that of the full-length published sequence HCV-J, referred to as the "wild-type", were non-responders (NR). All those with more than 3 amino acid differences in this region were sustained responders, "mutant type". The majority of patients with an HCV ISDR with 1-3 differences from HCV-J, "intermediate type", did not respond to IFN treatment. To determine whether the putative ISDR amino acid sequence, or any other region of the NS5A gene could be used to predict response to IFN therapy, a prospective study of 35 HCV-infected individuals was initiated. HCV RNA was extracted from pretreatment serum samples and almost the whole NS5A gene amplified in 2 sections by reverse transcription and nested polymerase chain reaction. The amplified cDNAs were analysed by direct nucleotide sequencing. The data obtained did not provide any evidence for the existence of an ISDR at the region identified by Enomoto et al, nor in the remaining portion of the carboxy terminus of the NS5A gene analysed. Amino acid sequence of the NS5A gene could not be used to predict response to IFN treatment in genotype lb, la or 3a HCV-infected individuals. Statistical analysis of other possible prognostic factors identified a relationship between genotype of HCV and an initial response to IFN treatment (virus not detectable after 12 weeks of therapy). This relationship did not hold true for the long term response (virus not detectable after 6 months after therapy stopped). Additional evidence against the presence of a predictive amino acid sequence in the NS5A gene of HCV was provided from sequence data from pre-treatment, during and after IFN treatment serum samples. Sequence variation of the NS5A gene in the absence of therapeutically administered IFN from a control group of 10 patients showed that the majority amino acid sequence of the ISDR and the larger region of the NS5A gene analysed showed no selection during the normal course of HCV infection. Analysis of the study patients who had received IFN therapy again suggested that therapeutically administered IFN does not provide a selective pressure for change in NS5A. SSCP, an electrophoresis technique for the detection of nucleotide sequence variation, was optimised and employed in this study for the analysis of the HCV quasispecies population in vivo. Single stranded DNA was generated from the ISDR PCR products from before, during and after IFN treatment serum samples from the 35 study patients and 10 controls. SSCP analysis showed that HCV quasispecies diversity did not correspond to the outcome of IFN treatment. However, it did indicate that, both in the absence and presence of IFN therapy, the actual variants which make up the quasispecies in an infected individual vary. This provided additional evidence that IFN does not exert selection pressure on NS5A. In an attempt to identify any "hidden" IFN resistant "wild-type" sequences within the quasispecies, to confirm the SSCP data and to provide reagents for functional studies, the entire HCV NS5A gene from a subset of 9 patients was amplified and cloned into the vector pACT2. The ISDR region of 5-11 clones from each patient sample was sequenced. No IFN resistant sequences, as defined by Enomoto et al, were identified and the SSCP data were confirmed, reinforcing the conclusion that the ISDR is not a determinant of response to IFN therapy in the HCV-infected patients studied here.
Notlar:
School code: 0547
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
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XX(684807.1) | 684807-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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