- Email: [email protected]
The delivery of antiviral genes to hepatitis B virus infected liver cells by baculovirus vectors
14.5
Genetics, genetic diseases, gene therapy
1 P/C10/013 1
) P/C10/015 1
PREVALENCE OF HFE MUTATIONS lN PATIENTS WITB IRON OVERLOADAND IN CONTROLSUBJBCI’SFROMTHE GENERAL BELGIANPOPULATION.
HEMOCHROMATOSIS
K. Dahan’. Y. Horsmans*. V. Lambot’. A. Geubel*. Ch. VerellenDumoulin’. Centre de GCdlique rkdicale’, and Service de Gastroent&ologie2,UCL, St.-LucUniversityHospital,Belgium. From the two mutations discovered in the HFE gene, only C282Y is involved in patienls with genetic hemochromatosis(GH) while the other (H63D) remainsof unclearsignificance.The aims were lo appreciatethe prevalenceof the two identifiedHFE mutationsin hvo groups-ofpatients with or without iron overload. Patients and methods: 2 I I unrelated patients of northernEuropeanorigin were diagnosed as having iron overloadon the basis of serum iron studies(serum transferrinsaturation > 45 % 9grJserum fenitin > 300 pg/L). The group of controls was composed of 140 randomly chosen subjects from the general Belgian population.The C282Y and H63D mutationswere identifiedby PCR and restrictionenzyme cleavage. Results. The prevalenceof lhe 2 mutations was as follows: bon overload Controls Genotype N (140) % C282Y - - H63D N(211) % +I+ -/115 54.5 0 0 __ I +I+ I 3.3 5 3.6 +/+t8 3.8 2 1.4 +/-I13 6.2 15 LO.7 __ I +I28 13.3 46 32. 8 12 51.4 J% 40 19 Conclusions. 1) The first cause of iron overload is associated lo the homozygous C282Y genotype. 2) Concerningthe impact of the double heterozygosity, our data didn’t suggest a clear relationship with iron storage. However, more data must be collected in a new group of patienls, mostly hemochromatosispatient relatives, to correlate the ability of iron overload development with their genotype. Yet, these preliminary dam show the dillkulty but also the need of a commrm attitudein presymptomaticdiagnosis.
EFFECTS OF THERAPEUTIC PHLEBOTOMIES ON GENETIC EPIDEMIOLOGIC
REGARDING
A FRENCH NATIONAL
STUDY ON A SAMPLE OF 498 CATIENTS.
JM Didelot. P Blanc. P Martinez. D Larrev. H Michel. HBpital Saint Eloi. 2, avenue Bertin Sam. 34295 Montpellier Cedex 5 France BACKGROUND AND AIM OF THE STUDY : Since 1947, the only beatment of genetic hemochromatosis (GH) is therapeutic phlebotomy. We studied its efficiency on different complications of GH determined by the first national epidemiologic study ever carried out in France on patients with GH. -Two questionnaires about the clinical symptoms at the onset of the disease were sent in 1997 to 1500 patients with GH of the French Association of Hemochromatosis.498 patients were selected among 754 who answered the first questionnaire because the diagnosis of GH was definied and performed by either therapeutic phlebotomies during more than one year or liver biopsy or serum transferrin saturation > 60% in men and > 50% in women and serum ferritin concentration > 200 pg/l. Among these 498 patients, 371 answered a second questionnaire. more complete about the outset of the disease.The statistical study used a monofactorial analysis of variance followed by a Student’s two-tailed t-test. USULTS; Before treatment, 85% of patients had arthralgia, 80% asthenia, 67% skin abnormalities, 53% hepatomegaly, 39% heart dysfunctions. GH treatment was performed with desferrioxamine in 9% of cases and in 92% with therapeutic phlebotomies. The mean volume of phlebotomies were 413 ml for all, 422 ml in male and 389 ml in female (p=O.OOOl). They were performed in mean 10.5 days for all, 9.6 days in male and 12.7 in female (p=O,OO3).During a mean time of treatment of 7 yrs, 91% of the patients had no complications of liver disease but 1% had ascitis, 1% jaundice, 2.5% bleeding esophageal varices and 4.5% hepatocellular carcinoma. Therapeutic phlebotomies improved overall condition in 65% of patient.. Only 5% of patients worsened. After therapeutic phlebotomies, melanodermia improved in 66%, hepatomegaly in 55% and asthenia in 51%. Arthralgia were not improved by this treatment and were even worse than before treatment. CONCLUSION :Therapeutic phlebotomies allow substantial improvement of symptoms of GH particularly asthenia, melanodermia and hepatomegaly. However, athralgia remain unchanged
1 P/C10/014 1
MUTATIONS IN THE HEMOCHROMATOSIS GENE AND RESPONSE TO INTERFERON IN CHRONIC HEPATITIS C M.Sampietro, N.Corbetta. M.Amato, A.Casatta. L.Lupica, M.Mattioli, M Del Vitto, V Moltem, A L l+racanzam, S largion, G.l+iorelli. l&to dz Mediczna Interni k iWopatologzziMt?dzca, lRU3
Ospedale Maf,giore- Universitd di Milano, Italy
THE DELIVERY OF ANTIVIRAL GENES TO HEPATITIS B VIRUS INFECTED LIVER CELLS BY BACULOVIRUS VECTORS W Errinnton, E.D.G. McIntosh, H.C.Thomas and McGarvev MJ Department of Medicine A, Imperial College School of Medicine at St Mary’s, London, UK (e-mail: [email protected]) Recombinant baculoviruses which contain the CAT reporter gene, under the control of a CMV promoter, using the shuttle vector PCRBAC, were previously shown to give a large increase in CAT activity when they were used to infect Huh-7 and HepG2 cells. Non-hepatocyte cell lines showed no increase in the level of CAT activity, and no CAT protein could be detected when they were infected with these baculovirus constructs. Baculovirus constructs which contain the CAT reporter gene and upstream genetic elements (including the NF-El3 sequence), which can be stimulated by the hepatitis B virus (HBV) X protein, have also been made. Co-expression of the HBV X protein in cells infected by the NF-RB CAT recombinant baculovims showed a five-fold stimulation of the CAT activity. To evaluate the ability of this system to deliver genes which may have an anti-viral effect, we have made baculovimses which contain the herpes simplex virus thymidine kinase (HSV-TK) gene which are under the control a CMV promoter or genetic elements which can be stimulated by the HBV X protein. These baculovimses have been used to infect hepatocyte cell lines which express the HBV X protein. A biological assay, based on the inhibition of vaccinia virus replication, has been used to demonstrate the conversion of the anti-viral pro-drug ganciclovir (GCV), to the more potent phosphorylated form in cells which have been infected by recombinant bacnlovimses that express the TK gene. The effects of TE mediated activation of GCV on HBV replication in infected liver cells is currently being investigated.
Genetics, genetic diseases, gene therapy
1 P/C10/013 1
) P/C10/015 1
PREVALENCE OF HFE MUTATIONS lN PATIENTS WITB IRON OVERLOADAND IN CONTROLSUBJBCI’SFROMTHE GENERAL BELGIANPOPULATION.
HEMOCHROMATOSIS
K. Dahan’. Y. Horsmans*. V. Lambot’. A. Geubel*. Ch. VerellenDumoulin’. Centre de GCdlique rkdicale’, and Service de Gastroent&ologie2,UCL, St.-LucUniversityHospital,Belgium. From the two mutations discovered in the HFE gene, only C282Y is involved in patienls with genetic hemochromatosis(GH) while the other (H63D) remainsof unclearsignificance.The aims were lo appreciatethe prevalenceof the two identifiedHFE mutationsin hvo groups-ofpatients with or without iron overload. Patients and methods: 2 I I unrelated patients of northernEuropeanorigin were diagnosed as having iron overloadon the basis of serum iron studies(serum transferrinsaturation > 45 % 9grJserum fenitin > 300 pg/L). The group of controls was composed of 140 randomly chosen subjects from the general Belgian population.The C282Y and H63D mutationswere identifiedby PCR and restrictionenzyme cleavage. Results. The prevalenceof lhe 2 mutations was as follows: bon overload Controls Genotype N (140) % C282Y - - H63D N(211) % +I+ -/115 54.5 0 0 __ I +I+ I 3.3 5 3.6 +/+t8 3.8 2 1.4 +/-I13 6.2 15 LO.7 __ I +I28 13.3 46 32. 8 12 51.4 J% 40 19 Conclusions. 1) The first cause of iron overload is associated lo the homozygous C282Y genotype. 2) Concerningthe impact of the double heterozygosity, our data didn’t suggest a clear relationship with iron storage. However, more data must be collected in a new group of patienls, mostly hemochromatosispatient relatives, to correlate the ability of iron overload development with their genotype. Yet, these preliminary dam show the dillkulty but also the need of a commrm attitudein presymptomaticdiagnosis.
EFFECTS OF THERAPEUTIC PHLEBOTOMIES ON GENETIC EPIDEMIOLOGIC
REGARDING
A FRENCH NATIONAL
STUDY ON A SAMPLE OF 498 CATIENTS.
JM Didelot. P Blanc. P Martinez. D Larrev. H Michel. HBpital Saint Eloi. 2, avenue Bertin Sam. 34295 Montpellier Cedex 5 France BACKGROUND AND AIM OF THE STUDY : Since 1947, the only beatment of genetic hemochromatosis (GH) is therapeutic phlebotomy. We studied its efficiency on different complications of GH determined by the first national epidemiologic study ever carried out in France on patients with GH. -Two questionnaires about the clinical symptoms at the onset of the disease were sent in 1997 to 1500 patients with GH of the French Association of Hemochromatosis.498 patients were selected among 754 who answered the first questionnaire because the diagnosis of GH was definied and performed by either therapeutic phlebotomies during more than one year or liver biopsy or serum transferrin saturation > 60% in men and > 50% in women and serum ferritin concentration > 200 pg/l. Among these 498 patients, 371 answered a second questionnaire. more complete about the outset of the disease.The statistical study used a monofactorial analysis of variance followed by a Student’s two-tailed t-test. USULTS; Before treatment, 85% of patients had arthralgia, 80% asthenia, 67% skin abnormalities, 53% hepatomegaly, 39% heart dysfunctions. GH treatment was performed with desferrioxamine in 9% of cases and in 92% with therapeutic phlebotomies. The mean volume of phlebotomies were 413 ml for all, 422 ml in male and 389 ml in female (p=O.OOOl). They were performed in mean 10.5 days for all, 9.6 days in male and 12.7 in female (p=O,OO3).During a mean time of treatment of 7 yrs, 91% of the patients had no complications of liver disease but 1% had ascitis, 1% jaundice, 2.5% bleeding esophageal varices and 4.5% hepatocellular carcinoma. Therapeutic phlebotomies improved overall condition in 65% of patient.. Only 5% of patients worsened. After therapeutic phlebotomies, melanodermia improved in 66%, hepatomegaly in 55% and asthenia in 51%. Arthralgia were not improved by this treatment and were even worse than before treatment. CONCLUSION :Therapeutic phlebotomies allow substantial improvement of symptoms of GH particularly asthenia, melanodermia and hepatomegaly. However, athralgia remain unchanged
1 P/C10/014 1
MUTATIONS IN THE HEMOCHROMATOSIS GENE AND RESPONSE TO INTERFERON IN CHRONIC HEPATITIS C M.Sampietro, N.Corbetta. M.Amato, A.Casatta. L.Lupica, M.Mattioli, M Del Vitto, V Moltem, A L l+racanzam, S largion, G.l+iorelli. l&to dz Mediczna Interni k iWopatologzziMt?dzca, lRU3
Ospedale Maf,giore- Universitd di Milano, Italy
THE DELIVERY OF ANTIVIRAL GENES TO HEPATITIS B VIRUS INFECTED LIVER CELLS BY BACULOVIRUS VECTORS W Errinnton, E.D.G. McIntosh, H.C.Thomas and McGarvev MJ Department of Medicine A, Imperial College School of Medicine at St Mary’s, London, UK (e-mail: [email protected]) Recombinant baculoviruses which contain the CAT reporter gene, under the control of a CMV promoter, using the shuttle vector PCRBAC, were previously shown to give a large increase in CAT activity when they were used to infect Huh-7 and HepG2 cells. Non-hepatocyte cell lines showed no increase in the level of CAT activity, and no CAT protein could be detected when they were infected with these baculovirus constructs. Baculovirus constructs which contain the CAT reporter gene and upstream genetic elements (including the NF-El3 sequence), which can be stimulated by the hepatitis B virus (HBV) X protein, have also been made. Co-expression of the HBV X protein in cells infected by the NF-RB CAT recombinant baculovims showed a five-fold stimulation of the CAT activity. To evaluate the ability of this system to deliver genes which may have an anti-viral effect, we have made baculovimses which contain the herpes simplex virus thymidine kinase (HSV-TK) gene which are under the control a CMV promoter or genetic elements which can be stimulated by the HBV X protein. These baculovimses have been used to infect hepatocyte cell lines which express the HBV X protein. A biological assay, based on the inhibition of vaccinia virus replication, has been used to demonstrate the conversion of the anti-viral pro-drug ganciclovir (GCV), to the more potent phosphorylated form in cells which have been infected by recombinant bacnlovimses that express the TK gene. The effects of TE mediated activation of GCV on HBV replication in infected liver cells is currently being investigated.