Serum anti-glycan-antibodies in relatives of patients with inflammatory bowel disease
Serum anti-glycan-antibodies in relatives of patients with inflammatory bowel disease
Florian Kamm 0 1
Ulrike Strauch 0 1
Frauke Degenhardt 1
Rocio Lopez 1
Claudia Kunst 0 1
Gerhard Rogler 1
Andre Franke 1
Frank Klebl 0 1
Florian Rieders 0 1
☯ These authors contributed equally to this work. 1
0 Department of Internal Medicine I, University of Regensburg , Regensburg, Germany , 2 Institute of Clinical Molecular Biology, Christian-Albrechts-University of Kiel , Kiel, Germany , 3 Department of Quantitative Health Sciences, Cleveland Clinic Foundation, Cleveland, United States of America, 4 Department of Gastroenterology and Hepatology, University Hospital ZuÈ rich, ZuÈ rich, Switzerland, 5 Zurich Center for Integrative Human Physiology, University of ZuÈ rich, ZuÈ rich, Switzerland, 6 Department of Pathobiology, Lerner Research Institute, Cleveland Clinic Foundation , Cleveland , United States of America
1 Editor: John Green, University Hospital Llandough , UNITED KINGDOM
Serum anti-glycan antibodies are a promising tool for differential diagnosis, disease stratification and prediction of Crohn's disease (CD). To investigate possible heritability of the markers we assessed the presence of serum anti-glycan antibodies in affected and unaffected relatives of patients with CD.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: This work was supported by the Ministry
of Science and Education (Bundesministerium fuÈr
Bildung und Forschung), Kompetenznetz chronisch
entzuÈndliche Darmerkrankungen (Competence
Network "Inflammatory Bowel Disease"). FR
received grants from the NIH, European Crohn's
and Colitis Organization, Crohn's and Colitis
Foundation and German Research Foundation. The
Serum samples of 169 IBD patients of the German inflammatory bowel disease (IBD)
network (140 CD & 29 Ulcerative colitis (UC)), 349 relatives of CD patients, 63 relatives of UC
patients and 46 healthy controls were tested for the presence of anti-glycan antibodies by
ELISA in a blinded fashion. Clinical data of the IBD patients and controls were available.
A higher proportion of non-affected CD relatives was positive for anti-glycan antibodies
compared to healthy subjects. No inheritance of a specific pattern of anti-glycan antibodies
could be detected. No difference in marker expression depending on the degree of
relationship in the non-affected relatives was noted and the presence of family history did not lead
to a difference in marker levels in the affected CD subjects.
Non-affected CD relatives had a higher frequency of anti-glycan antibodies compared to
healthy subjects. This difference was mild and was found to be true for the overall reactivity
to glycan antigens, but not for specific patterns. This may indicate an inherited mechanism
resulting in a non-specific increased reactivity to microbial antigens in IBD.
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
Competing interests: F.R. is on the advisory board
and/or speakers' bureau of AbbVie, UCB and
consultant to UCB, Celgene, Pliant, Roche,
Sansung and Thetis. G.R. has consulted to Abbvie,
Augurix, Boehringer, Calypso, FALK, Ferring,
Fisher, Genentech, Essex/MSD, Novartis, Pfizer,
Phadia, Roche, UCB, Takeda, Tillots, Vifor, Vital
Solutions and Zeller; Gerhard Rogler has received
speaker's honoraria from Astra Zeneca, Abbott,
Abbvie, FALK, MSD, Phadia, Tillots, UCB, and Vifor;
Gerhard Rogler has received educational grants
and research grants from Abbot, Abbvie,
Ardeypharm, Augurix, Calypso, Essex/MSD, FALK,
Flamentera, Novartis, Roche, Takeda, Tillots, UCB
and Zeller. This does not alter our adherence to
PLOS ONE policies on sharing data and materials.
Abbreviations: ACCA, Anti-chitobioside
carbohydrate antibody; ALCA, Anti-laminaribioside
carbohydrate antibody; AMCA, Anti-mannobioside
carbohydrate antibody; Anti-C, Anti-chitin;
AnticBir1, Antibodies against the bacterial flagellin
cBir1; Anti-I2, Antibodies against
Pseudomonasassociated sequence I2; Anti-L, Anti-laminarin;
Anti-OmpC, Anti-outer membrane porin C of
Escherichia coli; BMI, Body mass index; CD,
Crohn's disease; ELISA, Enzyme linked
immunosorbent assay; EU, ELISA units; gASCA,
Anti-Saccharomyces cerevisiae antibody; IBD,
Inflammatory bowel disease; UC, Ulcerative colitis.
Aside the environment and a dysregulated immune system, genetic factors are a critical
component in the pathogenesis of Crohn's disease (CD) [
]. The heritable nature of this disease
has been determined in twin studies and by investigating familial aggregation, with observed
concordance rates of 20±40% in monozygotic twins and 0±7% in dizygotic twin pairs, but also
by ethnic/racial differences in the prevalence of CD [
]. The host immune response to
commensal bacteria is crucial in maintaining mucosal homeostasis. It has become a well-accepted
concept that an abnormality of this response is a key contributing factor to disease
1, 4, 5
In concordance with this notion CD patients exert a measurable immune response to
different microbial factors leading to serologic antibodies directed against microbial components
[6±8]. Examples for these antibodies are anti-Saccharomyces cerevisiae antibodies (ASCA),
antibodies against Pseudomonas-associated sequence I2 (anti-I2), outer membrane porin C
(OmpC) of Escherichia coli and against the bacterial flagellin cBir1 (Anti-cBir1) [
6, 9, 10
different set of serum antibodies directed against microbial antigens are
anti-glycan-antibodies, consisting of anti-Saccharomyces cerevisiae antibodies (gASCA), anti-mannobioside
antibodies (AMCA), anti-laminaribioside antibodies (ALCA), anti-chitobioside antibodies
(ACCA), anti-laminarin antibody (Anti-L) and anti-chitin antibody (Anti-C). This set of
biomarkers is highly specific for the diagnosis of CD and associated with and predictive of
complicated CD courses, signified by the earlier development of stricture, internal penetrating disease
or need for surgery [7, 11±15].
The association of the anti-glycan-antibody panel with CD could represent an inherited
increased immune response which may be a primary phenomenon or secondary to e.g.
increased permeability of the gut [
]. This hypothesis led us to investigate whether there is
an increased response to anti-glycan antibodies in affected and unaffected family members of
patients with CD.
IBD relatives. IBD relatives, defined as related family members of IBD patients (either
CD or UC), were recruited in the year 2007 through the IBD patients from the IBD center
of the University Hospital of Regensburg. All relatives of our IBD patients were asked to
participate. Only Caucasian families were included in this study. There were between 1 and 8
relatives per reference IBD subject with a median of 2 relatives [25th percentile (P25), 75th
percentile (P75): 2, 3]. Family clusters were taken into account for any comparison involving the
reference IBD patients vs. their relatives. Of the 349 relatives of CD patients 330 did not have
IBD, 13 had CD and 6 had UC. Of the 65 UC relatives 63 were healthy and 2 had UC. The two
UC relatives having UC were excluded from this study due to their number being too low for a
meaningful analysis. 66.7% of the CD and 74.6% of the UC relatives were first degree relatives
(parents, siblings and children) and the remainder more distant relatives (uncles, nieces,
grandparents, etc.). The demographic information can be found in Table 1.
IBD and control cohort. The IBD relatives were related to 140 CD and 29 UC patients,
which are part of the previously described cross-sectional IBD cohort [
]. An additional
set of 46 apparently healthy controls was also investigated. Collection of this cohort occurred
between 2000 and 2006. The healthy controls were not related to the IBD patients and had no
family history of IBD. The diagnosis of CD and UC was made based on clinical, radiographic,
endoscopic and histopathological criteria [
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The following demographic data was included: body mass index (BMI), gender, date of sample
procurement, age at diagnosis, occurrence of complications, surgery and disease location.
Collected data were transferred and stored in a secure coded anonymized database for analysis. The
procurement of the clinical data points mentioned above occurred in a blinded fashion to the
antibody values. The presence of IBD in the unaffected IBD relatives was excluded by evaluating
their history and the absence of typical symptoms. Signed informed consent was obtained from
all participants. The ethics committee of the University of Regensburg approved the study.
Serum was separated from whole blood by centrifugation and kept frozen at -80ÊC until use.
Serum was analyzed for levels of gASCA IgG, ALCA IgG, ACCA IgA, AMCA IgG, Anti-L IgA
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and Anti-C IgA in a blinded fashion and as previously described [
]. We used enzyme
linked immunosorbent assay (ELISA) following the manufacturer's protocol (Glycominds,
Ltd; Lod, Israel). The cut-off values were chosen based on a previous cross sectional analysis
]: gASCA 50 ELISA unit (EU), ACCA 90 EU, ALCA 60 EU, AMCA 100 EU, Anti-L 120
EU, Anti-C 50 EU. Samples with measurements exceeding the above cut-off values for the respective antibody were called antibody positive, samples with measurements below the cutoff were called antibody negative.
Descriptive statistics were computed for all clinical variables and the antibody measurements.
These include the mean, standard deviation, percentiles for continuous and frequencies for
categorical variables. Within each analyzed clinical group we determined the number of
samples with 0, 1, 2, 3, 4, 5 or 6 positive antibodies (see above). To assess the overall antibody
response within the clinical groups quartile scores for each serological marker were calculated,
as described previously [
9, 12, 14, 21
]. By adding individual quartile scores for each glycan
antigen, a semi-quantitative quartile sum score (range 6±24) representing the cumulative
quantitative immune response towards all six antigens for each patient was obtained.
Comparisons between healthy controls and non-IBD relatives as well as comparisons between
reference UC patients and CD relatives with UC were done using Student's t-tests or the
nonparametric Wilcoxon rank sum tests for continuous or ordinal variables and Pearson's
chisquare tests or Fisher's Exact tests for categorical factors. Comparisons between relatives and
reference IBD patients were done using generalized linear mixed models with family cluster as
a random effect. SAS version 9.2 software (The SAS Institute, Cary, NC) and R version 2.4.1
software (The R Institute for Statistical Computing, Vienna, Austria) were used for all analyses.
A p<0.05 was considered statistically significant.
Status and level of anti-glycan antibodies in CD and UC patients and their
We analyzed the prevalence of anti-glycan antibodies in the reference CD and UC patients
(Table 2). The measured levels of the anti-glycan markers in the IBD patients was comparable
to previously published studies, including our own [
], indicating that no selection bias
compared to the whole IBD patient cohort of the University Hospital of Regensburg was present.
We first compared the reference CD patients to their affected relatives: No difference was
noted in quality (defined as positive or negative for a certain antibody) or quantity (level of a
certain antibody) of the antibodies between CD relatives affected by CD compared to the
reference CD subjects. In the CD relatives affected by UC compared to the reference CD subjects
only Anti-L levels and the quartile sum score were lower. Interestingly, in the CD relatives
affected by UC compared to the reference UC subjects a trend towards higher levels of the
glycan markers was detected. More reference UC subjects were negative for all markers
compared to the UC affected CD relatives and the frequency of gASCA was higher in the UC
affected CD relatives compared to the reference UC patients. This indicates an overall stronger
immune response in UC relatives of CD patients compared to UC patients with no CD
relatives (Table 2).
Secondly, we compared the non-affected CD relatives with their affected CD family mem
bers: The levels of gASCA, ALCA, Anti-L and Anti-C, the frequency of gASCA, ALCA and
AMCA and the quartile sum score were lower in the non-affected CD relatives. More non
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CD: Crohn's disease, UC: Ulcerative colitis, non IBD: non-inflammatory bowel disease affected, EU: ELISA units, SD: Standard deviation, gASCA: anti-Saccharomyces
cerevisiae antibodies, ACCA: anti-chitobioside carbohydrate IgA antibodies, ALCA: anti-laminaribioside carbohydrate IgG antibodies, AMCA: anti-mannobioside
carbohydrate IgG antibodies, Anti-L: anti-laminarin carbohydrate antibody, Anti-C: anti-chitin carbohydrate antibody
Comparisons between Non IBD relatives and controls were done using chi-square or Fisher's Exact tests for positivity, Wilcoxon rank sum test for number of positive
markers and t-tests for marker levels and sum of quartiles.
a: significantly different from Non IBD CD relative
b: significantly different from Non IBD UC relative
Comparisons between reference UC patients and UC relatives of CD were done using chi-square or Fisher's Exact tests for positivity, Wilcoxon rank sum test for #
positve markers and t-test for marker levels and sumo of quartiles.
²: significantly different from reference UC patient
affected CD relatives were negative for all markers compared to the reference CD patients.
This confirms that CD patients have higher immune responses to glycan antigens than their non-affected family members (Table 2).
Thirdly, the non-affected UC relatives were tested against the reference UC subjects: Nonaffected UC relatives had lower levels of ALCA and Anti-L as well as a lower quartile sum score than reference UC subject, while there was no difference in the overall marker frequency or the number of positive markers (Table 2).
Fourthly, we compared the apparently healthy controls (no intestinal inflammation or dis
ease, none of the healthy controls were related to each other) to the non-affected IBD relatives:
Levels of ACCA, AMCA, ALCA and Anti-L as well as frequency of ACCA and AMCA were
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lower in the healthy controls compared to the CD relatives. There was a trend towards a higher
quartile sum score in the non-affected CD relatives compared to healthy controls. More
healthy controls were negative for all markers compared to the non-affected relatives of CD
patients (Table 2), indicating a more profound immune response towards glycan antigens in
the non-affected CD relatives.
Glycan marker expression by degree of relationship
To further elucidate a possible hereditary pattern of the anti-glycan antibodies, we investigated
whether the degree of relationship with the reference CD subject influences the anti-glycan
antibody expression. For this purpose, we divided the non-affected relatives of CD patients
into first-degree relatives and non-first degree relatives. No difference in anti-glycan antibody
expression was noted between these two groups (S1 Table), indicating that a relation to a CD
patient appears to influence the marker expression, but the degree of relation does not.
Expression of the glycan markers in CD patients based on family history
Since the degree of relationship did not make a difference in the glycan marker expression in
the non-affected CD relatives we investigated whether the presence of a family history in the
affected CD subjects influences their immune response towards glycan antigens. For this
purpose, we divided the reference CD patients into two groups: CD subjects with IBD relatives
and those without. ACCA was the only marker with a higher frequency in the CD subjects that
have a family history of IBD compared to no family history of IBD. All other markers, the
number of positive markers per patient or the quartile sum score were approximately the same
Expression levels of the glycan markers in IBD relatives based on the
quartile sum score or the ASCA status of the reference IBD population
To assess if the overall strength of the immune response is inherited we tested if the level of the
quartile sum score in the reference IBD patients is linked to the quartile sum score (QSS) or
the number of positive markers in the non-affected IBD relatives. For this purpose, we divided
our IBD reference cohort into two equal numbered parts according the QSS ( 15 or <15).
There was no difference in marker expression in the relatives of CD patients with a high versus a low QSS (Table 3).
Influence of levels of anti-glycan antibodies on the later development of
To assess whether an increased level of anti-glycan antibodies in the relatives of CD patients
predisposes to the development of IBD we contacted all relatives 5 years after sample
procurement to determine, if they developed IBD. In the UC healthy relatives group information was
available on 52 subjects and none of them developed IBD. In the CD healthy relatives group
we received 246 responses. A total of 3 patients developed IBD during follow-up (2 CD with
an initial QSS of 6 and 9; 1 UC with an initial QSS of 11). The median QSS of the CD relatives
remaining healthy was 9 (P25, P75: 7,11).
The underlying data set for this study can be downloaded as S3 Table.
We herein show that the frequency of anti-glycan antibodies in non-affected relatives of CD patients is higher compared to healthy controls. The strength of the immune response based
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Marker positivity n (%)
Number positive markers
Sum of Quartiles
on the quartile sum score was not inherited. The degree of relationship did not influence the
anti-glycan antibody levels.
This is the to date largest study examining anti-glycan antibodies in relatives of IBD
patients. Other serum markers linked to CD have been studied in families, including ASCA
and anti-OmpC. In CD ASCA has been shown to be familial in both affected and non-affected
relatives of CD patients. This was true for a qualitative (positive or negative for a certain
marker) and a quantitative (levels of a certain marker) relationship [22±27]. The prevalence in
relatives of ASCA positive CD patients was higher compared to the prevalence in relatives of
ASCA negative patients , which has been shown in independent cohorts from Europe,
USA and Tunisia [26±28]. Mei et al. found comparable results for anti-OmpC [
] with a
qualitative and quantitative increase in unaffected relatives of patients with CD. The phenomenon
of heritability is not specific for anti-microbial antibodies. Goblet cell antibodies and
antibodies against exocrine pancreas are found in increased prevalence in first degree relatives of
patients with CD [
]. Interestingly familiarity of ASCA occurs independently of the
diagnosis of CD and vertical transmission of the marker from mother to child has been suggested
. In CD patients, the presence of a family history was not associated with different levels of
ASCA . One report investigated the anti-glycan antibodies ASCA, AMCA, ALCA and
ACCA among others in multiple-affected families with CD, but no detailed analysis compar
ing unaffected relatives of IBD patients and healthy controls was performed [
Our study uses a more recent panel of anti-microbial antibodies, so called anti-glycan anti
bodies, in relatives of IBD patients. We confirmed prior findings of a stronger immune
response towards microbial components in non-affected IBD relatives, also with glycans as
antigens. This was found to be true for the overall reactivity to glycan antigens. However, the
degree of relationship did not make a difference in the expression of the markers, the presence
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of a family history did not lead to a difference in marker levels in the affected CD subjects and
the strength of the immune response was not inherited. These findings argue against a strong
inheritable nature of the anti-glycan antibodies in IBD relatives, but may rather present an
epiphenomenon due to e.g. increased intestinal permeability, an unidentified inherited risk factor
or shared environmental exposures. Nevertheless, they might serve as a risk marker for CD
and indicate a family member with an increased risk for developing CD in the future. Our
follow-up may have been too short to detect this pattern. A longitudinal study could help clarify
The present study has several limitations: We did not have access to household members of
the affected patients and their relatives as a control population. Therefore, we cannot exclude
an environmental contribution to the familial expression of these markers, such as domestic
hygiene, diet, other environmental factors or life habits [
]. However, studies of other serum
markers, such as ASCA and anti-OmpC [
] suggested that a shared household in
adulthood cannot explain the familial aggregation. The low number of non-affected IBD relatives
that developed IBD over time does not allow a meaningful analysis of the glycan markers
predicting disease development. The detected antibodies may not be specific for the `microbial'
glycans, but may be directed against self glycans  and this study is not able to answer this
definitively. This study is exploratory and no comparison for multiple testing has been
performed. The findings in this study need to be externally validated. We do not have information
about potential IgA deficieny in our subjects. Three of the anti-glycan antibodies are of the
IgA class and this could have influenced the results.
In summary, a higher frequency of non-affected CD relatives was positive for anti-glycan antibodies compared to healthy subjects. This was found to be true for the overall reactivity to glycan antigens, but not for the strength of the response. This could indicate an inherited increased reactivity to microbial antigens in IBD.
We would like to thank the study nurses and physicians of the Department of Internal Medicine I for their contribution to this work. This work was supported by the Ministry of Science and Education (Bundesministerium fuÈr Bildung und Forschung), Kompetenznetz chronisch entzuÈndliche Darmerkrankungen (Competence Network ¹Inflammatory Bowel Diseaseª).
F.R. is on the advisory board and/or speakers' bureau of AbbVie, UCB and consultant to UCB,
Celgene, Pliant, Roche, Sansung and Thetis. G.R. has consulted to Abbvie, Augurix, Boehrin
ger, Calypso, FALK, Ferring, Fisher, Genentech, Essex/MSD, Novartis, Pfizer, Phadia, Roche,
UCB, Takeda, Tillots, Vifor, Vital Solutions and Zeller; Gerhard Rogler has received speaker's honoraria from Astra Zeneca, Abbott, Abbvie, FALK, MSD, Phadia, Tillots, UCB, and Vifor;
8 / 11
Gerhard Rogler has received educational grants and research grants from Abbot, Abbvie,
Ardeypharm, Augurix, Calypso, Essex/MSD, FALK, Flamentera, Novartis, Roche, Takeda, Tillots, UCB and Zeller. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
Conceptualization: Florian Kamm, Claudia Kunst, Gerhard Rogler, Andre Franke, Frank
Klebl, Florian Rieders.
Data curation: Ulrike Strauch, Rocio Lopez, Florian Rieders.
Formal analysis: Rocio Lopez.
Investigation: Florian Kamm, Frauke Degenhardt, Claudia Kunst, Gerhard Rogler, Florian
Methodology: Florian Kamm, Ulrike Strauch, Frauke Degenhardt, Frank Klebl, Florian
Project administration: Florian Rieders.
Resources: Gerhard Rogler, Andre Franke.
Supervision: Ulrike Strauch, Rocio Lopez, Florian Rieders.
Visualization: Frauke Degenhardt.
Writing ± original draft: Florian Kamm, Ulrike Strauch, Florian Rieders.
Writing ± review & editing: Florian Kamm, Frauke Degenhardt, Rocio Lopez, Claudia Kunst,
Gerhard Rogler, Andre Franke, Frank Klebl, Florian Rieders.
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