- Email: [email protected]
3020insC mutation of the NOD2 gene in familial multiplex Crohn's disease
M1566
were reviewed. DL~easelocalization and behaviour determined by the Vienna Classification. AJ and SJ ethnicities were assigned on basis of the birth place of the four grandparents. DNA samples were extracted and genotyped for the three mutations, using allele-specific PCR and Taqman system (Applied Biosystems). Comparisons were carriedsout between 78 'pure'AJ patients and 37 'pure'SJ patients. Results: AJ patients had significantly higher prevalence of the Gly908Arg and Leu 1007fsinsC alleles and higher CARD 15 mutation carriage. SJ patients had significantly more stricturing disease and more extra-intestinal manifestations.Differences in disease extent were not significant. Conclusion: CARD15 may play a greater role in the pathogenesis of CD in Ashkenazi patients. Alternatively, additional polymorphisms in the CARD15 gene might be associated with susceptibility to CD in Sephardic patients.
Agnkenazl CD (n-78) Sephardlr CD (n=37) Arg7O2Trp* 9/156 (5.7%) 5/66 (7.5%) Glyg06Arg* 24/156 (15.4%) 4/66 (8%) Leul0OlfslnsC* 12/156 (7.69%) 1/66 (1.5%) % CARD15 carriage 38/78 (48.7%) 9/33 (27.3%) Behavior:. Inflammatory 38/72 (52.8%) 14/37 (37.8%) Stdc'tudmg 18/72 (25%) 17/37 (46%) PenelmtJng 16/72 (22.2%) 6/37 (16.2%) Exttatnte~lnat menifenta. 12/78 (15.4%) 14/37 (37.8%) tions *allele frequency -p value were calculated with a z lest for equality of proportions
Sequence variation in the CARD15 (NOD2) gene and susceptibility to Crohn's disease Cive Onine, K King, M. Mirza, S. Fisher, A. Cuthbert, J. Hampe, J. Mansfield, Alistair Forbes, Jeremy Sanderson, Katherine Lewis, Chris Mathew Introduction: Crohn's disease (CD), a subtype of inflammatory bowel disease (IBD), is a complex disorder, with both genetic and environmental aetiology. Three sequence variants in the CARD15 gene have recently been shown to be associated with susceptibihty to CD. There is also evidence of an excess of rare CARD15 variants in CD, but whether these are true disease susceptibility alleles (DSAs) is unknown. Clarification of the status of rare variants would facilitate a more accurate assessment of the extent of the contribution of CARD15 to Crohn's disease, and of the likely genetic model for the effect of CARD15. Methods and results: In order to investigate the contribution of rare variants to CD we have selected 100 Crohn's patients who are heterozygous for one of the three associated DSAs (R702W, G908R or 1007fs) for comprehensive mutation screening. The coding sequence of CARD15 was screened by denaturing HFLC for mutations to identify both common polymorphisms and rare variants. 21 patients had one or more additional rare CARD15 mutations, 10 of which were non-synonymous. One of these was a novel nonsense mutation that would produce a truncated protein (R896X), and four (P,235C, $431L, A612T, R10IgG) were amino acid substitutions that were predicted to alter the function of the protein. Conclusion: The finding that only a minority of patients who are heterozygous for common disease susceptibihty alleles in CARD15 have a second mutation is consistent with the gene dosage model for CARD15 in Crohn's disease.
p.value~ 0,5 0.06 0.05 0.05 0.14 0.03 0.46 0,007
M1564 M1567
Phosphoinositide Signalling In Inflammation And Colorectal Cancer Caroline Osborne, Olagnnju Ogunbiyi, Satish Keshav
A Higher Allele Frequency of the lO07fs/3020insC Mutation of the NOD2 Gene in Familial Multiplex Crohn's Disease Wen Jie Zhang, Jennifer L Thompson, Lisa S. Poritz, Walter A. Kohun
Background and Aims Phosphoinositide-3-kinase gamma (PI-3-K~) is a lipid kinase activated by G-protein coupled receptors (GPCR) to regulate such cellular functions as chemotaxis, cell proliferation and apoptosis. Transgeinc mice that lack PI-3-K~ are immune suppressed and develop colorectal cancer (CRC), and mice lacking the G-protein subunit Gai2, that may interact with P1-3-K'y develop ulcerative colitis (UC) and CRC. Patients with UC have a 30% lifetime risk of CRC, and UC-associated CRC(UC-CRC) develops by a distinct pattern of genetic changes to sporadically occuring CRC. We investigated the expression of PI-3K',/in colonic tissue from patients with UC, UC-CRC and CRC. Methods PI(3)K~/expression was determined in normal colon, UC, CRC and UC-CRC by immunohistochemistry and in situ hybridisation. Using RT-PCR and western blotting, PI-3-K~ expression was determined in normal colonic mucosa, colonic epithelial cells and a colon cancer cell fne(Caco2). Methylation specific PCR was performed on Caco2 cell, sporadic CRC and UC-CRC DNA to determine if silencing of PI-3-K~/was associated with hypermethylation. Results PI-3-K'y expression was demonstrated in leukocytes, colonic epithelial cells and Caco2 cells. All UC specimens expressed PI-3-K'y protein at higher levels than normal tissue, and expression was lost in the majority of UC-CRC. PI-3-K'y mRNA was detected in normal tissue, CRC and UC, hut was absent in UC-CRC. In Caco2 cells treated with stimulated lymphocyte conditioned medium, expression of PI-3-K~ was increased, and as the gene is unmethylated in these cells, it is probably upregulated by inflammation, and subsequent loss of expression may be mediated by hypermethylauon. Conclusion Loss of expression of PI(3)K'y is seen more frequently in UC-CRC than sporadic CRC and our data suggest that it plays a critical role in intestinal epithelial cell function in the context of inflammation, where its expression is increased. We hypothesise that subsequent loss of PI-3-K~ expression, possibly through hypermethylation, promotes neoplastic transformation in the inflamed colonic epithelium.
Background: Several mutations of the NOD2/CARD15 gene have been identifed to be a predisposing genetic factor for Crohn's disease (CD) but not ulcerative colitis (UC). Later studies in different geographic Caucasoid CD populations have suggested various mutation rates for 3 common mutations of the gene, namely, lO07fs (frame shift due to a C insertion or 3020insC), G908R and R702W. Japanese CD patients do not carry these 3 mutations indicating the importance of ethnicity in CD susceptibility genes. We have investigated two such mutations in CD patients from central Pennsylvania and show that lO07fs mutation rate is higher in familial multiplex CD. Methods: SSP-PCR was employed to genotype 1007fs mutation and PCR-RFLP was used to investigate G908R variants. A total of 27 multiplex (2 to 5 patients per family) Caucasoid 1BD families were derived from a central Pennsylvania IBD patient registry, including families of 9 CD, 8 UC and 10 CD + UC mixed, with 35 CD patients, 24 UC patients and 73 non-IBD relatives. Results: High frequencies of 1007fs mutation carriers were observed in familial CD (11/35 or 31.4%), UC (3/24 or 12.5%) and non-IBD (14/73 or 19.2%) relatives. A comparison among 3 geographical IBD populations revealed that allele frequencies of 1007fs were higher in all categories in our multiplex IBD families (Table). No homozygous 1007fs carriers were observed. For Gg08R variants, we only detected 4 heterozygous mutation carriers, two of which were CD patients and the other two were non-IBD relatives. Conclusions: Familial multiplex CD families present a higher rate of the 1007fs mutation of the NOD2, which may explain high prevalence or clustering of CD patients in these families. Since our 1BD population is located in rural Pennsylvania area with limited migration, its genetic makeup may be more homogeneous than those in large metropolitan areas, which would contribute to the higher mutation rate observed. In addition, a large number of non-lBD relatives (19.2%) carry the 1007fs but do not develop disease suggesting that this mutation, if in linkage to or combination with other CD susceptibility gene(s), could be phenotypically more penetrating.
M1565 Association Analysis of a SNP Polymorphism within the 3'UTR of the STAT6 Gene in Familial Crohn's Disease Wen Jie Zhang, Jennifer L. Thompson, Lisa S. Poritz, Eva Galka, Walter A. Kokun
A Comparison of Nlele Fraquendes for 1007fs Mut~
AGA Abstracts
Chlcego/P#tsburghlBat~imora
France
Family Type
Multiplex IBD
Possibly Simplex IBD
DiseaseType Frequency (%)
CD / UC IBD1 Non-
CD t UC / Non-IBD
Simplex.,-Multiplex IBD CD / UC / Non-IBD
15.7 / 6.3 / 9.6
8.2 / 3.0 / 4.0
12.0 / 1.0 / 2.0
Region/Country
Background: The Stat6 signaling pathway is active in a variety of cell types, including immune cells and cancer cells, and plays an important role in the regulation of gene expression, such as proinflammatory cytokines ILl2 and TNFa, which are involved in mucosal tissue injury. We have previously identified a defective Stat6 activational phenotype, Star6~"i~,in some familial IBD (inflammatory bowel disease) patients. In this study, we have investigated possible association of familial 1BD with a SNP polymorphism (single nucleotide polymorphism) in the 3'UTR (untranslated region) of the STAT6 gene. Methods: A PCRRFLP genotyping methodology was employed to investigate G2964A SNP pofymorphism of the STAT6 gene. Multiplex (more than one patient per family) Caucasoid IBD families (24) were derived from a central Pennsylvania [BD patient registry, including 32 patients with Crohn's disease (CD), 23 patients with ulcerative colitis (UC) and 73 non-diseased family members. Results: The majority of healthy family members were GG homozygotes (51/73 or 69.9%). Genotype A was rarer in our population and there were 30.1% (22/73) of healthy individuals carrying genotype A (GA heterozygutes). Thus far, we only detected one homozygons AA carrier (1.4%) among healthy individuals, which was in sharp contrast to a British blood donor population with 10% AA homozygosity. In CD patients, however, there was an increased frequency of genotype A carriers (13/32 or 40.6%), which was 10.5% higher than that of healthy controls (30.1%). A moderately higher frequency (8/23 or 36.4%) of heterozygous A carriers was observed in UC patient group. Conclusions: A skewed distribution of the rarer A2964 SNP allele of the STAT6 gene has been observed in familial CD patients from multiplex IBD families. More such families (>60) are currently being tested for this SNP polymorphism, which will then be analyzed by a statistical method for linkage analysis (transmission disequilibrium test or TDT) to confirm this association. Since polymorphisms within the 3'UTR of genes may affect mRNA stability therefore affecting protein production, the G2964A SNP polymorphism may be implicated in Stat6 protein levels and its IL4-induced activational phenotypes (Stat6"", Stat6I~ and Stat6 h~gh,respectively) we have observed previously.
in Three Coucasold IBD Studies
Central Penn. sylvania
M1568 Correlation Between Mutations of NOD2/CARD15 Gene and Clinical Patterns of Crohn's Disease (CD) Alicia M Sambueni, Laura Murillo, Silvia Negreira, Anibal Gil, Sergio Huernos, Sllvina Goncalves, Horacio Vazquez, Eduardo Maurino, Julio C. Bai, Amado S. Pena BACKGROUND/AIMS: The clinical heterogeneity of CD may reflect a different genetic/ environmental background. We aimed to assess the frequency of three major NOD2 mutations associated with CD susceptibility in a cohort of Argentine CD patients and to determine their relation with the different clinical patterns. MATERIAL/METHODS: 143 CD, median duration from disease onset 10.9 yrs (range 1-57) and 121 controls (58 healthy individual, 63 celiac disease) were analyzed by PCR for the presence of 3020insC frameshif (SNP13), Gly908Arg (SNP 12) and Arg702trp (SNP8) mutations. CD was categorized according location in small bowel enterocolonic, colonic and based on behavior in: inflammatory, stricturing fistulizing. Variables significantly associated from univariate analyses (c2, Fisher) were treated by logistic regression. RESULTS: Carriage rates for SNP13, SNP12, SNP8 were 12%, 7%, 12% in CD and 2%, 4%, 4% in controls, respectively. Compared with controls, CD had significantly higher allelic frequencies of SPN13 (OR 7.4 95% C1 1.7-32.6; p =0.004) and SNP8 (OR 3.0, 95%CI 1.1-8.3; p = 0.0421), but not for SPN12 (4% vs. 2%, p=NS). There were no differences between healthy controls and disease controls. Univariate analyses detected diverse genotypic-phenotypic associations. SNP13 carriage was highly associated
A-374
were reviewed. DL~easelocalization and behaviour determined by the Vienna Classification. AJ and SJ ethnicities were assigned on basis of the birth place of the four grandparents. DNA samples were extracted and genotyped for the three mutations, using allele-specific PCR and Taqman system (Applied Biosystems). Comparisons were carriedsout between 78 'pure'AJ patients and 37 'pure'SJ patients. Results: AJ patients had significantly higher prevalence of the Gly908Arg and Leu 1007fsinsC alleles and higher CARD 15 mutation carriage. SJ patients had significantly more stricturing disease and more extra-intestinal manifestations.Differences in disease extent were not significant. Conclusion: CARD15 may play a greater role in the pathogenesis of CD in Ashkenazi patients. Alternatively, additional polymorphisms in the CARD15 gene might be associated with susceptibility to CD in Sephardic patients.
Agnkenazl CD (n-78) Sephardlr CD (n=37) Arg7O2Trp* 9/156 (5.7%) 5/66 (7.5%) Glyg06Arg* 24/156 (15.4%) 4/66 (8%) Leul0OlfslnsC* 12/156 (7.69%) 1/66 (1.5%) % CARD15 carriage 38/78 (48.7%) 9/33 (27.3%) Behavior:. Inflammatory 38/72 (52.8%) 14/37 (37.8%) Stdc'tudmg 18/72 (25%) 17/37 (46%) PenelmtJng 16/72 (22.2%) 6/37 (16.2%) Exttatnte~lnat menifenta. 12/78 (15.4%) 14/37 (37.8%) tions *allele frequency -p value were calculated with a z lest for equality of proportions
Sequence variation in the CARD15 (NOD2) gene and susceptibility to Crohn's disease Cive Onine, K King, M. Mirza, S. Fisher, A. Cuthbert, J. Hampe, J. Mansfield, Alistair Forbes, Jeremy Sanderson, Katherine Lewis, Chris Mathew Introduction: Crohn's disease (CD), a subtype of inflammatory bowel disease (IBD), is a complex disorder, with both genetic and environmental aetiology. Three sequence variants in the CARD15 gene have recently been shown to be associated with susceptibihty to CD. There is also evidence of an excess of rare CARD15 variants in CD, but whether these are true disease susceptibility alleles (DSAs) is unknown. Clarification of the status of rare variants would facilitate a more accurate assessment of the extent of the contribution of CARD15 to Crohn's disease, and of the likely genetic model for the effect of CARD15. Methods and results: In order to investigate the contribution of rare variants to CD we have selected 100 Crohn's patients who are heterozygous for one of the three associated DSAs (R702W, G908R or 1007fs) for comprehensive mutation screening. The coding sequence of CARD15 was screened by denaturing HFLC for mutations to identify both common polymorphisms and rare variants. 21 patients had one or more additional rare CARD15 mutations, 10 of which were non-synonymous. One of these was a novel nonsense mutation that would produce a truncated protein (R896X), and four (P,235C, $431L, A612T, R10IgG) were amino acid substitutions that were predicted to alter the function of the protein. Conclusion: The finding that only a minority of patients who are heterozygous for common disease susceptibihty alleles in CARD15 have a second mutation is consistent with the gene dosage model for CARD15 in Crohn's disease.
p.value~ 0,5 0.06 0.05 0.05 0.14 0.03 0.46 0,007
M1564 M1567
Phosphoinositide Signalling In Inflammation And Colorectal Cancer Caroline Osborne, Olagnnju Ogunbiyi, Satish Keshav
A Higher Allele Frequency of the lO07fs/3020insC Mutation of the NOD2 Gene in Familial Multiplex Crohn's Disease Wen Jie Zhang, Jennifer L Thompson, Lisa S. Poritz, Walter A. Kohun
Background and Aims Phosphoinositide-3-kinase gamma (PI-3-K~) is a lipid kinase activated by G-protein coupled receptors (GPCR) to regulate such cellular functions as chemotaxis, cell proliferation and apoptosis. Transgeinc mice that lack PI-3-K~ are immune suppressed and develop colorectal cancer (CRC), and mice lacking the G-protein subunit Gai2, that may interact with P1-3-K'y develop ulcerative colitis (UC) and CRC. Patients with UC have a 30% lifetime risk of CRC, and UC-associated CRC(UC-CRC) develops by a distinct pattern of genetic changes to sporadically occuring CRC. We investigated the expression of PI-3K',/in colonic tissue from patients with UC, UC-CRC and CRC. Methods PI(3)K~/expression was determined in normal colon, UC, CRC and UC-CRC by immunohistochemistry and in situ hybridisation. Using RT-PCR and western blotting, PI-3-K~ expression was determined in normal colonic mucosa, colonic epithelial cells and a colon cancer cell fne(Caco2). Methylation specific PCR was performed on Caco2 cell, sporadic CRC and UC-CRC DNA to determine if silencing of PI-3-K~/was associated with hypermethylation. Results PI-3-K'y expression was demonstrated in leukocytes, colonic epithelial cells and Caco2 cells. All UC specimens expressed PI-3-K'y protein at higher levels than normal tissue, and expression was lost in the majority of UC-CRC. PI-3-K'y mRNA was detected in normal tissue, CRC and UC, hut was absent in UC-CRC. In Caco2 cells treated with stimulated lymphocyte conditioned medium, expression of PI-3-K~ was increased, and as the gene is unmethylated in these cells, it is probably upregulated by inflammation, and subsequent loss of expression may be mediated by hypermethylauon. Conclusion Loss of expression of PI(3)K'y is seen more frequently in UC-CRC than sporadic CRC and our data suggest that it plays a critical role in intestinal epithelial cell function in the context of inflammation, where its expression is increased. We hypothesise that subsequent loss of PI-3-K~ expression, possibly through hypermethylation, promotes neoplastic transformation in the inflamed colonic epithelium.
Background: Several mutations of the NOD2/CARD15 gene have been identifed to be a predisposing genetic factor for Crohn's disease (CD) but not ulcerative colitis (UC). Later studies in different geographic Caucasoid CD populations have suggested various mutation rates for 3 common mutations of the gene, namely, lO07fs (frame shift due to a C insertion or 3020insC), G908R and R702W. Japanese CD patients do not carry these 3 mutations indicating the importance of ethnicity in CD susceptibility genes. We have investigated two such mutations in CD patients from central Pennsylvania and show that lO07fs mutation rate is higher in familial multiplex CD. Methods: SSP-PCR was employed to genotype 1007fs mutation and PCR-RFLP was used to investigate G908R variants. A total of 27 multiplex (2 to 5 patients per family) Caucasoid 1BD families were derived from a central Pennsylvania IBD patient registry, including families of 9 CD, 8 UC and 10 CD + UC mixed, with 35 CD patients, 24 UC patients and 73 non-IBD relatives. Results: High frequencies of 1007fs mutation carriers were observed in familial CD (11/35 or 31.4%), UC (3/24 or 12.5%) and non-IBD (14/73 or 19.2%) relatives. A comparison among 3 geographical IBD populations revealed that allele frequencies of 1007fs were higher in all categories in our multiplex IBD families (Table). No homozygous 1007fs carriers were observed. For Gg08R variants, we only detected 4 heterozygous mutation carriers, two of which were CD patients and the other two were non-IBD relatives. Conclusions: Familial multiplex CD families present a higher rate of the 1007fs mutation of the NOD2, which may explain high prevalence or clustering of CD patients in these families. Since our 1BD population is located in rural Pennsylvania area with limited migration, its genetic makeup may be more homogeneous than those in large metropolitan areas, which would contribute to the higher mutation rate observed. In addition, a large number of non-lBD relatives (19.2%) carry the 1007fs but do not develop disease suggesting that this mutation, if in linkage to or combination with other CD susceptibility gene(s), could be phenotypically more penetrating.
M1565 Association Analysis of a SNP Polymorphism within the 3'UTR of the STAT6 Gene in Familial Crohn's Disease Wen Jie Zhang, Jennifer L. Thompson, Lisa S. Poritz, Eva Galka, Walter A. Kokun
A Comparison of Nlele Fraquendes for 1007fs Mut~
AGA Abstracts
Chlcego/P#tsburghlBat~imora
France
Family Type
Multiplex IBD
Possibly Simplex IBD
DiseaseType Frequency (%)
CD / UC IBD1 Non-
CD t UC / Non-IBD
Simplex.,-Multiplex IBD CD / UC / Non-IBD
15.7 / 6.3 / 9.6
8.2 / 3.0 / 4.0
12.0 / 1.0 / 2.0
Region/Country
Background: The Stat6 signaling pathway is active in a variety of cell types, including immune cells and cancer cells, and plays an important role in the regulation of gene expression, such as proinflammatory cytokines ILl2 and TNFa, which are involved in mucosal tissue injury. We have previously identified a defective Stat6 activational phenotype, Star6~"i~,in some familial IBD (inflammatory bowel disease) patients. In this study, we have investigated possible association of familial 1BD with a SNP polymorphism (single nucleotide polymorphism) in the 3'UTR (untranslated region) of the STAT6 gene. Methods: A PCRRFLP genotyping methodology was employed to investigate G2964A SNP pofymorphism of the STAT6 gene. Multiplex (more than one patient per family) Caucasoid IBD families (24) were derived from a central Pennsylvania [BD patient registry, including 32 patients with Crohn's disease (CD), 23 patients with ulcerative colitis (UC) and 73 non-diseased family members. Results: The majority of healthy family members were GG homozygotes (51/73 or 69.9%). Genotype A was rarer in our population and there were 30.1% (22/73) of healthy individuals carrying genotype A (GA heterozygutes). Thus far, we only detected one homozygons AA carrier (1.4%) among healthy individuals, which was in sharp contrast to a British blood donor population with 10% AA homozygosity. In CD patients, however, there was an increased frequency of genotype A carriers (13/32 or 40.6%), which was 10.5% higher than that of healthy controls (30.1%). A moderately higher frequency (8/23 or 36.4%) of heterozygous A carriers was observed in UC patient group. Conclusions: A skewed distribution of the rarer A2964 SNP allele of the STAT6 gene has been observed in familial CD patients from multiplex IBD families. More such families (>60) are currently being tested for this SNP polymorphism, which will then be analyzed by a statistical method for linkage analysis (transmission disequilibrium test or TDT) to confirm this association. Since polymorphisms within the 3'UTR of genes may affect mRNA stability therefore affecting protein production, the G2964A SNP polymorphism may be implicated in Stat6 protein levels and its IL4-induced activational phenotypes (Stat6"", Stat6I~ and Stat6 h~gh,respectively) we have observed previously.
in Three Coucasold IBD Studies
Central Penn. sylvania
M1568 Correlation Between Mutations of NOD2/CARD15 Gene and Clinical Patterns of Crohn's Disease (CD) Alicia M Sambueni, Laura Murillo, Silvia Negreira, Anibal Gil, Sergio Huernos, Sllvina Goncalves, Horacio Vazquez, Eduardo Maurino, Julio C. Bai, Amado S. Pena BACKGROUND/AIMS: The clinical heterogeneity of CD may reflect a different genetic/ environmental background. We aimed to assess the frequency of three major NOD2 mutations associated with CD susceptibility in a cohort of Argentine CD patients and to determine their relation with the different clinical patterns. MATERIAL/METHODS: 143 CD, median duration from disease onset 10.9 yrs (range 1-57) and 121 controls (58 healthy individual, 63 celiac disease) were analyzed by PCR for the presence of 3020insC frameshif (SNP13), Gly908Arg (SNP 12) and Arg702trp (SNP8) mutations. CD was categorized according location in small bowel enterocolonic, colonic and based on behavior in: inflammatory, stricturing fistulizing. Variables significantly associated from univariate analyses (c2, Fisher) were treated by logistic regression. RESULTS: Carriage rates for SNP13, SNP12, SNP8 were 12%, 7%, 12% in CD and 2%, 4%, 4% in controls, respectively. Compared with controls, CD had significantly higher allelic frequencies of SPN13 (OR 7.4 95% C1 1.7-32.6; p =0.004) and SNP8 (OR 3.0, 95%CI 1.1-8.3; p = 0.0421), but not for SPN12 (4% vs. 2%, p=NS). There were no differences between healthy controls and disease controls. Univariate analyses detected diverse genotypic-phenotypic associations. SNP13 carriage was highly associated
A-374