- Email: [email protected]
Medroxyprogesterone acetate (MPA) is effective in the treatment of ulcerative colitis (UC)
2315
D14S1070, DAD1,TCRA, D14S1003, D14S275). Conclusion: Our linkagedisequilibrium mapping results provide additional evidencefor a true susceptibility locus contributing to the risk of CD in the chromosome 14ql 1-12 region. This region contains several positional candidate genes which may be relevant 1o the pathogeoesisof CD.
Medroxyprogesterone Acetate (MPA) is Effective in the Treatment of Ulcerative Colitis (UC). Craig Aronchick, PennsylvaniaHosp, Philadelphia, PA; Douglas Dalke, Gastroenterology Specialties, PC, Lincoln, NE; Stephen Ionna, TOM Research,Cinncinati, OH; Dennis Rift, Assoc GastroenterologyMedical Group, Anaheim, CA; Michael Schiff, GI Assoc of St Augustine, St. Augustine, FL
2313
Background: MPA, a progestational agent, has shown efficacy in the treatment of Crohn's disease. Aims: This study was conducted to evaluateoral MPA as a treatment of acute flares in adult pts with mild to moderate UC. Methods: Pts with active UC, who had a baseline Disease Activity Index (DAI) between 3 and 10, with diary confirmation of increased stool frequency and/or presenceof blood, sigmoidoscopic confirmation of disease,and stool negative for enteric pathogens or C. difficile toxin were eligible. Cyclosporine, methotrexate, 6mercaptopurine, azathioprine, and aminosalicylatesprior to study entry were permitted but dose change was not permitted during the study. Systemic steroids -< 20 rag/day prednisone equivalent were permitted. Daily diaries were provided to record scores for stool frequency, blood in the stool, abdominal pain, and functional assessment (symptom awareness). Pts were instructed to take 1000 mg MPA every 6 hrs for 8 doses followed by a maintenance dose of 500 mg every 12 hrs through the end of the B week treatment period. Efficacy was assessedby the Investigator's GlobalAssessment(IGA) score after 2, 4 and 8 wks of therapy, and DAI score at Wk 8, Responsewas defined as any decrease in the IGA score or a -> 2point decreasein DAI comparedto baseline.The IGAwas basedon the investigator'sjudgement of the severity of diseaseactivity. The DAI total score was tabulated from the scores for stool frequency and blood in the stool (over the previous 7 days), and the IGA and sigmoidoscopy scores at baseline and Wk 8. Results: Fourteen pts were enrolled; 11 were evaluable for efficacy. Eight pts completed 8 wks; one pt is still on-study. Of the pts who completed Wk 8, 7 of 8 (88%) respondedby IGA and DAI. IGA responseswere establishedby Wk 2. Among pts who responded, 3(43%) showed improvement on sigmoidoscopy at Wk B; 3 pts had no change from baseline, and, for 1 pt (14%), the sigmoidoscopy score worsened. One pt discontinued due to allergic drug reaction; one pt discontined due to jaundice and increased abdominal pain. The latter pt was hospitalizedand confirmed to have pre-existing sclerosing cholangitis with symptom onset prior to study drug initiation. Other adverse events included weight gain and menstrualabnormalities.Serum hepatictransaminaselevelswere not elevated. Conclusion: Preliminary evidence suggests that MPA is safe and effective for the treatment of pts with active UC. Further studies are planned. This study was supported by InKine Pharmaceutical Company, Inc.
2316 Release of 5-ASA from Asacol Appears Independent of Gut Luminal pH in Ulcerative Colitis Sean Nugent, St Bartholomew's and the Royal London Sch of Medicine, London United Kingdom; David Rampton, Omar Obeid, David Evans, Devinder Kumar, St George's Hosp, London United Kingdom
The 1804Locus Shows Linkage Heterogeneity Between Crohn's Disease And
Ulcerative Colitis Richard H. Duerr, M Michael Barmada, Leilei Zhang, Univ of Pittsburgh, Pittsburgh, PA; Jean-Paul Achkar, ClevelandClin Fdn, Cleveland,OH; Robert N. Baldassano,Children's Hosp of Philadelphia,Philadelphia,PA; Daniel E. Weeks, Univ of Pittsburgh, Pittsburgh, PA Background & Aim: We found significant linkage between Crohn's disease(CO) and chromosome 14q11-12 in a genome scan, confirming suggestive linkage between CD and the same locus from a prior study and establishing this locus as the IBD4 locus (Duerr et al. Am J Hum Genet 2000;66:1857-1862). Subsequently,linkage to/BO4 was detected in a third COdominated dataset (Vermeire et al. Gastroenterology2000;118(Suppl 2):A338). Our aim was to determine whether there is linkage to IBD4 in ulcerativecolitis (UC) families or mixed IBD (CD and UC) families, and whether there is linkage heterogeneitybetweenCD and UC at IBD4. Methods: We genotyped 44 UC families containing 73 affected relative pairs, and 81 mixed IBD families containing 171 affected relative pairs, at four microsatellitas spanning 34 cM on chromosome 14ql 1-12. Nonparametriclinkageanalyseswere performedusing GENEHUNTERPLUS. 62 CD families containing 127 affected relative pairs were already genotyped at the four microsatellites in our CD genome scan. We formally tested for linkage heterogeneity between the CD and UC families, and between the CD and mixed IBD families, using two types of simulation studies. In the first simulation study design,we determinedthe significance of our observed Morton's M statistic by simulating its distribution under the null hypothesis of no linkage. The Morton's M statistic is a measure of the differences in the GENEHUNTERPLUS Iod scores between the various groups. In the second simulation study design, we compared the distribution of GENEHUNTER-PLUSIod scores obtained when the total group of families was randomly split into groups of similar size to our CO, UC, and mixed IBD family groups. Results: We found no linkage to chromosome 14q11-12 in either the UC families or the mixed IBD families (Iod = 0 in each group of families). When we compared the CD and UC families in the Morton's M test simulation study, only 7 of 10,000 replicates had a greater M test statistic value than the observedvalue (p = 0.007). When we compared the GENEHUNTER-PLUSIod differences between groups of randomly selected families that were similar in size to our CD and UC family groups, none of 10,0000 replicates had a Iod difference as great as the CD-UCdifferencethat we observed(p < 0.0001). When we compared the CD families and mixed IBD families, both simulation studies gave p values < 0.0001. Conclusions: IBD4 is probably a CD-specific locus and not a generic IBD locus. IBD4 shows significant linkage heterogeneitybetween CO and UC.
2319 Detection Of Possible Inflammatory Bowel Disease-Related Genes Using Microarray Analysis. Elizabeth E. Mannick, Maria-Stella Serrano, L S U Health Science Ctr, New Orleans, LA; Joseph C. Bonomolo, Johns Hopkins Univ, Baltimore, MD; Michael Lau, John N. Udall Jr, Zhiyun Liu, L S U Health Science Ctr, New Orleans, LA
Background:Releaseof 5-ASAfrom the pH-dependentmesalazinepreparation,Asacol, requires exposure of the capsule to pH > 7 for =4 hrs in vitro. It has been suggestedthat luminal pH may be reduced, at least in the colon, in patients with ulcerative colitis (UC), and that bioavailabilityof 5-ASA from Asacol capsules may consequentlybe impaired. Aims: To relate urinary excretion of 5-ASA and N-acetyl-5-ASA to intestinal luminal pH in patients taking Asacol for UC. Methods: In 8 patients with UC (4 active, 4 inactive), ambulatory gut pH was measured with a radiotelemetry pH-sensitiveglass capsule, aerial receiver and portable data recorder. 24 hour urinary excretion of 5-ASAand its metabolite N-acetyl-5-ASAwas analysed by HPLC and results expressedas a percentageof the total 5-ASA daily ingestion. Results: Median 24 hour urinary excretion of 5-ASA was 1.3(0 6.4)% and of N-acetyl-5-ASA 20(5 33)% of the ingested 5-ASA dose. Intestinal (small bowel and colon) pH was > 7 for < 4 hours in none of the patients and there was no correlation between total duration of pH > 7 and 24 hr urinary excretion of 5-ASAor N-acetyl-5-ASA.The same was true for small bowel pH alone. In 2 patients with very low N-acetyl-5-ASA excretion ( < 7% ingested 5-ASA), small bowel pH was > 7 for 6 and 8 hrs. Conclusion: In all patients with UC, gut luminal pH appearedhigh enough for a sufficient time to allow adequatereleaseof 5-ASAfrom Asacol capsules. The low urinary output of N-acetyl-5-ASA in 2 patients implies that other factors, such as rapid transit not reflected by that of the pH capsule, may reduce 5-ASA releasefrom Asacol in a minority of patients with UC.
BACKGROUND:Microarray technology enables analysis of the expression of large numbers of genes in small patient samples and can be used to screen for disease-relatedgenes. OBJECTIVES:The objective of this study was to analyzethe expression of 2400 genes on a glass-slide array (MICROMAX, NEN Life Sciences, Boston, MA) in peripheral blood mononuclear cells (PBMC)from patients with Crohn's disease,ulcerativecolitis, other gastrointestinal (GI) inflammatory illnesses as compared to healthy controls. METHODS:After obtaining consent, 6 ml of whole blood was drawn from 12 patients with biopsy-provenCrohn's disease, 7 patientswith biopsy-provenulcerativecolitis, 4 patientswith other GI inflammatory disorders (celiac disease,C. difficile colitis, H. pylorigastritis and food poisoning) and 23 asymptomatic, age- and sex-matchedcontrols. PBMC's were extracted using Ficoll and total RNA extracted using the Trisol method. RNAquantity and quality was assessedby agarosegel electrophoresis and spectrophotometry and RNA converted to cDNA using reverse transcriptase. During reverse transcription, patient samples were labeled with dinitrophenol (DNP) and control samples with biotin, cDNA was purified by phenol-chloroform and ethanol extraction and adequacy of labeling assessed by dot blot. cDNA from patient and control were pooled, denatured and hybridizedon a glass slide containing cDNA from 2400 genes overnight at 65 C. Slides wore r~nsed in SSC buffer, incubated with antibodies to DNP and streptavidin, conjugated to horseradish peroxidase and cyanine3-1abeledtyramide (patient) or cyanine5labeledtyramide (control). Using a laser scanner, ratios of cyanine3to cyanine5fluorescence were generated that quantitate differential gene expression in patients versus controls. RESULTS: A subset of 16 genes that could distinguish inflammatory bowel disease from other GI disorders in our patients with 100% sensitivity and 75% specificity were identified using cluster analysis(http: rana.stanford.edu).These genes are: beta-sarcoglycanA3b, ZIP kinase, dual-specificity phosphatase 1, MATS, enhancer of zests homolog 1, complement component C3, membrane glycoprotein M6, gamma glutamyl hydrolase, golgin-245, signal recognition particle SRP54, IEF SSP 9502, TAXREB3O2,doublecortin, vasopressin activated calcium mobilizing receptor-like protein, inositol polyphosphate 5-phosphatase, KIAA0373. CONCLUSIONS:Microarray analysis can be used to identify new IBD-related genes.
2317 Fine Mapping of the Chromosome 14q11-12 Region Reveals Associations Between Crohn's Disease and Multiple Markers and Haplotypes Huiying Yang, Kent D. Taylor, Ying-Chao Lin, Tieu D. Hang, Nathan FischeI-Ghodsian, Stephan R. Targan, Jerome I. Rotter, Cedars-Sinai Medical Ctr, IBD Ctr, Los Angeles, CA Background and Aims: Genetic epidemiological studies and animal models all suggest that inherited factors play important roles in the susceptibility to both forms of inflammatory bowel disease -- Crohn's disease (CO) and ulcerative colitis (UC). We first reported a suggestive linkage (LOD=2.8) between CD and chromosome 14q11-12 (Maet al. 1999). This linkage was later confirmed in an independentsample (LOD=3.6) (Duerr et al. 2000). The aim of this study was to identify genetic association by the use of denser markers in the linkage region. Methods: Seventeenpolymorphic markers were typed in a region of approximately 13.2Mb (average marker density O.83Mb) in 289 nuclear families with one or more sibs affected with CD. The transmission/disequilibrium test (TDT) was performed to evaluate associations betweeneach marker or haplotype with CD using the GeneHunter2(testing each allele individually) and TDTEX (testing multiple alleles simultaneously) programs. Results: Both analytic methods identified similar association patterns in the chromosome14q11-12 region, with positive associations observed for multiple markers. The best association was observed approximately4Mb distal from our linkage peak (D14S261), though many markers showed a significant association(p
A-455
D14S1070, DAD1,TCRA, D14S1003, D14S275). Conclusion: Our linkagedisequilibrium mapping results provide additional evidencefor a true susceptibility locus contributing to the risk of CD in the chromosome 14ql 1-12 region. This region contains several positional candidate genes which may be relevant 1o the pathogeoesisof CD.
Medroxyprogesterone Acetate (MPA) is Effective in the Treatment of Ulcerative Colitis (UC). Craig Aronchick, PennsylvaniaHosp, Philadelphia, PA; Douglas Dalke, Gastroenterology Specialties, PC, Lincoln, NE; Stephen Ionna, TOM Research,Cinncinati, OH; Dennis Rift, Assoc GastroenterologyMedical Group, Anaheim, CA; Michael Schiff, GI Assoc of St Augustine, St. Augustine, FL
2313
Background: MPA, a progestational agent, has shown efficacy in the treatment of Crohn's disease. Aims: This study was conducted to evaluateoral MPA as a treatment of acute flares in adult pts with mild to moderate UC. Methods: Pts with active UC, who had a baseline Disease Activity Index (DAI) between 3 and 10, with diary confirmation of increased stool frequency and/or presenceof blood, sigmoidoscopic confirmation of disease,and stool negative for enteric pathogens or C. difficile toxin were eligible. Cyclosporine, methotrexate, 6mercaptopurine, azathioprine, and aminosalicylatesprior to study entry were permitted but dose change was not permitted during the study. Systemic steroids -< 20 rag/day prednisone equivalent were permitted. Daily diaries were provided to record scores for stool frequency, blood in the stool, abdominal pain, and functional assessment (symptom awareness). Pts were instructed to take 1000 mg MPA every 6 hrs for 8 doses followed by a maintenance dose of 500 mg every 12 hrs through the end of the B week treatment period. Efficacy was assessedby the Investigator's GlobalAssessment(IGA) score after 2, 4 and 8 wks of therapy, and DAI score at Wk 8, Responsewas defined as any decrease in the IGA score or a -> 2point decreasein DAI comparedto baseline.The IGAwas basedon the investigator'sjudgement of the severity of diseaseactivity. The DAI total score was tabulated from the scores for stool frequency and blood in the stool (over the previous 7 days), and the IGA and sigmoidoscopy scores at baseline and Wk 8. Results: Fourteen pts were enrolled; 11 were evaluable for efficacy. Eight pts completed 8 wks; one pt is still on-study. Of the pts who completed Wk 8, 7 of 8 (88%) respondedby IGA and DAI. IGA responseswere establishedby Wk 2. Among pts who responded, 3(43%) showed improvement on sigmoidoscopy at Wk B; 3 pts had no change from baseline, and, for 1 pt (14%), the sigmoidoscopy score worsened. One pt discontinued due to allergic drug reaction; one pt discontined due to jaundice and increased abdominal pain. The latter pt was hospitalizedand confirmed to have pre-existing sclerosing cholangitis with symptom onset prior to study drug initiation. Other adverse events included weight gain and menstrualabnormalities.Serum hepatictransaminaselevelswere not elevated. Conclusion: Preliminary evidence suggests that MPA is safe and effective for the treatment of pts with active UC. Further studies are planned. This study was supported by InKine Pharmaceutical Company, Inc.
2316 Release of 5-ASA from Asacol Appears Independent of Gut Luminal pH in Ulcerative Colitis Sean Nugent, St Bartholomew's and the Royal London Sch of Medicine, London United Kingdom; David Rampton, Omar Obeid, David Evans, Devinder Kumar, St George's Hosp, London United Kingdom
The 1804Locus Shows Linkage Heterogeneity Between Crohn's Disease And
Ulcerative Colitis Richard H. Duerr, M Michael Barmada, Leilei Zhang, Univ of Pittsburgh, Pittsburgh, PA; Jean-Paul Achkar, ClevelandClin Fdn, Cleveland,OH; Robert N. Baldassano,Children's Hosp of Philadelphia,Philadelphia,PA; Daniel E. Weeks, Univ of Pittsburgh, Pittsburgh, PA Background & Aim: We found significant linkage between Crohn's disease(CO) and chromosome 14q11-12 in a genome scan, confirming suggestive linkage between CD and the same locus from a prior study and establishing this locus as the IBD4 locus (Duerr et al. Am J Hum Genet 2000;66:1857-1862). Subsequently,linkage to/BO4 was detected in a third COdominated dataset (Vermeire et al. Gastroenterology2000;118(Suppl 2):A338). Our aim was to determine whether there is linkage to IBD4 in ulcerativecolitis (UC) families or mixed IBD (CD and UC) families, and whether there is linkage heterogeneitybetweenCD and UC at IBD4. Methods: We genotyped 44 UC families containing 73 affected relative pairs, and 81 mixed IBD families containing 171 affected relative pairs, at four microsatellitas spanning 34 cM on chromosome 14ql 1-12. Nonparametriclinkageanalyseswere performedusing GENEHUNTERPLUS. 62 CD families containing 127 affected relative pairs were already genotyped at the four microsatellites in our CD genome scan. We formally tested for linkage heterogeneity between the CD and UC families, and between the CD and mixed IBD families, using two types of simulation studies. In the first simulation study design,we determinedthe significance of our observed Morton's M statistic by simulating its distribution under the null hypothesis of no linkage. The Morton's M statistic is a measure of the differences in the GENEHUNTERPLUS Iod scores between the various groups. In the second simulation study design, we compared the distribution of GENEHUNTER-PLUSIod scores obtained when the total group of families was randomly split into groups of similar size to our CO, UC, and mixed IBD family groups. Results: We found no linkage to chromosome 14q11-12 in either the UC families or the mixed IBD families (Iod = 0 in each group of families). When we compared the CD and UC families in the Morton's M test simulation study, only 7 of 10,000 replicates had a greater M test statistic value than the observedvalue (p = 0.007). When we compared the GENEHUNTER-PLUSIod differences between groups of randomly selected families that were similar in size to our CD and UC family groups, none of 10,0000 replicates had a Iod difference as great as the CD-UCdifferencethat we observed(p < 0.0001). When we compared the CD families and mixed IBD families, both simulation studies gave p values < 0.0001. Conclusions: IBD4 is probably a CD-specific locus and not a generic IBD locus. IBD4 shows significant linkage heterogeneitybetween CO and UC.
2319 Detection Of Possible Inflammatory Bowel Disease-Related Genes Using Microarray Analysis. Elizabeth E. Mannick, Maria-Stella Serrano, L S U Health Science Ctr, New Orleans, LA; Joseph C. Bonomolo, Johns Hopkins Univ, Baltimore, MD; Michael Lau, John N. Udall Jr, Zhiyun Liu, L S U Health Science Ctr, New Orleans, LA
Background:Releaseof 5-ASAfrom the pH-dependentmesalazinepreparation,Asacol, requires exposure of the capsule to pH > 7 for =4 hrs in vitro. It has been suggestedthat luminal pH may be reduced, at least in the colon, in patients with ulcerative colitis (UC), and that bioavailabilityof 5-ASA from Asacol capsules may consequentlybe impaired. Aims: To relate urinary excretion of 5-ASA and N-acetyl-5-ASA to intestinal luminal pH in patients taking Asacol for UC. Methods: In 8 patients with UC (4 active, 4 inactive), ambulatory gut pH was measured with a radiotelemetry pH-sensitiveglass capsule, aerial receiver and portable data recorder. 24 hour urinary excretion of 5-ASAand its metabolite N-acetyl-5-ASAwas analysed by HPLC and results expressedas a percentageof the total 5-ASA daily ingestion. Results: Median 24 hour urinary excretion of 5-ASA was 1.3(0 6.4)% and of N-acetyl-5-ASA 20(5 33)% of the ingested 5-ASA dose. Intestinal (small bowel and colon) pH was > 7 for < 4 hours in none of the patients and there was no correlation between total duration of pH > 7 and 24 hr urinary excretion of 5-ASAor N-acetyl-5-ASA.The same was true for small bowel pH alone. In 2 patients with very low N-acetyl-5-ASA excretion ( < 7% ingested 5-ASA), small bowel pH was > 7 for 6 and 8 hrs. Conclusion: In all patients with UC, gut luminal pH appearedhigh enough for a sufficient time to allow adequatereleaseof 5-ASAfrom Asacol capsules. The low urinary output of N-acetyl-5-ASA in 2 patients implies that other factors, such as rapid transit not reflected by that of the pH capsule, may reduce 5-ASA releasefrom Asacol in a minority of patients with UC.
BACKGROUND:Microarray technology enables analysis of the expression of large numbers of genes in small patient samples and can be used to screen for disease-relatedgenes. OBJECTIVES:The objective of this study was to analyzethe expression of 2400 genes on a glass-slide array (MICROMAX, NEN Life Sciences, Boston, MA) in peripheral blood mononuclear cells (PBMC)from patients with Crohn's disease,ulcerativecolitis, other gastrointestinal (GI) inflammatory illnesses as compared to healthy controls. METHODS:After obtaining consent, 6 ml of whole blood was drawn from 12 patients with biopsy-provenCrohn's disease, 7 patientswith biopsy-provenulcerativecolitis, 4 patientswith other GI inflammatory disorders (celiac disease,C. difficile colitis, H. pylorigastritis and food poisoning) and 23 asymptomatic, age- and sex-matchedcontrols. PBMC's were extracted using Ficoll and total RNA extracted using the Trisol method. RNAquantity and quality was assessedby agarosegel electrophoresis and spectrophotometry and RNA converted to cDNA using reverse transcriptase. During reverse transcription, patient samples were labeled with dinitrophenol (DNP) and control samples with biotin, cDNA was purified by phenol-chloroform and ethanol extraction and adequacy of labeling assessed by dot blot. cDNA from patient and control were pooled, denatured and hybridizedon a glass slide containing cDNA from 2400 genes overnight at 65 C. Slides wore r~nsed in SSC buffer, incubated with antibodies to DNP and streptavidin, conjugated to horseradish peroxidase and cyanine3-1abeledtyramide (patient) or cyanine5labeledtyramide (control). Using a laser scanner, ratios of cyanine3to cyanine5fluorescence were generated that quantitate differential gene expression in patients versus controls. RESULTS: A subset of 16 genes that could distinguish inflammatory bowel disease from other GI disorders in our patients with 100% sensitivity and 75% specificity were identified using cluster analysis(http: rana.stanford.edu).These genes are: beta-sarcoglycanA3b, ZIP kinase, dual-specificity phosphatase 1, MATS, enhancer of zests homolog 1, complement component C3, membrane glycoprotein M6, gamma glutamyl hydrolase, golgin-245, signal recognition particle SRP54, IEF SSP 9502, TAXREB3O2,doublecortin, vasopressin activated calcium mobilizing receptor-like protein, inositol polyphosphate 5-phosphatase, KIAA0373. CONCLUSIONS:Microarray analysis can be used to identify new IBD-related genes.
2317 Fine Mapping of the Chromosome 14q11-12 Region Reveals Associations Between Crohn's Disease and Multiple Markers and Haplotypes Huiying Yang, Kent D. Taylor, Ying-Chao Lin, Tieu D. Hang, Nathan FischeI-Ghodsian, Stephan R. Targan, Jerome I. Rotter, Cedars-Sinai Medical Ctr, IBD Ctr, Los Angeles, CA Background and Aims: Genetic epidemiological studies and animal models all suggest that inherited factors play important roles in the susceptibility to both forms of inflammatory bowel disease -- Crohn's disease (CO) and ulcerative colitis (UC). We first reported a suggestive linkage (LOD=2.8) between CD and chromosome 14q11-12 (Maet al. 1999). This linkage was later confirmed in an independentsample (LOD=3.6) (Duerr et al. 2000). The aim of this study was to identify genetic association by the use of denser markers in the linkage region. Methods: Seventeenpolymorphic markers were typed in a region of approximately 13.2Mb (average marker density O.83Mb) in 289 nuclear families with one or more sibs affected with CD. The transmission/disequilibrium test (TDT) was performed to evaluate associations betweeneach marker or haplotype with CD using the GeneHunter2(testing each allele individually) and TDTEX (testing multiple alleles simultaneously) programs. Results: Both analytic methods identified similar association patterns in the chromosome14q11-12 region, with positive associations observed for multiple markers. The best association was observed approximately4Mb distal from our linkage peak (D14S261), though many markers showed a significant association(p
A-455