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Is there genetic anticipation in IBD? A population-based study
April 2000
IBD phenotype. Linkage has been observed in IBD patients at chr6p and case control studies have demonstrated associations in the TNF promoter. Aim: To characterize genomic variation throughout the TNF gene in IBD patients and assess the functional impact of suggestive genetic variants. Methods: The TNF gene was sequenced in 12 unrelated IBD patients from families with greatest evidence for linkage at chr6p. Single nucleotide polymorphisms (SNPs) were typed in 135 multiply affected families and studied using the transmission/disequilibrium test (TOT). Allele-specific binding was tested with electromobility shift assays (EMSAs). Results: Promoter variants at -1031, -863, -308 and -238 were identified. The -857 variant reported in the Japanese IBD population was not observed. Two intronic variants were identified, SNPintrona and SNPintronb' No SNPs were present in coding or 3' -untranslated regions. The single locus TDT tests for linkage showed neutral or inverse transmission for the less common -863 and -308 A alleles. No significant linkage was observed with the less common A alleles at -1031 (62 transmissions, 51 non-transmissions) or -238 (17 transmissions, 13 non-transmissions). The -238 variant was in complete and significant (p = 1.3 x 10-6 ) linkage disequilibrium with SNPintrona and SNPintronb' respectively. The (C-A-G), (-1031, -238, SNP intronb] haplotype demonstrated evidence for association by TDT (using a single, randomly selected affected from 192 multiplex families), with 15 transmissions (both CD and UC) and 5 non-transmissions (p= 0.03, uncorrected). The -238 variant is contained within a known repressor element. EMSAs were performed using allele-specific oligos in this region incubated with nuclear extracts from U937 mononuclear cells stimulated with phorbol myristyl acetate. Three specific DNA-protein complexes were observed, but no allele-specific binding was present. Conclusions: No single TNF promoter polymorphism by itself is significantly associated with IBD as assessed by the TDT test in multiply affected families. Nominal evidence for association with the 3 locus, C-A-G (-1031, -238, SNPintronb) haplotype is observed. Linkage disequilibrium patterns throughout the TNF gene are complex, reflecting selection patterns throughout evolution. Consideration of haplotypes for both genetic and functional studies is required.
1797 LIMITED GENETIC EVIDENCE FOR MDRI AS A CANDIDATE GENE FOR INFLAMMATORY BOWEL DISEASE. Steven R. Brant, Richard Ramos, Denise K. Bonen, Stuart M. Lubinski, Geoffrey Ravenhill, Patrick M. Rohal, Sinda Lee, Judy H. Cho, Johns Hopkins Univ, Baltimore, MD; Univ of Chicago, Chicago, IL. Introduction: The multidrug resistance gene MORI is expressed in intestinal epithelial cells and lymphoid cells. Recently, MORI knock-out mice have been shown to develop spontaneous colitis when maintained in pathogen-free conditions (Panwala et al., J Irnmunol, 1998). The human MDRI gene maps to chromosome 7q, and thus is a candidate gene for the 7q putative inflammatory bowel disease (IBD)locus identifiedby Satsangi et al., Nat Gen, 1996. Linkage analysis in sibling pair pedigrees from our previous reported genome screen (Cho et al., PNAS, 1998) was maximal at 91 cM on chromosome 7q (Mlod = 2.81, Z=3.6, p=1.59 x 10-4), between markers D7S2204 and D7S820 and within 10 cM of the MORI gene (Brant & Cho, unpublished results).We thus evaluated MORI as a candidate gene. Methods: The genomic sequences of the 27 MOR I coding exons, containing over 4000 bp, and > 20 bp of each flanking intron sequence were determined by PCR amplification with MORI specific primers, PCR product purification and then fluorescent sequencing, using the DNA purified from 18 individuals with IBD and two controls. These individuals were probands from pedigrees having maximal linkage evidence to the MDRI mapping location in our genome screen. Two polymorphisms were genotyped in 169 multiplex IBD pedigrees (36% Ashkenazi Jewish) and in 20 additional pedigrees with DNA samples obtained from both parents and a single affected child. 103 pedigrees were Crohn' s, 24 were UC and 62 were mixed. Tests for linkage using Genehunter-Plus and linkage disequilibrium (TOn by Chi-Square analysis were performed. Results: Six polymorphisms were identified.Two resulted in amino acid changes. Addition of these two amino acid polyrnorphisms did not significantly alter linkage evidence. TOT was minimally significant (p= 0.029). No specific association was observed with either Crohn's disease or ulcerative colitis, or with Ashkenazi Jewish ethnicity. Conclusion:There is preliminary evidence that MDRI may have coding changes specific to lBD. Genotyping of additional pedigrees and functional studies will be necessary for more conclusive evidence.
1798 SEX SPECIFIC ASSOCIATION OF OSTEOPOROSIS WITH VITA· MIN D RECEPTOR POLYMORPHISM (FOKI).
AGAA831
statistically significant association between the genotype and the bone mineral density in female patients with inflammatory bowel disease. Male IBD patients with the ff genotype had an significantly (1 SD) lower bone mineral density of the lumbar spine compared with the FF group (p=0.037). None of the men with the FF-genotype had osteoporosis. Conclusions: The FokI polymorphism seems to be a genetic risk factor for developing osteoporosis in male patients with inflammatory bowel disease
1799 PASSIVE SMOKING IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE - AN ISRAELI MULTICENTER CASE CONTROLSTUDY. Rami Eliakim, Shimon Reif, Alexandra Lavy, Daniel Keter, Shmuel Odes, Aharon Halak, Efrattt Broide, Yaron Niv, Yishai Ron, Julian Paz, Alexander Fich, Yael Villa, Tuvia Gilet, Hadassah Univ Hosp, Jerusalem, Israel; Rambam Hosp, Israel; Kaplan Hosp; Tel-Aviv Sourasky Med Ctr; Assaf Harofeh, Zerifin, Israel; Beilinson Hosp, Petach Tikva, Israel; Wolfson Med Ctr, Holon, Israel; Shaare Zedek Hosp, Jerusalem, Israel; Soroka Hosp, Beersheba, Israel. Background: The association between smoking and inflarumatory bowel disease (IBD) is well-established. There are no large scale studies of passive smoking in inflammatory bowel disease (IBD) Aim: To study the passive smoking exposure of Jewish IBD patients in Israel in a large scale multicenter study. Methods: Patients with established IBD aged 18-70 years, were interviewed. Two controls, clinic and neighborhood, matched by age, sex, community group, and education, were sought. Results: 534 patients [273 ulcerative colitis (UC) and 261 Crohn's disease (CD»), 478 clinic controls and 430 community controls were interviewed. There were no significant differences in the passive smoking habits between IBD patients and their controls. 51% of UC patients, 50% of the clinic controls and 58% of the community controls were exposed to passive smoking at home (NS); similar results were found among crn patients. When a quantitative exposure index was used UC patients were significantly less exposed to passive smoking than their community controls (7.46 :!:8.40 versus 9.36 :!: 9.46, n=229, p<0.031). No differences in exposure to passive smoking were found when DC patients who had never smoked were compared with their controls. Conclusion: There is a lack of association between passive smoking and IBD in Jewish patients in Israel. When a quantitative exposure index was used DC patients were found to be less exposed to passive smoking than their community controls.
1800 IS THERE GENETIC ANTICIPATION IN mD? A POPULATIONBASED STUDY. Elisa M. Faybush, James F. Blanchard, Patricia Rawsthorne, Charles N. Bernstein, Univ of Manitoba, Winnipeg, MB, Canada. Background: Case ascertainment bias may be responsible for the observation from tertiary referral centers that affected offspring of parents with IBD are diagnosed at a younger age. We aimed to determine if genetic anticipation existed in a population-based database. Methods: In creating the population-based University of Manitoba IBD Database (1996) we collected questionnaires from 2759 subjects. We contacted those subjects who reported having family members (first or second degree) with IBD and their family members for verification of diagnosis & age at diagnosis (AAD). Results: Of 475 subjects reporting positive family histories, 300 (63%) were reachable by phone (161 Crohn s (CD) and 139 (UC). There were 153 cases with 2: 1 1 relative, and 179 cases with 2: 1 2 relative. There were no gender specific trends for either older or younger members of pairings & no differences for UC vs CD. Mean age of onset in familial cases=33.4 :!: 14.3y, non-familial cases=34.1 :!: 15.3y (p=0.49). A similar number of familial cases had appendectomies prior to disease diagnosis for UC (11.5%) as for CD (14.9%) (p=0.49). Conclusions: In a populationbased database we found that anticipation for AAD existed similarly for CD & UC, and there were no gender differences for pairings. There is a similar effect examining pairs of child-parent or child-aunt/uncle & a near doubling of effect when examining child-grandparent. Familial cases on average do not have lower AAD than non-familial cases. It is likely that the anticipation in years for AAD reflects an environmental factor associated with a period of increased incidence. Furthermore, if appendectomy is protective in UC it may only be true in non genetically-linked cases. 0
0
Nicole Bregenzer, Schaeffler Andreas, Dept ofInternal Medicine I, Univ of Regensburg, Regensburg, Germany. Background & Aims: We examined the association of bone mineral density of patients with inflammatory bowel disease with a polymorphism in the gene encoding for the vitamin D receptor. The thyminelcytosine (TIC) polymorphism in the first of two start codons can be defined by a restriction fragment length polymorphism using the restriction endonuclease FokI. Vitamin D receptor alleles containing this polymorphism were denoted by f and alleles lacking the site by F. Methods: The FokI genotype was determined in 138 Caucasian patients with inflammatory bowel disease, 62 were men and 76 women, 92 had Crohn's disease and 46 ulcerative colitis. Bone mineral density of the lumbar spine and the femoral neck was measured by dual-energy X-ray absorptiometry. Results: There was no
Ox inolder case
UC +CD (n=68) UC +CD (n=72) UC+CD(20) data inmean yrs
ParentAAD
Offspring AAD
Anticipation
45.3
28,9
16,4
Aunt/uncle AAD
Nlecelneph AAD
45.0
26.8
Grnomlgdad AAD 62,5
GchlldAAD
22.9
182 29.6
IBD phenotype. Linkage has been observed in IBD patients at chr6p and case control studies have demonstrated associations in the TNF promoter. Aim: To characterize genomic variation throughout the TNF gene in IBD patients and assess the functional impact of suggestive genetic variants. Methods: The TNF gene was sequenced in 12 unrelated IBD patients from families with greatest evidence for linkage at chr6p. Single nucleotide polymorphisms (SNPs) were typed in 135 multiply affected families and studied using the transmission/disequilibrium test (TOT). Allele-specific binding was tested with electromobility shift assays (EMSAs). Results: Promoter variants at -1031, -863, -308 and -238 were identified. The -857 variant reported in the Japanese IBD population was not observed. Two intronic variants were identified, SNPintrona and SNPintronb' No SNPs were present in coding or 3' -untranslated regions. The single locus TDT tests for linkage showed neutral or inverse transmission for the less common -863 and -308 A alleles. No significant linkage was observed with the less common A alleles at -1031 (62 transmissions, 51 non-transmissions) or -238 (17 transmissions, 13 non-transmissions). The -238 variant was in complete and significant (p = 1.3 x 10-6 ) linkage disequilibrium with SNPintrona and SNPintronb' respectively. The (C-A-G), (-1031, -238, SNP intronb] haplotype demonstrated evidence for association by TDT (using a single, randomly selected affected from 192 multiplex families), with 15 transmissions (both CD and UC) and 5 non-transmissions (p= 0.03, uncorrected). The -238 variant is contained within a known repressor element. EMSAs were performed using allele-specific oligos in this region incubated with nuclear extracts from U937 mononuclear cells stimulated with phorbol myristyl acetate. Three specific DNA-protein complexes were observed, but no allele-specific binding was present. Conclusions: No single TNF promoter polymorphism by itself is significantly associated with IBD as assessed by the TDT test in multiply affected families. Nominal evidence for association with the 3 locus, C-A-G (-1031, -238, SNPintronb) haplotype is observed. Linkage disequilibrium patterns throughout the TNF gene are complex, reflecting selection patterns throughout evolution. Consideration of haplotypes for both genetic and functional studies is required.
1797 LIMITED GENETIC EVIDENCE FOR MDRI AS A CANDIDATE GENE FOR INFLAMMATORY BOWEL DISEASE. Steven R. Brant, Richard Ramos, Denise K. Bonen, Stuart M. Lubinski, Geoffrey Ravenhill, Patrick M. Rohal, Sinda Lee, Judy H. Cho, Johns Hopkins Univ, Baltimore, MD; Univ of Chicago, Chicago, IL. Introduction: The multidrug resistance gene MORI is expressed in intestinal epithelial cells and lymphoid cells. Recently, MORI knock-out mice have been shown to develop spontaneous colitis when maintained in pathogen-free conditions (Panwala et al., J Irnmunol, 1998). The human MDRI gene maps to chromosome 7q, and thus is a candidate gene for the 7q putative inflammatory bowel disease (IBD)locus identifiedby Satsangi et al., Nat Gen, 1996. Linkage analysis in sibling pair pedigrees from our previous reported genome screen (Cho et al., PNAS, 1998) was maximal at 91 cM on chromosome 7q (Mlod = 2.81, Z=3.6, p=1.59 x 10-4), between markers D7S2204 and D7S820 and within 10 cM of the MORI gene (Brant & Cho, unpublished results).We thus evaluated MORI as a candidate gene. Methods: The genomic sequences of the 27 MOR I coding exons, containing over 4000 bp, and > 20 bp of each flanking intron sequence were determined by PCR amplification with MORI specific primers, PCR product purification and then fluorescent sequencing, using the DNA purified from 18 individuals with IBD and two controls. These individuals were probands from pedigrees having maximal linkage evidence to the MDRI mapping location in our genome screen. Two polymorphisms were genotyped in 169 multiplex IBD pedigrees (36% Ashkenazi Jewish) and in 20 additional pedigrees with DNA samples obtained from both parents and a single affected child. 103 pedigrees were Crohn' s, 24 were UC and 62 were mixed. Tests for linkage using Genehunter-Plus and linkage disequilibrium (TOn by Chi-Square analysis were performed. Results: Six polymorphisms were identified.Two resulted in amino acid changes. Addition of these two amino acid polyrnorphisms did not significantly alter linkage evidence. TOT was minimally significant (p= 0.029). No specific association was observed with either Crohn's disease or ulcerative colitis, or with Ashkenazi Jewish ethnicity. Conclusion:There is preliminary evidence that MDRI may have coding changes specific to lBD. Genotyping of additional pedigrees and functional studies will be necessary for more conclusive evidence.
1798 SEX SPECIFIC ASSOCIATION OF OSTEOPOROSIS WITH VITA· MIN D RECEPTOR POLYMORPHISM (FOKI).
AGAA831
statistically significant association between the genotype and the bone mineral density in female patients with inflammatory bowel disease. Male IBD patients with the ff genotype had an significantly (1 SD) lower bone mineral density of the lumbar spine compared with the FF group (p=0.037). None of the men with the FF-genotype had osteoporosis. Conclusions: The FokI polymorphism seems to be a genetic risk factor for developing osteoporosis in male patients with inflammatory bowel disease
1799 PASSIVE SMOKING IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE - AN ISRAELI MULTICENTER CASE CONTROLSTUDY. Rami Eliakim, Shimon Reif, Alexandra Lavy, Daniel Keter, Shmuel Odes, Aharon Halak, Efrattt Broide, Yaron Niv, Yishai Ron, Julian Paz, Alexander Fich, Yael Villa, Tuvia Gilet, Hadassah Univ Hosp, Jerusalem, Israel; Rambam Hosp, Israel; Kaplan Hosp; Tel-Aviv Sourasky Med Ctr; Assaf Harofeh, Zerifin, Israel; Beilinson Hosp, Petach Tikva, Israel; Wolfson Med Ctr, Holon, Israel; Shaare Zedek Hosp, Jerusalem, Israel; Soroka Hosp, Beersheba, Israel. Background: The association between smoking and inflarumatory bowel disease (IBD) is well-established. There are no large scale studies of passive smoking in inflammatory bowel disease (IBD) Aim: To study the passive smoking exposure of Jewish IBD patients in Israel in a large scale multicenter study. Methods: Patients with established IBD aged 18-70 years, were interviewed. Two controls, clinic and neighborhood, matched by age, sex, community group, and education, were sought. Results: 534 patients [273 ulcerative colitis (UC) and 261 Crohn's disease (CD»), 478 clinic controls and 430 community controls were interviewed. There were no significant differences in the passive smoking habits between IBD patients and their controls. 51% of UC patients, 50% of the clinic controls and 58% of the community controls were exposed to passive smoking at home (NS); similar results were found among crn patients. When a quantitative exposure index was used UC patients were significantly less exposed to passive smoking than their community controls (7.46 :!:8.40 versus 9.36 :!: 9.46, n=229, p<0.031). No differences in exposure to passive smoking were found when DC patients who had never smoked were compared with their controls. Conclusion: There is a lack of association between passive smoking and IBD in Jewish patients in Israel. When a quantitative exposure index was used DC patients were found to be less exposed to passive smoking than their community controls.
1800 IS THERE GENETIC ANTICIPATION IN mD? A POPULATIONBASED STUDY. Elisa M. Faybush, James F. Blanchard, Patricia Rawsthorne, Charles N. Bernstein, Univ of Manitoba, Winnipeg, MB, Canada. Background: Case ascertainment bias may be responsible for the observation from tertiary referral centers that affected offspring of parents with IBD are diagnosed at a younger age. We aimed to determine if genetic anticipation existed in a population-based database. Methods: In creating the population-based University of Manitoba IBD Database (1996) we collected questionnaires from 2759 subjects. We contacted those subjects who reported having family members (first or second degree) with IBD and their family members for verification of diagnosis & age at diagnosis (AAD). Results: Of 475 subjects reporting positive family histories, 300 (63%) were reachable by phone (161 Crohn s (CD) and 139 (UC). There were 153 cases with 2: 1 1 relative, and 179 cases with 2: 1 2 relative. There were no gender specific trends for either older or younger members of pairings & no differences for UC vs CD. Mean age of onset in familial cases=33.4 :!: 14.3y, non-familial cases=34.1 :!: 15.3y (p=0.49). A similar number of familial cases had appendectomies prior to disease diagnosis for UC (11.5%) as for CD (14.9%) (p=0.49). Conclusions: In a populationbased database we found that anticipation for AAD existed similarly for CD & UC, and there were no gender differences for pairings. There is a similar effect examining pairs of child-parent or child-aunt/uncle & a near doubling of effect when examining child-grandparent. Familial cases on average do not have lower AAD than non-familial cases. It is likely that the anticipation in years for AAD reflects an environmental factor associated with a period of increased incidence. Furthermore, if appendectomy is protective in UC it may only be true in non genetically-linked cases. 0
0
Nicole Bregenzer, Schaeffler Andreas, Dept ofInternal Medicine I, Univ of Regensburg, Regensburg, Germany. Background & Aims: We examined the association of bone mineral density of patients with inflammatory bowel disease with a polymorphism in the gene encoding for the vitamin D receptor. The thyminelcytosine (TIC) polymorphism in the first of two start codons can be defined by a restriction fragment length polymorphism using the restriction endonuclease FokI. Vitamin D receptor alleles containing this polymorphism were denoted by f and alleles lacking the site by F. Methods: The FokI genotype was determined in 138 Caucasian patients with inflammatory bowel disease, 62 were men and 76 women, 92 had Crohn's disease and 46 ulcerative colitis. Bone mineral density of the lumbar spine and the femoral neck was measured by dual-energy X-ray absorptiometry. Results: There was no
Ox inolder case
UC +CD (n=68) UC +CD (n=72) UC+CD(20) data inmean yrs
ParentAAD
Offspring AAD
Anticipation
45.3
28,9
16,4
Aunt/uncle AAD
Nlecelneph AAD
45.0
26.8
Grnomlgdad AAD 62,5
GchlldAAD
22.9
182 29.6