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  • C12Q1/708—Specific hybridization probes for papilloma
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• the present invention relates to methods of predicting the outcome of persistent HBV infection in a subject, as well as the outcome of cytokine therapy, particulariy interferon therapy, in patients suffering from Chronic Hepatitis infection, particularly chronic HBV infection.
• HBV hepatitis B virus
• Viral elimination may be induced by treatment with interferon alpha or lymphoblastoid interferon.
• the response rate for this therapy is limited, eg only around 40% in the case of chronic HBV.
• interferon therapy can also be unpleasant as the majority of patients suffer influenza-type symptoms. More severe side effects include suppression of bone marrow, abnormalities of thyroid function and endogenous depression. Interferon treatment is also expensive and thus there are pressures to rationalise the use of such treatment within the healthcare community.
• TNF alpha gene There is a bi-allelic polymorphism of the promoter for the TNF alpha gene.
• the mutation is located at position -308 icldLive to che transcription initiation sequence of the TNF alpha gene (Mcguire, et al , Nature, 371:508- 510 (1994)) .
• the rare TNF-2 allele is associated with higher constitutive and inducible transcription of the TNF alpha gene.
• the TNF-2 allele is found to be in linkage disequilibrium with the B8-DR3 haplotype m
• TNF2 the rarer allele, known as TNF2
• TNF mhiDits the expression of HBV in HBV transgenic mice (Daniels et al , Lancet , 335:875 (1990) ; Guilhot et al, J. virol . , 67 (12) :7444-9 (1993) ) and TNF production is higher m patients with chronic HBV infection who respond successfully to interferon therapy This phenomenon occurs towards the end of a period of treatment and is therefore not of use in identifying, prior to treatment, patient ⁇ likely to respond to interferon.
• Polymorphism of any cytokine or cytokine promoter including Alpha interferon subtypes, gamma interferon, IL2 , IL4, IL5, IL6 , IL10 and IL12, could also be expected to influence outcome, not only of hepatitis B infection, but also hepatitis C, hepatitis G, human papilloma virus, human immunodeficiency virus and other persistent virus infections. Thus, sensible targeting of cytokine therapy should be possible.
• the present invention provides a method for assessing and/or predicting the probable outcome of treating a subject suffering from a persistent virus infection with a cytokine comprising the step of determining whether the subject carries one or more alleles associated with an improved prooability of a therapeutic response when treated with said cytokine.
• the persistent virus infection is a hepatitis virus infection, particularly hepatitis B.
• the method comprises determining whether the subject carries the TNF-2 allele.
• the present invention provides a method for assessing and/or predicting the probable outcome of hepatitis virus infection m a subject comprising the step of determining whether the subject carries the TNF-2 allele.
• the hepatitis virus infection is hepatitis B virus (HBV) infection
• HBV hepatitis B virus
• the preferred method of carrying out the determination for both these aspects of the invention is to analyse a sample of the subject's DNA.
• a sample can conveniently be obtained from a biological sample, eg a blood sam ⁇ le.
• the DNA obtained from the biological sample will be amplified using techniques well .no n to those skilled n the art, eg PCR techniques.
• the TNF alpha gene region, and more particularly, the TNF alpha promoter region can be amplified.
• Such techniques will involve the use of at least one pair of suitable primers. Suitable primers can be chosen on the basis of the DNA sequence of the cytokine gene in question , eg the TNF alpha gene.
• TNF alpha gene region can mean the whole of the TNF alpha gene or, alternatively, a part thereof. Clearly, however, if only a part is amplified it should include the promoter for the gene since this is where the TNF-2 allele will be found , if it is present .
• the present invention provides a kit for use in a method for assessing and/or predicting the probable outcome of treating a subject suffering from a persistent virus infection with a cytokine, which comprises at least one pair of primers suitable for PCR amplification of at least a portion of the gene coding for the cytokine.
• the present invention provides a kit for use n a method for assessing and/or predicting the probable outcome of hepatitis virus infection in a subject, which comprises at least one pair of primers suitable for PCR amplification of at least a portion of the gene coding for the TNF alpha.
• a group of 14 patients suffering from chronic hepatitis B virus infection were studied. All the patients had been undergoing treatment with either alpha interferon or lymphoblastoid interferon for at least three months.
• TNF-2 allele confers an increased probability of response to interferon.
• HBV core antibody anti-HBc
• HBsAg HBV surface antigen
• HBsAg HBV surface antigen
• People with persistent HBV infection were IgG anti-HBc and HBsAg positive.
• People with acute HBV infection who may not have had sufficient opportunity to eliminate HBsAg ⁇ HBsAg positive and IgM anti-HBc positive) were excluded from the analysis.
• 1 07 people wno ha ⁇ been vaccinated against HBV ( ⁇ 10%) were classified m the uninfected group and were therefore not included in the analysis of the TNF allele frequencies. The five individuals with HIV antibodies ( ⁇ 1%) were also excluded.
• Plasma samples were taken from all participants and stored at -20°C.
• Anti-HBc (total) , anti-HBc (IgM) , HBsAg status and anti-HBs concentration were dtermined cy ELISA according to the manufacturers instructions (Boehringer Mannheim, Kunststoff, Germany) .
• HIV antibodies were dtermined by Wellcozyme ELISA (Wellcome, Beckenham, UK) Positive results were confirmed by western blot .
• the polymerase cnam reaction was used to amplify a 519 base pair fragment of the promoter from position -502 to +17.
• the primer sequences were: 5' -CAAACACAGGCCTCAGGACTC-3 ' 3 ' -AGGGAGCGTCTGCTGGCTG-5'
• the reaction mix was heated to 95°C for 10 minutes then 1.5U Taq DNA polymerase was added.
• the reaction mix was then given 35 cycles of 95°C for 1 minute, 60°C for 1 minute and 72°C for 1 minute m a Perkm-Elmer 9600 Thermocycler with heated lid.
• the PCR product was finally left at 72°C for 10 minutes.
• the PCR product was denatured with 0.4M NaOH, dotted on to nylon membranes (Amersham High-Bond N) using a vacuum manifold and fixed for 1 minute with UV light. Allele- specific oligonucleotides were end labelled with digoxigenm-ddUT? and hybridized to the membrane with 3M TMAC (tetramethyiammonium chloride) solution at 55°C for 1 hour. Excess probe was removed by washing m 2 x SSPE/0.1% SDS at room temperature and subsequently in 3M TMAC at 58°C.
• 3M TMAC tetramethyiammonium chloride
• the membranes were treated with anti- digoxigenm Fab fragments and, after washing to remove excess antibody, they were incubated with Lumigen PPD and exposed to X-ray film for 15-30 minutes. Allele type was scored by two independent observers. The accuracy of this method was confirmed by DNA sequencing.
• oligonucleotide probes wild type 5 ' -AGGGGCATGGGGACGGG-3 ' -308 variant 5 ' -AGGGGCATGAGGACGGG-3 '
• the -238g allele was typed by amplification refractory mutation system PCR using a conserved primer (5'- AAGACCCCCCTCGGAATCA-3 ' ) together with sequence speciric primers for either the -238g allele (5'- AGACCCCCCTCGGAATCG-3' ) or the 238a allele (5'- AAGACCCCCCTCGGAATCA-3' ) to generate a 459bp product.
• each reaction mixture contained the following primers:
• Each 25 ⁇ i reaction mixture contained lOOng genomic DNA, 67mM Tris HCl, 16mM ammonium sulphate, 2mM magnesium chloride, lOO ⁇ M of each dNTP, 0.5 units of Taq polymerase (Biolme) , 0. l ⁇ M of each TNF primer and 0.2 ⁇ M of each c.2 microglobulm primer.
• the mixture was incubated at 95°C for 5 mmutes followed by 5 cycles of (1 minute at 95°C, 1 minute at 67°C, 1 minute at 72°C) , then 25 cycles of (1 minute at 95°C, 1 minute at 62°C, 1 minute at 72°C) and finally 10 mmutes at 72°C.
• the products were resolved on a 2% agarose gel stained with ethidium oromide and visualised under UV light.
• Table 1 Allele frequencies for the TNF ⁇ promoter -308 variants supjects with persistent HBV 'HBsAg positive anti-HBc dgM) negative) and with self limiting HBV infection (anti-HBc (total) positive, HBsAg negative) .
• Table 2 Allele frequencies for the TNF ⁇ promoter -238 variants in subjects with persistent HBV (HBsAg positive anti-HBc (IgM) negative) and with self limiting HBV infection (anti-HBc (total) positive, HBsAg negative) .

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