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CARD15 mutations in a central area of Spanish Crohns diseaseA population: Prevalence and association with clinical phenotypes
with both, the stricturing behavior (at least one allele 31%) (vs. inflammatory: 3%, fistulizing: 7%, OR 7.8 95%CI 2.6-23.2; p = 0.0002) and the small bowel location (25%) (vs. colonic 4%, enterocolonic 15%; OR 3.8 95%CI 1.3-10.8; p=0.015). SNP12 was associated with enterocolonic location (19%) (vs. colonic 2%, small bowel 0%, OR 21.0 95%CI 2.5-174.3; p=0.0004). SNP8 carriers presented a trend for extensive colonic compromise (21% vs. other 8%, p = 0.06). While SNP13 phenotype was protective for perianal fistulas (p = 0.035), the SNP12 was associated with abdominal fistulas (OR 5.7 95% CI 1.4-22.7; p = 0.018). The logistic regression analyses demonstrated independent associations of SNP13 with the stricturirtg pattern (p=0.0024), SNP12 with enterocolonic location (p=O.01) and SNP8 with the fistulizing (p = 0.036). CONCLUSIONS: Our data confirm the association of SNP13 and SNP8 NOD2 variants with CD. Diverse modalities of genotypic-phenotypic relations were detected. The most relevant associations were between SNP13 and stricntring behavior, SNP12 and enterocolonic location and finally, SPN8 with a global flatulizing pattern, on one hand, and a trend for extensive colonic compromise, on the other.
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NOD2/CARD15 Mutations in a Central Area of Spanish Crohn S Disease Population: Prevalence and Assodation with Clinical Phenotypes Juan Luis Mendoza, Laura Murillo, Laura Fernandez, Raquel Lana, Dulce Maria CruzSantamaria, Francisca Cuenca, Emilio Gomez de La Concha, Manuel Diaz-Rubio, Julio Garcia-Paredes, A. Salvador Pena Background and aims: Genetic predisposition for Crohn s disease (CD) has been demostrated by epidemiological and genetic linkage analysis. The NOD2/CARD15 gene has been identified and is thought to play an important role in the susceptibility to CD. Different mutations in this gene are associated with different clinical patterns in western population. We report in the present communication the prevalence of the 3 major NOD2/CARD15 mutations in Spanish CD patients and their relation with clinical phenotypes performed in two independent laboratories (Netherlands and Spain) with different techniques. Methods: All 204 patients and 140 control subjects were genotyped by PCR for CARD15 gene mutations: 3020insC frameshift (SNP13), Gly908Arg (SNP12) and Arg702trp (SNP8). The few disagreements in typing in the laboratories were resolved by DNA sequencing analyses of the samples. The following clinical data were extracted for all patients: the sex, family history, age at diagnosis, location, behaviour disease, extra-intestinal manifestations, smoking habits, appendectomy and therapeutic management. Variables significantly associated from univariate analyses (X2, Fisher) were treated by logistic regression. Results: A total of 204 Spanish Caucasian CD patients, with median follow-up 9.9 years (range: 1-44). The frequencies of heterozygosity for the 3 mutations (SNP8, SNP12 and SNP13) were 13,7%, 8,3%, 14,2% in CD and 4,29%, 2.14%, 4.29% in controls, respectively, demonstrating that of the three variants were positively associated with CD OR= 4.08 (1C 95%; 2.21-7.50). The logistic regression analyses demonstrated that CD patients who carried at least one of mutations were positively associated with ileal location (L1) (p= 0,001; RR= 1.61 (95% CI; 1.21-2.15)) and a less frequent with colonic involvement (L2) (p = 0.007; RR= 0,29 (95% CI: 0,11-0,80)). SPN12 was associated with both, stricturing behaviour (B2) (p = 0.002; RR= 3.38 (95% CI; 1.866.28)), previous appendectomy (p= 0.02; RR= 2.54 (95% CI: 1.22-5.30)), and surgery (p = 0,0005; RR= 2.4 (95% CI; 1.64-3.51)). However, no correlation was observed between the CARD15 polymorphism and the rest of clinical characteristics. Conclusions: Our present data confirm the association of three mutations in the CARD15 gene with Spanish Castilian CD patients mainly with ileal disease. The patients with SNP12 mutation in the CARD15 are characterized by a strictnring behaviour, previous appendectomy and surgery.
M1569 CD14 Expression on Monocytes and Soluble CD14 Plasma Levels in Correlation to the Promotor Polymorphism of the Endotoxin Receptor CD14 Gene in Patients with Inactive Cruhn's disease Thomas Griga, Wolfram Klein, Joerg T. Epplen, Ute Hebler, Axel Stachon, Wolff H. Schmiegei
Background: The association of the single nucleotide polymorphism in the promotor of the lipopolysaccharide receptor CD14 gene (T/C at position -159) with Crohn's disease has recently been demonstrated. This CD14 polymorphism is a potential predisposition factor responsible for interindividual differing inflammatory reactions involving the CD 14 receptor. We studied the correlation between the CD14 genotype (CC, CT, TT) and the membranebound CD14 monocyte expression and soluble CD14 in patients with inactive Crohn's disease. Methods: In 23 patients and 29 healthy volunteers the membrane-bound CD14 density on unstimufated monocytes and soluble CD14 plasma levels were examined using quantitative flow cytometry and enzyme-linked immunosorbent assay. Results: In normal controls membrane-bound CD14 monocyte density did not differ significantly between the genotypes CC, CT, or "fT. In contrast, patients with inactive Crohn's disease and genotype TT showed a significantly lower membrane-bound CD14 density on monocytes compared to patients with genotype CC. Soluble CD14 plasma levels were significantly higher in patients with inactive Crohn's disease compared to the same genotype of healthy controls, but there was no significant difference between the genotypes CC, CT, and TT. Conclusions: Our data show that the membrane-bound CD14 monocyte expression and the soluble CD14 plasma levels in patients with inactive Crohn's disease completely differ from that in healthy individuals. In order to develop individualised therapy strategies further studies should be carned out to evaluate whether the TT genotype is associated with differences in the clinical course of Crohn's disease and in the response to antibacterial treatment.
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A 295T-to-C Promotor Polymorphism of the IL-16 Gene is Associated with Crohn's Disease, Regardless of the NOD2/CARD15 Status or the Clinical Phenotype Juergen Glas, Helga-Paula Torok, Hermann Unterhuber, Mi~am Radlmayr, Christian Folwaczny BACKGROUND: Recently, a T-to-C polymorphism at position -295 in the promotor region of the human interleukin-16 (IL-16) gene has been reported. IL-16 is a proinflammatory cytokine first described as lymphocyte chemoattractant factor in 1982 (LCF). The main receptor for IL-16 is CD4, it recruits CD4+ T ceils niducing a Th-I immune response and its expression is dependent on the NFKB pathway. The expression of IL-16 is increased in inflammatory bowel disease, in particular in Crohn's disease. AIM: Data concerning the ILl6 promotor polymorphism in inflammatory bowel disease are lacking. Thus, the current study aimed at the assessment of this polymorphism in Crohn's disease and ulcerative colitis. METHODS: 103 patients with Crohn's disease, 100 patients with ulcerative colitis and 120 healthy unrelated controls were genotyped for the promotor polymorphism. Furthermore, patients with Crohn's disease were stratified according to the 3020insC mutation within the NOD2/CARD 15 gene, the disease localization and the respective clinical phenotype (fistulizing, fibrostenotic or inflammatory). Genotyping was performed by PCRintroducing a recognition site for the restriction enzyme AhdI, subsequent digestion and followed by agarose gel electrophoresis. RESULTS: The frequencies of the T-allele (p<0.01) and the TT genotype (p<0,01) were significantly increased in patients with Crohn's disease when compared to the controls, regardless of the NOD2/CARD15 mutation, the disease phenotype or the site of intestinal involvement. An association with ulcerative colitis was not observed. CONCLUSIONS: Herein, a new association between a prnmotor polymorphism of the IL-16 gene and Crohn's disease was observed and correlates with the previously described increased mucosa[ expression of IL-]6 in inflammatory bowel disease
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Multiple Drug Resistance Gene (MDRI) Polymorphisms and Steroid Resistance in Inflammatory Bowel Disease (IBD) Megan Dring, Nassir Mahmud, Maria O'Sullivan, Colm O'Morain, Richard Farrell, Carol Goulding, Dermot Kellehor, Ross McManus Introduction: Medical therapy for IBD relies mainly on steroid treatment. However, failed medical therapy from glucocorticoid resistance can be as high as 30% of patients. One cause for this may relate to inherited variations in the MDRI gene. The MDR1 gene encodes Pgp170, an ATP-dependent drug effhix pump which transports many therapeutic steroids. Its expression and activity are crucial in determining if sufficiently high intracellular steroid concentrations can be achieved. Allelic differences in the MDR1 gene may be associated with, or even causative for, different expression levels. We have previously shown that Pgp170 expression is increased in lymphocytes from steroid resistant IBD patients. Aims: To determine whether the MDR1 SNPs in exon 21 (21-2677) and exon 26 (26-3435) are associated with steroid resistance in IBD. Methods: DNA was extracted from 92 IBD patients, 50 of which were Crohns disease (CD) patients (29 were resistant to steroid treatment and 21 were responsive to steroid treatment) and 42 of which were Ulcerative Colitis (UC) patients (29 resistant and 13 responsive to steroid treatment). The 26-3435 SNP was genotyped using a Taqman PCR assay and allele specific probes, and the 21-2677 SNP using a PCR-RFLP assay. The results were analysed with respect to steroid response (as defined by Farre[l et al, 2000) and disease type. Results: The allele frequencies of 21-2677 were found to be significantly different (p<0.01) between the steroid resistant and responsive groups in the UC population, using Markov Chain Monte Carlo simulation, with the T allele frequency significantly higher in the responsive group. No other significant differences were found. Conclusion: The 21-2677 G allele has been associated with upregulation of Pgp-170, while the T and A alleles have been associated with lower expression. Our results indicate that 21-2677 is an important marker for steroid resistance in UC but is not associated with steroid resistance in CD, suggesting that the mechanism of steroid resistant in Ulcerative Colitis and Crohns disease may differ significantly. Hence, the G21-2677 allele may help identify a subset of UC patients who fail to respond to steroid treatment.
M1573 Nod2/Card15 Mutations and Phenotypic Correlation in Pediatric Crohn's Disease (Cd) Subra Kugathasan, Nicole Collins, Karen Maresso, Andrea Matter, Karen Sherry, Colin Rudolph, David Binion, Ulrich Broeckel
Background and Aims: Mutations in the NOD2 gene have been associated with susceptibility to Crohn's disease (CD) in adult patients. In addition, NOD2 mutations have been independently associated with CD phenotypes characterized by ileal location and fibrostenosing behavior, a longtenn complication of chronic intestinal inflammation. The prevalence of NOD2 mutations in pediatric CD and attempts to correlate genotype with early disease phenotype, prior to the emergence of longterm chronic complications have not been described. We sought to characterize rates of NOD2 mutations in a cohort of pediatric CD patients and their biologic parents. Methods: We prospectively recruited both affected children and their parents for genetic analysis from a pediatric statewide IBD registry from the state of Wisconsin, which is an ethnically and demographically diverse patient cohort. We analyzed patients with pediatric-onset CD for the presence of mutations in the NOD2 gene, including previously identified mutations by direct sequencing (Arg702Trp, Gly908Arg, and Leul007fsnisC). Relevant clinical data including ethnicity, age of disease onset, disease location and disease behavior, and response to therapy were gathered from these patients. Results: There were 352 CD pediatric patients who were eligible for genotyping. Out of the
21-2677 allele frequ~mclesin the IBD populMion. Allele G Allele T Allele A P UC-NR 0.556 0.444 0.000 UC-R 0.143 0.785 0.071 p<0.01 Cl)-NR 0.550 0.450 0.000 Cd-R 0.500 0.500 0.000 n,s. UC=UlcetalJveColitis,CD=Crohns Disease, NR=Non Responsive, R=Responsive.
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AGA Abstracts
M1571
NOD2/CARD15 Mutations in a Central Area of Spanish Crohn S Disease Population: Prevalence and Assodation with Clinical Phenotypes Juan Luis Mendoza, Laura Murillo, Laura Fernandez, Raquel Lana, Dulce Maria CruzSantamaria, Francisca Cuenca, Emilio Gomez de La Concha, Manuel Diaz-Rubio, Julio Garcia-Paredes, A. Salvador Pena Background and aims: Genetic predisposition for Crohn s disease (CD) has been demostrated by epidemiological and genetic linkage analysis. The NOD2/CARD15 gene has been identified and is thought to play an important role in the susceptibility to CD. Different mutations in this gene are associated with different clinical patterns in western population. We report in the present communication the prevalence of the 3 major NOD2/CARD15 mutations in Spanish CD patients and their relation with clinical phenotypes performed in two independent laboratories (Netherlands and Spain) with different techniques. Methods: All 204 patients and 140 control subjects were genotyped by PCR for CARD15 gene mutations: 3020insC frameshift (SNP13), Gly908Arg (SNP12) and Arg702trp (SNP8). The few disagreements in typing in the laboratories were resolved by DNA sequencing analyses of the samples. The following clinical data were extracted for all patients: the sex, family history, age at diagnosis, location, behaviour disease, extra-intestinal manifestations, smoking habits, appendectomy and therapeutic management. Variables significantly associated from univariate analyses (X2, Fisher) were treated by logistic regression. Results: A total of 204 Spanish Caucasian CD patients, with median follow-up 9.9 years (range: 1-44). The frequencies of heterozygosity for the 3 mutations (SNP8, SNP12 and SNP13) were 13,7%, 8,3%, 14,2% in CD and 4,29%, 2.14%, 4.29% in controls, respectively, demonstrating that of the three variants were positively associated with CD OR= 4.08 (1C 95%; 2.21-7.50). The logistic regression analyses demonstrated that CD patients who carried at least one of mutations were positively associated with ileal location (L1) (p= 0,001; RR= 1.61 (95% CI; 1.21-2.15)) and a less frequent with colonic involvement (L2) (p = 0.007; RR= 0,29 (95% CI: 0,11-0,80)). SPN12 was associated with both, stricturing behaviour (B2) (p = 0.002; RR= 3.38 (95% CI; 1.866.28)), previous appendectomy (p= 0.02; RR= 2.54 (95% CI: 1.22-5.30)), and surgery (p = 0,0005; RR= 2.4 (95% CI; 1.64-3.51)). However, no correlation was observed between the CARD15 polymorphism and the rest of clinical characteristics. Conclusions: Our present data confirm the association of three mutations in the CARD15 gene with Spanish Castilian CD patients mainly with ileal disease. The patients with SNP12 mutation in the CARD15 are characterized by a strictnring behaviour, previous appendectomy and surgery.
M1569 CD14 Expression on Monocytes and Soluble CD14 Plasma Levels in Correlation to the Promotor Polymorphism of the Endotoxin Receptor CD14 Gene in Patients with Inactive Cruhn's disease Thomas Griga, Wolfram Klein, Joerg T. Epplen, Ute Hebler, Axel Stachon, Wolff H. Schmiegei
Background: The association of the single nucleotide polymorphism in the promotor of the lipopolysaccharide receptor CD14 gene (T/C at position -159) with Crohn's disease has recently been demonstrated. This CD14 polymorphism is a potential predisposition factor responsible for interindividual differing inflammatory reactions involving the CD 14 receptor. We studied the correlation between the CD14 genotype (CC, CT, TT) and the membranebound CD14 monocyte expression and soluble CD14 in patients with inactive Crohn's disease. Methods: In 23 patients and 29 healthy volunteers the membrane-bound CD14 density on unstimufated monocytes and soluble CD14 plasma levels were examined using quantitative flow cytometry and enzyme-linked immunosorbent assay. Results: In normal controls membrane-bound CD14 monocyte density did not differ significantly between the genotypes CC, CT, or "fT. In contrast, patients with inactive Crohn's disease and genotype TT showed a significantly lower membrane-bound CD14 density on monocytes compared to patients with genotype CC. Soluble CD14 plasma levels were significantly higher in patients with inactive Crohn's disease compared to the same genotype of healthy controls, but there was no significant difference between the genotypes CC, CT, and TT. Conclusions: Our data show that the membrane-bound CD14 monocyte expression and the soluble CD14 plasma levels in patients with inactive Crohn's disease completely differ from that in healthy individuals. In order to develop individualised therapy strategies further studies should be carned out to evaluate whether the TT genotype is associated with differences in the clinical course of Crohn's disease and in the response to antibacterial treatment.
M1572
A 295T-to-C Promotor Polymorphism of the IL-16 Gene is Associated with Crohn's Disease, Regardless of the NOD2/CARD15 Status or the Clinical Phenotype Juergen Glas, Helga-Paula Torok, Hermann Unterhuber, Mi~am Radlmayr, Christian Folwaczny BACKGROUND: Recently, a T-to-C polymorphism at position -295 in the promotor region of the human interleukin-16 (IL-16) gene has been reported. IL-16 is a proinflammatory cytokine first described as lymphocyte chemoattractant factor in 1982 (LCF). The main receptor for IL-16 is CD4, it recruits CD4+ T ceils niducing a Th-I immune response and its expression is dependent on the NFKB pathway. The expression of IL-16 is increased in inflammatory bowel disease, in particular in Crohn's disease. AIM: Data concerning the ILl6 promotor polymorphism in inflammatory bowel disease are lacking. Thus, the current study aimed at the assessment of this polymorphism in Crohn's disease and ulcerative colitis. METHODS: 103 patients with Crohn's disease, 100 patients with ulcerative colitis and 120 healthy unrelated controls were genotyped for the promotor polymorphism. Furthermore, patients with Crohn's disease were stratified according to the 3020insC mutation within the NOD2/CARD 15 gene, the disease localization and the respective clinical phenotype (fistulizing, fibrostenotic or inflammatory). Genotyping was performed by PCRintroducing a recognition site for the restriction enzyme AhdI, subsequent digestion and followed by agarose gel electrophoresis. RESULTS: The frequencies of the T-allele (p<0.01) and the TT genotype (p<0,01) were significantly increased in patients with Crohn's disease when compared to the controls, regardless of the NOD2/CARD15 mutation, the disease phenotype or the site of intestinal involvement. An association with ulcerative colitis was not observed. CONCLUSIONS: Herein, a new association between a prnmotor polymorphism of the IL-16 gene and Crohn's disease was observed and correlates with the previously described increased mucosa[ expression of IL-]6 in inflammatory bowel disease
M1570
Multiple Drug Resistance Gene (MDRI) Polymorphisms and Steroid Resistance in Inflammatory Bowel Disease (IBD) Megan Dring, Nassir Mahmud, Maria O'Sullivan, Colm O'Morain, Richard Farrell, Carol Goulding, Dermot Kellehor, Ross McManus Introduction: Medical therapy for IBD relies mainly on steroid treatment. However, failed medical therapy from glucocorticoid resistance can be as high as 30% of patients. One cause for this may relate to inherited variations in the MDRI gene. The MDR1 gene encodes Pgp170, an ATP-dependent drug effhix pump which transports many therapeutic steroids. Its expression and activity are crucial in determining if sufficiently high intracellular steroid concentrations can be achieved. Allelic differences in the MDR1 gene may be associated with, or even causative for, different expression levels. We have previously shown that Pgp170 expression is increased in lymphocytes from steroid resistant IBD patients. Aims: To determine whether the MDR1 SNPs in exon 21 (21-2677) and exon 26 (26-3435) are associated with steroid resistance in IBD. Methods: DNA was extracted from 92 IBD patients, 50 of which were Crohns disease (CD) patients (29 were resistant to steroid treatment and 21 were responsive to steroid treatment) and 42 of which were Ulcerative Colitis (UC) patients (29 resistant and 13 responsive to steroid treatment). The 26-3435 SNP was genotyped using a Taqman PCR assay and allele specific probes, and the 21-2677 SNP using a PCR-RFLP assay. The results were analysed with respect to steroid response (as defined by Farre[l et al, 2000) and disease type. Results: The allele frequencies of 21-2677 were found to be significantly different (p<0.01) between the steroid resistant and responsive groups in the UC population, using Markov Chain Monte Carlo simulation, with the T allele frequency significantly higher in the responsive group. No other significant differences were found. Conclusion: The 21-2677 G allele has been associated with upregulation of Pgp-170, while the T and A alleles have been associated with lower expression. Our results indicate that 21-2677 is an important marker for steroid resistance in UC but is not associated with steroid resistance in CD, suggesting that the mechanism of steroid resistant in Ulcerative Colitis and Crohns disease may differ significantly. Hence, the G21-2677 allele may help identify a subset of UC patients who fail to respond to steroid treatment.
M1573 Nod2/Card15 Mutations and Phenotypic Correlation in Pediatric Crohn's Disease (Cd) Subra Kugathasan, Nicole Collins, Karen Maresso, Andrea Matter, Karen Sherry, Colin Rudolph, David Binion, Ulrich Broeckel
Background and Aims: Mutations in the NOD2 gene have been associated with susceptibility to Crohn's disease (CD) in adult patients. In addition, NOD2 mutations have been independently associated with CD phenotypes characterized by ileal location and fibrostenosing behavior, a longtenn complication of chronic intestinal inflammation. The prevalence of NOD2 mutations in pediatric CD and attempts to correlate genotype with early disease phenotype, prior to the emergence of longterm chronic complications have not been described. We sought to characterize rates of NOD2 mutations in a cohort of pediatric CD patients and their biologic parents. Methods: We prospectively recruited both affected children and their parents for genetic analysis from a pediatric statewide IBD registry from the state of Wisconsin, which is an ethnically and demographically diverse patient cohort. We analyzed patients with pediatric-onset CD for the presence of mutations in the NOD2 gene, including previously identified mutations by direct sequencing (Arg702Trp, Gly908Arg, and Leul007fsnisC). Relevant clinical data including ethnicity, age of disease onset, disease location and disease behavior, and response to therapy were gathered from these patients. Results: There were 352 CD pediatric patients who were eligible for genotyping. Out of the
21-2677 allele frequ~mclesin the IBD populMion. Allele G Allele T Allele A P UC-NR 0.556 0.444 0.000 UC-R 0.143 0.785 0.071 p<0.01 Cl)-NR 0.550 0.450 0.000 Cd-R 0.500 0.500 0.000 n,s. UC=UlcetalJveColitis,CD=Crohns Disease, NR=Non Responsive, R=Responsive.
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AGA Abstracts