- Email: [email protected]
Mutation analysis of the HFE gene in hereditary hemochromatosis
Poster Sessions
220
production occurs both in hepatocytes and Kupffer cells, its suppression in the both ceils is clinically important. Rac, a Rho-GTPase, plays a major role in ROS production, and NFkB, a pivotal transcription factor, can be activated by ROS. In this study, we examined whether adenovirusmediated gene transfer with dominant-negative Rac I or super-repressor IkB to the both cells in vivo could attenuate hepatic ischemia-reperfusion injury. Methods and Results: Male SD rats were infected with Ad5RaclN17 or Ad5IkB (S32A/S36A) [5 x 109 pfu/rat] by intravenous injection. At 72 hr after the infections, rats were underwent total clamp of hepatic blood flow for 20 min, followed by reperfusion. Ad5LacZ was used as a control virus. Gene transfer to both hepatocytes and Kupffer cells was confirmed by immunohistochemistry. After reperfusion, prominent increase in s-AST and s-ALT observed in control animals was significantly attenuated in both Ad5Racl- or Ad5IkB-infected rats. Necrosis around the central veins observed by histology was almost blocked by RaclN17 and IkB (S32A/S36A). RaclN17 expression significantly blocked ROS production and nuclear translocation of NFkB. Induction of iNOS mRNA was also attenuated by RaclN17 and IkB (S32A/S36A). Conclusions: Adenovirus-mediated gene transfer with RaclN17 and IkB (S32A/S36A) efficiently blocked hepatic reperfusion injury. Manipulation of Rac 1-NFkB signaling cascade in both hepatocyte and Kupffer cells could be therapeutically useful.
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NEW APPROACHES TO TREAT LECITHIN-CHOLESTEROL ACYLTRANSFERASE (LCAT) DEFICIENCY IN LIVER DISEASE: PROTEIN THERAPY AND MICROENCAPSULATION OF RECOMBINANT CELLS
J.K. Low 1, K.A. Heald 2, S. Schepelmann 1, D.V. Vinogradov 1, A.D. Tagalakis 1'3, I. Graham 3, J.G. Dickson 3, M.C. Winslet 1, J.S. Owen 1. ] Royal Free & University College Medical School;
2 WorcesterRoyal Infirmary," 3Royal Holloway University of London, UK New approaches to treat lecithin-cholesterol acyltransferase (LCAT) deficiency in liver disease: protein therapy and microencapsulation of recombinant cells Low, JK (1); Heald, KA (2); Schepelmann, S (1); Vinogradov, DV (1); Tagalakis, AD (1, 3); Graham, I (3); Dickson, JG (3); Owen, JS (1) (1) Royal Free & University College Medical School, UK; (2) Worcester Royal Infinrmry, UK; (3) Royal Holloway University of London, UK. Plasma LCAT is secreted by hepatocytes; its failure to esterify cholesterol in liver disease causes many cellular and metabolic disturbances. Here, we have produced stably-transfected CHO cells secreting human LCAT and then injected enzyme purified from media, or encapsulated recombinant cells, into the peritioneal cavity of L C A T - / mice. LCAT was detectable in plasma following enzyme injection and it increased cholesterol esterification and normalised lipoprotein pattern. Cells were encapsulated to produce biocompatible and semipermeable alginate-polylysine microcapsules. In vitro, after an initial lag phase, LCAT (1.5/zg/ml/24 h) was secreted for over 90 days and agarose gel electrophoresis showed a mobility pattern similar to the LCAT secreted by non-encapsulated cells. We implanted these microencapsulated cells into peritoneal cavities of L C A T - / - mice; enzyme activity was detected (1.3 /zg/ml) in plasma after 3 days and the relative amount of unesterified cholesterol, with the potential to damage cell membranes, was reduced. We conclude that injection of recombinant enzyme or of encapsulated LCAT-secreting cells are feasible therapies for acquired LCAT deficiency.
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"HOMOZYGOUS" VARIEGATE PORFYRIA
E. Malonova, A.M. Robreau 1, j. Zeman, H. Puy 1, V. Da Silva 1, R. Rosipal, P. Martasek, J.C. Deybach I . Department of Pediatrics, 1st
Medical Faculty, Charles University Prague, Czech Republic; z Centre Franfais des Porphyries, Colombes, France Variegate porphyria (VP) is an autosomal dominant acute hepatic porphyria, characterized by skin and/or neurovisceral symptomatology. The disease is caused by reduced activity of protoporphyrinogen oxidase (PPO). The human PPO gene contains 13 exons and is located on chromosome lq23. In 1984 we published the first case of VP with clinical manifestations in early childhood in Czech siblings, which was recently shown to be caused by compound heterozygosity in the PPO gene. Now we refer another case of VP in a child of Czech origin. The boy, of small posture with shorter fingers and slight mental retardation, developed severe photosensitivity with at the age of two. The plasma fluorescence emission maximum of 626 nm (excitation 400 nm) is pathognomonic for VP, and the increased protoporphyrin (PP-IX) levels in erythrocytes are in agreement with our previous description of increased P-IX levels in all homozygous cases with acute hepatic porphyrias. Denaturing gradient gel electrophoresis (DGGE) of the whole coding region of PPO gene and subsequent sequencing of atypical DGGE pattern led to the description of two abnormal alleles, 1072G > A (Gly358Arg) in exon 10 and IVS10+4 At > G in intron 10. The Gly358Arg mutation is identical with the mutation in siblings originally found and described in the first "homozygous" Czech VP family. Expression of this mutation in E. coli showed 9.5% residual activity as compared to wt PPO. [Supported by grant GAUK 5/2000/C from Charles University, and by INSERM, France]
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MUTATION ANALYSIS OF THE HFE GENE IN HEREDITARY HEMOCHROMATOSlS
O. Kenneth, C.A. Buettner, J. Genschel, M. Steiner, H. Gerl, Wemer P. Schuff, H. Lochs, H.H.J. Schmidt. Med. Klinik Gastroenterologie,
Hepatologie, Endokrinologie, Campus Charite Mine, Germany Homozygosity for the C282Y mutation and compound heterozygosity for the C282Y/H63D mutation account for 80-90% and approximately 7% cases of hereditary hemochromatosis, respectively. For the remainder a number of sequence variants in the HFE gene have been identified. We screened 482 patients in our outpatient clinic with liver or endocrine disorder for the presence of the Cys282Tyr and His63Asp mutations. We identified 13 C282Y homozygous, 18 C282Y/H63D compound heterozygous, 36 C282Y heterozygous and 106 H63D heterozygous. Eight patients presenting hereditary hemochromatosis with neither C282Y nor H63D mutation were further evaluated by sequence analysis of the HFE gene. One Patient (Asian), revealed the new reported homozygous mutation IVS5+I G/A. Interestingly, this patient presented with tertiary adrenal cortical insufficiency. The second (Caucasian) had the previously reported $65C mutation and a RsaI polymorphism (IVS2+4 T/C). The additional analysis of the IVS2+4 T/C polymorphism in 132 unrelated subjects revealed, that 53 patients had either a C282Y or H63D mutation and in 42 of these the polymorphism (IVS2+4 T/C) was identified; 5 in combination with C282Y heterozygosity, 5 with compound C828Y/H63D heterozygosity, and 31 with H63D he. The additional tool of genetic diagnostics in hereditary hemochromatosis is helpful in identifying patients already at a very early stage. The IVS5+I T/C and the combination of a mutation within HFE with IVS2+4 T/C may represent for some of the genetic changes associated with the non-C282Y-mutation in hereditary hemochromatosis. The new IVS5+ 1 G/A mutation may have broader implications in the mutation analysis of hereditary hemochromatosis in asian subjects.
220
production occurs both in hepatocytes and Kupffer cells, its suppression in the both ceils is clinically important. Rac, a Rho-GTPase, plays a major role in ROS production, and NFkB, a pivotal transcription factor, can be activated by ROS. In this study, we examined whether adenovirusmediated gene transfer with dominant-negative Rac I or super-repressor IkB to the both cells in vivo could attenuate hepatic ischemia-reperfusion injury. Methods and Results: Male SD rats were infected with Ad5RaclN17 or Ad5IkB (S32A/S36A) [5 x 109 pfu/rat] by intravenous injection. At 72 hr after the infections, rats were underwent total clamp of hepatic blood flow for 20 min, followed by reperfusion. Ad5LacZ was used as a control virus. Gene transfer to both hepatocytes and Kupffer cells was confirmed by immunohistochemistry. After reperfusion, prominent increase in s-AST and s-ALT observed in control animals was significantly attenuated in both Ad5Racl- or Ad5IkB-infected rats. Necrosis around the central veins observed by histology was almost blocked by RaclN17 and IkB (S32A/S36A). RaclN17 expression significantly blocked ROS production and nuclear translocation of NFkB. Induction of iNOS mRNA was also attenuated by RaclN17 and IkB (S32A/S36A). Conclusions: Adenovirus-mediated gene transfer with RaclN17 and IkB (S32A/S36A) efficiently blocked hepatic reperfusion injury. Manipulation of Rac 1-NFkB signaling cascade in both hepatocyte and Kupffer cells could be therapeutically useful.
~0-~
NEW APPROACHES TO TREAT LECITHIN-CHOLESTEROL ACYLTRANSFERASE (LCAT) DEFICIENCY IN LIVER DISEASE: PROTEIN THERAPY AND MICROENCAPSULATION OF RECOMBINANT CELLS
J.K. Low 1, K.A. Heald 2, S. Schepelmann 1, D.V. Vinogradov 1, A.D. Tagalakis 1'3, I. Graham 3, J.G. Dickson 3, M.C. Winslet 1, J.S. Owen 1. ] Royal Free & University College Medical School;
2 WorcesterRoyal Infirmary," 3Royal Holloway University of London, UK New approaches to treat lecithin-cholesterol acyltransferase (LCAT) deficiency in liver disease: protein therapy and microencapsulation of recombinant cells Low, JK (1); Heald, KA (2); Schepelmann, S (1); Vinogradov, DV (1); Tagalakis, AD (1, 3); Graham, I (3); Dickson, JG (3); Owen, JS (1) (1) Royal Free & University College Medical School, UK; (2) Worcester Royal Infinrmry, UK; (3) Royal Holloway University of London, UK. Plasma LCAT is secreted by hepatocytes; its failure to esterify cholesterol in liver disease causes many cellular and metabolic disturbances. Here, we have produced stably-transfected CHO cells secreting human LCAT and then injected enzyme purified from media, or encapsulated recombinant cells, into the peritioneal cavity of L C A T - / mice. LCAT was detectable in plasma following enzyme injection and it increased cholesterol esterification and normalised lipoprotein pattern. Cells were encapsulated to produce biocompatible and semipermeable alginate-polylysine microcapsules. In vitro, after an initial lag phase, LCAT (1.5/zg/ml/24 h) was secreted for over 90 days and agarose gel electrophoresis showed a mobility pattern similar to the LCAT secreted by non-encapsulated cells. We implanted these microencapsulated cells into peritoneal cavities of L C A T - / - mice; enzyme activity was detected (1.3 /zg/ml) in plasma after 3 days and the relative amount of unesterified cholesterol, with the potential to damage cell membranes, was reduced. We conclude that injection of recombinant enzyme or of encapsulated LCAT-secreting cells are feasible therapies for acquired LCAT deficiency.
[
•
7
]
"HOMOZYGOUS" VARIEGATE PORFYRIA
E. Malonova, A.M. Robreau 1, j. Zeman, H. Puy 1, V. Da Silva 1, R. Rosipal, P. Martasek, J.C. Deybach I . Department of Pediatrics, 1st
Medical Faculty, Charles University Prague, Czech Republic; z Centre Franfais des Porphyries, Colombes, France Variegate porphyria (VP) is an autosomal dominant acute hepatic porphyria, characterized by skin and/or neurovisceral symptomatology. The disease is caused by reduced activity of protoporphyrinogen oxidase (PPO). The human PPO gene contains 13 exons and is located on chromosome lq23. In 1984 we published the first case of VP with clinical manifestations in early childhood in Czech siblings, which was recently shown to be caused by compound heterozygosity in the PPO gene. Now we refer another case of VP in a child of Czech origin. The boy, of small posture with shorter fingers and slight mental retardation, developed severe photosensitivity with at the age of two. The plasma fluorescence emission maximum of 626 nm (excitation 400 nm) is pathognomonic for VP, and the increased protoporphyrin (PP-IX) levels in erythrocytes are in agreement with our previous description of increased P-IX levels in all homozygous cases with acute hepatic porphyrias. Denaturing gradient gel electrophoresis (DGGE) of the whole coding region of PPO gene and subsequent sequencing of atypical DGGE pattern led to the description of two abnormal alleles, 1072G > A (Gly358Arg) in exon 10 and IVS10+4 At > G in intron 10. The Gly358Arg mutation is identical with the mutation in siblings originally found and described in the first "homozygous" Czech VP family. Expression of this mutation in E. coli showed 9.5% residual activity as compared to wt PPO. [Supported by grant GAUK 5/2000/C from Charles University, and by INSERM, France]
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MUTATION ANALYSIS OF THE HFE GENE IN HEREDITARY HEMOCHROMATOSlS
O. Kenneth, C.A. Buettner, J. Genschel, M. Steiner, H. Gerl, Wemer P. Schuff, H. Lochs, H.H.J. Schmidt. Med. Klinik Gastroenterologie,
Hepatologie, Endokrinologie, Campus Charite Mine, Germany Homozygosity for the C282Y mutation and compound heterozygosity for the C282Y/H63D mutation account for 80-90% and approximately 7% cases of hereditary hemochromatosis, respectively. For the remainder a number of sequence variants in the HFE gene have been identified. We screened 482 patients in our outpatient clinic with liver or endocrine disorder for the presence of the Cys282Tyr and His63Asp mutations. We identified 13 C282Y homozygous, 18 C282Y/H63D compound heterozygous, 36 C282Y heterozygous and 106 H63D heterozygous. Eight patients presenting hereditary hemochromatosis with neither C282Y nor H63D mutation were further evaluated by sequence analysis of the HFE gene. One Patient (Asian), revealed the new reported homozygous mutation IVS5+I G/A. Interestingly, this patient presented with tertiary adrenal cortical insufficiency. The second (Caucasian) had the previously reported $65C mutation and a RsaI polymorphism (IVS2+4 T/C). The additional analysis of the IVS2+4 T/C polymorphism in 132 unrelated subjects revealed, that 53 patients had either a C282Y or H63D mutation and in 42 of these the polymorphism (IVS2+4 T/C) was identified; 5 in combination with C282Y heterozygosity, 5 with compound C828Y/H63D heterozygosity, and 31 with H63D he. The additional tool of genetic diagnostics in hereditary hemochromatosis is helpful in identifying patients already at a very early stage. The IVS5+I T/C and the combination of a mutation within HFE with IVS2+4 T/C may represent for some of the genetic changes associated with the non-C282Y-mutation in hereditary hemochromatosis. The new IVS5+ 1 G/A mutation may have broader implications in the mutation analysis of hereditary hemochromatosis in asian subjects.