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Anti-Saccharomyces cerevisiae mannan antibodies in familial crohn’s disease
THE AMERICAN JOURNAL OF GASTROENTEROLOGY Copyright © 1998 by Am. Coll. of Gastroenterology Published by Elsevier Science Inc.
Vol. 93, No. 8, 1998 ISSN 0002-9270/98/$19.00 PII S0002-9270(98)00296-2
Anti-Saccharomyces cerevisiae Mannan Antibodies in Familial Crohn’s Disease Boualem Sendid, M.D., Jean-Franc¸ois Quinton, M.D., Gwe´nae¨le Charrier, M.D., Olivier Goulet, M.D., Antoine Cortot, Bruno Grandbastien, M.D., Daniel Poulain, Ph.D., and Jean-Fre´de´ric Colombel, M.D. Service d’He´pato-Gastroente´rologie et Registre des Maladies Inflammatoires du Tube Digestif du Nord-Ouest de la France (EPIMAD); Laboratoire de Parasitologie-Mycologie, CHRU Lille; INSERM U 42, Lille; Service de Gastroente´rologie et Nutrition, De´partement de Pe´diatrie, Hopital Necker, Paris, France
Objective: Anti-Saccharomyces cerevisiae mannan antibodies (ASCA) are associated with Crohn’s disease. The aim of this study was to determine the prevalence of ASCA in families in which at least two members were affected with Crohn’s disease. Methods: A total of 20 families including two (n 5 15) or more (n 5 5) patients with Crohn’s disease were tested for ASCA with use of an ELISA method. Overall, 51 affected members, 66 healthy first degree relatives, and 163 healthy control subjects were studied. Results: ASCA were detected in 35 of 51 (69%) patients with Crohn’s disease and in 13 of 66 (20%) healthy relatives versus one of 163 healthy control subjects (p < 0.0001 and p < 0.001). ASCApositive relatives were distributed in 12 of 20 families. ASCA were present in eight healthy parents and four healthy siblings. The prevalence of ASCA in relatives did not depend on the ASCA status of affected members. Conclusion: ASCA in 20% of healthy first degree relatives of patients with Crohn’s disease suggest that these antibodies might be a subclinical marker for Crohn’s disease in families. Whether ASCA reflect environmental or genetic factors or a combination of both is unknown. (Am J Gastroenterol 1998;93: 1306 –1310. © 1998 by Am. Coll. of Gastroenterology)
that may be insufficient: clinically unaffected individuals bearing the variant genotype will not be recognized (1). Such unaffected individuals could be better identified by the use of subclinical markers that could detect the abnormal genotype in the absence of the full phenotype (1). Several candidate markers have been proposed thus far, including lymphocytotoxic antibodies, intestinal permeability, C3 dysfunction and obligate anaerobic flora in CD (5– 8), colonic mucins, mucosal Ig distribution and antineutrophil cytoplasmic antibodies (ANCA) in UC (9 –11), and antibodies to colonic epithelial cells in both CD and UC (12). To date, the results remain conflicting and none of these markers has been definitively established as a subclinical marker of either CD or UC. Systemic antibodies against the yeast Saccharomyces cerevisiae have been described in sera from patients with CD but not with UC (13–15). This serologic response mainly concerns sequences of mannose residues expressed in the cell wall mannan of an S. cerevisiae strain designated as Su1 (16). By using the crude Su1 strain mannan as an antigen, we have developed an ELISA procedure that allows an easy serological screening for anti-mannan of Saccharomyces cerevisiae antibodies (ASCA). We have then confirmed in a large series of patients including IBD and various intestinal inflammatory disorders that ASCA had a high specificity for CD (17). ASCA-positive status did not correlate with disease extent and activity and was not influenced by previous intestinal resection. This suggests that occurrence of ASCA is not an epiphenomenon secondary to bowel inflammation but could be a marker of an immunoregulatory disturbance associated with CD. This hypothesis led us to investigate whether ASCA might serve as a subclinical marker in CD by studying their prevalence in patients with familial CD and in their healthy first degree relatives.
INTRODUCTION Evidence for genetic predisposition including ethnic differences in disease frequency, familial aggregation, familiality in clinical characteristics, and higher monozygotic twin concordance has been gathered for several years in inflammatory bowel diseases (IBD) (1). An important step forward in research of genetic susceptibility to IBD has been the recent disclosure, using systematic genome screening, of susceptibility loci for Crohn’s disease (CD) on chromosome 16 (2, 3) and both for CD and ulcerative colitis (UC) on chromosomes 3, 7, and 12 (4). However, case ascertainment in genetic studies relied on clinical and endoscopic findings
MATERIALS AND METHODS Study population Families with at least two first degree relatives having CD were included in the study. Methods used for collecting information within families and the criteria for ascertaining
Received Sep. 17, 1997; accepted Apr. 10, 1998. 1306
AJG – August 1998 the diagnosis of CD were similar to those previously described (18). The probands were contacted and the family pedigrees were then established. Twenty families were screened, of which 15 had two affected members, two had three, one had four, one had six, and one had seven. Serum samples were not available from two of 53 patients (one was dead; one lived abroad). Among 51 sampled, there were 29 men and 22 women, median age 29.6 yr (range, 12– 67 yr). Disease location was the small bowel in 15 patients, the colon in 10 patients, and both the small bowel and the colon in 26 patients. Of 88 healthy relatives, 66 (75%) were studied. Their health status was assessed by an interviewer practitioner who visited the families. None of them had a previous history or symptoms of IBD. There were 26 men and 40 women, with a median age of 38 yr (range, 13– 68). There were 12 fathers, 17 mothers, 14 brothers, and 23 sisters. Of the 22 kindred not studied, four were dead, 11 refused to participate in the study, and seven lived abroad. Sera from 163 healthy hospital staff members and blood donors without any history of gastrointestinal disease or familial history of IBD were used as control subjects. Twenty-nine control subjects were matched for age and sex with 29 members of four families. All sera were stored at 240°C until assayed and blinded for diagnosis. Detection of ASCA by ELISA Antigens consisted of mannan extracted from yeast cells of S. cerevisiae Su1 strain grown in bioreactors. ELISA was performed as previously described. Briefly, plates were coated with 100 ml of PPM at a concentration of 1 mg/ml in sodium carbonate buffer (60 mmol/L pH 9.6) for 1 h at 37°C and overnight at 4°C, in moist chambers, and then washed four times in TNT (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.05% Tween 20, pH 7.5). Patients’ sera were diluted 1:1000 in TNT. Alkaline phosphatase-labeled goat antihuman immunoglobulin (G, A, M) (H and L chains) (Zymed, Biosoft, Paris, France) was diluted 1:3000 in TNT. A color reaction was rendered by using substrate Biotrol EIA 405 (Biotrol, Paris, France) for alkaline phosphatase. Plates were read at 405 nm on an Immunotech (Luminy, France) automatic reader. Pools of sera strongly reacting with S. cerevisiae mannan were used for standardizing the tests. Reactivities of individual sera were expressed as a percentage of the highest reactivity of the standard, arbitrarily defined as 100%. The upper limit of a normal result, which gave the best compromise between sensitivity and specificity for CD, was determined according to receiver operating characteristics (ROC) curves. Sera that showed reactivity above this upper limit were considered as positive. C-reactive protein was measured in sera obtained at the same time from 47 of 51 patients and 64 of 66 healthy relatives. Statistical analysis Statistical comparisons of prevalence of ASCA in the study groups were performed using the x2 test. Concordance for ASCA was defined as two or more affected members of
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the same family having positive ASCA. In families with more than two affected members, the percentage of concordance was calculated as the sum of concordant patients divided by the total number of patients. To establish the statistical significance of concordance for ASCA positivity, we compared observed concordant numbers to theoretical numbers using a x2 test. Theoretical numbers were calculated from values recorded in sporadic cases using a binomial theorem (19). They represented the expected values if the distribution of sporadic cases was applied to familial cases. RESULTS The detection of ASCA in the various groups is shown in Figure 1. A significant increased prevalence of positive ASCA was found not only in the probands (69%, 35 of 51, p , 0.0001) but also in their unaffected relatives (20%, 13 of 66, p , 0.001) as compared with controls (0.61%, one of 163). Percentages of ASCA positivity were 54%, 60%, and 76% in patients with small bowel involvement, pure colonic disease, and both small bowel and colon involvement, respectively. Presence of ASCA was independent from C-reactive protein in patients with CD. Two of 64 relatives had a C-reactive protein .15 mg/L. One was ASCA-positive and one was ASCA-negative. Pedigrees of families are given in Figure 2. ASCA-positive healthy relatives were distributed in 12 of 20 families. They were evenly distributed among families and did not cluster within a small number of multiplex families. Eight of 13 of the ASCA-positive healthy relatives were parents. In five families, children with CD were born from parents of whom one at least had CD. In 15 other families, CD occurred in children born from healthy parents and in eight of 15 of these families, at least one of both parents was ASCApositive. Among relatives of families with two affected members, 50% were concordant for ASCA positivity. This percentage reached 68% within families with more than two affected members. When comparing observed concordances for ASCA positivity within families with theoretical numbers based on sporadic cases, there was no overall significant difference but there was an excess of concordance for ASCA positivity in families with more than two affected members (Table 1). Finally, the prevalence of ASCA in healthy relatives was not higher when at least one affected relative was ASCApositive than when all affected relatives were ASCA-negative: nine of 53 (17%) versus four of 13 (31%) (NS). DISCUSSION Using a simple ELISA procedure involving the mannan of a S. cerevisiae strain, we have previously shown that antibodies against S. cerevisiae oligomannose residues, designated as ASCA, have a high degree of specificity for CD compared with UC and other colitides (16). The results of
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FIG. 1. Detection of ASCA by ELISA in study groups. CD: Crohn’s disease. HR: healthy relatives. Results are expressed as the logarithm of the percentage of reactivity. The horizontal dotted line represents log of 3.09%, which is the cut-off value of the test (16, 17). Values above this line were regarded as positive.
FIG. 2. Pedigrees of 20 families with Crohn’s disease. h: healthy man; E: healthy woman; ■ F: patient with Crohn’s disease; 1: ASCA-positive; 2: ASCA-negative; †: deceased.
the present study confirm the increased frequency of ASCA in patients with familial CD with an almost similar prevalence (68.5%) than that previously observed in sporadic cases (66%) (16, 17). Most interestingly, the frequency of ASCA was also increased in healthy relatives of patients with CD. This observation was strengthened by the fact that presence of ASCA in healthy relatives was observed in 12 of 20 families and was not restricted to a few particular multiplex families. The presence of ASCA in sera of healthy relatives may first indicate that a subclinical phase of CD process is
occurring in these subjects, which may or not eventuate in clinical disease. However, most ASCA-positive healthy relatives were parents with a median age of 54 yr, who were unlikely to further develop CD: in our region, 85% of incident cases of CD occur between the ages of 15 and 40 yr, with no second peak of incidence around 50 yr (20). Children with CD were born from parents having CD themselves in five cases and from ASCA positive healthy parents in eight cases. This led us to suppose that ASCA in parents is at least as potent as a risk factor for children to develop CD as parental CD.
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TABLE 1 Comparison Between Observed and Theoretical Concordances for ASCA Positivity Within Familial Cases
Total number of patients Observed numbers of concordant patients within families Theoretical numbers of concordant patients within families
All Families (n 5 19)*
Families With Two Affected Members (n 5 14)
Families With More Than Two Affected Members (n 5 5)
50 29 21.2
28 14 13.1
22 15* 8.1
* Concordance study was not possible in one family (n° 10);*: p , 0.01 vs theoretical number.
Whether ASCA reflect environmental or genetic factors remains unknown. Family occurrence of ASCA positive status in healthy relatives appears to be more an intergeneration phenomenon than an intrageneration phenomenon, as 62% of the ASCA-positive healthy relatives were parents. No significant difference in prevalence of ASCA was found in families with two or more affected members. These data suggest, rather, that family clustering of ASCA might be more secondary to genetic than to environmental factors. This is supported by a study from Sweden in which 26 monozygotic twin pairs with IBD (14 with CD and 12 with UC) and 52 healthy control subjects were investigated for serum antibodies against ovalbumin, betalactoglobulin, gliadin, whole yeast (Saccharomyces cerevisiae) and yeast cell wall mannan (21). Five of 14 CD twin pairs were concordant for the disease. Patients with UC were indistinguishable from healthy twins and control subjects for the response to yeast. Twins who had developed CD displayed higher antibody titers of all types toward yeast cell wall mannan and whole yeast. Also, healthy monozygotic twins of CDdiseased patients had a raised level of IgA to yeast cell wall mannan and whole yeast compared with control subjects. A negative finding in our study was that we found no difference in the frequency of ASCA in the relatives of probands whose sera were ASCA-positive compared with the relatives of probands whose sera were ASCA-negative. This suggests that ASCA are not a marker of disease heterogeneity in CD. In our previous evaluations of CD characteristics in relation to the presence of ASCA, the only distinguishing clinical feature was a younger age at diagnosis in ASCA-positive patients than in ASCA-negative patients (17). On the other hand, the pattern of distribution of positive results within some pedigrees including multiple affected members of the same generation (16, 19, 20), and the excess of concordance for ASCA positivity in families with more than two affected members might be in favor of an environmental contribution. Along this line, a possible link between the presence of ASCA and an increased intestinal permeability might be discussed. Although the results remain controversial, the hypothesis that an increased intestinal permeability serves as a possible subclinical marker for CD has been supported by the finding that a subgroup of 10 –25% of healthy relatives had an increased intestinal permeability expressed by the lactulose-mannitol index (22,
23). This permeability defect might in turn lead to an increased stimulation by yeast antigens. Although no definitive conclusions can yet be drawn, several arguments suggest that the immune response raised against mannooligosaccharides present in Saccharomyces cerevisiae mannan does not merely reflect an enhanced intestinal permeability. The humoral response was far more specific than that directed against dietary antigens or various yeasts including Candida albicans, which is a normal component of the endogenous microflora (16, 21). Factors such as disease location and activity, medication, and surgery, which are known to influence permeability, did not affect ASCA status in Crohn’s patients (17). In conclusion, the presence of ASCA in 20% of healthy relatives of patients with CD suggests that they represent a new marker of genetic/environmental susceptibility in CD. ACKNOWLEDGEMENTS This work was supported by a grant from the INSERM (CRI 4UOO4B and Registre 92/R2) and the Centre Hospitalier et Universitaire de Lille (Contrat 96/38/9713). Reprint requests and correspondence: J.F. Colombel, M.D., Clinique des maladies de l’appareil digestif, CHRU de Lille, Hopital Claude Huriez, 59037 Lille Cedex, France.
REFERENCES 1. Yang H, Rotter JL. Genetics of inflammatory bowel disease. In: Targan SR, Shanahan F, eds. Inflammatory bowel disease: From bench to bedside. Baltimore: Williams & Wilkins, 1994:32– 64. 2. Hugot JP, Laurent-Puig P, Gower-Rousseau C, et al. Mapping of a susceptibility locus for Crohn’s disease on chromosome 16. Nature 1996;379:821–3. 3. Ohmen JD, Yang HY, Yamamoto KK, et al. Susceptibility locus for inflammatory bowel disease on chromosome 16 has a role in Crohn’s disease, but not in ulcerative colitis. Hum Mol Genet 1996;5:1679 – 83. 4. Satsangi J, Parkes M, Louis E, et al. Two stage genome-wide search in inflammatory bowel disease provide evidence for susceptibility loci on chromosomes 3, 7 and 12. Nature Genet 1996;14:199 –202. 5. Korsmeyer SJ, Wiliams RC Jr, Wilson ID, et al. Lymphocytotoxic antibody in inflammatory bowel disease—a family study. N Engl J Med 1975;293:1117–20. 6. Hollander D, Vadheim CM, Brettholtz E, et al. Increased intestinal permeability in Crohn’s patients and their relatives: An etiological factor? Ann Intern Med 1986;105:883–95. 7. Elmgreen J, Both H, Binder V. Familial occurrence of complement dysfunction in Crohn’s disease: Correlation with intestinal symptoms and hypercatabolism of complement. Gut 1985;26:151–7. 8. Van de Merwe JP, Schroder AM, Wensinck F, et al. The obligate
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9. 10. 11. 12. 13. 14.
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anaerobic faecal flora of patients with Crohn’s disease and their firstdegree relatives. Scand J Gastroenterol 1988;23:1125–31. Tysk C, Riedesel H, Lindberg E, et al. Colonic glycoprotein in monozygotic twins with inflammatory bowel disease. Gastroenterology 1991;100:419 –23. Helgeland L, Tysk C, Ja¨rnerot G, et al. IgG subclass distribution in serum and rectal mucosa of monozygotic twins with or without inflammatory bowel disease. Gut 1992;33:1358 – 64. Shanahan F, Duerr RH, Rotter JI, et al. Neutrophil autoantibodies in ulcerative colitis: Familial aggregation and genetic heterogeneity. Gastroenterology 1992;103:456 – 61. Fiocchi C, Roche JK, Michener WM. High prevalence of antibodies to intestinal epithelial antigens in patients with inflammatory bowel disease and their relatives. Ann Intern Med 1989;110:786 –94. Main J, McKenzie H, Yeaman GR, et al. Antibody to Saccharomyces cerevisiae (baker’s yeast) in Crohn’s disease. Br Med J 1988;297: 1105– 6. Barnes RMR, Allan S, Taylor-Robinson CH, et al. Serum antibodies reactive with Saccharomyces cerevisiae in inflammatory bowel disease. Is IgA antibody a marker for Crohn’s disease? Int Arch Allergy Appl Immunol 1990;92:9 –15. Giaffer MH, Clark A, Holdsworth CD. Antibodies to Saccharomyces cerevisiae in patients with Crohn’s disease and their possible pathogenic importance. Gut 1992;33:1071–5.
AJG – Vol. 93, No. 8, 1998 16. Sendid B, Colombel JF, Jacquinot PM, et al. Specific antibody response to oligomannosidic epitopes in Crohn’s disease. Clin Diag Lab Immunol 1996;3:219 –26. 17. Quinton JF, Sendid B, Reumaux D, et al. Anti-Saccharomyces cerevisiae mannan combined with antineutrophil antibodies in inflammatory bowel disease: Prevalence and diagnostic role. Gut 1998;42:788 –91. 18. Colombel JF, Grandbastien B, Gower-Rousseau C, et al. Clinical characteristics of Crohn’s disease in 72 families. Gastroenterology 1996;111:604 –7. 19. Colton T. Statistics in medicine. Boston: Little Brown, 1974. 20. Gower-Rousseau C, Salomez JL, Dupas JL, et al. Incidence of inflammatory bowel disease in northern France (1988 –1990). Gut 1994;35: 1433– 8. 21. Lindberg E, Magnusson KE, Tysk C, et al. Antibody (IgG, IgA, and IgM) to baker’s yeast (Saccharomyces cerevisiae), yeast mannan, gliadin, ovalbumin and betalactoglobulin in monozygotic twins with inflammatory bowel disease. Gut 1992;33:909 –13. 22. May GR, Sutherland LR, Meddings JB. Is small intestinal permeability really increased in relatives of patients with Crohn’s disease? Gastroenterology 1993;104:1627–32. 23. Peeters M, Geypens B, Claus D, et al. Clustering of increased small intestinal permeability in families with Crohn’s disease. Gastroenterology 1997;113:802–7.
Vol. 93, No. 8, 1998 ISSN 0002-9270/98/$19.00 PII S0002-9270(98)00296-2
Anti-Saccharomyces cerevisiae Mannan Antibodies in Familial Crohn’s Disease Boualem Sendid, M.D., Jean-Franc¸ois Quinton, M.D., Gwe´nae¨le Charrier, M.D., Olivier Goulet, M.D., Antoine Cortot, Bruno Grandbastien, M.D., Daniel Poulain, Ph.D., and Jean-Fre´de´ric Colombel, M.D. Service d’He´pato-Gastroente´rologie et Registre des Maladies Inflammatoires du Tube Digestif du Nord-Ouest de la France (EPIMAD); Laboratoire de Parasitologie-Mycologie, CHRU Lille; INSERM U 42, Lille; Service de Gastroente´rologie et Nutrition, De´partement de Pe´diatrie, Hopital Necker, Paris, France
Objective: Anti-Saccharomyces cerevisiae mannan antibodies (ASCA) are associated with Crohn’s disease. The aim of this study was to determine the prevalence of ASCA in families in which at least two members were affected with Crohn’s disease. Methods: A total of 20 families including two (n 5 15) or more (n 5 5) patients with Crohn’s disease were tested for ASCA with use of an ELISA method. Overall, 51 affected members, 66 healthy first degree relatives, and 163 healthy control subjects were studied. Results: ASCA were detected in 35 of 51 (69%) patients with Crohn’s disease and in 13 of 66 (20%) healthy relatives versus one of 163 healthy control subjects (p < 0.0001 and p < 0.001). ASCApositive relatives were distributed in 12 of 20 families. ASCA were present in eight healthy parents and four healthy siblings. The prevalence of ASCA in relatives did not depend on the ASCA status of affected members. Conclusion: ASCA in 20% of healthy first degree relatives of patients with Crohn’s disease suggest that these antibodies might be a subclinical marker for Crohn’s disease in families. Whether ASCA reflect environmental or genetic factors or a combination of both is unknown. (Am J Gastroenterol 1998;93: 1306 –1310. © 1998 by Am. Coll. of Gastroenterology)
that may be insufficient: clinically unaffected individuals bearing the variant genotype will not be recognized (1). Such unaffected individuals could be better identified by the use of subclinical markers that could detect the abnormal genotype in the absence of the full phenotype (1). Several candidate markers have been proposed thus far, including lymphocytotoxic antibodies, intestinal permeability, C3 dysfunction and obligate anaerobic flora in CD (5– 8), colonic mucins, mucosal Ig distribution and antineutrophil cytoplasmic antibodies (ANCA) in UC (9 –11), and antibodies to colonic epithelial cells in both CD and UC (12). To date, the results remain conflicting and none of these markers has been definitively established as a subclinical marker of either CD or UC. Systemic antibodies against the yeast Saccharomyces cerevisiae have been described in sera from patients with CD but not with UC (13–15). This serologic response mainly concerns sequences of mannose residues expressed in the cell wall mannan of an S. cerevisiae strain designated as Su1 (16). By using the crude Su1 strain mannan as an antigen, we have developed an ELISA procedure that allows an easy serological screening for anti-mannan of Saccharomyces cerevisiae antibodies (ASCA). We have then confirmed in a large series of patients including IBD and various intestinal inflammatory disorders that ASCA had a high specificity for CD (17). ASCA-positive status did not correlate with disease extent and activity and was not influenced by previous intestinal resection. This suggests that occurrence of ASCA is not an epiphenomenon secondary to bowel inflammation but could be a marker of an immunoregulatory disturbance associated with CD. This hypothesis led us to investigate whether ASCA might serve as a subclinical marker in CD by studying their prevalence in patients with familial CD and in their healthy first degree relatives.
INTRODUCTION Evidence for genetic predisposition including ethnic differences in disease frequency, familial aggregation, familiality in clinical characteristics, and higher monozygotic twin concordance has been gathered for several years in inflammatory bowel diseases (IBD) (1). An important step forward in research of genetic susceptibility to IBD has been the recent disclosure, using systematic genome screening, of susceptibility loci for Crohn’s disease (CD) on chromosome 16 (2, 3) and both for CD and ulcerative colitis (UC) on chromosomes 3, 7, and 12 (4). However, case ascertainment in genetic studies relied on clinical and endoscopic findings
MATERIALS AND METHODS Study population Families with at least two first degree relatives having CD were included in the study. Methods used for collecting information within families and the criteria for ascertaining
Received Sep. 17, 1997; accepted Apr. 10, 1998. 1306
AJG – August 1998 the diagnosis of CD were similar to those previously described (18). The probands were contacted and the family pedigrees were then established. Twenty families were screened, of which 15 had two affected members, two had three, one had four, one had six, and one had seven. Serum samples were not available from two of 53 patients (one was dead; one lived abroad). Among 51 sampled, there were 29 men and 22 women, median age 29.6 yr (range, 12– 67 yr). Disease location was the small bowel in 15 patients, the colon in 10 patients, and both the small bowel and the colon in 26 patients. Of 88 healthy relatives, 66 (75%) were studied. Their health status was assessed by an interviewer practitioner who visited the families. None of them had a previous history or symptoms of IBD. There were 26 men and 40 women, with a median age of 38 yr (range, 13– 68). There were 12 fathers, 17 mothers, 14 brothers, and 23 sisters. Of the 22 kindred not studied, four were dead, 11 refused to participate in the study, and seven lived abroad. Sera from 163 healthy hospital staff members and blood donors without any history of gastrointestinal disease or familial history of IBD were used as control subjects. Twenty-nine control subjects were matched for age and sex with 29 members of four families. All sera were stored at 240°C until assayed and blinded for diagnosis. Detection of ASCA by ELISA Antigens consisted of mannan extracted from yeast cells of S. cerevisiae Su1 strain grown in bioreactors. ELISA was performed as previously described. Briefly, plates were coated with 100 ml of PPM at a concentration of 1 mg/ml in sodium carbonate buffer (60 mmol/L pH 9.6) for 1 h at 37°C and overnight at 4°C, in moist chambers, and then washed four times in TNT (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.05% Tween 20, pH 7.5). Patients’ sera were diluted 1:1000 in TNT. Alkaline phosphatase-labeled goat antihuman immunoglobulin (G, A, M) (H and L chains) (Zymed, Biosoft, Paris, France) was diluted 1:3000 in TNT. A color reaction was rendered by using substrate Biotrol EIA 405 (Biotrol, Paris, France) for alkaline phosphatase. Plates were read at 405 nm on an Immunotech (Luminy, France) automatic reader. Pools of sera strongly reacting with S. cerevisiae mannan were used for standardizing the tests. Reactivities of individual sera were expressed as a percentage of the highest reactivity of the standard, arbitrarily defined as 100%. The upper limit of a normal result, which gave the best compromise between sensitivity and specificity for CD, was determined according to receiver operating characteristics (ROC) curves. Sera that showed reactivity above this upper limit were considered as positive. C-reactive protein was measured in sera obtained at the same time from 47 of 51 patients and 64 of 66 healthy relatives. Statistical analysis Statistical comparisons of prevalence of ASCA in the study groups were performed using the x2 test. Concordance for ASCA was defined as two or more affected members of
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the same family having positive ASCA. In families with more than two affected members, the percentage of concordance was calculated as the sum of concordant patients divided by the total number of patients. To establish the statistical significance of concordance for ASCA positivity, we compared observed concordant numbers to theoretical numbers using a x2 test. Theoretical numbers were calculated from values recorded in sporadic cases using a binomial theorem (19). They represented the expected values if the distribution of sporadic cases was applied to familial cases. RESULTS The detection of ASCA in the various groups is shown in Figure 1. A significant increased prevalence of positive ASCA was found not only in the probands (69%, 35 of 51, p , 0.0001) but also in their unaffected relatives (20%, 13 of 66, p , 0.001) as compared with controls (0.61%, one of 163). Percentages of ASCA positivity were 54%, 60%, and 76% in patients with small bowel involvement, pure colonic disease, and both small bowel and colon involvement, respectively. Presence of ASCA was independent from C-reactive protein in patients with CD. Two of 64 relatives had a C-reactive protein .15 mg/L. One was ASCA-positive and one was ASCA-negative. Pedigrees of families are given in Figure 2. ASCA-positive healthy relatives were distributed in 12 of 20 families. They were evenly distributed among families and did not cluster within a small number of multiplex families. Eight of 13 of the ASCA-positive healthy relatives were parents. In five families, children with CD were born from parents of whom one at least had CD. In 15 other families, CD occurred in children born from healthy parents and in eight of 15 of these families, at least one of both parents was ASCApositive. Among relatives of families with two affected members, 50% were concordant for ASCA positivity. This percentage reached 68% within families with more than two affected members. When comparing observed concordances for ASCA positivity within families with theoretical numbers based on sporadic cases, there was no overall significant difference but there was an excess of concordance for ASCA positivity in families with more than two affected members (Table 1). Finally, the prevalence of ASCA in healthy relatives was not higher when at least one affected relative was ASCApositive than when all affected relatives were ASCA-negative: nine of 53 (17%) versus four of 13 (31%) (NS). DISCUSSION Using a simple ELISA procedure involving the mannan of a S. cerevisiae strain, we have previously shown that antibodies against S. cerevisiae oligomannose residues, designated as ASCA, have a high degree of specificity for CD compared with UC and other colitides (16). The results of
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FIG. 1. Detection of ASCA by ELISA in study groups. CD: Crohn’s disease. HR: healthy relatives. Results are expressed as the logarithm of the percentage of reactivity. The horizontal dotted line represents log of 3.09%, which is the cut-off value of the test (16, 17). Values above this line were regarded as positive.
FIG. 2. Pedigrees of 20 families with Crohn’s disease. h: healthy man; E: healthy woman; ■ F: patient with Crohn’s disease; 1: ASCA-positive; 2: ASCA-negative; †: deceased.
the present study confirm the increased frequency of ASCA in patients with familial CD with an almost similar prevalence (68.5%) than that previously observed in sporadic cases (66%) (16, 17). Most interestingly, the frequency of ASCA was also increased in healthy relatives of patients with CD. This observation was strengthened by the fact that presence of ASCA in healthy relatives was observed in 12 of 20 families and was not restricted to a few particular multiplex families. The presence of ASCA in sera of healthy relatives may first indicate that a subclinical phase of CD process is
occurring in these subjects, which may or not eventuate in clinical disease. However, most ASCA-positive healthy relatives were parents with a median age of 54 yr, who were unlikely to further develop CD: in our region, 85% of incident cases of CD occur between the ages of 15 and 40 yr, with no second peak of incidence around 50 yr (20). Children with CD were born from parents having CD themselves in five cases and from ASCA positive healthy parents in eight cases. This led us to suppose that ASCA in parents is at least as potent as a risk factor for children to develop CD as parental CD.
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TABLE 1 Comparison Between Observed and Theoretical Concordances for ASCA Positivity Within Familial Cases
Total number of patients Observed numbers of concordant patients within families Theoretical numbers of concordant patients within families
All Families (n 5 19)*
Families With Two Affected Members (n 5 14)
Families With More Than Two Affected Members (n 5 5)
50 29 21.2
28 14 13.1
22 15* 8.1
* Concordance study was not possible in one family (n° 10);*: p , 0.01 vs theoretical number.
Whether ASCA reflect environmental or genetic factors remains unknown. Family occurrence of ASCA positive status in healthy relatives appears to be more an intergeneration phenomenon than an intrageneration phenomenon, as 62% of the ASCA-positive healthy relatives were parents. No significant difference in prevalence of ASCA was found in families with two or more affected members. These data suggest, rather, that family clustering of ASCA might be more secondary to genetic than to environmental factors. This is supported by a study from Sweden in which 26 monozygotic twin pairs with IBD (14 with CD and 12 with UC) and 52 healthy control subjects were investigated for serum antibodies against ovalbumin, betalactoglobulin, gliadin, whole yeast (Saccharomyces cerevisiae) and yeast cell wall mannan (21). Five of 14 CD twin pairs were concordant for the disease. Patients with UC were indistinguishable from healthy twins and control subjects for the response to yeast. Twins who had developed CD displayed higher antibody titers of all types toward yeast cell wall mannan and whole yeast. Also, healthy monozygotic twins of CDdiseased patients had a raised level of IgA to yeast cell wall mannan and whole yeast compared with control subjects. A negative finding in our study was that we found no difference in the frequency of ASCA in the relatives of probands whose sera were ASCA-positive compared with the relatives of probands whose sera were ASCA-negative. This suggests that ASCA are not a marker of disease heterogeneity in CD. In our previous evaluations of CD characteristics in relation to the presence of ASCA, the only distinguishing clinical feature was a younger age at diagnosis in ASCA-positive patients than in ASCA-negative patients (17). On the other hand, the pattern of distribution of positive results within some pedigrees including multiple affected members of the same generation (16, 19, 20), and the excess of concordance for ASCA positivity in families with more than two affected members might be in favor of an environmental contribution. Along this line, a possible link between the presence of ASCA and an increased intestinal permeability might be discussed. Although the results remain controversial, the hypothesis that an increased intestinal permeability serves as a possible subclinical marker for CD has been supported by the finding that a subgroup of 10 –25% of healthy relatives had an increased intestinal permeability expressed by the lactulose-mannitol index (22,
23). This permeability defect might in turn lead to an increased stimulation by yeast antigens. Although no definitive conclusions can yet be drawn, several arguments suggest that the immune response raised against mannooligosaccharides present in Saccharomyces cerevisiae mannan does not merely reflect an enhanced intestinal permeability. The humoral response was far more specific than that directed against dietary antigens or various yeasts including Candida albicans, which is a normal component of the endogenous microflora (16, 21). Factors such as disease location and activity, medication, and surgery, which are known to influence permeability, did not affect ASCA status in Crohn’s patients (17). In conclusion, the presence of ASCA in 20% of healthy relatives of patients with CD suggests that they represent a new marker of genetic/environmental susceptibility in CD. ACKNOWLEDGEMENTS This work was supported by a grant from the INSERM (CRI 4UOO4B and Registre 92/R2) and the Centre Hospitalier et Universitaire de Lille (Contrat 96/38/9713). Reprint requests and correspondence: J.F. Colombel, M.D., Clinique des maladies de l’appareil digestif, CHRU de Lille, Hopital Claude Huriez, 59037 Lille Cedex, France.
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