Twin Studies in Inflammatory
Bowel Disease- A Review
G. Järnerot, J. Halfvarson, C. Tysk
Division of Gastroenterology, Dept of Medicine, Örebro University Hospital, S-701 85 Örebro, Sweden
The cause of the inflammatory bowel diseases (IBD),
ulcerative colitis (UC) and Crohn's disease (CD) is still
unknown. Clinically, it is generally accepted that UC and
CD are two distinct diseases, although it is sometimes difficult
to separate the two diagnosis and in such cases the term
indeterminate colitis is often used.1, 2 However, a strong
association exists between UC and CD. Relatives to patients
with CD have an increased risk of UC and vice versa.3
Geographic areas with a high incidence of one of the diseases
usually have a high incidence of the other.4
Whether CD and UC are two separate diseases or the
clinician's blunt classification of a complex of disorders with
multifactorial aetiology remains to be proven. A prevailing
hypothesis is that the intestinal inflammation represents an
inappropriate immune response to the normal gut bacterial
flora in genetically susceptible individuals.5 This abnormal
response might be facilitated by defects in the intestinal
epithelium and mucosal immune response.
Twin studies provide a powerful tool to disentangle the
relative contribution of genetics and environmental
factors. Monozygotic twins (MZ) are genetically identical,
share the intrauterine environment and to a high extent
other, but necessarily not all, external factors during
childhood. Dizygotic twins (DZ) share environment to
the same extent as monozygotic pairs but, on average,
only half of the genes.
CONCORDANCE
In 1988 we published the first study of an unselected group
of twins with IBD, showing a higher pair concordance rate
for MZ twins with CD than for DZ twins, strongly suggesting
that genetics cause this difference.6 In addition, in MZ twins
the concordance rate was higher for CD than for UC,
reflecting a more pronounced genetic component in CD
than in UC. Since then, British7, 8 and a Danish twin study9
have been published. In total, these three studies enrolled
428 twin pairs with known zygosity, and combined data
show a pair concordance rate for CD of 30/76 (39%) and 9/
134 (7%) in MZ and DZ twin pairs, respectively. The
corresponding rates for UC were 18/89 (15%) and 5/129
(4%).
A follow up in the year 2000 of the Swedish twin cohort
Correspondence to: Dr. G. Järnerot, Division of Gastroenterology, Dept of Medicine, Örebro University Hospital, S-701 85 Örebro, Sweden
Austral - Asian Journal of Cancer ISSN-0972-2556, Vol. 6, No.1, January 2007
29
G. Järnerot
published in 19886 showed that in UC the pair concordance
had increased from 6% to 18.8% and in CD from 44.4% to
50%10 However, none of the cases diagnosed between 1988
and 2000 were really new as all had clinical symptoms already
at the first survey, but the evidence for a definite diagnosis
was considered insufficient at that time.
among the total number of 9 MZ twin pairs in which both
twins had CD was 0.000076. Thus, in MZ twins concordant
for CD the genetic impact on disease characteristics seems
to be extremely high. The number of concordant MZ twins
with UC was to small for a similar comparison.
GENETIC MARKERS
Thus, the evidence is still supporting that genetics has a
more pronounced influence in CD than in UC.
CLINICAL CHARACTERISTICS
Comparison of clinical characteristics in MZ twin pairs,
where both twins in each pair are affected, can give an
estimate of the importance of genetics. In the Swedish twin
cohort followed up in the year 200010 only three MZ pairs
were concordant for UC. The location of the disease at
diagnosis was similar in two pairs, but differed slightly in the
third. In 2 of 3 pairs concordance for age at diagnosis was
observed. One twin had a proctocolectomy, but the
remaining five twins had not needed surgery. However, it is
obvious that the number of twin pairs with UC was to small
for conclusions.
Nine pairs of MZ twins were concordant for CD. In 6 pairs,
the diagnosis was made two years or less apart in each twin,
but in three pairs the time interval was considerably longer.
The onset of symptoms showed similar time aspects. The
location of disease at diagnosis according to the Vienna
classification11 was identical in 7 of 9 pairs, but differed
slightly in 2. During the observation period, the extent of
the inflammation in each twin could progress or remain
within the same part of the gastrointestinal tract. In 8 pairs,
the twins showed the same pattern in this respect. The
twins whose extent of inflammation increased also showed
a remarkable similarity in disease progress.
The Vienna classification11 divides disease behaviour into
stricturing, penetrating or non-stricturing non-penetrating.
Six of the 9 pairs were concordant for disease behaviour. In
2 other pairs, one twin had stricturing and the other
penetrating disease. It may be questioned whether
penetrating disease occurs without a certain degree of
stricturing influence. There seemed to be a definite
discordance in disease behaviour in only one pair.
In 7 pairs, the smoking pattern at the time of diagnosis
was identical within each pair. The occurrence or lack
of extraintestinal manifestations was similar in 5 of the 9
pairs.
The P value for observing 7 pairs (or more) with
concordance in at least 3 of the 4 clinical characteristics
Austral - Asian Journal of Cancer ISSN-0972-2556, Vol. 6, No.1, January 2007
In recent years, the understanding of genetics in CD has
made considerable progress. The most important finding
has perhaps been identification of the CARD15/NOD2
gene polymorphisms associated with CD.12, 13 However, there
are big geographic variations in the presence of CARD15/
NOD2 mutations.14, 15 No data had been published from
Sweden at the time of our investigation. Fifty-eight MZ
twins were studied, 38 with CD. CARD15/NOD2
polymorphisms were identified in 3 of 29 twin pairs. Within
each pair, both twins carried one and the same variant. Five
of the 38 (13%) twins with CD carried any of the CARD15/
NOD2 mutations, corresponding to a total allele frequency
for CARD15/NOD2 polymorphisms of 6.6%. One healthy
twin sibling carried a Arg702Trp variant.16
Seven of 9 concordant twin pairs did not carry any of the
three CARD15/NOD2 polymorphisms. In spite of the fact
that the total allele frequency of theses mutations was 4.4
times higher (95.5 CI 1.0 - 21.5) in twins in concordant pairs
than in twins in discordant pairs, 11,1% vs 2.5%, CARD15/
NOD2 polymorphisms do not seem to be of major genetic
importance in Sweden. It is also worth noticing that the
CARD15/NOD2 polymorphism frequency in discordant
MZ pairs with CD is almost identical to the 2.6% in the
healthy Swedish population.
MUCOSAL LEVEL
It has been hypothesized that especially UC might, at least
in part, be due to a defect in the colonic mucus barrier. This
was supported by findings of a selective depletion in a
subgroup of mucus glycoproteins as defined by ion-exchange
chromatography.17 This technique defined six different
species (I-VI) of chromatographically distinguishable
glycoprotein populations in normal colonic mucosa.17 In UC,
a reduction in species IV was found both during active and
inactive disease, which was present both in inflamed and in
uninvolved parts of the colon. Patients with CD, infectious
colitis, ischemic colitis or radiation colitis had a normal
glycoprotein composition, indicating that the finding was
not a non-specific reflection of inflammatory activity.18
However, it remained unclear whether these alterations
precede onset of inflammation and thus, might be
genetically determined or were a subtle biochemical residue
of past activity.
30
Twin Studies in Inflammatory Bowel Disease- A Review
A study of rectal biopsies from identical UC and CD twins
showed a significant reduction of glycoprotein species IV
both in twins affected by UC and in their unaffected twin
siblings in comparison to normal controls.19 Patients with
UC had an increase in species V as described in patients in
remission.17 Their healthy twin partners also showed the
same findings. Twins with CD and their healthy twin
partners had a similar glycoprotein composition as the control
subjects.19
The technique used in this study has been criticized as it
depended on ion-exchange chromatography performed
with a discontinuous salt gradient to separate mucin fractions
by charge. Mucus glycoproteins are notoriously difficult to
purify free from contaminating glycoproteins and
proteoglycans and it is possible that the depletion of mucin
fraction IV seen in UC twins in fact represents overall mucus
depletion. However, the same finding was made in healthy
twins and this would be compatible with subclinical
inflammation in the unaffected twins since the goblet cell
depletion that occurs in UC is probably driven by
inflammation.
Rectal biopsies from the same set of twins have later been
investigated in another study assessing the mucosal
expression of the oncofetal Thomsen-Friedenreich (TF)
carbohydrate antigen (galactose β 1,3 N acetyl
galactosamine α).20 Furthermore, immunohistochemistry
for activated NF kappa B was performed.
Positive staining for TF-antigen was an invariable finding
amongst all the 22 affected MZ twins with IBD, irrespective
of whether the patient had UC or CD. Fifteen of the 16
unaffected twin siblings of IBD patients showed a similarly
high prevalence of positivity and they also had a significantly
higher staining score than healthy control subjects. Only 5/
29 of healthy controls had positive TF-antigen staining. In
twins with IBD and their unaffected twin siblings staining
was generally localised to the supranuclear region plus a
variable degree of staining of the glycocalyx and of surface
secretory material. In healthy controls staining was
exclusively confined to the supranuclear cytoplasmic
region.21
Immunohistochemistry for activated NF kappa B showed
evidence of activation in the surface epithelium in 18 of 22
affected IBD twins and in 13 of 14 unaffected IBD twins.
Some activated NF kappa B was also seen in some
histologically normal controls but less frequently (7 of 22)
and at a significantly lower intensity.21
Thus, in this study the same colonic mucosal glycosylation
abnormality was found in both MZ twin with IBD,
Austral - Asian Journal of Cancer ISSN-0972-2556, Vol. 6, No.1, January 2007
irrespective of if they had UC or CD, and their unaffected
twin sibling. It was predominantly seen in the surface
epithelium whereas genetic changes in glycosalation would
be expected to originate from the stem cell compartment
and therefore to affect whole crypts.
Rectal biopsies from the unaffected twins had been
evaluated over and over again without showing evidence
of inflammation within the crypt or surface epithelium or
the lamina propria. In spite of that activated NF kappa B
could be identified in all but one of the unaffected IBD
identical twins, which was largely confined to the surface,
implying a result of interaction between the surface
epithelium and some intra-luminal component (s) such as
bacteria or bacterial products22 or even immunoglobulin.23
Thus, at present the conclusion must be that the mucin
alterations seen in these MZ twins are acquired by a
subclinical inflammation and not genetic. One may wonder
why the unaffected twins have not developed disease. To
explain that one would have to postulate that some
additional trigger would be needed to induce clinical disease
or that these twins have developed some kind of defence
system which the affected twins lack.
The finding of NF kappa B activation both in diseased
twins with IBD and their unaffected twin siblings may also
explain the findings in an earlier twin study24 which found
an increased per centage of Ig G1 producing cells and a
decrease of IgG2 producing cells in IBD. This finding was
most consistent in UC, but the same trend was seen also in
CD.
PERMEABILITY
CD is associated with increased permeability,25, 26 but it is
unclear whether this is a primary or a secondary event.27, 28
An early finding of disrupted intestinal barrier in relatives
of patients with CD29 led to the hypothesis of a genetic
permeability disorder of aetiological importance. However,
this finding could not be reproduced.30, 31 We tried to
evaluate permeability in MZ twins with CD and their
unaffected twin siblings,32 without finding any differences.
A subsequent study using provocation with acetylsalicylic
acid did not show an augmented response in healthy twins,
but in twins affected by CD an abnormal response was seen
in 28%, which differed significantly from healthy controls.33
However, so far twin studies have not been of decisive help
when it comes to permeability.
SEROLOGY
Food antigens have long been implicated in the aetiology
of IBD. Higher serum antibody titres to Saccharomyces
31
G. Järnerot
cerevisiae (baker's yeast) in patients with CD than in UC or
healthy controls evoked much interest.34 A serologic twin
study of antibodies (IgA, IgG, IgM) to yeast cell mannan, a
whole yeast (Saccharomyces cerevisiae) preparation,
betalactoglobulin, gliadin and ovalbumin was performed.35
Two striking observations were made: first individuals with
UC were indistinguishable from healthy twins and controls
except for the response to gliadin. Both healthy and diseased
twins with UC had higher IgA levels to gliadin than controls.
Second twins who had developed CD displayed higher
antibody titres towards yeast cell mannan in particular, but
also to whole yeast (Saccharomyces cerevisiae), of all
antibody types (IgA, IgG, IgM) This suggested that mannan,
or some antigen rich in mannose and cross reacting with
mannan, may play an aetiological role in CD, but not in
UC. This was in fact the first study which showed that cell
wall mannan was the main antigen producing the ASCA
antibody.
Anti-Saccharomyces cerevisiae antibodies (ASCA) are
a serological marker of CD, especially associated with young
age at onset,36, 37 ileal disease36, 38, 39 and stricturing as well as
penetrating disease behaviour.37-39 Several studies suggested
that ASCA reflects the load of occurrence of CD in
families.40-43 Whether ASCA is a familial trait due to a
genetic factor or to increased exposure to an environmental
factor is unknown. Ninety-eight twin pairs were
investigated. ASCA were found in 57% of twins with CD,
12% of twins with UC, 17% of healthy twin siblings to twins
with CD and 14% of healthy twin siblings to twins with UC.
Mean ASCA titres in these four groups were 15.8, 4.2, 5.4,
and 4.3 U/ml, respectively. There was no increased
occurrence of ASCA in healthy twin siblings in discordant
MZ twin pairs (5%) with CD compared with DZ twins
(26%).44 These findings contradicts the hypothesis that
ASCA may be a genetic susceptibility marker for CD and
the results rather suggest that ASCA in healthy family
members is a marker of shared environment.
In concordant MZ twin pais with CD a high degree of
similarity in the level of ASCA titres was observered within
each pair. This suggests that ASCA is associated with CD
and a marker of response to an environmental antigen and
that a specific gene(s) other than CARD15/NOD2
determines the level of response.44
concordant or discordant for UC could not support this
hypothesis .46
CONCLUSION
As shown in this review twin studies are of value in
evaluation of the genetic vs the environmental factors in
the aetiology of IBD. The drawback is the relatively small
number of twins with IBD, which underlines the importance
of collaboration between countries which have national twin
registries in order to increase numbers.
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