- Email: [email protected]
Inhibition of transthyretin-met30 expression with inosine15.1-hammerhead ribozymes in cell culture
April 2000
following novel SNPs were detected: C-730T, G-646A, A2132G, G2164A, G2490A, G3045A and C3648T. High-throughput genotyping of a subset of these and published SNPs was performed using the TaqMan 7700 Sequence Detection System for di-allelic discrimination. Genotypes in a panel of 350 IBD families were analysed for association with either the CD or UC phenotypes, using the transmission disequilibrium test. No significant association of any of the NRAMP2 SNPs with the IBD phenotype was observed. Linkage disequilibrium analysis between the SNPs found them to fit into 3 distinct groups. Physical proximity of SNPs to each other was not indicative of the extent of disequilibrium between them. These findings suggest high density SNP typing may be required to detect associations between candidate genes and IBD. 3080 THE ROLE OF THE PRIMER SELECTION ON THE DETECTION RATE OF THE HFE GENE MUTATION IN PATIENTS WITH LIVER DISEASE. Kenneth Ocran, Janine Genschel, Guido Schachschal, Bettina Bochow, Heiko Mix, Carsten A. Buettner, Jens Jetschmann, Matthias Plauth, Hartmut H-J Schmidt, Herbert Lochs, Campus Charite Mitte, Berlin, Germany. The homozygous mutation of the Cys282Tyr within the HFE gene is primarily responsible for hereditary hemochromatosis. Molecular diagnosis of this mutation is increasingly being used especially for presymptomatic screening of individuals with suspected disease or of affected family members. Recently Jeffrey et al. (Nature Genet, 22:325; 1999) suggested a possible overestimation of Cys282Tyr homozygosity in hereditary hemochromatosis using the primers initially reported by Feder et al. (Nature Genet 13:399-408;1996). We screened 218 patients in our outpatient clinic with elevated liver enzymes for the presence of the Cys282Tyr and the His63Asp mutation. PCR was first performed using primers described by Feder et al. Restriction digests were performed directly in the PCR mixes by the addition of Rsa I (codon 282) or Bel I (codon 63). We identified 18 subjects with a heterozygosity for the Cys282Tyr mutation, six patients were compound heterozygote for Cys282TyrIHis63Asp, and five subjects were homozygous for the Cys282Tyr mutation. The prevalence of homozygotes in our selected population was I in 44. In addition, subjects found to be Cys282Tyr heterozygous, homozygous or C282Y1H63D compound heterozygous were re-examined using the new primer set published by Jeffrey et al. All 34 studied alleles were confirmed by this reaction. Thus we did not observe any false positive results. We conclude that in our selected patient population the prevalence of Cys282Tyr homozygous subject was higher than recently reported. The polymorphism 5569G/A within the HFE gene had no effect on the frequency of detection of the Cys282Tyr mutation in this studied population. 3081 NEURAL CREST CELL (NCC) RECEPTORS AND NEUROTRO· PHINS IN THE DEVELOPING MURINE ENTERIC NERVOUS SYSTEM. Jolanta E. Pitera, Virpi V. Smith, Peter 1. Milia, Institute of Child Health, London, United Kingdom. The enteric nervous system is derived from cells which colonise the fetal gut, migrating in a rostro-caudal direction from the vagal and trunkal neural crest. Mesenchymal neurotrophic factorslret coreceptor Gdnf/GFRa I, neuturinlGFRa2 and their NCC receptor c-ret as well as Sox-IO and hoxb-5 are crucial for survival/function/differentiation of NCC derived enteric neurons. Gdnf/GFRal and neuturinlGFRa2 complexes bind to c-ret which activates a downstream signal transduction pathway. It has been suggested that receptor/ligand expression is developmentally regulated and mutations in genes encoding them result in enteric nervous system abnormalities. We have studied the temporal and spatial distribution of ligand and receptor transcripts during gut development and compared that with the expression of enteric neural crest markers employing mRNA in situ hybridisation in whole mounts of dissected gut primordia from mouse embryos 10.5-16.5 days post coitum (dpc). The expression showed dynamic rostro-caudal gradients with unique complementary and overlapping patterns along the gut. Sox-lO was expressed in early neural crest cells migrating rostrocaudally along the lateral and dorsal oesophagus, colonising the stomach at 10.5dpc. This migration pathway and foregut colonisation by neural crest cells expressing Sox-lO seemed to be shared by NCC expressing c-ret but not hoxb-S, which appeared more caudal, in the stomach at lO.5dpc. Gdnf/GFRa 1 and neuturinlGRRa2 were expressed in the mesenchyme and developing muscle. Gdnf expression began in the oesophagus, and postcaecal gut at 1O.5dpcand from 11.5-13.5dpc expression became stronger in the caudal gut. Neuturin expression however began in the stomach. Such nested caudal expression seemed to correlate with the expression of Hox genes from paralogous groups 4 and 5 important in gut patterning. This suggests a functional relationship between neurotrophic factors and these developmental control genes.
AGAA595
3082 INHIBITION OF TRANSTHYRETIN·MET30 EXPRESSION WITH INOSINE1s.1·HAMMERHEAD RIBOZYMES IN CELL CULTURE. Marcus 1. Proepsting, Janine Genschel, Carsten A. Buettner, Stefan Kubicka, Peter Baier, Michael P. Manns, Herbert Lochs, Hartmut H. Schmidt, Campus Charite Mitte, Berlin, Germany; Med Hochschule Hannover, Hannover, Germany. The most common cause of hereditary amyloidosis is the val30met mutation in the transthyretin protein (TTR-met30). The mutation is caused by a mononucleic substitution from G to A (GVC to AVC) at position 173 in the transthyretin gene resulting in the exchange for the amino acids valine to methionine in the corresponding protein sequence. The devastating course of clinical amyloidosis is caused by the aggregation of proteins in clear structures forming extracellular fibrils containing f3-sheet components of TTR-met30. The primarily involved organs in this process represent the nervous system, kidney, and heart resulting in the phenotype of HA. At present liver transplantation at the time of occurrence of first clinical pathological symptoms is the only therapeutic option. Therefore, the establishment of new gene therapeutic concepts will be a promising option. The aim of our work was the development of a specific cleavage of TTR-met30 mRNA using hammerhead ribozymes. We chemically modified nuclease stable Inosine' 5"-Hammerhead ribozymes to target the TIRmet30 mRNA with hi¥h specificity Q3BRC 260, 313-7;1999). The exchange of adenosine IS. with inosine I .1 in the catalytic core of the hammerhead ribozvme resulted in a change of the cleavable target sequence from NI6.2Ulo.IHI7 to NI6.2CI6.1HI7 without loss in ribozymal activity (Nucl. Acids Res. 1998;26:2279-85). Subsequently, we used as cell culture model the wildtype human normal hnTTR expressing cell line HepG2. For TTR-met30 experiments we used the stable transfected cell line 293-TTRmet30. We cleaved the TTR-met30 and hnTTR mRNA with specific nuclease stable chemically modified Inosine1s.1-Hammerhead ribozymes and analyzed the protein after immunoprecipitation and subsequent western-blotting. These experiments revealed the downregulation of the TTR protein using this approach. We were able specifically to target the TTRmet30 and hnTTR expression, respectively, also in the cell culture system. The ribozymes were given to cell culture medium with and without liposomes as transfection system. The transfection rate and thus the therapeutic effect was improved using cationic liposomes by 2 fold. In HepG2 cells the nhTTR concentration was downregulated to a level of 39% and the TTR-met30 concentration in 293-met30 cells to a level of 68%. The successful employment of InosineI 5.I-Hammerhead ribozymes in cell culture is therefore a promising tool for the development of a gene therapeutic strategy for hereditary amyloidosis. 3083 BONE MINERAL DENSITY MEASUREMENTS IN FEMALE HETEROZYGOTES FOR CYSTIC FmROSIS. Miklos Toth, Rita Ujhelyi, Pal Miheller, Miklos Szathmari, Monika Horvath, Tivadar Tulassay, Zsolt Tulassay, 2nd Dept of Medicine, Semmelweis Vniv of Medicine, Budapest, Hungary; Heim Pal Childrens Hosp, Budapest, Hungary; 1st Dept of Medicine, Semmelweis Univ of Medicine, Budapest, Hungary; 1st Dept of Pediatric, Semmelweis Vniv of Medicine, Budapest, Hungary. Background/aims: Children and young adults with cystic fibrosis (CF) are frequently reported to have low bone mass and osteoporotic fractures. The pathomechanism of reduced bone mass in these patients is not fully understood. CF heterozygotes are reported to have altered cellular functions linked to active ion transport. Osteoclasts are actively acidifying cells and do have a number of ion channels. There are no reports on bone mass measurements in CF heterozygotes. Therefore the aim of the present study was to examine whether the CF heterozygote status is related to bone mass. Patients and methods: 42 mothers of CF patients diagnosed by measurement of increased sweat chloride concentration and molecular genetic techniques as well as 120 healthy females were studied. The mean ages of CF mothers and that of the healthy females were 38.4 and 43.5 years, resp. There were no significant differences in height (163.1 ±6.0 vs. l63.0±6.0 em), weight (65.5±12.5 vs. 66.6±1!.8 kg) and body mass index (24.7±4.9 vs. 25.1 ±4.3 kg/m2) between the CF mothers and healthy controls. BMD measurements were performed by DEXA at the lumbar spine (L2-L4), left femoral neck, trochanteric and intertrochanteric regions and the distal third of nondominant radius using a Hologic QDR 4500C DEXA instrument. Results: see Table below Conclusions: These data indicate that CF gene carriership is associated with decreased mineral content of axial bones and may have implication in relation to the pathophysiology of osteoporosis. The pathomechanism by which the CF gene product (CFTR protein) determine bone mass should be investigated. BMD z-scores ofCF heterozygote mothers and healthy controls (mean±SD)
Lumbar spine Femoral neck Trochanteric region Intertroch. region Radius distal 1/3
CF mothers
Healthy controls
pvalue
-0.473 ± 1.189 -0.321 ± 1.134 -0.137± 1037 -0.321 ± 1.014 -0.253 ± 0638
-0.034 ± 1.188 +O.142± 1.058 +0.293 ± 1.026 +0.113 ± 0.962 -0.216 ± 1.007
p < 0.05 p < 0.05 p < 0.05 p < 0.05 NS
following novel SNPs were detected: C-730T, G-646A, A2132G, G2164A, G2490A, G3045A and C3648T. High-throughput genotyping of a subset of these and published SNPs was performed using the TaqMan 7700 Sequence Detection System for di-allelic discrimination. Genotypes in a panel of 350 IBD families were analysed for association with either the CD or UC phenotypes, using the transmission disequilibrium test. No significant association of any of the NRAMP2 SNPs with the IBD phenotype was observed. Linkage disequilibrium analysis between the SNPs found them to fit into 3 distinct groups. Physical proximity of SNPs to each other was not indicative of the extent of disequilibrium between them. These findings suggest high density SNP typing may be required to detect associations between candidate genes and IBD. 3080 THE ROLE OF THE PRIMER SELECTION ON THE DETECTION RATE OF THE HFE GENE MUTATION IN PATIENTS WITH LIVER DISEASE. Kenneth Ocran, Janine Genschel, Guido Schachschal, Bettina Bochow, Heiko Mix, Carsten A. Buettner, Jens Jetschmann, Matthias Plauth, Hartmut H-J Schmidt, Herbert Lochs, Campus Charite Mitte, Berlin, Germany. The homozygous mutation of the Cys282Tyr within the HFE gene is primarily responsible for hereditary hemochromatosis. Molecular diagnosis of this mutation is increasingly being used especially for presymptomatic screening of individuals with suspected disease or of affected family members. Recently Jeffrey et al. (Nature Genet, 22:325; 1999) suggested a possible overestimation of Cys282Tyr homozygosity in hereditary hemochromatosis using the primers initially reported by Feder et al. (Nature Genet 13:399-408;1996). We screened 218 patients in our outpatient clinic with elevated liver enzymes for the presence of the Cys282Tyr and the His63Asp mutation. PCR was first performed using primers described by Feder et al. Restriction digests were performed directly in the PCR mixes by the addition of Rsa I (codon 282) or Bel I (codon 63). We identified 18 subjects with a heterozygosity for the Cys282Tyr mutation, six patients were compound heterozygote for Cys282TyrIHis63Asp, and five subjects were homozygous for the Cys282Tyr mutation. The prevalence of homozygotes in our selected population was I in 44. In addition, subjects found to be Cys282Tyr heterozygous, homozygous or C282Y1H63D compound heterozygous were re-examined using the new primer set published by Jeffrey et al. All 34 studied alleles were confirmed by this reaction. Thus we did not observe any false positive results. We conclude that in our selected patient population the prevalence of Cys282Tyr homozygous subject was higher than recently reported. The polymorphism 5569G/A within the HFE gene had no effect on the frequency of detection of the Cys282Tyr mutation in this studied population. 3081 NEURAL CREST CELL (NCC) RECEPTORS AND NEUROTRO· PHINS IN THE DEVELOPING MURINE ENTERIC NERVOUS SYSTEM. Jolanta E. Pitera, Virpi V. Smith, Peter 1. Milia, Institute of Child Health, London, United Kingdom. The enteric nervous system is derived from cells which colonise the fetal gut, migrating in a rostro-caudal direction from the vagal and trunkal neural crest. Mesenchymal neurotrophic factorslret coreceptor Gdnf/GFRa I, neuturinlGFRa2 and their NCC receptor c-ret as well as Sox-IO and hoxb-5 are crucial for survival/function/differentiation of NCC derived enteric neurons. Gdnf/GFRal and neuturinlGFRa2 complexes bind to c-ret which activates a downstream signal transduction pathway. It has been suggested that receptor/ligand expression is developmentally regulated and mutations in genes encoding them result in enteric nervous system abnormalities. We have studied the temporal and spatial distribution of ligand and receptor transcripts during gut development and compared that with the expression of enteric neural crest markers employing mRNA in situ hybridisation in whole mounts of dissected gut primordia from mouse embryos 10.5-16.5 days post coitum (dpc). The expression showed dynamic rostro-caudal gradients with unique complementary and overlapping patterns along the gut. Sox-lO was expressed in early neural crest cells migrating rostrocaudally along the lateral and dorsal oesophagus, colonising the stomach at 10.5dpc. This migration pathway and foregut colonisation by neural crest cells expressing Sox-lO seemed to be shared by NCC expressing c-ret but not hoxb-S, which appeared more caudal, in the stomach at lO.5dpc. Gdnf/GFRa 1 and neuturinlGRRa2 were expressed in the mesenchyme and developing muscle. Gdnf expression began in the oesophagus, and postcaecal gut at 1O.5dpcand from 11.5-13.5dpc expression became stronger in the caudal gut. Neuturin expression however began in the stomach. Such nested caudal expression seemed to correlate with the expression of Hox genes from paralogous groups 4 and 5 important in gut patterning. This suggests a functional relationship between neurotrophic factors and these developmental control genes.
AGAA595
3082 INHIBITION OF TRANSTHYRETIN·MET30 EXPRESSION WITH INOSINE1s.1·HAMMERHEAD RIBOZYMES IN CELL CULTURE. Marcus 1. Proepsting, Janine Genschel, Carsten A. Buettner, Stefan Kubicka, Peter Baier, Michael P. Manns, Herbert Lochs, Hartmut H. Schmidt, Campus Charite Mitte, Berlin, Germany; Med Hochschule Hannover, Hannover, Germany. The most common cause of hereditary amyloidosis is the val30met mutation in the transthyretin protein (TTR-met30). The mutation is caused by a mononucleic substitution from G to A (GVC to AVC) at position 173 in the transthyretin gene resulting in the exchange for the amino acids valine to methionine in the corresponding protein sequence. The devastating course of clinical amyloidosis is caused by the aggregation of proteins in clear structures forming extracellular fibrils containing f3-sheet components of TTR-met30. The primarily involved organs in this process represent the nervous system, kidney, and heart resulting in the phenotype of HA. At present liver transplantation at the time of occurrence of first clinical pathological symptoms is the only therapeutic option. Therefore, the establishment of new gene therapeutic concepts will be a promising option. The aim of our work was the development of a specific cleavage of TTR-met30 mRNA using hammerhead ribozymes. We chemically modified nuclease stable Inosine' 5"-Hammerhead ribozymes to target the TIRmet30 mRNA with hi¥h specificity Q3BRC 260, 313-7;1999). The exchange of adenosine IS. with inosine I .1 in the catalytic core of the hammerhead ribozvme resulted in a change of the cleavable target sequence from NI6.2Ulo.IHI7 to NI6.2CI6.1HI7 without loss in ribozymal activity (Nucl. Acids Res. 1998;26:2279-85). Subsequently, we used as cell culture model the wildtype human normal hnTTR expressing cell line HepG2. For TTR-met30 experiments we used the stable transfected cell line 293-TTRmet30. We cleaved the TTR-met30 and hnTTR mRNA with specific nuclease stable chemically modified Inosine1s.1-Hammerhead ribozymes and analyzed the protein after immunoprecipitation and subsequent western-blotting. These experiments revealed the downregulation of the TTR protein using this approach. We were able specifically to target the TTRmet30 and hnTTR expression, respectively, also in the cell culture system. The ribozymes were given to cell culture medium with and without liposomes as transfection system. The transfection rate and thus the therapeutic effect was improved using cationic liposomes by 2 fold. In HepG2 cells the nhTTR concentration was downregulated to a level of 39% and the TTR-met30 concentration in 293-met30 cells to a level of 68%. The successful employment of InosineI 5.I-Hammerhead ribozymes in cell culture is therefore a promising tool for the development of a gene therapeutic strategy for hereditary amyloidosis. 3083 BONE MINERAL DENSITY MEASUREMENTS IN FEMALE HETEROZYGOTES FOR CYSTIC FmROSIS. Miklos Toth, Rita Ujhelyi, Pal Miheller, Miklos Szathmari, Monika Horvath, Tivadar Tulassay, Zsolt Tulassay, 2nd Dept of Medicine, Semmelweis Vniv of Medicine, Budapest, Hungary; Heim Pal Childrens Hosp, Budapest, Hungary; 1st Dept of Medicine, Semmelweis Univ of Medicine, Budapest, Hungary; 1st Dept of Pediatric, Semmelweis Vniv of Medicine, Budapest, Hungary. Background/aims: Children and young adults with cystic fibrosis (CF) are frequently reported to have low bone mass and osteoporotic fractures. The pathomechanism of reduced bone mass in these patients is not fully understood. CF heterozygotes are reported to have altered cellular functions linked to active ion transport. Osteoclasts are actively acidifying cells and do have a number of ion channels. There are no reports on bone mass measurements in CF heterozygotes. Therefore the aim of the present study was to examine whether the CF heterozygote status is related to bone mass. Patients and methods: 42 mothers of CF patients diagnosed by measurement of increased sweat chloride concentration and molecular genetic techniques as well as 120 healthy females were studied. The mean ages of CF mothers and that of the healthy females were 38.4 and 43.5 years, resp. There were no significant differences in height (163.1 ±6.0 vs. l63.0±6.0 em), weight (65.5±12.5 vs. 66.6±1!.8 kg) and body mass index (24.7±4.9 vs. 25.1 ±4.3 kg/m2) between the CF mothers and healthy controls. BMD measurements were performed by DEXA at the lumbar spine (L2-L4), left femoral neck, trochanteric and intertrochanteric regions and the distal third of nondominant radius using a Hologic QDR 4500C DEXA instrument. Results: see Table below Conclusions: These data indicate that CF gene carriership is associated with decreased mineral content of axial bones and may have implication in relation to the pathophysiology of osteoporosis. The pathomechanism by which the CF gene product (CFTR protein) determine bone mass should be investigated. BMD z-scores ofCF heterozygote mothers and healthy controls (mean±SD)
Lumbar spine Femoral neck Trochanteric region Intertroch. region Radius distal 1/3
CF mothers
Healthy controls
pvalue
-0.473 ± 1.189 -0.321 ± 1.134 -0.137± 1037 -0.321 ± 1.014 -0.253 ± 0638
-0.034 ± 1.188 +O.142± 1.058 +0.293 ± 1.026 +0.113 ± 0.962 -0.216 ± 1.007
p < 0.05 p < 0.05 p < 0.05 p < 0.05 NS